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                        STUDIES ON FERMENTATION




                        STUDIES ON FERMENTATION

                          THE DISEASES OF BEER

             THEIR CAUSES, AND THE MEANS OF PREVENTING THEM


                                   BY

                             LOUIS PASTEUR

    MEMBER OF INSTITUTE OF FRANCE, THE ROYAL SOCIETY OF LONDON, ETC.


          _A TRANSLATION, MADE WITH THE AUTHOR’S SANCTION, OF
         “ÉTUDES SUR LA BIÈRE,” WITH NOTES, INDEX, AND ORIGINAL
                             ILLUSTRATIONS_


                                   BY

                             FRANK FAULKNER

                  AUTHOR OF “THE ART OF BREWING,” ETC.

                                  AND

                        D. CONSTABLE ROBB, B.A.

                LATE SCHOLAR OF WORCESTER COLL., OXFORD


                                 London

                             MACMILLAN & CO

                                  1879




PARDON AND SONS, PRINTERS, PATERNOSTER ROW, LONDON.




                                   TO

                             THE MEMORY OF

                               MY FATHER,

               FORMERLY A SOLDIER UNDER THE FIRST EMPIRE,

                                  AND

                    KNIGHT OF THE LEGION OF HONOUR.



     THE LONGER I LIVE, THE BETTER DO I UNDERSTAND THE KINDNESS OF
               THY HEART AND THE EXCELLENCE OF THY MIND.

       TO THY EXAMPLE AND COUNSELS DO I OWE THE EFFORTS THAT HAVE
       BEEN DEVOTED TO THESE STUDIES, AS WELL AS TO ALL THE WORK
        I HAVE EVER DONE. AND NOW, HOW CAN I BETTER HONOUR THESE
     FILIAL REMEMBRANCES THAN BY DEDICATING MY BOOK TO THY MEMORY?

                              L. PASTEUR.




                           AUTHOR’S PREFACE.


Our misfortunes inspired me with the idea of these researches. I
undertook them immediately after the war of 1870, and have since
continued them without interruption, with the determination of
perfecting them, and thereby benefiting a branch of industry wherein we
are undoubtedly surpassed by Germany.

I am convinced that I have found a precise, practical solution of the
arduous problem which I proposed to myself—that of a process of
manufacture, independent of season and locality, which should obviate
the necessity of having recourse to the costly methods of cooling
employed in existing processes, and at the same time secure the
preservation of its products for any length of time.

These new studies are based on the same principles which guided me in my
researches on wine, vinegar, and the silkworm disease—principles, the
applications of which are practically unlimited. The etiology of
contagious diseases may, perhaps, receive from them an unexpected light.

I need not hazard any prediction concerning the advantages likely to
accrue to the brewing industry from the adoption of such a process of
brewing as my study of the subject has enabled me to devise, and from an
application of the novel facts upon which this process is founded. Time
is the best appraiser of scientific work, and I am not unaware that an
industrial discovery rarely produces all its fruit in the hands of its
first inventor.

I began my researches at Clermont-Ferrand, in the laboratory, and with
the help, of my friend M. Duclaux, professor of chemistry at the Faculty
of Sciences of that town. I continued them in Paris, and afterwards at
the great brewery of Tourtel Brothers, of Tantonville, which is admitted
to be the first in France. I heartily thank these gentlemen for their
extreme kindness. I owe also a public tribute of gratitude to M. Kuhn, a
clever brewer of Chamalières, near Clermont-Ferrand, as well as to M.
Velten, of Marseilles, and to MM. de Tassigny, of Reims, who have placed
at my disposal their establishments and their products, with the most
praiseworthy willingness.

L. PASTEUR.

PARIS, _June 1, 1876_.




                      PREFACE TO ENGLISH EDITION.


My first idea of placing before English brewers a translation of “Études
sur la Bière” was meagre in the extreme, compared with the final
realization of it as it appears in the following pages.

Seeing the vast importance of Pasteur’s work from a practical point of
view, after writing a review of it for the _Brewers’ Journal_, I
determined to procure, at any rate for the use of my pupils, a literal
translation, illustrated by photo-lithographic copies of the original
plates, the thankless task of executing this preliminary translation for
so limited a number of readers being most kindly and generously carried
out for me by my friend Mr. Frank U. Waite, who, being engaged with me
at the time in practical brewing operations, shared my views as to the
value of the original work.

It was on the completion of this translation that my views and desires
expanded. The more I studied the work, the more I was convinced of its
immense value to the brewer as affording him an intelligent knowledge of
the processes and materials with which he deals, but over and above all
this, it was impossible not to feel that the researches of such a
devoted and accomplished _savant_ as Pasteur, possessed a scientific
interest much wider than their mere relation to the art of brewing would
imply. As the work of a skilful chemist and a laborious and accurate
observer, such a protracted and careful study of the lowest and simplest
forms of life, must necessarily be of first importance to the
biologist—to the beginner as an admirable introduction to the study of
practical physiology in general, as well as to the more advanced
student, from the suggestive light which it throws on the nature of
analogous phenomena in more complex organisms.

I determined accordingly _to publish_ the work if I could secure the
consent of its distinguished author, but at the same time I felt that
the publication of M. Pasteur’s “Studies” in the form in which Mr. Waite
had, at my request, translated it, and illustrated only with inferior
copies of the original plates, would not be either advisable or just;
but that I was bound rather to put the book before the English public in
as satisfactory and complete a form as lay within my power. Under these
circumstances I was induced to seek the aid of Mr. D. Constable Robb,
B.A., of The Oxford University Museum, who, in taking Mr. Waite’s
version as a basis, has so elaborated, annotated, and recast it, that I
feel bound to say that much of the value of “Studies on Fermentation,”
as it now appears, is due to the care that Mr. Robb has bestowed upon
the revision that he so kindly undertook; a revision the result of which
has created a feeling of confidence in the success of the translation as
it now stands, which I could not have had in any mere literal version.

To the practical worker the original illustrations alone, which appear
in this version, cannot but be of immense value in the microscopical
study of the changes in the liquids with which he deals; whilst the many
notes and additions, which are a feature peculiar to the English
edition, more particularly the rendering into the equivalents, with
which, unfortunately, practical men on this side of the channel are
still most at home, of the metric weights and measures and centigrade
temperatures, as well as the Index which Mr. Robb has compiled, will, I
trust, render the book of still greater service than it otherwise would
have been to many of those who may favour it with their attention.

The debt which we English brewers owe to M. Pasteur can hardly be
over-estimated, and I must be allowed here to express my personal
obligations to that distinguished worker for the permission which allows
this translation; and to the French publishers for their help with
regard to the interleaved illustrations.

The author’s preface and dedication are, of course, reproduced, the
former making it unnecessary for me to refer more in detail to the
contents of the translation.

FRANK FAULKNER.

THE BREWERY, ST. HELEN’S, LANCASHIRE,
_September, 1879_.




                           TABLE OF CONTENTS.


AUTHOR’S DEDICATION v

AUTHOR'S PREFACE vii

TRANSLATOR’S PREFACE ix


CHAPTER I.

ON THE INTIMATE RELATION EXISTING BETWEEN THE DETERIORATION OF BEER, OR
THE WORT FROM WHICH IT IS MADE, AND THE PROCESS OF BREWING 1


CHAPTER II.

ON THE CAUSES OF THE DISEASES WHICH AFFECT BEER AND WORT.

§ I. Every unhealthy change in the quality of beer coincides with a
development of microscopic germs which are alien to the pure ferment of
beer 19

§ II. The absence of change in wort and beer coincides with the absence
of foreign organisms 25


CHAPTER III.

ON THE ORIGIN OF FERMENTS PROPERLY SO CALLED.

§ I. On the conditions which cause variations in the nature of the
organized products existing in infusions 33

§ II. Experiments on blood and urine taken in their normal state, and
exposed to contact with air that has been deprived of the particles of
dust which it generally holds in suspension 40

§ III. Experiments on the juice contained in grapes 54

§ IV. Wort and must exposed to common air 59

§ V. New comparative studies on the germs held in suspension by the air
of different places which are near each other, but subjected to
different conditions affecting the production and diffusion of the
particles of dust found in them 72

§ VI. Yeast may become dry and be reduced to dust without losing its
faculty of reproduction 81


CHAPTER IV.

THE GROWTH OF DIFFERENT ORGANISMS IN A STATE OF PURITY: THEIR AUTONOMY.

§ I. Growth of _Penicillium glaucum_ and _Aspergillus glaucus_ in a
state of purity.—Proofs that these fungoid growths do not become
transformed into the alcoholic ferments of beer or wine.—Preliminary
inquiry into the cause of fermentation 86

§ II. Growth of _Mycoderma vini_ in a state of purity.—Confirmation of
our original conjectures as to the cause of fermentation.—_Mycoderma
vini_ does not change into yeast, although it may give rise to
fermentation 108

§ III. Growth of _Mycoderma aceti_ in a state of purity 121

§ IV. Growth of _Mucor racemosus_ in a state of purity.—Example of life
more active and lasting when removed from the influence of air 127


CHAPTER V.

THE ALCOHOLIC FERMENTS.

§ I. On the origin of ferment 143

§ II. On “spontaneous” ferment 182

§ III. On “high” and “low” ferments 186

§ IV. On the existence and production of other species of ferment 196

§ V. On a new race of alcoholic ferments: Aërobian ferments 205

§ VI. The purification of commercial yeasts 219


CHAPTER VI.

THE PHYSIOLOGICAL THEORY OF FERMENTATION.

§ I. On the relations existing between oxygen and yeast 235

§ II. Fermentation in saccharine fruits immersed in carbonic acid gas
266

§ III. Reply to certain critical observations of the German naturalists,
Oscar Brefeld and Moritz Traube 279

§ IV. Fermentation of dextro-tartrate of lime 284

§ V. Another example of life without air.—Fermentation of lactate of
lime 292

§ VI. Reply to the critical observations of Liebig, published in 1870
316


CHAPTER VII.

NEW PROCESS FOR THE MANUFACTURE OF BEER.

§ I. Preliminary experiments 338

§ II. Method of estimating the oxygen held in solution in wort 353

§ III. On the quantity of oxygen existing in a state of solution in
brewers’ worts 364

§ IV. On the combination of oxygen with wort 371

§ V. On the influence of oxygen in combination on the clarification of
wort 381

§ VI. Application of the principles of the new process of brewing with
the use of limited quantities of air 387


APPENDIX 396

INDEX 403




                            INDEX TO PLATES.


I. Principal disease-ferments met with in wort and beer 6

II. Appearance under the microscope of the deposit from “turned” beer 20

III. _Torulæ_ in process of development 72

IV. _Mycoderma vini_ functioning as an alcoholic ferment.—Right half,
showing appearance of spores just sown; left half, their appearance
after an interval of submerged life 116

V. _Mucor_, vegetating submerged, in deficit of air 136

VI. Ferment of _mucor_ 138

VII. Yeast-cells—worn out and dissociated (left), after revival in a
sweet wort (right) 148

VIII. Fertile mould-cells from the outer surface of grapes 152

IX. Various examples of the mode of growth of mould-cells from the outer
surface of grapes 154

X. One of the ferments of acid fruits at the commencement of
fermentation in its natural _medium_ 166

XI. _Saccharomyces pastorianus_, in course of regular growth 168

XII. Ferment-cells from a spontaneous fermentation just starting 184




                               CHAPTER I.
On the Intimate Relation existing between the Deterioration of Beer, or
      the Wort from which it is Made, and the Process of Brewing.


At the outset of these “Studies,” let us briefly consider the nature of
beer and the methods of its manufacture.

Beer is a beverage which has been known from the earliest times. It may
be described as an infusion of germinated barley and hops, which has
been caused to ferment after having been cooled, and which, by means of
“settling” and racking, has ultimately been brought to a high state of
clarification. It is an alcoholic beverage, vegetable in its origin—a
_barley wine_, as it is sometimes rightly termed.[1]

Beer and wine, however, differ widely in their composition. Beer is less
acid and less alcoholic than wine; it holds more ingredients in
solution, and the nature of these ingredients is by no means similar to
that of those which are found in wine.

These differences in the component parts of wine and beer give rise to
corresponding differences in the keeping qualities of the two liquids.
The small amount of acidity in beer, its poverty in alcohol, and the
presence of matter that is saccharine, or liable to become so, all
operate in imparting to beer a tendency to change, which wine does not
possess. That this unequal resistance to the aggression of diseases is
due to such differences, may be proved by the fact that wine could be
made much more liable to change than it actually is, by a diminution of
its acidity and its usual proportion of alcohol, or by increasing the
proportion of viscid or saccharine matters,[2] modifications which would
tend to assimilate its composition to that of beer.

We have remarked elsewhere that the pains devoted to the rearing of
vines, and to the ordinary operations of vinification, such as
_ouillage_,[3] sulphuring, and repeated rackings, as well as the use of
cellars and vessels hermetically closed, are entailed by the necessity
of counteracting and preventing the diseases to which wine is liable.
The same may be said, _a fortiori_, of beer, inasmuch as it is more
liable to change than wine. Manufacturers and retailers of this beverage
have to strive constantly with the difficulty of preserving it, or the
wort used in its manufacture. We may readily be convinced of this by
reviewing the usual processes of the art of brewing.

When the infusion of malt and hops, which is termed wort, is completed,
it is left to cool. It is next put into one or more casks or vats, in
which it is made to undergo alcoholic fermentation—the most important of
all the processes in brewing.

The cooling must be as rapid as possible. This is a condition of
success; otherwise, the wort may deteriorate, which will necessarily
lead to deterioration in the quality of the beer. As long as the wort is
at a high temperature it will remain sound; when under 70° C. (158° F.),
and particularly when at a temperature of from 25° C. to 35° C. (77° F.
to 95° F), it will be quickly invaded by lactic and butyric ferments.
Rapidity in cooling is so essential that to secure it recourse is had to
special apparatus.[4] Even in the preparation of wort, especially when
it is effected by successive mashings, in summer, deterioration is
imminent: in fact, it is not rare to see the wort becoming acid during
the _mashes_, if these are not accomplished with all possible celerity.

After the wort has been cooled, it is mixed with yeast. This is obtained
from a previous fermentation, and, after being thoroughly pressed, is
added at the rate of from one to two thousandth parts of the weight of
the wort, that is, from 100 to 200 grammes per hectolitre (about 4 oz.
to 8 oz. average for every 25 gallons). At first sight, this yeast seems
free from the possible diseases of the wort and beer; but this is by no
means the case.

Now, why do we add yeast to our wort? This practice is unknown in the
art of vinification. The must is always left to spontaneous
fermentation. Why should we not leave the wort to operate in the same
manner?

It would be a mistake to suppose that in the brewing of beer yeast is
added with the sole object of accelerating fermentation, and making it
more rapid. Rapidity in fermentation is a very questionable advantage,
and one which is not desired by brewers, who rather agree in
pronouncing it injurious to the quality of beer. It is in the easy
deterioration of the wort, or what is tantamount to it, in the
facility it affords to various spontaneous fermentations, that we find
an answer to these questions. The must, through its acidity, due to
the presence of bitartrate of potash—which seems to promote alcoholic
fermentation—through its proportion of sugar, and perhaps in
consequence of some other peculiarity of its composition, always
undergoes regular alcoholic fermentation. The diseases of wine, at the
commencement of its manufacture, show themselves, so to say, in a
latent state only. Therefore a vintage can be left, without
inconvenience, to spontaneous fermentation.

With wort the case is quite different. Under certain accidental
circumstances it is possible that alcoholic fermentation alone may take
place in a wort left to ferment spontaneously, and the quality of the
beer remain unimpaired, but such an event would be exceptional, and of
very rare occurrence. In most cases we should obtain an acid or putrid
liquid resulting from the production and multiplication of alien
ferments.

The addition of yeast is made in consequence of the necessity of
exciting through the whole bulk of wort, as soon as it is cold, a single
fermentation—viz., the alcoholic, the only one that can produce beer
properly so called.

The alcoholic ferments concerned in the production of beer will be found
represented in several of the engravings in this work. Other ferments we
may term “diseased”; these include all those that may occur
spontaneously—that is, whose germs have not been directly and
intentionally introduced—amongst the actual alcoholic ferments.

The expression, “diseased ferments,” is justified by the circumstance
that the propagation of these ferments is always accompanied by the
production of substances which are acid, putrid, viscous, bitter, or
otherwise unpalatable, a consideration of commercial rather than
scientific importance. From a physiological point of view, all these
ferments are of equal interest and importance. The botanist, as a man of
science, in contemplating nature, must give equal attention to all
plants, whether useful or noxious, since they are all governed by the
same natural laws, among which no order of merit could be established.
The exigencies of industry and health require, however, wide
distinctions.

[Illustration: Plate 1. Principal Disease-ferments met with in Wort and
Beer.]

The first engraving (Plate I.) represents the different diseased
ferments, together with some cells of alcoholic yeast, to show the
relative size of these organisms.

No. 1 of the engraving represents the ferments of _turned_ beer, as it
is called. These are filaments, simple or articulated into chains of
different size, and having a diameter of about the thousandth part of a
millimetre (about 1/25000 inch). Under a very high power they are seen
to be composed of many series of shorter filaments, immovable in their
articulations, which are scarcely visible.

In No. 2 are given the lactic ferments of wort and beer. These are
small, fine and contracted in their middle. They are generally detached,
but sometimes occur in chains of two or three. Their diameter is a
little greater than that of No. 1.

In No. 3 are given the ferments of putrid wort and beer. These are
mobile filaments whose movements are more or less rapid, according to
the temperature. Their diameter varies, but is for the most part greater
than that of the filaments of Nos. 1 and 2. They generally appear at the
commencement of fermentation, when it is slow, and are almost invariably
the result of very defective working.

In No. 4 are given the ferments of viscous wort, and those of ropy beer,
which the French call _filante_. They form chaplets of nearly spherical
grains. These ferments rarely occur in wort, and still less frequently
in beer.

No. 5 represents the ferments of pungent, sour beer, which possesses an
acetic odour. These ferments occur in the shape of chaplets, and consist
of the _mycoderma aceti_, which bears a close resemblance to lactic
ferments (No. 2), especially in the early stages of development. Their
physiological functions are widely different, in spite of this
similarity.

The ferments given in No. 7 characterize beer of a peculiar acidity,
which reminds one more or less of unripe, acid fruit, with an odour _sui
generis_. These ferments occur in the form of grains which resemble
little spherical points, placed two together, or forming squares. They
are generally found with the filaments of No. 1, and are more to be
feared than the latter, which cause no very great deterioration in the
quality of beer, when alone. When No. 7 is present, by itself or with
No. 1, the beer acquires a sour taste and smell that render it
detestable. We have met with this ferment existing in beer,
unaccompanied by other ferments, and have been convinced of its fatal
effects.

No. 6 represents one of the deposits belonging to wort. This must not be
confounded with the deposits of diseased ferments. The latter are always
visibly organized, whilst the former is shapeless, although it would not
always be easy to decide between the two characters, if several samples
of both descriptions were not present. This shapeless deposit interferes
with wort during its cooling. It is generally absent from beer, because
it remains in the backs, or on the coolers; or it may get entangled in
the yeast during fermentation and disappear with it.

Among the shapeless granulations of No. 6 may be discerned little
spheres of different sizes and perfect regularity. These are balls of
resinous and colouring matter that are frequently found in old beer, at
the bottom of bottles or casks; sometimes they occur in wort preserved
after Appert’s method. They resemble organized products, but are nothing
of the kind. We have remarked before, in “Studies on Wine,” that the
colouring matter of wine would settle, in course of time, in that form.

It is evident that the different ferments delineated in Plate I. are
worthy of thorough study, in consequence of the fermentations to which
they may give rise. Care must be taken to isolate the action of each of
them in fermentations which we may call pure—a condition of some
difficulty, but one that may be carried out by an adoption of the
methods explained in this work.

All these diseased ferments have a common origin. Their germs,
infinitesimal and hardly perceptible as they are, even with the aid of
the microscope, form a part of the dust conveyed through the air. This
dust the air is continually taking from or depositing upon all objects
in nature, so that the dust that clings to the ingredients from which
our beer is manufactured, may teem with the germs of diseased ferments.

During the process of fermentation, the occult power of diseased
ferments, although it may escape the observation of the brewer, is
manifested in a high degree.

During the last thirty years, or so, the art of brewing has undergone a
radical change, at least in Europe. This change has been effected by a
partial abandonment of the process of fermentation formerly used. Thirty
years ago only one kind of beer was known; there are nowadays two
distinct kinds—beer fermented at a high temperature, and beer fermented
at a low temperature. Each of these is subdivided into many varieties,
to which different names are given, according to their strength or
colour. This is the case in England, where we find porter, ale, pale
ale, stout, bitter beer, and other varieties of beer, although, as a
matter of fact, the English have but one kind of beer, all the English
beers being fermented at a high temperature.

Let us briefly examine the differences existing between the two kinds of
beer.

Formerly all beer was fermented at a high temperature. The wort, after
having been cooled in the backs, was run into a large vat, at a
temperature of about 20° C. (68° F.). Yeast was then added to it, and
when the fermentation began to show itself, in the formation of a light,
white froth, upon the surface of the liquid, the wort was run into
casks, having a capacity of from 50 to 100 litres (11 to 22 gallons)—75
litres being the commonest size. These casks were placed in cellars,
having a temperature of from 18° C. to 20° C. (64° F. to 68° F.). The
activity of fermentation soon produced a froth that grew thicker and
more and more viscous in proportion to the quantity of yeast it
contained. The yeast worked out of the bung-holes and dropped into a
vessel placed under the casks; there it was gathered for subsequent
operations. It always exceeded the quantity used in the first instance,
the ferment increasing greatly during the process of fermentation. The
increase in its weight varied with the weight of yeast used and the
composition of the wort. Under the ordinary conditions of brewing, where
the weight of the pitching yeast was about one thousandth part of the
weight of the wort, the increase is said to have been from five to seven
times the weight of the yeast; but such increase must naturally have
been determined by the quality of the wort, the quantity of hops used,
the action of oxygen, and the proportion of barm employed. The process
of fermentation lasted from three to four days. By that time the beer
was finished, and had become limpid, the fermentation having been
completed. The bungs could then be placed in the casks, and the beer be
delivered to the customer.[5] A certain amount of yeast still remained
in the casks, and caused the beer to become thick, in transit; but a few
days’ rest sufficed to restore its brilliancy, and render it fit for
drinking or bottling.

Here we have an explanation of the term “_high_ fermentation,” which has
been applied to the foregoing process. This process is conducted at a
high temperature, which, commencing at 19° C. or 20° C. (66° F. or 68°
F.), is raised to 20° C. or 21° C. (68° or 70° F.) by the action of
fermentation, which is always accompanied by an increase of heat.[6]

This is not, however, the only reason for the use of the term “_high_
fermentation.” We have just seen that the fermenting casks were so
arranged that most of the yeast produced during the process of
fermentation would rise to the upper part of the casks and work out of
the bung-holes. In this practical fact we have the actual origin of the
expressions “_high fermentation_” and “_high beer_,” which are used to
distinguish this peculiar fermentation and the quality of beer derived
from it.

As we have already observed, all beer was formerly produced by this mode
of fermentation, which even at the present time is still practised in
the breweries of Great Britain, where beer fermented at a low
temperature is absolutely unknown.

“Low fermentation” is a slow process, effected at a low temperature,
during which the yeast sinks to the bottom of the vats or casks. The
wort, after cooling, is run into open wooden vats. In cooling, the wort
is brought to as low a temperature as 8° C. (47° F.), or even 6° C. (43°
F.), at which point it is maintained by cones or cylinders (styled
_nageurs_, i.e., floats, by the French) floating in the fermenting vats.
These floats may be filled with ice if the outside temperature requires
it, as is invariably the case in summer.

The duration of this fermentation is ten, fifteen, or even twenty days.
The yeast, which is produced less abundantly than in the case of beer
fermented at a high temperature, is gathered after the beer has been
drawn off, and is partly used in subsequent fermentations.

The term “_low_ fermentation” is derived partly from the lowness of the
temperature during the fermenting process, and partly from the fact that
the yeast is gathered at the bottom, and not at the upper part of the
fermenting vessels.[7]

Beer fermented at a low temperature, of which there are several
varieties, differing in colour and quality, is of Bavarian origin.[8]
The preference of the public for this kind of beer, and the increased
facilities that such a beer affords the trade, are the two reasons why
its manufacture has so greatly increased. In Austria, Bavaria, Prussia,
and other Continental countries this new method of brewing is almost
exclusively adopted.

In the _Moniteur de la Brasserie_ of the 23rd April, 1871, may be found
the following significant remarks on the increase in the production of
beer fermented at a low temperature on the Continent: “The number of
breweries manufacturing _high_ beer is rapidly decreasing, whilst the
number of those producing _low_ beer is still more rapidly increasing.
There were in Bohemia, in 1860, 281 breweries in which high fermentation
was practised; in 1865, only 81 of these remained; in 1870, the number
had declined to 18. On the other hand, the number of breweries
practising _low_ fermentation increased from 135 in 1860 to 459 in 1865,
and in 1870 had risen to 831. In 1860 there were 620 breweries in which
the two methods were employed; in 1865 there were 486; in 1870 only 119
remained. The number of breweries at present existing in Bohemia amounts
to 968.”

In France, we are still in a period of transition; but year by year the
manufacture of “low beer” is increasing, to the evident detriment of its
competitor.

It is unnecessary to dwell upon certain differences existing between the
two kinds of beer, such as may be traced to the preparation and
composition of their respective worts. The brewing of “high beer,” by
hand or machinery, is effected in one operation; the brewing of “low
beer” is accomplished by successive mashings, the temperatures of which
are gradually raised. These differences, and others that result from the
longer boiling of the wort, in the “high fermentation” process, give
rise to diversities in the composition and colour of the worts, from
which circumstance “low beers” are sometimes termed _white beers_, in
contradistinction to the others, which have a deeper colour, and are
known as _dark beers_ (_bruns_). The name of Strasburg is generally
given to “low beer” in France, but sometimes it is called German beer.

It is easy to account for the changes introduced into the construction
and working of breweries by the new process of “low fermentation.” A low
temperature is essential not only to the manufacture but also to the
preservation of “low beer,” and must be secured by the use of
ice-cellars in which the temperature may be maintained at 5° R. or 6° R.
(43° F. or 45° F.), and even at 1°, 2°, or 3° R. (35° to 39° F.)
throughout the year. This necessitates an accumulation of ice and the
construction of cellars of enormous extent, for the storage of the beer.
“Low beer” is essentially a stock beer, especially if brewed in winter,
when due advantage is taken of the low temperature of the season. It is
kept in cold cellars until the spring or summer, when beer is consumed
in larger quantities. It is calculated that 100 kilos. (1·96 cwt.) of
ice is the average quantity used per hectolitre (22 gallons) of good
beer, between the cooling of the wort and the day of sale.[9]

In the manufacture of “high beer” we find none of these complications,
nor have we in that manufacture any similar difficulty of working or
expense of construction to contend against. The whole process of
brewing, including the delivery of the beer, does not take more than
eight days. Why should a mode of brewing so simple, so rapid, and
comparatively so inexpensive, have been abandoned by the greater part of
Europe in favour of a system disadvantageous to the brewer in so many
respects? It would be a mistake to suppose that the sole reason for such
a change might be found in the superior quality of “low beer.” That such
a superiority does exist is admitted as a fact by the majority of beer
drinkers; but taken by itself, this fact is not sufficient to account
for the radical transformation that has taken place in the manufacture
of beer, as is proved by the example of England, which, we believe, does
not possess as yet one single “low beer” brewery, from which
circumstance we may fairly suppose that the English have a decided
preference for “high beer.”

The principal advantage of working at a low temperature lies in the fact
that “low beer” is less liable to deterioration, and is less prone to
contract diseases than “high beer,” especially whilst it remains in the
brewery—a circumstance that places the brewer in a position vastly
superior to that which he occupied in former times. With the help of ice
the brewer can manufacture beer during winter and the early part of
spring, for consumption in summer.[10] “High beer,” on the other hand,
must be consumed within a short time of its production. The brewer is
thus compelled to manufacture it as it is wanted, and as orders are sent
in, the demand for it being in a great measure dependent upon the state
of the weather.

Conditions so unfavourable as these must necessarily operate
prejudicially against trade. Industry requires more stability and
uniformity, both in the production and the sale of its goods. “Low beer”
can be brewed in large quantities at any time to be delivered at any
other time, according to requirements; its manufacture, therefore, is
unattended by the inconveniences which we have just noticed.[11]

How is it that the use of ice and yeast operating at a low temperature
so greatly facilitates the preservation of our beer and enables us to
secure such striking advantages? The explanation is simple: the diseased
ferments, which we have pointed out, rarely appear at a lower
temperature than 10° C. (50° F.), and at that temperature their germs
cease to be active. The adoption of low temperatures by brewers is
mainly due to this physiological fact. On one occasion only have we met
with active _vibrios_ (No. 3, Plate I.), at a very low temperature;
these were forming with great difficulty in wort fermenting at 5° C.
(41° F.).

From this we see that the changes which the manufacture of beer has
undergone during the present century have been based mainly on the
diseases to which beer is liable, either during or after the process of
brewing. The fact that English brewers have not as yet adopted “low
fermentation” may be accounted for, in a great measure, by the
difficulty of enlarging existing breweries, in cities like London, to
the extent required for the new method of manufacture. Even in the event
of public taste demanding a “low beer,” English brewers will hesitate a
long time before converting their breweries. Such conversion would
impose upon them expenses and difficulties of a very serious nature. If
ever such a change should take place, it will probably be inaugurated
out of London. It is, however, worthy of remark that English brewers,
without adopting “low fermentation,” have introduced considerable
improvements in brewing, especially in the management of the temperature
during fermentation; this must be preserved within narrow and exact
limits, for fear of injury to the product. It might easily be shown that
these improvements have resulted from the liability of the beer to
contract diseases, although this fact may not have been recognized by
the brewers who have introduced them.

Besides the yeasts which belong to the two principal kinds of
fermentation, there exist many varieties of alcoholic ferment that
produce, each of them, a special kind of beer. Among these special beers
some are deficient in taste, others in aroma, others in brilliancy. Let
us suppose that in the manufacture of a beer with one of these yeasts,
from which a peculiar flavour is derived, a different and inferior
variety is accidentally mixed with that which we intend to use; in such
a case, the inferior variety, the product of which will possess an
inferior quality, will exercise such an influence on the brewing as to
induce the belief that disease must be present. The microscope, if
consulted, will reveal no special organism, nor any of those diseased
ferments of which we have given specimens. It is in the study of yeast
that we must endeavour to find the cause of the results we observe. This
point, which is of the greatest importance to brewers, will become
clearer as we proceed.

If we examine the practices of the beer trade, in its retail as well as
in its export branches, we shall find that many of them afford evidence
of the liability of the beer to deteriorate. We may cite some of these.
When taken out of the ice-cellars, the beer is kept in casks of small
capacity, that it may be the sooner consumed; when exposed to a high
temperature, the beer will not keep sound for any length of time, but
will speedily effloresce with _mycoderma vini_ or _mycoderma aceti_.

Beer which is intended for bottling should not be kept for more than a
month or six weeks. Even in bottling we may perceive the tendency of the
liquid to deteriorate.[12] It is necessary that the bottles, immediately
after being filled, should be laid on their sides for twenty-four or
forty-eight hours; they may then be placed upright; the reason for this
is that the air left between the cork and the beer might give rise to
the production of efflorescence. If we lay the bottles down on their
sides, the oxygen of this air will be absorbed by the oxidizable
substances in the liquid, and there will be little fear of germs
developing themselves when the bottles are placed upright. The bottles
should not, however, be left on their sides longer than forty-eight
hours; otherwise the supplementary fermentation may force the corks out.
Moreover, when the bottles stand upright the products of fermentation
collect at the bottom, and not at the sides.

Beer which is intended for keeping, if exported or conveyed some
distance off, must be surrounded with ice. Without this precaution it
will ferment too much or contract some disease.

“High beer” cannot stand travelling. This kind of beer should not be
exported unless the ordinary proportion of hops has been greatly
increased—hop oil acting in some respects as an antiseptic, and
preventing the beer from contracting diseases.[13] The export of English
beer to India and the Continent has fallen off of late years, or rather,
has not increased to the extent that was anticipated; in fact, this
trade has entailed great losses upon those engaged in it. It is said
that an English firm lost as much as £48,000 on one consignment, which
on its arrival in India was found to be all turned.

There are no breweries in hot countries, where beer would command a very
large sale. It is a well-known fact that beer is a remarkably pleasant
drink in tropical climates, provided its temperature be a few degrees
below that of the atmosphere, but the expenses of its production would
be enormous, on account of the immense quantity of ice that would be
required in its manufacture and for its preservation. It is in hot
countries that beer is most liable to deterioration.

Beer is said to be the beverage of northern regions, which are deprived
of the vine, by the rigour of their climate. In these regions man has
sought in the abundance of grain-fruit a substitute for grapes. To a
certain extent this is true; nevertheless, it is an undoubted fact that
beer was first brewed in Egypt, a very hot country, whence its
manufacture has spread over Europe. It was called Pelusian wine, from
Pelusium, a city on the banks of the Nile, which produced a beer that
was held in high esteem.[14]

Beyond a doubt, hot countries, even those in which the vine is
cultivated, would consume much beer, could it but stand their high
temperature.[15] A considerable quantity of beer is now brewed in
British India, but its manufacture entails an enormous outlay for ice.

The complications which result from the tendency of wort and beer to
deteriorate, underlie almost all the details of the process of brewing
and the sale of beer, and have been the cause of most of the changes and
improvements that have been effected in brewing, during the present
century.

Footnote 1:

  This expression is found for the first time, it would appear, in
  Theophrastus, B.C. 371. [See, however, Herodotus, Bk. II., chap. 77.
  Speaking of some Egyptians he says, “They drink a kind of wine made
  from barley (ὄινῳ δ’ ἐκ κριθέων πεποιημένῳ), for the grape does not
  grow in that part of the country.” Herodotus wrote about 450 B.C.
  Æschylus (480 B.C.) has a similar expression, Suppl. 953.—D. C. R.]

Footnote 2:

  One of these modifications is a real source of serious danger to the
  preservation of wine; for instance, during rainy years, at the time of
  vintage, the grapes may happen to be covered with earthy matter,
  consisting principally of carbonate of lime; this will dissolve in
  wine and partly neutralize its acidity, and the wine will thus become
  more liable to disease.

Footnote 3:

  Transferring from one cask to another for the purpose of clarifying
  the wine.

Footnote 4:

   We shall hereafter revert to this rapidity in cooling, to show that
  it is also of use in the subsequent clarification of beer.

Footnote 5:

  In some breweries (at Lyons especially) fermentation at a high
  temperature is practised in large vats at about 15° C. (59° F.). The
  yeast which covers the surface of the liquid is skimmed off and stored
  in flat tubs.

Footnote 6:

  The initial temperature of the wort must be regulated by the quantity
  of wort subjected to fermentation. In English breweries, where large
  quantities are brewed at a time, the heat created by the action of
  fermentation would produce a temperature sufficiently high to affect
  the quality of the beer, if the yeast were added at 19° C. or 20° C.

  The following are the temperatures at which the worts are pitched, in
  the principal London breweries:—For common ale, 60° F. or 15·5° C.;
  for pale ale, 58° F. or 14·4° C.; for porter, 64° F. or 17·8° C. The
  fermentation is commenced in large vats; from these the beer is run
  into vessels of a much smaller capacity, in which it completes its
  fermentation by working off the yeast and cleansing itself.

  For white beer of superior quality, the temperature during
  fermentation must not rise beyond 72° F. or 22·2° C.; some brewers
  never allow it to exceed 18°C. (65° F.). The temperature is lowered by
  means of a current of cold water, which circulates through a coil
  fixed in the vats or other fermenting vessels.

  In the case of porter, the initial heat of which is 64° F. or 17·8°
  C., the temperature in the vats sometimes rises to 78° F. or 25·5° C.;
  but such an increase in temperature excites considerable apprehension.

  We have seen a tun for pale ale, containing 200 barrels of 36 gallons,
  pitched with 600 lbs. of fairly solid yeast. In forty-six hours the
  attenuation was considered sufficient, and the beer, which from an
  initial heat of 58·1° F. or 14·5° C., had risen to 72° F. or 22·2° C.,
  was cleansed to working casks. The large vats in which the
  fermentation is started may be considered as equivalent to the _cuves
  guilloires_ of French breweries, the casks in which it is completed
  and the yeast thrown off representing their 75-litre vessels,
  improperly called _quarts_. Notwithstanding the enormous English beer
  manufacture, and although the fermenting vat, as in making porter, for
  instance, sometimes attains the capacity of 2,000 to 3,000 litres (400
  to 600 gallons), the casks into which it is run are never larger than
  15 to 20 hectolitres (300 to 400 gallons); and even at Burton, in the
  celebrated breweries of Allsopp and Bass, the pale ale is finished in
  casks of a capacity less than 10 hectolitres, and yet the average
  turn-out of these immense works reaches 3,000 to 4,000 hectolitres
  (60,000 to 80,000 gallons) of beer per day.

Footnote 7:

  [The expressions “high” and “low” fermentation or beer strictly refer
  to temperature, whereas the other expressions used (“top” or “bottom”)
  refer to the behaviour of the yeast. The French words _haute_ and
  _basse_ seem to look both ways.—D. C. R.]

Footnote 8:

  It is said that the floating cones or cylinders filled with ice, which
  enable brewers to manufacture beer at a low temperature, even in
  summer, were first used in Alsace.

Footnote 9:

  45 million kilos. of ice are annually consumed in the brewery of M.
  Dreher, in Vienna. The brewery of Sedlmayer, at Munich, uses about 10
  million kilos. (_Journal des Brasseurs_, 22nd June, 1873.)

Footnote 10:

  It should, however, be borne in mind that these remarks on the
  relative preservative powers of the two beers hold true on account of
  three things—differences in the respective modes of brewing,
  artificial cooling during the process of fermentation, and the storing
  of the “low beer” in ice-cellars. In itself, perhaps, “low beer” is
  more liable to change than “high beer;” that this does not actually
  take place, is due to the employment of artificial cooling. A brewery
  which has an average annual production of 10,000 hect. will use 8,000
  cwt. of ice. If we add to this the ice used during the retail of the
  beer, which is best drunk at 12° C. (54° F.), we shall arrive at the
  total of 100 kilos. per hectolitre.

Footnote 11:

  [In connection with the comparison here instituted by M. Pasteur
  between the drinking and keeping qualities of the two kinds of beer,
  it may be useful to draw the reader’s attention to a review by Dr.
  Charles Graham of the French edition of this work, published in
  _Nature_ for January 11th, 1877, page 216. At the same time we must
  remark that Dr. Graham appears to have overlooked M. Pasteur’s
  footnote, page 12, English edition:—“His assertion, that by bottom
  fermentation store beers can be produced, whereas those produced by
  top fermentation must be consumed at once and cannot be transported,
  are certainly strange to an Englishman. So far from these unfavourable
  comparisons being true in all cases, the exact opposite is generally
  the case. Bavarian and other bottom fermentation beers are in fact
  those which can neither be preserved nor transported without the
  liberal employment of ice; even that sent from Vienna to London must
  be kept cold artificially, in order to avoid rapid destruction. As
  regards flavour, there are many who think a glass of Burton pale ale,
  or of good old College rent ale, to be superior to any Bavarian beer.
  The chief cause of the decline in the production of top fermentation
  beers on the Continent has been the want of attention in the
  fermentation process; whereas the English brewer, especially the
  brewer of high-class ales, has been unremitting in his attention to
  the temperature in fermentation and to the perfect cleansing of the
  ale. Now, where such attention is given, it is not difficult to obtain
  ales which will keep a few years. While objecting to our English
  produce being so hastily depreciated by M. Pasteur, our brewers will
  be the first to avail themselves of his biological researches, in
  order to render their produce more stable and better flavoured,
  without having recourse to the general adoption of the vastly more
  costly system of bottom fermentation.”—D. C. R.]

Footnote 12:

  To preserve bottled beer from deterioration, some bottlers employ, at
  the moment of filling, a small quantity of bisulphite of lime. Others
  heat the bottles to a temperature of 55° C. (131° F.) In the north of
  Germany and in Bavaria, this practice has been widely adopted since
  the publication of the author’s “Studies on Wine,” and some of M.
  Velten’s writings. The process has been termed _pasteurization_ in
  recognition of the author’s discovery of the causes of deterioration
  in fermented liquors, and of the means of preserving such liquors by
  the application of heat. Unfortunately this process is less successful
  in the case of beer than in that of wine, for the delicacy of flavour
  which distinguishes beer is affected by heat, especially when the beer
  has been manufactured by the ordinary process. This effect would be
  less felt in beer manufactured by the process which is advocated in
  this work.

Footnote 13:

  A convincing proof of the influence of hops on the ferment organisms
  is contained in the fact that beer, even after being raised to 60° C.
  or 70° C. (140° or 160° F.), will, if unhopped, readily take on the
  butyric fermentation, from which, if hopped, it would remain perfectly
  free.

Footnote 14:

  For historical details, see _l’Encyclopédie_, Art. _Bière_.

Footnote 15:

  As a wine-producing country France has been highly favoured by nature,
  but the consumption of beer in France is increasing every year. In
  1873 the quantity of beer, paying excise duties, amounted to 7,413,190
  hect., which yielded to the Treasury the sum of 20,165,136 fr. These
  figures are taken from a report published in 1875 by M. Jacquème,
  inspector of finances, who remarks that the quantity of beer upon
  which excise duties are paid represents, probably, not more than
  one-third of the total production: two-thirds of the quantity brewed
  evades the duties.




                              CHAPTER II.
       On the Causes of the Diseases which affect Beer and Wort.


From our preceding observations it will be evident that the manufacture
of beer, the arrangement of breweries, and all the processes practised
by the brewer immediately depend upon this fact, that beer and wort are
fluids essentially liable to change. Thus it becomes a matter of extreme
importance that we should have an exact knowledge of the causes and
nature of the changes which affect our produce, and it may be that this
knowledge will lead us to regard the conditions of the brewing industry
from a novel point of view, and bring about important modifications in
the practices of the trade. We might vainly search the numerous works
which have been written on brewing for information respecting the
proposed subject of these studies. At the most we should find the
diseases to which beer is liable in the course of its manufacture, or
afterwards, vaguely hinted at; perhaps we might be favoured with certain
empirical recipes for disguising the evil effects of those diseases.

It will be our endeavour to demonstrate the truth of the proposition we
have already laid down, that every change to which wort and beer are
liable is brought about solely by the development of organic ferments,
whose germs are being perpetually wafted to and fro in the dust floating
through the air, or distributed over the surface of the different
materials and utensils used in brewing, such as malt, yeast, water,
coolers, vats, tubs, casks, shovels, workmen’s clothes, and innumerable
other things.

It is evident that this proposition bears a marked resemblance to the
one which we have demonstrated concerning the diseases of wine.[16]

By the expression _diseases_ of wort and beer, we mean radical changes
which so affect the nature of those liquids as to make them unpalatable,
especially if they are kept; such changes produce beer which is sharp,
sour, turned, oily, putrid, and otherwise bad. It would be unreasonable
to apply the term _disease_ to certain modifications in the quality of
beer, which may be produced by practices more or less commendable. Such
modifications, too, may result from want of skill in the brewer, from
the composition of the wort, from the specific nature of the yeast, or
from the inferior quality of ingredients. It is a well-known fact that
“low beer,” if manufactured according to the ordinary process, has not
that same delicacy of flavour which characterizes beer fermented at a
lower temperature than 10° C. (50° F.). Fermented at 10° C. (50° F.), or
12° C. (53° F.) or at a higher temperature, it loses the peculiar
properties which consumers prize. Nevertheless, in point of soundness it
may be as good a beer as one which has been fermented at 6° C. (43° F.),
or 8° C. (46° F.). One might say of the former beer that it is inferior
to the latter in estimation; but we could not rightly call it diseased,
for we are supposing a case in which disease does not actually exist.

  § I.—Every Unhealthy Change in the Quality of Beer coincides with a
Development of Microscopic Germs which are alien to the Pure Ferment of
                                 Beer.


Our proposition concerning the causes of the diseases of wort and beer
might be demonstrated in several ways. The following is one of the
simplest:—Take a few bottles of sound beer, say, for instance, that
which is known in Paris as Tourtel’s, Grüber’s, or Dreher’s, from the
name of the brewer who manufactures it. Place some of these bottles in a
hot-water bath and raise the temperature to about 60° C. (140° F.).
Permit them to cool, and then place them by the side of the other
bottles that have not been heated. In every case, especially if we
conduct this experiment in summer, we shall find that in the course of a
few weeks—the length of time varying according to the temperature and
the quality of the beer—all the bottles which have not been heated will
have become diseased, in some cases even to the extent of being
undrinkable. Let us next examine, by way of comparison, the deposits in
the heated and non-heated bottles. We shall find associated with the
pure alcoholic ferment other organisms, filiform and for the most part
very slender, and either simple or articulated, as represented in Plate
II., the design of which is taken from actual deposits occurring in beer
that had been kept for some time at the ordinary temperature. A number
of bottles of beer which had been heated on October 8th, 1871, were
compared with those of an equal number of bottles of the same beer which
had not been heated. The examination took place on July 27th, 1872. The
beer, which had been heated to 55° C. (131° F.), was remarkably sound,
well flavoured, and still in a state of fermentation. As a matter of
fact, we have proved by exact experiments that alcoholic ferments,
heated in beer, can endure a temperature of 55° C. (131° F.), without
losing the power of germination; but the action is rendered somewhat
more difficult and slower. Diseased ferments, however, existing in the
same medium, perish at this temperature, as they do in the case of wine.
The beer which had not been heated, had undergone changes which rendered
it quite undrinkable. Its acidity, due to volatile acids, was higher
than that of the other beer in the proportion of 5 to 1. The beer which
had been heated contained ½ per cent. of alcohol more than the other.

[Illustration: Plate 2. Appearance under the Microscope of the Deposit
from “Turned” Beer.]

The deposits in the heated bottles also showed filaments of disease, but
in such minute quantity that it was necessary to search many fields of
the microscope to discover their existence. Those which we found after
the heating must have existed in the beer before that operation; the
heat had destroyed them, without sensibly altering their shape or size;
they could neither multiply nor continue to exert any influence upon the
components of the beer.[17]

From these experiments we may easily perceive, on the one hand, that
beers apparently sound to the taste do not contain these or any other
filiform ferments, save in a scarcely appreciable quantity; and, on the
other hand, that these same ferments appear with the first unfavourable
change in the quality of the beers, and that they exist more or less
abundantly in proportion to the intensity of disease.

In certain extremely rare cases it may happen—so, at least, we have been
assured, but we have not proved the fact ourselves—that beer may keep
sound in bottle, even without the preliminary heating. This exception
can only occur in the case of certain beers of a peculiar composition,
which are highly hopped, and are made during the favourable months of
November or December, out of the choicest materials, and fermented with
yeast that happens to be pure. In the deposit of such beer, even after a
lapse of several months or several years, we should find only the
ordinary alcoholic ferment, the slow action of which would merely cause
a gradual increase in the quantity of alcohol existing in the beer, and
a diminution in the proportion of dextrine. This beer might grow old, as
wine does, and remain perfectly sound.

Very often the whole work of the brewer is jeopardized by the
unsuspected presence of diseased ferments, a remedy for which is only
devised after the evil has evoked the complaints of customers. In such a
case the brewer avails himself of the kindness of some other brewer to
obtain a change of yeast—a custom which is recognized and valued in the
trade, since all managers of breweries have an interest in keeping it
up. The brewer whose produce is most satisfactory recognizes the fact
that unforeseen circumstances may compel him at any moment to change his
yeast.

We have frequently had occasion to show that this necessity for a change
of yeast depends, in most cases, on some change brought about by the
presence of diseased ferments, the multiplication of which has resulted
fortuitously from some unconscious neglect during the process of
brewing, or from climatic influences. When we reflect that yeast is a
living being, and that the medium which serves as its aliment, and the
water in which it lives, are remarkably well adapted for the development
of a vast number of other microscopic beings, the comparative purity of
yeast should surprise us even more than its deterioration does.

Now by means of microscopical observations we might often detect the
existence of the evil long before we are warned of it by a defective
working, which invariably entails great losses.[18]

In proof of this remark we may cite the following facts. In the month of
September, 1871, we were permitted to go through a large London brewery,
in which the microscopical study of yeast was altogether unknown. We
were allowed to make certain experiments in the presence of the managers
of the establishment. We first examined porter yeast, which was
collected in a channel that received the yeast as it worked out of the
fermenting vessels. One of the ferments of disease abounded in this
yeast, as may be seen in the accompanying sketch, which was taken on the
spot (Fig. 1). It was evident that the working of the porter was
extremely unsatisfactory, and had, perhaps, been so for a long time;
indeed, we were told that they had obtained a change of yeast from
another London brewery that same day. We made a point of examining this
yeast with the microscope. It was beyond comparison purer than the
preceding yeast.

[Illustration: Fig. 1.]

It is evident that if these brewers had been in the habit of using the
microscope they might have detected the unsoundness of their produce
before the time when they actually made the discovery, which, no doubt,
was forced upon them by the complaints of their customers, or some other
annoying circumstance, that led to their obtaining a change of yeast.

We next obtained permission to examine the yeast of the other beers
undergoing fermentation, especially those of white beers, such as ale
and pale ale.

In the sketch which we made of these yeasts one may detect the presence
of the filaments peculiar to turned beer (Fig. 2).

[Illustration: Fig. 2.]

We examined with much interest the ales which had immediately preceded
those undergoing fermentation, the yeast of which we had just
inspected. We were furnished with two kinds, both in casks, the one
fined, the other not fined. The latter was visibly turbid, and,
examining a drop of it, we discovered three or four filaments present
in every field of the microscope. The ale which had been fined was
nearly clear, but wanting in brilliancy; it contained about one
filament to the field. We asserted in the presence of the head brewer,
who had been summoned, that these ales were extremely liable to
change, that it was highly necessary to dispose of them without delay,
and that they were necessarily already faulty in flavour—a fact which
all admitted after some hesitation—attributable, of course, to the
natural reluctance which every manufacturer feels to own that his
produce is not above reproach. We were shown some of the finings used
in the brewery; they were swarming with the same filaments of
disease-organisms.

We then propounded to the managers certain questions on the subject of
the losses which a brewery may sustain from changes in its beer. We had
heard from several brewers that the selling price of beer differed so
greatly from the cost of its production solely in consequence of the
losses which the unavoidable waste of large quantities of beer was
constantly causing; several brewers have in our presence estimated these
losses at 20 per cent. of the total production, on the average.

At first the English brewers returned somewhat vague answers to our
questions; however, after what had taken place, they doubtless
recognized the fact that a mutual understanding between a savant and a
practical man may often be of considerable benefit to the latter, and in
the end they confessed to us that they had stowed away in their brewery
a large quantity of beer which had gone bad in cask a fortnight or so
after it was brewed. Having avowed thus much they expressed their great
anxiety to learn the cause of so serious a change in their beer, which
was quite undrinkable. We examined it under the microscope, without
being able to detect immediately any diseased ferments, but being aware
that the beer had probably been clarified, by remaining undisturbed for
a very long time, and that these ferments might have become inert and
precipitated to the bottom of the enormous vats containing the beer, we
examined the deposits which had formed at the bottom of these vats. They
were composed solely of filaments of disease-organisms, without even the
least trace of the globules of alcoholic yeast. The supplementary
fermentation of this beer had evidently been nothing but a diseased
fermentation.

The resemblance between these filaments and those which, in considerably
smaller proportion, accompanied the globules of alcoholic ferment in our
preceding observations, the change in the beer, which was almost as bad
as beer could possibly be, along with an abundance of filaments, and the
change, to a minor extent, in that beer which only presented a few
filaments in a field of the microscope, impressed those managers of the
brewery who were present with an entire belief in the theory which we
had been endeavouring to impress on their minds concerning the causes of
the badness of their beer. Some eight days afterwards we paid another
visit to this same brewery, and learnt that the directors had lost no
time in acquiring a microscope, and in procuring changes of yeast for
all the varieties of beer, which they had put in working since our first
visit.

There are some periods of the year—early spring, summer, and autumn, for
instance—when the working of a brewery is a matter of great difficulty.
The preservation of yeast becomes a subject requiring the most delicate
treatment, in consequence of the increase in the temperature. In the
early part of autumn the most important ingredients used in brewing are
of inferior quality; the deteriorating influences which have been at
work have covered them with a variety of parasites. All these
circumstances contribute to facilitate the development of diseased
ferments.


§ II.—The Absence of Change in Wort and Beer Coincides with the Absence
                         of Foreign Organisms.


The method which we have just pursued in demonstrating the existence of
a relation between the diseases of beer and certain microscopic
organisms can scarcely leave a doubt, it seems to us, as to the
correctness of the principles which we are advocating. In every case
where the microscope reveals in a yeast, especially a yeast which is in
a state of activity, products which are foreign to the composition of
alcoholic ferment, properly so called, the flavour of the beer is more
or less unsatisfactory, according to the abundance or nature of these
minute organisms. Moreover, when a finished beer of superior quality
loses in the course of time its agreeable flavour, and becomes sour, it
may readily be shown that the alcoholic ferment in the deposit existing
in bottles or casks, although originally pure, at least in appearance,
becomes gradually intermixed with these same filiform ferments or other
ones. These facts may be deduced from what precedes; nevertheless, some
prejudiced minds might perhaps urge that these foreign ferments are the
consequence of some diseased condition, produced by circumstances of
which we know nothing.

Although this gratuitous supposition may be difficult to sustain, we
shall endeavour to corroborate our preceding observations by the method
of experiment which will be seen to be the more decisive.

This method consists in proving that beer never possesses any unpleasant
flavour, so long as the alcoholic ferment, properly so called, is not
associated with foreign ferments; that this also holds good in the case
of wort, and that wort, liable to change as it is, may be preserved in a
state of purity, if it is kept under conditions that protect it from the
invasion of microscopic parasites, to which it presents not only
favourable nutriment, but also a field for development.

By employing this second method we shall, moreover, have the advantage
of proving with certainty a proposition that we just advanced, and
showing that the germs of these organisms proceed from the particles of
dust which the common air wafts about and deposits on every object, or
which are spread over the utensils and materials used in a brewery,
materials that are naturally charged with microscopic germs, which
various changes in the store-houses and maltings may multiply to an
indefinite extent.[19]

[Illustration: Fig. 3.]

Let us take a glass flask having a long neck (Fig. 3 A), and holding
from 250 c.c. to 300 c.c. (_i.e._, about 9 or 10 fl. oz.); let us put
into it some wort, hopped or not, and then draw out the neck of the
flask in the flame of a lamp, so as to give to it the shape B (Fig. 3);
let us next heat the liquid to the boiling point, when the steam will
rush with a hissing sound out of the curved end. We may then, without
further precaution, permit our flask to cool, or, as an additional
safeguard, we may introduce a small quantity of asbestos into the open
extremity, at the very moment when the flame is taken away from the
flask. Before introducing the asbestos, we may pass it through the
flame, and we may repeat this after it has been placed in the end of the
tube.[20] The air which first re-enters the flask must come in contact
with the heated glass and the hot liquid, and these will destroy the
vitality of any germs existing in such particles of dust as this air may
introduce. The re-entrance of the air will be effected very
gradually—sufficiently so to enable the drop of water which the air, as
it enters, forces up the curved tube, to catch all particles of dust.
Ultimately, the tube will become dry, but then the passage of the air
will proceed so slowly that every foreign particle will get deposited on
its interior sides.

Experience tends to prove that external particles of dust cannot find
their way into flasks of this pattern, having free communication with
the air, at all events within ten or twelve years—the longest time that
has been devoted to experiments of this kind; the liquid in the flasks,
if originally clear, will not become in the least degree contaminated,
either upon its surface or throughout its bulk, although the outside of
the flasks may be covered with a thick coat of dust. This is an
undeniable proof of the impossibility of particles of dust finding their
way inside such flasks.

Wort treated thus will preserve its purity for an indefinite time,
notwithstanding its extreme liability to rapid change when exposed to
the air, under conditions which cause it to come in contact with the
particles of dust that air contains. This also holds good in the case of
wine, beef-tea, the must of grapes, and, generally speaking, of all
liquids which are subject to putrefaction or fermentation, and which
possess the faculty, when their temperature is raised to about 100°C.
(212°F.), of destroying the vitality of those microscopic germs that are
found in dust.

A flask such as we have described (Fig. 3) is all that we require when
we have to demonstrate the facts that we have just reviewed, a more
detailed account of which may be found in our Memoir published in the
_Annales de Chimie et de Physique_, for 1862, under the title _Mémoire
sur les corpuscules organisés en suspension dans l’atmosphère. Examen de
la doctrine des générations spontanées._ The shape of the flask
represented in Fig. 4 only differs from that of the preceding one in
having a second little tube attached to the globular part of the flask;
this presents great advantages for different objects of study, and it
was adopted in the subsequent investigations detailed in this work.

This flask will permit us to study without difficulty every separate
kind of microscopic organism in the liquid best adapted to it without
fear, if we take reasonable precautions, that the subject of our study
may become associated with other organisms, the accidental presence of
which cannot fail to seriously affect the results of our observations.

Let us use one of those flasks in our experiment on yeast, at the same
time expressly assuming that the minute germs of yeast are free from all
contamination by foreign germs, an object which we shall learn how to
realize by a variety of methods in a subsequent chapter.

[Illustration: Fig. 4.]

Let us introduce some wort into our flask (Fig. 4); then, after we have
fitted an india-rubber tube to the little supplementary tube, let us
boil the liquid; the steam, finding an easier exit through the
india-rubber opening than through the drawn-out tube, will rush out
through the india-rubber tube, and thus, as it passes, destroy any germs
which may be adhering to the sides of the little supplementary tube. If
we close the india-rubber tube by means of a glass stopper, the steam
will immediately issue through the bent tube. On permitting the flask to
cool, it will then be ready for impregnation. If there were any fear
that some foreign germ of an unknown nature might have effected an
entrance during cooling, or had not been destroyed by the steam (which
is always a little super-heated, in consequence of the resistance which
it meets in escaping), we need only place the flasks on a warm stove,
and leave them there for a few days or a few weeks to ascertain whether
the liquid in them has undergone any change. We should then only use
those flasks which contain a sound liquid.

At the same time we must warn our readers that this cause of error does
not exist once in a thousand times, especially if we use an asbestos
stopper to prevent the entrance of little insects which are attracted by
the odour of the liquid, and which instinctively seek to enter at the
extremity of the tube, and to pass through it into the flask. In going
so far, and incurring such labour in search of their food, they condemn
themselves to certain death, for they are sure to be drowned, since it
would require an intelligence superior to that which they possess to
enable them to get out of the flask; the liquid, morever, could not fail
to undergo some change, in consequence of the particles of dust that the
insects would introduce into it.

After having passed the flame of a spirit-lamp quickly over the
india-rubber tube, the glass stopper, the curved tube, and even over the
fingers of the operator, we may withdraw the glass stopper, and
introduce the pure yeast by means of a glass pipette that has been
previously heated. This yeast is kept in a vessel also free from the
dust floating in the air. However few globules of yeast the glass tube
may take up, it is sure to introduce a hundred or a thousand times more
than is necessary for the impregnation of the liquid. The glass stopper
must then be replaced immediately, after having been again quickly
passed through the flame. In transferring our yeast from the vessel
containing it to the flask, by means of a glass pipette, it is exposed
to another cause of impurity, since we cannot avoid bringing it in
contact with the common air. If this risk frequently troubled us in our
experiments, we might banish it or minimize it by some new arrangement;
but this is unnecessary. We have suffered no inconvenience from this
cause, as there does not exist in the atmosphere anything like a
continuous supply of that from which the so-called _spontaneous_
generation arises, as was erroneously believed to be the case before the
publication of our Memoir in 1862, to which we have already alluded.

The following are the results of the experiments conducted in the manner
just described.

The yeast which we sowed, in ever so small a quantity, seemed to acquire
vigour, to bud, and to multiply. Soon, that is to say in the course of
twenty-four or forty-eight hours or longer, according to the
temperature, and, more especially, the degree of vitality in the
globules, we found that the sides of our flasks were covered with a
white yeasty deposit, and noticed on the surface of the liquid a fine
froth, which at first appeared like little islets formed by groups of
bubbles so minute that they would have been imperceptible had they not
been joined together. These patches increased in size, and gradually
attached themselves to each other, finally forming a thick froth. In the
course of two or three days this froth fell, the fermentation proceeded
less rapidly, and then ceased completely. The beer was finished. This
beer might be preserved for an indefinite period in the flask without
undergoing any change. The external air passes freely into and out of
the flask, as the pressure of the atmosphere and the temperature vary,
and the beer in the course of time becomes flat; it acquires age in much
the same manner as wine does, but it never contracts any taste of
disease, it never becomes sour, or sharp, or bitter, or putrid; it does
not even become covered with _mycoderma vini_ as is usually the case
with all beer exposed to the common air in the course of trade.

After some weeks, or perhaps months, a white ring may show itself on the
surface of the liquid on the glass. This is a crown formed by a mass of
young yeast globules, which grow there like a mould, by absorbing the
oxygen of the air as it enters the flask. The bulk of the yeast which
has been fermenting remains at the bottom of the liquid in the form of
an inert deposit. This inertness, however, is apparent rather than real;
the globules may be internally active, without any development of new
buds, and the effects of this working may cause them to become more and
more languid, and in the course of time may even destroy them.

The case is quite different when the yeast with which our wort is
impregnated, instead of being pure, is mixed to any extent whatsoever
with diseased ferments. Should there be any of these in the yeast with
which we impregnate our wort, even though their quantity were so
infinitesimal that the most skilful observer could scarcely discover
them with the microscope, they would multiply in the flask after the
beer had been finished, especially if the beer were left for a short
time on a stove. In this manner we may secure an excellent test of the
original purity of the yeast which we employ in the impregnation of our
wort.

Thus it may be seen that the absence of microscopic organisms that are
foreign to the nature of pure yeast may invariably be noticed in the
case of a beer which is sound, and which will remain sound for any
length of time, when in contact with pure air, at any temperature. We
may see, too, that the presence of these organisms may invariably be
detected in an unsound beer, the peculiar unsoundness of which depends
upon the peculiar species of the organisms contained in it. It would be
difficult to adduce clearer proofs than those which we have given as to
the intimate relation existing between these organisms and the
deterioration of beer. The relation between cause and effect, in the
succession of physical phenomena in general, is established by proofs
that are by no means more decisive.

Footnote 16:

  A statement of this proposition, as far as it concerns beer, appeared
  first in outline in the author’s _Etudes sur le vin_, published in
  1866.

Footnote 17:

  As the deposit in the heated bottles is, as a rule, inconsiderable, it
  is necessary to exercise some precaution in collecting it. The bottles
  are taken up; after some days’ rest they are decanted very carefully,
  with as little shaking as possible, until not more than one or two
  cubic centimetres (about a tea-spoonful) of the liquid remains at the
  bottom. The bottles are then shaken vigorously, with the object of
  collecting the whole of the deposit from the bottom and the sides into
  this small quantity of liquid; a drop of this is then examined under
  the microscope.

Footnote 18:

  Since the publication of the author’s “Studies on the Diseases of
  Wine, and the Dangers resulting to Wine and Beer from the Microscopic
  Parasites found therein,” some intelligent brewers have derived
  considerable profit from the application of the theories laid down in
  that work.

Footnote 19:

  If we put a handful of germinating barley from a maltster’s cistern
  into a little water, and examine drops of the liquid, after it has
  become turbid, under a microscope, we shall be amazed at the wonderful
  number of strange microscopic organisms that swarm on the surface of
  the grains and on the sides of the cistern. There is no doubt that
  their presence is injurious to germination, inasmuch as they absorb
  much oxygen; moreover, they acidify the grain and cause it to
  deteriorate.

Footnote 20:

  In these experiments the asbestos is only introduced by way of extra
  precaution. Originally, in his early experiments in connection with
  the subject of spontaneous generation, the results of which were
  published in 1860-62, the author did not use it, and he observed no
  ill effects resulting from the omission; now, however, he constantly
  makes use of it. In studies of this kind novel precautions are never
  thrown away; moreover, the presence of this asbestos is a sure bar to
  the entrance of insects. The author has preserved for a long time a
  flask, in the slender neck of which an insect is contained; he killed
  it with a flame just as it was approaching the liquid. Quite recently,
  M. Calmettes, a young engineer from the École Centrale, when engaged,
  at Tantonville, in Tourtel’s brewery, in carrying out certain
  practical experiments in connection with the process that will he
  described in one of the later chapters of this work, wrote complaining
  that his flasks had been suddenly invaded by a swarm of aphides,
  scarcely larger than phylloxeras, and that many of them had even
  penetrated into the inside of the curved tubes.




                              CHAPTER III.
             On the Origin of Ferments properly so called.


The new process of brewing, which it is the principal object of this
work to explain, and which will follow as an immediate and inevitable
deduction from the novel facts herein demonstrated, cannot be fully
understood without a knowledge of all the principles upon which it is
founded. One of the most essential of these has reference to the purity
of our fermentation. We should gain but little from the use of a yeast
uncontaminated by any foreign germs if natural organic substances had
the power of organizing themselves by means of spontaneous generation,
or by some transformation which took place amongst them, or even by a
conversion of certain microscopic beings into certain others. Theories
of this kind are still warmly advocated, but, to our thinking, rather
from sentimental considerations or prejudice than from any basis of
serious experimental proofs.

Be this as it may, we must free our minds from all suppositions which
might qualify the exactness of the principles upon which our new process
is founded, or cause any doubt in the minds of our readers as to the
possibility of its application and the benefits to be derived from its
adoption.


   § I.—On the Conditions which cause Variations in the Nature of the
               Organized Products existing in Infusions.


We have proved by experiment, in the preceding chapter, that a few
minutes’ boiling renders liquids, and more especially wort, absolutely
free from liability to change when in contact with pure air, that is,
air which contains none of the germs of organisms that are continually
floating about in the atmosphere.

What is true in the case of wort is equally so in the case of all
organic liquids; there is not a single one that could not be rendered
inaccessible to any subsequent change if it were brought, first of all,
to a suitable temperature, which would vary with the nature of the
liquids. Amongst them there are some which, like vinegar, lose their
tendency to change after having been rapidly raised to a temperature of
not more than 50° C. (122° F.); others, like wine, require a greater
heat. Wort, to which no hops have been added, should be subjected to a
temperature of not more than 90° C. (194° F.); milk to about 110° C.
(230° F.).[21]

It is easy to show that these differences in temperature, which are
required to secure organic liquids from ultimate change, depend
exclusively upon the state of the liquids, their nature, and, above all,
on the conditions that affect their neutrality, whether towards acidity
or alkalinity; for it is not difficult to observe that the least changes
in these respects lead to considerable variations in the temperatures
which we must employ.[22] We could adduce many examples of this. The
only difference between the nature of must and that of wine is caused by
fermentation. We may say the same thing of wort and beer, and, better
still, of new milk and sour milk. Must, to be secured from change,
requires a much higher temperature than wine does; similarly, wort
requires a much higher temperature than beer. Milk must be heated to
about 110° C. (230° F.), as we have just stated, but sour milk would
require 20° C. or 30° C. (36° F. or 54° F.) less. Wine, when fresh,
ceases to be liable to change after it has been brought to a temperature
below 100° C. (212° F.); it requires a temperature of more than 100° C.
in the presence of carbonate of lime.

As regards the explanation of the influence which acidity or alkalinity
exerts in diminishing or increasing the temperature required to protect
infusions and organic matters from ultimate change, although this is a
subject which claims special study, we are inclined to believe that
acidity permits, and alkalinity prevents, the penetration of moisture
into the interior of the cells of the germs belonging to infusions, so
that in heating the outer cases of these cells in an alkaline medium we
heat the germs in a dry state; and in heating the outer cases in an acid
medium we heat the germs when they are moist. We all know that these
conditions make a great difference in the resistance which bodies offer
to the action of heat. A particle of mould which, in a moist state,
cannot survive a temperature of from 60° C. to 100° C. (140° to 212°
F.), will preserve its fecundity even if heated to 120° C. (248° F.), if
it has been previously well dried.[23]

The nature of the spontaneous productions which we see appear in organic
liquids is affected to a remarkable extent by the smallest change in the
compositions of those liquids. Generally speaking, as we have often
proved in our former works, a feeble acidity is unfavourable to the
development of bacteria and infusoria, and, on the other hand,
favourable to the growth of mould. A liquid which is neutral or of
feeble alkalinity behaves in an exactly inverse manner.

Those who support the theory of spontaneous generation seek to find in
the natural differences between the organic productions of various
liquids which are simultaneously exposed to the same atmosphere, an
argument in favour of their doctrine. These differences, however, are
only an effect of the greater or less fitness of a peculiar liquid for a
certain kind of growth. When an acid organic liquid, such as the must of
grapes for example, is exposed freely to the air, it is besieged
simultaneously by spores of _mucedines_, as well as germs of _bacteria_,
_leptothrix_, _vibrios_, _&c._, but the latter germs are checked in
their development, if, indeed, they are not actually destroyed, by the
acidity of the liquid; and we should never find them there in their
adult state.

Most moulds, on the other hand, thrive in acid liquids, and therefore
they are generally found alone. If we began by saturating must with
carbonate of lime, previously dried or otherwise, we should observe
opposite phenomena; bacteria, lactic ferments and butyric vibrios would
invade our fields long before the spores of mould had time to grow,
since the germination of these proceeds very languidly in neutral or
alkaline liquids, and an infusion once occupied by a living organism has
much trouble in nourishing others; the early developments consume the
alimentary substances, especially the oxygen.

Differences as marked as these in the adaptability of certain liquids to
certain growths give rise to innumerable illusions, and are one of the
chief sources of error in the study of this subject. If we sow in an
acid liquid, such as must, an alcoholic ferment, the development of
which is not arrested by the acid character of our medium, it will
multiply there, and we shall have no difficulty in growing it over and
over again in the same acid medium. This being the case, let us suppose
that our alcoholic ferment is impure—let us say, mixed with filaments of
turned wine, which are due to a ferment checked in its development by
the peculiar qualities of the must. In repeating the growth of this
alcoholic ferment in the must the filaments which were, by supposition,
present in our first sowing, and which cannot reproduce themselves in
the must, or do so with great difficulty, will become very scarce in the
fields of our microscope; they will not, however, cease to exist, for
the repetition of our attempts to grow them only serves to spread the
original germs over a larger surface; and, although the eye fails to
detect them, they will only have become more difficult to discover. At
this point the experimentalist is in danger of falling into error, for
when he no longer perceives the foreign ferment he will be inclined to
believe that it has vanished, and that he may regard the alcoholic
ferment as being free from all impurity, without testing for the purity
by a direct experiment.

An example of this mistake is to be found in a recent work by M. Jules
Duval. This writer has published a theory according to which yeast
becomes transformed into lactic ferment, and likewise into other
ferments—that of urea, for example—the only condition of change,
according to M. Jules Duval, being that we should cultivate it in
suitable mediums. The proofs by which he supports his conclusion are
altogether inadmissible, and a simple glance at his experiments enables
us to detect innumerable causes of error. M. Duval believes that yeast
becomes transformed into lactic ferment from the fact that he obtained a
fermentation which furnished lactate of lime and lactic ferment from
some sour milk to which he had added some glucose, chalk, and phosphate
of ammonia before impregnating it with yeast; but he took no steps to
secure himself against introducing into his medium—which was, as a
matter of fact, well adapted to lactic fermentation, inasmuch as it was
a little alkaline—an alcoholic ferment containing impurities. This was
the crucial point in his experiments. M. Duval recognizes this, but he
deceives himself when he says, without proof:—“My alcoholic ferment is
pure, for I have grown it over and over again in must, preserved in
flasks prepared after the manner of those which M. Pasteur uses in his
experiments.” Here we have merely a simple assertion; a direct
experimental test might have proved that the yeast was impure.

Yeast cannot transform itself into lactic ferment. No matter what the
medium may be in which it is sown, _if it is actually pure_ it will
never present the least trace of lactic ferment or of any other
ferments. By certain changes in the nature of the medium, the
temperature, and other conditions, the cells of the ferment may become
oval, elongated, spherical, and larger or smaller in size, but they will
never produce the most minute quantity of lactic ferment or lactic acid.
The whole theory of the transmutation of ferments, which M. Duval has
published, is imaginary.[24]

We have asserted that the source from which we obtain our supply of the
substances that we expose to contact with the air may likewise furnish a
reason for the development of vegetable or animal organisms which make
their appearance in our infusions. This may be easily understood. If we
expose the infusions to temperatures more or less high, with the object
of destroying the vitality of the germs which they may contain, we
completely suppress all germs originating from two different
sources—those which the infusions may have acquired directly from the
atmospheric air, or from the dust upon our utensils, and those which may
have been introduced by the materials used in manufacturing our
infusions or decoctions, which materials must have been brought from
some distance. An infusion, after having been heated to a sufficient
degree, can harbour and nourish only such germs as are conveyed to it by
the air after the heating. These floating germs are far from being as
varied in their nature as it pleases those to believe who are tied down
by their arbitrary and faulty interpretation of the knowledge that we
have acquired, and the discussions in which we have taken part, during
the last fifteen years. Air, unless in violent agitation, can hold only
the most minute particles in suspension. The observations which have
been recorded concerning organisms of spontaneous growth found in
infusions, have always been made in sheltered places—in rooms or
laboratories, the atmosphere of which is, relatively speaking, very
still. For this reason, in liquids that have been subjected to heat, the
_flora_ and _fauna_—if we may use such an expression—are very poor, and
all the more so because, as we have recently had occasion to remark, a
great number of the organisms which would spring into existence of their
own accord, if they were allowed sufficient time for germination, are
kept back by others of a more rapid development. The truth of this is
evident from the fact that we may observe greater variations in the
nature and number of species of living organisms, if we divide one
infusion amongst several vessels which are immediately closed up again,
than if we leave our infusion in contact with an unlimited volume of
surrounding air. By this means we expose each portion of the liquid to
no other germs than those existing in a state of suspension in the
volume of air introduced into the vessels; and it so frequently happens
that we obtain a variety of germs which, coming into contact with a
liquid adapted to their nutrition, without having mixed with others,
finally multiply there, in consequence of no other organisms of greater
activity occurring to impede their slow and laborious propagation.

The nature of the products resulting from raw infusions—that is, those
obtained, without heating, from the maceration of organic substances,
such as leaves, fruits, grains, or the organs of plants or animals—is
much more varied. The reason of this is that such substances generally
carry with them not only the particles of dust existing in air that is
in motion, but also microscopic parasites, which find a congenial
resting-place on their surface or its vicinity. We may cite a few
accurate observations on this point, for the subject is one of great
interest.

If we boil an infusion of hay, and then expose it to contact with the
air in a room, all its productions will be derived from such germs as a
comparatively still air can carry about; thus, we shall very rarely find
any _colpoda_ in our infusion, for the germs of these infusoria,
consisting of rather large cells, can scarcely exist in a state of
suspension in motionless air, in spite of their extraordinary diffusion
in nature. On the other hand, we almost invariably find _colpoda_ in
macerations of raw hay. This difference is easily accounted for; the
particles of dust adhering to the surface of hay, especially that which
comes from marshy districts, contain the germ-cells of _colpoda_ in
abundance. The reason is obvious; rain falls on a meadow and forms
little pools of water about the roots of the herbs that grow there; this
water remains for some time, and very soon swarms with a multitude of
infusoria, especially _colpoda_. Dryness follows; the _colpoda_ becomes
encysted, and forms a dust that is wafted by the winds on to the blades
of grass, so that the mower will carry away not only his hay, but also
myriads of _colpoda_, as well as spores of _mucedines_ and other
organisms.[25]

The maceration of pepper will give us some infusoria hardly ever found
in other infusions, the reason of this being that such infusoria exist
where the pepper grows—in other words, their germs are exotic. That the
infusion of a special plant should give us special infusoria, is
scarcely more surprising than the discovery of a particular parasite or
insect existing on a particular plant, and not on others of a different
species that grow near it. Thus it happens that the ferment-germs of
must exist on the surface of grapes, whether detached or in clusters. It
is only natural that we should find the organ or the plant that is
destined later on to become the food of a parasite serving as a
habitation for the germs of that parasite.


 § II.—Experiments on Blood and Urine taken in their Normal State, and
 Exposed to Contact with Air that has been Deprived of the Particles of
              Dust which it Generally holds in Suspension.


Recourse to the application of heat, in the first place, is an excellent
means, as we have just seen, of procuring organic liquids free from all
disturbing germs; but there is a still more remarkable and instructive,
we may even say more unlooked for, method of securing this result, which
may be described as in some measure borrowed from the nature of things.
It consists in seeking purity in the natural liquids of animals and
plants. It is difficult to understand how the liquids circulating in the
organs of animal bodies, such as blood, urine, milk, amniotic fluid, and
so on, can possibly secrete the germs of microscopic organisms. There
would be excellent opportunities for these germs to propagate themselves
if they actually did exist in the liquids appertaining to the animal
economy. Life in all probability would become impossible in the presence
of such guests. A proof of this is to be found in the multitude of
diseases which many of the greatest minds of modern times attribute to
parasitic developments of this nature. Medical men of high authority
agree in thinking that the questions of contagion and infection will
find solutions from the obscurity in which they are now involved in a
careful study of ferments, and that hygienists and physicians should
labour to secure by every possible means the destruction of the germs of
ferments, and should strive to check _their_ development, and prevent
the evils which _they_ cause when developed. Great progress has already
been made in this direction, and we deem it a signal honour that our
researches on the subject have been considered, even by those by whom
this progress has been accomplished, as the source from which they
derived their first inspirations. We shall, doubtless, be excused by our
readers if we recall this fact in relating certain historical details
which are especially necessary in order that they may comprehend the
principles that we are endeavouring to explain in these “studies.”

It is a matter of regret to us, however, that the facts which we have
established should have been accredited with any importance beyond that
which is their due. The exaggeration of novel ideas invariably leads to
a reaction, which, again, overshooting the mark, brings into disrepute
even those points in which such ideas are perfectly just, or, at all
events, worthy of serious consideration. There are certain symptoms of
such a reaction in the case of our theories: they are evident in the
tendency of unreflecting minds to give a total denial to the fact that
certain diseases may be derived from certain ferments—organized and
living ferments—of the nature of those which have been discovered in the
course of the last twenty years. We should be guided by facts, whichever
side of the question we espouse, and by facts alone we should test the
truth of doubtful discoveries. We are but on the threshold of the
exploration of our subject, and we should strive to discover new facts
in connection with it, and should deduce from these, whatever they may
be, only such conclusions as they may strictly warrant. Unfortunately,
there is amongst physicians a tendency to generalize by anticipation.
Many of them are men of rare natural or acquired talent, endowed with
keen powers of intellect, and the art of expressing themselves fluently
and persuasively; but the more eminent they are, the more they are
occupied by the duties of their profession, and the less leisure they
have for the work of investigation. Urged on by that thirst for
knowledge which belongs to superior minds, and perhaps, in some measure,
through associating with the upper classes of society, which are
becoming more and more interested in science, they eagerly seize upon
easy and plausible theories, readily adapted for statement which is
general and vague just in proportion to the unsoundness of the facts on
which they are based. When we see beer and wine undergo radical changes,
in consequence of the harbour which those liquids afford to microscopic
organisms that introduce themselves invisibly and unsought into it, and
swarm subsequently therein, how can we help imagining that similar
changes may and do take place in the case of man and animals? Should we,
however, be disposed to think that such a thing must hold true, because
it seems both probable and possible, we must, before asserting our
belief, recall to mind the epigraph of this work: _the greatest
aberration of the mind is to believe a thing to be, because we desire
it_.

One of the most distinguished members of the Academy of Medicine, M.
Davainne, who was the first to give his attention to rigorous
experiments on the influence that organic ferments exercise on the
production and propagation of infectious diseases, declares that the
idea of his researches on _splenic fever_ and _malignant pustule_ was
suggested to him by his perusal of our work on butyric fermentation,
published in 1861. In 1850 this gentleman and M. Rayer discovered in the
blood of animals attacked by these diseases minute filiform bodies, to
which they paid little attention, and which M. Davainne recollected,
when he came across our Memoir. He had the foresight to conjecture—a
conjecture that was soon confirmed most decisively by his
researches—that the former disease, known under the name of _sang de
rate_, might be the production of a fermentation analogous to the
butyric, in which the minute filiform bodies observed by Rayer and
himself, in 1850, played the part which vibrios fill in butyric
fermentation. Within two years of this the first works of Messrs. Coze
and Feltz appeared. These clever and courageous experimentalists avowed
that their beautiful researches had been suggested to them by the
perusal of my work on putrefaction, published in 1863. We might also
quote the striking and admirably conceived experiments of Dr. Chauveau,
on castration.[26] We cannot, however, refrain from reproducing here a
letter addressed to us in 1874 by the celebrated Edinburgh surgeon, Mr.
Lister:—

    “Edinburgh, Feb. 10, 1874.

    “DEAR SIR,—Will you permit me to beg your acceptance of a pamphlet
    which I forward to you by this post, and which describes certain
    inquiries into a subject upon which you have thrown so much
    light—the theory of germs and fermentation? It gives me pleasure to
    think that you will peruse with some interest what I have written
    about an organism that you were the first to study in your Memoir on
    lactic fermentation. I do not know if you ever see the records of
    British surgery. If you have perused them you may have observed from
    time to time notices of the antiseptic system which I have been
    endeavouring for the last nine years to bring to perfection. Permit
    me to take this opportunity of offering you my most hearty thanks
    for having demonstrated by your brilliant researches the truth of
    the theory of putrefactive germs, and for having afforded me in this
    manner the sole means of perfecting the antiseptic system.

    “Should you ever come to Edinburgh I am sure that you will be truly
    gratified to see in our hospital the extent to which the human race
    has profited by your work. I need hardly add, that I should have
    great satisfaction in showing you how greatly surgery is indebted to
    you.

    “Excuse the freedom which I have taken in addressing you, on the
    grounds of our common love for science, and believe in the profound
    esteem of yours, very sincerely, JOSEPH LISTER.”

The wad dressing of Dr. Alphonse Guérin, surgeon at the Hôtel-Dieu,
Paris, which has already rendered great assistance to surgery, and has
been the subject of a very favourable report at the Academy of Sciences
in Paris, was invented by its author in consequence of certain
reflections suggested to him by the perusal of our researches. The
commission which framed the report made, through M. Gosselin, some wise
reservations in the case of certain theoretical ideas which M. Guérin
had not sufficiently proved by experiment. We have no doubt, however,
that when the matter is thoroughly examined, facts will confirm the
truth of the wide views entertained by the surgeon of the Hôtel-Dieu.

Dr. Déclat has founded quite a new system for the treatment of
infectious diseases, which is based upon the use of one of the best
known antiseptics, phenic acid. His theory, which he affirms was
suggested to him by our studies on fermentations, is, that the diseases
which transmit themselves are, each of them, the product of a special
ferment, and that both medical and surgical professors of therapeutics
should make it their study to prevent exterior ferments from penetrating
into the liquids of our economy, or in the event of these ferments
having found their way into the system, to discover _antiferments_ for
their destruction, without effecting any change in the vitality of the
histological elements of the liquid or tissues.

There is no doubt that extreme caution must be exercised in dealing with
questions of this kind, as M. Sédillot has authoritatively remarked; but
at the same time it cannot be denied that the more such questions are
discussed with exactness, the more those celebrated practitioners who
originated them are confirmed in the ideas which first guided them. We
may give another example of this.

In 1874, in consequence of a communication addressed to the Academy of
Sciences by Messrs Gosselin and A. Robin, on the subject of ammoniacal
urine, we made the observation that we should endeavour to ascertain if,
in all such cases, the urinary fluids were not rendered ammoniacal by
the presence of the little ferment of the urea, which we have previously
noticed,[27] and which has since then been discussed with remarkable
intelligence by M. Van Tieghem, in the thesis which he maintained for
his doctor’s degree. The suggestion and the considerations that
justified it led to a discussion before the Academy of Medicine, in
which contrary opinions were maintained. We lost no time in submitting
these to the test of facts. We could not find a single person suffering
from ammoniacal urine, in whose case the little ferment which we
mentioned was not to be detected. Our predictions were thus completely
justified.

As early as 1864 the _Gazette Hebdomadaire de Médecine et de Chirurgie_
published an account of urine made ammoniacal in the bladder, the author
of which, Dr. Traube, makes the following observations:—“It was believed
that, in consequence of the retention of the liquid, and the resulting
distension of the bladder, that organ became irritated and produced a
larger quantity of mucus, and that this mucus was the ferment which
caused the decomposition of the urea, by virtue of a chemical action
peculiar to itself. This belief can no longer be held in presence of M.
Pasteur’s discoveries. This observer has demonstrated, in the most
conclusive manner, that ammoniacal fermentation, like alcoholic and
acetic, is produced by living beings, whose pre-existence in the
fermentable liquid is a necessary condition of the process. The
preceding fact offers a remarkable proof of M. Pasteur’s theory. In
spite of the long duration of the retention, alkaline fermentation was
not produced by an excessive secretion of the vesical mucus or of pus:
it only began from the moment when the germs of vibrios passed from the
outside into the bladder.”[28]

Finally, we may conclude with perfect truth that the liquids of our
economy, blood and urine, for instance, may afford a harbour to
different ferments, even in the inmost parts of the organs, when
external causes enable such ferments to find their way into those
liquids, and that diseases of greater or less gravity result from this
cause. On the other hand, it must be admitted that the bodies of animals
in a state of health afford no means of entrance to these external
germs. At the same time, direct experiment alone can convince the mind
as to the truth of this latter assertion. Let us take some of the
substances that are to be found inside living animals in perfect health,
and expose them, in the same condition in which life has formed them, to
contact with pure air.

[Illustration: Fig. 5.]

[Illustration: Fig. 6.]

For this purpose we must provide ourselves with a glass flask, joined to
a copper tap by means of an india-rubber tube, as shown in Fig. 5. The
two branches of the tap should be about twelve centimetres long (about
½-in.), the one which is free tapering off like the end of a pipe. In
order to destroy all germs which may exist in the flask, we must join
the free end of the copper tube to a platinum tube kept at a very high
temperature, after having carefully introduced into the flask a small
quantity of water, and expelled all the air by converting the water into
steam. Then as we allow the flask to cool, the air which re-enters it
will necessarily pass through the hot tube (Fig. 6).

[Illustration: Fig. 7.]

We may cause the water in the flask to boil at a temperature of more
than 100° C. (212° F.) by fitting to the free end of the platinum tube a
glass tube bent at right angles, which we plunge to any depth in a deep
vessel filled with mercury (Fig. 7). Whilst the water is boiling under
pressure, we must separate the tube which is plunged in the mercury; the
water in the flask will continue to boil at the ordinary pressure. We
must then leave the flask to cool. It will gradually become filled with
air that has been heated to a high temperature, more than sufficient to
burn up all the organic particles of dust which that air could have
contained. When the flask is cold we must close the tap and detach it,
and proceed to prepare other similar flasks. It will be advisable to
close the tap when the temperature of the flask is still a few degrees
above that of the surrounding atmosphere: this precaution will cause the
air in the cooled flask to have a lower pressure than that of the
external atmosphere.

During the interval which must elapse between the preparation and the
use of a flask, it is a good thing to keep the free branch of the tap
inclined towards the ground, to secure the inside of its tube from the
deposit of external particles of dust. Whether this precaution be
adopted or not, we must take care to heat this branch in the flame of a
spirit lamp just before we bring our flask into requisition.

If we have to study blood, we must take it from a living animal—a dog,
for example. We must expose a vein or an artery of the animal, and make
an incision into which the end of the free tap-branch, which has
previously been heated and allowed to cool, must be introduced and fixed
by a ligature in the vein or artery. On opening the tap, the blood will
rush into the flask; it must then be closed, and the flask placed in an
oven at a certain temperature. We have successfully accomplished these
manipulations, thanks to the kind help of our illustrious colleague and
friend, M. Claude Bernard.

The operation is nearly the same in the case of urine. The end of the
free branch of the tap is introduced into the passage of the urethra;
the tap is turned at the moment when the urine is emitted, which is then
allowed to pass into the flask, until it is a third or half filled.

The following were the results of our experiments:—Blood underwent no
putrefactive change even at the highest temperatures of the atmosphere,
but retained the odour of fresh blood, or acquired the smell of lye.
Contrary to what we might have expected, the direct oxidation of the
constituents of blood by slow combustion was rather sluggish. After
subjecting our flasks to a temperature of 25° C. or 30° C. (77° F. to
80° F.) in an oven for several weeks, we observed an absorption of not
more than 2 or 3 per cent. of oxygen, which was replaced by a volume of
carbonic acid gas of about an equal bulk.[29]

Nearly the same results were obtained in the case of urine; it underwent
no radical change; its colour merely assumed a reddish brown tint; it
formed some small deposits of crystals, but without becoming at all
turbid or putrefying in any way. The direct oxidation of the urinary
substances was likewise very sluggish. An analysis of the air in one of
the flasks, made forty days after the commencement of the experiment,
gave the following results:—

                      Oxygen                 19·2
                      Carbonic acid           0·8
                      Nitrogen               80·0
                                              ——-
                                            100·0

These experiments on blood and urine which we have just mentioned date
from 1863.[30] Ten years afterwards, in 1873, they were confirmed in an
important and striking manner by the results of a very able series of
experiments, which were carried out in our laboratory, by M. Gayon, who
was formerly a pupil in the _École Normale Supérieure_. M. Gayon proved
that what held good in the case of blood and urine, also held good in
the case of the substances contained in eggs. He found that the whites
of eggs might be exposed for any length of time to contact with air, as
also might the yolks, or the white and yolks mixed, without any
putrefactive change or fermentation resulting, and without the smallest
microscopic germ showing itself, the sole condition being that the air
must be freed from all organic particles of dust, germs of mould,
_bacteria_, _vibrios_, and other organisms which it holds in suspension.
This was only a part of the important facts brought to light by M.
Gayon. Amongst other things, he proved that spontaneous putrefaction in
eggs is invariably caused by the development of organized ferments,
thereby correcting the opposite statements announced by M. Donné and M.
Béchamp, who were led by their observations to believe that the change
in eggs took place quite independently of the action of _vibrios_ and
_mucedines_.[31]

It is almost superfluous to remark how greatly the results of these
experiments on blood, urine, and the components of the egg are opposed
to the doctrine of spontaneous generation, as also to most modern
theories on the generation of ferments. As long as experiments relating
to the question of so-called _spontaneous_ generation were made on
heated substances, the advocates of _heterogenesis_ had some grounds for
asserting that such materials could not satisfy the conditions of
spontaneous life, and that we should obtain different results by using
natural organic liquids, which, if exposed to contact of pure air would
doubtless serve for the production of new beings which did not issue
from parents which resembled them. This novel enunciation of the
hypothesis of spontaneous generation, the only one, we think, that could
be defended after the publication of our Memoir, in 1862, is condemned
by the preceding facts.

The same facts completely upset the hypothesis recently maintained by
Messrs. Fremy and Trécul on the subject of the causes of fermentation.

“Side by side with the immediate, definite principles which may be
formed by synthesis,” says M. Fremy, “such as glucose, oxalic acid, and
urea, other substances of greatly inferior stability exist, the
constitution of which is considerably more complicated, containing all
the elements of living organs, such as carbon, hydrogen, oxygen,
nitrogen, and even phosphorus and sulphur; and often salts of lime and
of the alkalies besides. These bodies are albumen, fibrin, casein, the
congeners of vitellin and others. Chemical synthesis cannot reproduce
them. It is impossible, in my opinion, to regard them as immediate,
definite principles. I designate them by the general name of
_semi-organized_ bodies, because they hold an intermediate place between
the immediate principle and the organized tissue.

“These semi-organized bodies, which contain all the elements of organs,
have the power, like a dry seed-grain, of existing in a state of organic
immobility, and of becoming active under circumstances which favour
organic development. By reason of the vital energy that they possess
they undergo a succession of decompositions, giving origin to new
derivatives, and to the advent of ferments, not by any process of
_spontaneous generation_, but by a _vital energy_, which pre-exists in
the semi-organized bodies, and is simply carried on, when this energy
manifests itself, in these most varied organic changes.”

After having expressed these hypothetical and confused opinions, M.
Fremy continues:—“I do not consider, then, that these semi-organized
bodies serve merely as nourishment for certain animal and vegetable
organisms, which may be the sole agents of fermentations, but I give
them a direct _rôle_ and admit that, under the influences which I have
already cited[32] they may assume a real and complete organization, and
produce ferments which are not derived, as we have seen, from a germ or
an ovum but from a semi-organized body, the vital energy of which has
become active.[33] It will be seen that these opinions are quite
different from those which M. Pasteur has maintained in his works, since
they attribute the origin of alcoholic and lactic ferments to an
albuminous substance. Taking the case of alcoholic fermentation alone, I
assert that, in the production of wine, it is the juice of the grape
itself which, in contact with air, produces grains of yeast, by the
transformation of the albuminous substances. M. Pasteur, on the
contrary, maintains that the grains of yeast are produced by certain
germs.”[34]

We have combated these propositions, so extraordinary and unsupported by
any rigorous experiments, before the Academy of Sciences, where they
were first enunciated. On that occasion we related the facts in
connection with blood and urine, which we have just discussed. Could
there be any more forcible argument against the theory of our honourable
colleague than those facts? Here we had natural albuminous substances,
forming part of matter eminently liable to putrefactive change and
fermentation, which produced no ferments of any sort whatsoever when
brought into contact with air deprived of its organic particles of dust.

Under no known circumstances is albuminous matter transformed into
grains of yeast or any other organized ferment, and, to our thinking,
nothing can be more chimerical than the gratuitous hypothesis of
_hemi-organism_.

We shall proceed to new proofs of this, dealing this time with a liquid
formed by the life of a vegetable.


          § III.—Experiments on the Juice contained in Grapes.


In the course of the discussion which took place, at the Academy, on the
subject of the generation of ferments, properly so called, much was said
about the oldest known fermentation, that of wine. We at once resolved
to demolish M. Fremy’s theory, by a decisive experiment on the juice of
grapes.

[Illustration: Fig. 8.]

We prepared forty flasks, capable of holding from 250 c.c. to 300 c.c.
(from 9 to 11 fl. oz.) and shaped as represented in Fig. 8. These we
filled with filtered must, which was perfectly bright, and which, like
all acid liquids, would remain sound, after having been boiled for a few
seconds, although the ends of the long curved necks of the flasks
containing the must might remain constantly open for months or years.

We washed, in a few cubic centimetres of water, part of a bunch of
grapes, washing the grapes separately, or the grapes and the wood
together, or even the wood of the bunch alone. This washing was easily
accomplished by means of a perfectly clean badger’s-hair brush, the
water receiving all the particles of dust adhering to the surface of the
grapes and the wood of the bunch. By means of a microscope we easily
proved that this water held in suspension an infinite number of
organized corpuscles, some of them bearing a very close resemblance to
the spores of fungoid growths, others to alcoholic ferment, others to
_mycoderma vini_, and so on.[35]

[Illustration: Fig. 9.]

We next proceeded to put on one side ten of our forty flasks, to serve
for subsequent corroboration; in ten others, by means of the tube which
is represented on the right hand side of the flask (Fig. 8), we put a
few drops of the water in which the bunch of grapes had been washed; in
a third series of ten flasks we put a few drops of the same liquid,
after having previously boiled it. Lastly, we introduced into the ten
remaining flasks a drop of grape-juice, taken from the inside of an
uninjured grape. To do this we had to bend the right-hand tube of each
of our last ten flasks, drawing it out to a fine point and closing it in
the flame, as represented in Fig. 9 A. This fine closed point was filed
round near its extremity and then thrust, as represented in Fig. 9 B,
into a grape placed on a hard substance; when the point _b_ was felt to
touch the substance supporting the grape it was broken off by a slight
pressure sideways at the point _a_, where the file marks had been made.
We had taken care to secure a slight vacuum in the flask beforehand;
this now caused a drop of the juice to be drawn into the flask. We then
drew out the fine point, and closed it immediately in the flame of a
spirit lamp. The vacuum was produced by heating the flasks in our hands,
or over the flame of a lamp, thus causing a little air to be forced out
through the end of the bent tube, which we then closed up with the lamp.
When the flask was cool, the slight difference of pressure sufficed to
force into it some of the juice contained in the grape, as we have just
described. The drop of juice that is sucked into the flask generally
remains in the curved part of the fine tube; to mix it with the must we
must incline the flask so as to bring the must in contact with the drop;
after that we may replace the flask in its natural position.

The following are the results presented by our four series of
comparative experiments in the different cases. The first ten flasks—our
standard flasks, containing must boiled in contact with pure air—showed
no signs of organized products; the must might have remained in them for
any number of years without change. Our second series of flasks, which
contained the water in which the grapes, separately and in bunch, had
been washed, had undergone alcoholic fermentation in every instance;
this had manifested itself in all the flasks in the course of about
forty-eight hours, the temperature being at about summer heat. At the
same time that the yeast made its appearance in the form of little white
lines, which gradually joining together formed a deposit on the sides of
the flask, we perceived minute flakes of _mycelium_ forming; sometimes
as a single fungoid growth, sometimes combined with another, or with
many together—these growths being quite independent otherwise of the
yeast or alcoholic ferment. In several cases, too, _mycoderma vini_
showed itself on the surface of the liquid in the course of a few days.
_Vibrios_ and lactic ferments, properly so called, could not make their
appearance, on account of the nature of the liquid.

The flasks of our third series, containing the water in which the bunch
of grapes had been washed, and which we boiled before our experiment,
remained as free from change as the flasks of our first series had done.

Lastly, our fourth series of flasks, containing the drops of juice taken
from inside the grapes, remained equally free from change, although we
could not be certain of having removed, in every case, without
exception, all causes of error which must inevitably occur sometimes in
so delicate an experiment.

These experiments cannot leave the least doubt on our minds:

That must, if boiled, will never ferment when in contact with air that
has been freed from the germs which exist in it in a state of
suspension.

That must may be fermented, after boiling, by introducing into it a very
small quantity of water, in which a bunch of grapes has been washed.

That must will not ferment if we introduce into it some of this same
water which has been boiled and afterwards cooled.

That must will not ferment if we introduce into it a small quantity of
the juice contained in a grape.[36]

It follows, then, that the ferment which causes grapes to ferment in the
vintage tub must come from the exterior, and not the interior of the
grapes. Thus, the hypothesis of MM. Trécul and Fremy, according to which
albuminous substances transform themselves into grains of yeast by the
action of a peculiar vital force, is annihilated; _à fortiori_, there
can no longer be a question concerning Liebig’s theory, on the
transformation of albuminous substances into ferments, by a process of
oxidation.

Our readers may be curious to know what M. Fremy has been able to oppose
to such crucial experiments; they could scarcely have imagined the
following:—

“In my experiments, which I have varied in every possible manner,” says
that gentleman, “I have found that it is almost impossible to discover
alcoholic fermentation, appreciable by its results, in a single drop of
grape juice, and I may add that this fermentation must be still more
difficult to discover when this drop has been drowned in a large
quantity of juice that has been previously boiled.”[37]

It will be admitted that we were justified in saying, at the
commencement of this paragraph, that we should demolish the theory which
was opposed to ours, and which its advocates have been constrained to
defend by hypotheses manifestly false.

At the meeting following the one in which M. Fremy declared _that minute
quantities do not ferment_, we had the malicious satisfaction of showing
a great many very small closed flasks, into each of which we had caused
a single drop of the must of crushed grapes to be introduced by suction.
We broke the thin points of many of them, in the presence of the
Academy, and every one of them showed by a sharp hissing, which was
audible at a distance, that fermentation was proceeding in the drop of
liquid they contained. M. Fremy was there, but he made no remark.

We may cite some very curious facts on the subject of the period at
which the germs that develop yeast are in a condition to be able to
cause fermentation.

On July 25th, 1875, in the neighbourhood of Arbois (Jura), the grapes
were still green and of the size of peas. We went to a vine that was far
from paths and roads, and there, with a pair of small scissors, cut some
grapes from off a bunch and let them fall with their short stalks into
tubes half filled with gooseberry must, previously rendered unalterable
by boiling. These tubes we closed again with all possible precautions,
using corks which had been passed through the flame of a spirit-lamp and
carried them to our laboratory, where we left them to themselves. Some
days afterwards we saw diverse fungoid growths appear in most of the
tubes, but not one of them then, or subsequently, presented the least
appearance of fermentation. The germs of yeast at that period of the
year did not exist either upon the woody part of the bunch, or upon the
grapes. In Chapter V. we shall return to observations on this subject.


               § IV.—Wort and Must Exposed to Common Air.


If the principles which we have laid down possess all the value that we
attribute to them, if the cause of change in natural or artificial
organic liquids does not exist in those liquids themselves, if change
considered in itself depends upon the nature and number of the particles
of dust in various places, if it is, besides, radically affected by the
composition of the liquids, it must necessarily follow that wort or
must, whilst, under certain circumstances of exposure to air, it remains
absolutely free from life and its results, will, under other
circumstances, give rise to a variety of organisms and their
corresponding fermentations. This is, in fact, the lesson which direct
proofs will teach us. Before entering upon these new observations in
detail, we must call the reader’s attention to the difficulty, as
experience has shown, of interpreting correctly the facts connected with
the spontaneous impregnations of organic liquids.

Gay-Lussac crushed some grapes under a bell-jar filled with mercury,
after having washed them in hydrogen, to expel the air adhering to the
grapes and the sides of the jar. Having waited for several weeks without
detecting any signs of fermentation, he introduced some bubbles of
oxygen, and fermentation showed itself the following day. Gay-Lussac
concluded that the fermentation of must could not commence without the
help of oxygen.[38]

Under the conditions of his experiment nothing could be truer, and we
must admire the diffidence with which this great natural philosopher
interpreted the fact that he had observed. Another French natural
philosopher, however, M. Cagniard-Latour, observed that the ferment of
alcoholic fermentation was a little cellular plant. What was its origin
in Gay-Lussac’s experiment?

The advocates of the doctrine of spontaneous generation were ready with
their explanation, and we have seen how MM. Trécul and Fremy, following
many others, did not hesitate to maintain that the little plant with all
its particles was produced by the action of oxygen on the albuminous
substances contained in the juice of the grapes. The experiments, which
we have given in the preceding paragraph, show us positively that germs
of the ferment of must exist on the surface of the grape, and that,
consequently, Gay-Lussac’s experiment has a more simple and natural
explanation. The germs of the ferment existing on the surface of the
grape become mixed with the juice of the grape when the latter is
crushed; these germs remain inactive in the presence of hydrogen; they
vegetate as soon as oxygen is introduced to them.

Moreover, the results of our labours in connection with _spontaneous_
generation, in 1862, teach us that in Gay-Lussac’s experiment the germ
of the ferment might also have had its origin either in certain
particles of dust adhering to the sides of the glass bell, or upon the
mercury; and, in a laboratory where alcoholic fermentation is studied,
dust invariably contains dry cells of ferment. The necessity of oxygen
for the success of the experiment is surprising, when we reflect that
alcoholic fermentation often takes place in liquids that are not exposed
to contact with air; but we shall prove by experiment that,
notwithstanding what may happen during fermentation, oxygen has the
greatest influence on the readiness with which ferment develops itself,
and that this gas is indispensable to the revival of withered cells, and
still more so to the germination of special cells, which we may consider
to be the true germs of the little plant.

The advocates of the doctrine of _spontaneous_ generation have based
most of the objections which they vainly urge against their opponents
upon erroneous interpretations of certain facts relating to the
spontaneous impregnation of organic infusions. Taking a very wrong view
of the essential conditions of the phenomena, they require that the
assertors of the diffusion of the germs of microscopic organisms should
be compelled to place at any one point of space, so to say, all the
germs of the products of infusions; a demand which really borders on
absurdity. They believe, or feign to believe, that we are bound to admit
the existence of germs of must in all places and at all times, on the
banks of rivers and on the loftiest mountains, and so on.
“Fermentations,” said one of these gentlemen one day, before the
Academy, “cannot depend upon chance particles of atmospheric dust. How
is it possible that germs of yeast can be present everywhere throughout
the universe, ever ready to fall upon must?” It is an established fact
that grapes crushed in any part of the globe whatsoever, even on a
glacier or at the highest elevations, can set up a fermentation. The
explanation of this pretended impossibility is most simple, for we know,
from the facts related in the preceding paragraph, that grapes carry on
their skins the germs of their own ferments.

In experiments relating to the kind of organisms which we are discussing
we must never fail to take into account the action of the particles of
dust spread over the articles that are used. Very often an effect that
should be attributed to germs adhering to the vessels and utensils used
in experiments, the origin of which may be altogether special, is
erroneously imputed to the dust-forming germs—that is to say, to those
germs which exist in a state of suspension in the air.

In our Memoir of 1862, which we have quoted several times, we have
explained that it is almost impossible to draw any serious conclusions
from experiments made in a basin of mercury, because of the organic
particles of dust which always exist in that metal, and which, without
the knowledge of the operator, pass into the interior of the vessel,
where they produce certain changes which one is tempted to impute to
_heterogenesis_.

In all classical works an experiment of Appert’s, reproduced by
Gay-Lussac, is given. This, through a faulty interpretation, led to the
hypothesis of the continuity of the causes of fermentation, if we may
use such a term, in the atmospheric air.[39]

When we decant bottles of must, which has been preserved by Appert’s
method, into other bottles, all the latter soon set up a fermentation:
this constitutes the experiment. If it were proved that the must, whilst
being decanted, came in contact with atmospheric air alone, as
Gay-Lussac believed, we should be compelled to admit, according to the
theory of germs, that the must had come in contact with some particles
of ferment in the air during decanting. And again, if it were shown that
the experiment could succeed in any place whatsoever, we must come to
the conclusion that germs of ferment exist everywhere in a state of
suspension in the air.

“I have taken,” writes Gay-Lussac, “a bottle of must that had been
preserved for a year and was perfectly transparent, and have decanted
the must into another bottle, which I then carefully corked and exposed
to a temperature of 15° C. to 30° C. (59° F. to 86° F.). Eight days
afterwards the must has lost its transparency; fermentation has taken
place in it, and soon my must has become transformed into a vinous
liquor, sparkling like the best champagne. A second bottle of must that
had been preserved for a year, like the preceding one, but which had not
been brought into contact with the air, has presented no signs of
fermentation, although placed under conditions most favourable to its
development.”

The result of this experiment, when roughly made, is correctly described
by Gay-Lussac; in other words, it may be proved that if, at the time of
vintage, we prepare some bottles of must, after Appert’s process, and,
in the course of time, open them and decant their contents into other
bottles, we shall soon see the must ferment and deposit yeast. It is,
nevertheless, equally certain that the inferences which have been drawn
from this celebrated experiment have been founded on error, and that the
germs of yeast are very rarely derived from the particles of dust
floating in the air with which the must comes in contact. The germs in
question are, in our opinion, generally derived, not from the air, but
from the sides of the bottles, from corks, from the string employed in
corking, from corkscrews, and from a variety of other things. The reason
for this is that any room, vault, cellar, or laboratory where the
grapes, or must, or vintage, are handled—unless special precautions, of
which Appert and Gay-Lussac certainly never thought, are taken—all the
utensils, as well as all articles of clothing, and all the sides of the
bottles which the hands have touched, are contaminated by cells of
ferment derived from must that has fermented, or by germs from the
surface of grapes and clusters. Thus, at the moment of decanting the
must, a thousand accidental circumstances may lead to the introduction
of those germs, the origin of which, as we have seen, may be actually
traced to the very grapes which served for the manufacture of the must.
In other words, we believe the inference that the germs of yeast which
cause the experiment to succeed, are derived from particles of dust
floating in the air of the place where we decant the contents of our
bottles, to be altogether an erroneous one.

Since the preceding remarks were written, we have endeavoured to repeat
this experiment of Gay-Lussac’s in such a manner as we believed would
confirm our views, by varying the conditions in such a way as would
cause it to succeed or fail, according to the circumstances of the
manipulation employed.

On December 7th, 1874, we took two bottles of must which we had
preserved, after Appert’s process, in our laboratory from the beginning
of October, 1873. Both of these were covered with dust—the dust that
floated about in our laboratory. We decanted them as follows:—One
bottle, which we handled without special precautions, we uncorked by
means of an ordinary corkscrew, and decanted into another bottle that
had been well washed, as bottles are washed when they are to be used
subsequently. This bottle was taken from a number that had been standing
upside down on a drainer for a fortnight. We took no precaution to
remove the dust which covered the exterior of the bottle of must, or to
purify the washed bottle. The second bottle of must, on the other hand,
was decanted after we had removed the dust that covered it; its cork was
cut off close to the string, and the flame of a spirit lamp was passed
over the string and the surface of the cork; and, as a final precaution,
the corkscrew was passed through the flame. As for the bottle into which
we subsequently decanted the must, we first plunged it in a hot-water
bath kept at 100° C. (212° F.), then took it into a garden to cool
upside down in the open air. After these precautions we removed it, and
immediately decanted into it the must from the second bottle.

The first bottle showed signs of growths, both on the surface and at the
bottom of the must, the day after the operation, and manifested the
first symptoms of alcoholic fermentation on December 16th. The contents
of the second bottle remained perfectly unchanged after being exposed to
the warmth of a stove for several months.

Can anything be more conclusive than these facts? They are in perfect
keeping with the views that we have recently expressed, and with the
principles that we have maintained for nearly twenty years, on the
subject of the causes of change in organic liquids.

It is by no means our intention to assert that in the atmospheric air
there exist no germs of ferment in a state of suspension, as fine dust.
Beyond all doubt they do so exist in that state; but, as a rule, in
comparatively small number, their abundance or scarcity being dependent
upon circumstances which control their multiplication, favouring or
restricting it, as we are about to prove.

On May 2nd, 1873, we uncorked two ordinary bottles filled with wort,
prepared in December, 1872, after Appert’s process. To avoid the causes
of error which we have mentioned, we uncorked the bottles in the
following manner:—The cork was cut off to the level of the neck; the
cork and string were next passed through flame, regardless of burning
and charring them; we then gently extracted the cork by means of a
corkscrew which also had been passed through the flame.

The bottles thus prepared were placed on the table of an underground
room, in which we were continually making experiments on alcoholic
fermentation.

_1st bottle._—On May 7th we observed little particles of fungoid growth
on the surface of the liquid, and at the bottom were large flakes of
_mycelium_.

On May 11th a veil of _mycoderma vini_ had formed: there were no signs
of fermentation.

On May 13th vigorous fermentation commenced; it lasted until May 23rd.
The microscope revealed yeast in globules of two sizes, the larger of
which were considerably less numerous than the others. There were no
signs of lactic or butyric ferment.

_2nd bottle._—May 7th, particles of fungoid growth on the surface of the
liquid, and also a veil of _mycoderma vini_. On May 11th, 13th, and up
to the 23rd, no signs of fermentation were visible. On May 30th
fermentation was active. The microscope showed us yeast mixed with
butyric _vibrios_.

In this case, alcoholic ferments had come into existence, and from the
precautions taken at the moment when the liquid was brought into contact
with the external air, it is certain that the advent of the germs of
those ferments, as also those of the other organisms which made their
appearance—the fungoid growths, _mycoderma vini_ and _vibrios_—could
only be accounted for by the fall of particles of dust floating about in
the room. It follows, then, that under certain circumstances germs of
alcoholic ferment may be found floating in the air; but we can readily
show that the peculiar conditions of the place had a large share in
bringing about the results obtained by the foregoing experiment.

The same day, May 2nd, 1873, we uncorked, with the precautions that we
have already described, four other bottles of the same must. These were
placed in a room which was used less frequently than the preceding one,
and in which experiments relating to fermentation were seldom conducted.

_1st bottle._—On May 8th we observed on the surface of the liquid large,
frothy pieces of mycelium (_mucor mucedo_ or _mucor racemosus_). The
liquid was perfectly bright.[40]

May 30th.—No signs of actual fermentation yet visible.

_2nd bottle._—On May 8th we noticed a thin, greasy-looking scum on our
liquid, which had become turbid and acquired a sour smell. The
microscope showed that this scum was formed of _mycoderma aceti_. On May
30th the scum had assumed a whitish appearance, and seemed to be dead;
there was a green spot of _penicillium glaucum_ upon it. No signs of
fermentation.

_3rd bottle._—May 8th, patches of fungoid growth on the surface of the
liquid. May 30th, thick and abundant fungoid growth, but no
fermentation.

_4th bottle._—May 8th, little patches of fungoid growth, and a scum of
_mycoderma vini_. May 30th, still no fermentation.

Up to the month of August, 1873, not one of these bottles gave the least
sign of alcoholic or other fermentation.

On December 16th, 1872, we uncorked four bottles of wort, which also had
been preserved by Appert’s process; these we placed on an oven, where
there were always vessels fermenting, at about 25° C. (77° F.), but
where none of the manipulations required for the starting or final study
of fermentations were practised. The next day a fungoid growth, but
unaccompanied by any signs of fermentation, made its appearance, and
this state of things lasted for five months, after which we ceased to
keep these bottles under observation.

On May 26th, 1873, we uncorked, with all the necessary precautions, ten
bottles of wort, which had been preserved from April 9th, and then left
them undisturbed in a room where we were constantly engaged in the study
of fermentation.

On the following day, some patches of fungoid growth appeared on the
surface of the liquids.

May 30th.—Fermentation commenced in one of the bottles.

May 31st.—A second bottle likewise began to ferment.

June 9th.—Four bottles, including the two preceding ones, were now in a
state of fermentation. The six bottles that had not fermented were
thereupon covered with caps of paper, taken from the centre of a ream of
paper and passed through a flame. After this, and up to August 1, when
our observations were discontinued, these six bottles underwent no
fermentation.

From these examples, which are confirmed by many others that we shall
have occasion to mention in the course of this work, it will be seen
that the germs of alcoholic ferment are not present in every little
point of space, constantly ready to fall upon any object, not even in
those places where one is perpetually dealing with that kind of
growth.[41] If we conduct our experiments with exactness, we very soon
learn that all that has been written on the facility with which
saccharine musts may be made to ferment, by being rapidly brought into
contact with the surrounding air, is greatly exaggerated.

The germs of ferments, especially of alcoholic ferments—the yeast of
beer and the yeast of wine—are not nearly as abundant in atmospheric
air, or in the particles of dust spread over the surface of things, as
are the spores of fungoid growths. It is easy to understand this, for
spores are generally borne by aerial organs in a state of dryness, so
that the least breath of wind catches them up and carries them away,
whilst ferments are composed of moist cellules that do not readily
become dry.

The vacuous flasks, partly filled with organic liquids, which are opened
and closed again immediately, frequently give us fungoid growths, but
very rarely alcoholic fermentation, although in the latter respect they
may not be absolutely sterile. We may cite some proofs of this.

On June 19th, 1872, we prepared seven flasks of saccharine liquid,
impregnated with yeast—our flasks were of 300 c.c. capacity (about 10
fl. oz.)—and contained 100 c.c. of the liquid; we then drew out their
necks to a small opening, which was sealed during boiling, after the
steam had expelled all the air.

On June 29th we opened them in the principal room of our laboratory.

On July 9th, two of the seven flasks gave no sign of organized products;
the others were swarming with _mycelia_, submerged or fruiting on the
surface of the liquid, and either with or without _bacteria_ entangled
in their flakes. In two of the flasks there were visible at the bottom
of the liquid some white streaks, which is an indication sometimes of
the presence of alcoholic ferment, but much more frequently of a little
cellular plant resembling it in appearance, but purely aerial in its
growth, that is to say, taking no part in fermentation. Some days
afterward, we saw bubbles of gas rising from the bottom of one of these
flasks, and then fermentation proceeded so rapidly that we were obliged
to open the neck to avoid an explosion. We append a sketch of its
ferment (Fig. 10).

[Illustration: Fig. 10.]

The other flask with the white streaks showed no signs of any
fermentation.

In this kind of observation we rarely succeed in obtaining active
ferments, the reason being that we deal with volumes of air that are too
limited for the few germs of ferment that exist in a state of suspension
in it.

We are more sure of success if we expose a tolerably large surface of
saccharine liquid to the open air, because, under such circumstances,
even if the exposure is of short duration, a considerable volume of air
will pass over the surface of the liquid.

On May 29th, 1873, at five o’clock in the afternoon, we placed in the
underground room previously mentioned, at a height of about two feet,
ten porcelain dishes having surfaces of from thirty-five to forty square
inches. We had just taken them from boiling water, and after allowing
them to cool we placed in each quantities of wort to about one-third of
an inch deep, which we poured from bottles uncorked with every
precaution against the chance of the wort coming into contact with
anything besides the floating particles of dust. On May 30th, at five
o’clock, that is after twenty-four hours of exposure to the air of the
room, we emptied the contents of the basins separately into glass flasks
with long necks, which had been treated with boiling water and then
cooled, necks downwards. The beak of the basin, by means of which the
liquid was poured, and the funnels—for we used a separate funnel for
each flask—had been passed through the flame. The whole ten flasks were
then placed in an oven at a temperature of 25° C. (77° F.).[42]

On June 1st six of the flasks gave signs of fermentation, and next day
all the flasks were fermenting.

The following are some of the numerous microscopic observations which we
made on the liquids and their deposits.

On June 1st the liquid in one of the six flasks which had begun to
ferment was covered with a continuous scum of _mycoderma vini_, below
which appeared a filamentous network belonging to a _mycelium_, or other
fungoid growth. But neither in the liquid itself nor in the deposit
could we perceive any cells of the ferment of beer; the field, however,
was swarming with active butyric _vibrios_, rather thick and short,
their length being about twice their diameter. This fermentation was
exclusively butyric.

On June 2nd another of the flasks showed no _vibrios_, but alcoholic
ferment in small quantity, much lactic ferment, consisting of little
particles contracted at the middle and non-mobile, and, finally, some
slender filaments, resembling those represented in Plate I., Nos. 1 and
2.

On June 3rd we examined the liquid in the flask which showed the most
marked fermentation. In addition to the flakes of fungoid growths,
checked in their development through want of air, we found at least five
distinct productions, which are represented in the accompanying sketch
(Fig. 11).

    _aaa._—Thick cells of alcoholic ferment, the size of which is given
    in our sketch: thus, 13/450 indicates that the corresponding figure
    is 13/450 millimetre in length (rather more than 1/1000 inch).

    _bbb._—Small alcoholic ferment, such as we generally see in the must
    of acid and sugared fruits—especially in filtered grape must. Its
    dimensions varied from 1 to 1-½, or 2, 450ths of a millimetre.[43]

[Illustration: Fig. 11.]

    _ccc._—“Low” yeast, of a type resembling that existing in other
    preparations fermenting in the room.

    _ddd._—Enlarged, distended spores of _mucor racemosus_. These are
    scarce, and have an old appearance. We shall come to them again in a
    subsequent chapter, where we shall explain their real significance.

    _eee._—Short _vibrios_, occurring either contracted or not near the
    middle. Some were motionless, others vibrating to and fro and
    executing other movements. These forms belong to the butyric and
    lactic ferments shown on Plate I.

The preceding series of experiments shows us that, in the case of wort
exposed to the air, germs of divers organisms, amongst which various
ferments—butyric, lactic, and alcoholic—are to be found, fall
simultaneously from the particles of dust floating in the air. We must
observe, however, that we were dealing with the air of a laboratory in
which we were constantly studying analogous fermentations, and that a
different atmosphere would, most likely, give us different results. We
shall see a proof of this in the following paragraph, where we shall
also find some new facts tending to prove that the germs of alcoholic
ferment do not exist amongst the particles of dust floating in the air,
in anything like the quantity usually supposed.


§ V.—New Comparative Studies on the Germs held in Suspension by the Air
    of Different Places which are near each other, but Subjected to
   Different Conditions affecting the Production and Diffusion of the
                    Particles of Dust found in them.


We may compare the character and the greater or less abundance of
similar germs existing in neighbouring localities, by studying the
changes which take place in similar liquids exposed simultaneously to
the action of the air in those localities. To do this, we must prepare a
large number of flasks of the same size, free from air, and containing
about equal quantities of a particular liquid—the same being used for
all. We must open the same number of these flasks in each of the
localities we have selected, and permit the air with all its particles
of dust to rush into them; then we must close our flasks again, and
observe, day by day, the appearances they present. The results obtained
by these means will not furnish us with conclusions applicable to every
kind of germ that the air contains at any given moment, but only with
conclusions which apply to those germs which can develop in the
particular liquid employed. Thus, for example, we could draw no
inference as to the nature and relative number of _bacteria_ or
_vibrios_, in the case where we employ an acid liquid; for organisms of
that kind we must have recourse to neutral or slightly alkaline
infusions. On the other hand, liquids having a feeble acid reaction
would favour the growth of _mucedines_, _mycoderms_, and certain
ferments, as for example, the alcoholic.

On November 26th, 1872, we opened and reclosed thirty flasks containing
must kept from the last vintage.

Ten flasks were opened at the bottom of the garden of the _École
Normale_.

Ten on the landing of the second floor.

Ten in the principal room of our laboratory, which had been swept out
shortly before, by which operation the dust of the floor had been raised
and put in motion.

Different objects made their appearance in a certain number of the
thirty flasks on the following days; but from December 17th, things
remained stationary. The following observations were made at that date:—

Of the ten flasks from the bottom of the garden, only one had undergone
any change.

Of the ten flasks from the interior of the building, four had undergone
change.

Of the ten flasks from our laboratory, all had undergone change.

The difference in the number of germs held in suspension in the three
different places whence we had taken our air was, therefore,
considerable.

The difference in the nature of the germs was equally marked. Those
flasks of our first two series which had undergone change presented no
trace of _torulæ_, or anything besides fungoid growths, whilst three of
the last ten contained _torulæ_ associated with fungoid growths.[44]

[Illustration: Plate 3. Torulæ in Process of Development.]

On May 29th, 1873, eighteen flasks, free from air and with necks drawn
out to a fine point, containing must, were taken into one of our rooms
at the _École Normale_. A jet of gas from an ordinary burner was passed
over the surface of the glass down to the surface of the liquid, with
the object of burning any particles of dust that might have been
deposited from the atmosphere of the laboratory; the points of the
flasks were then broken with a pair of ordinary scissors that had been
passed through the flame of a spirit lamp; and, lastly, the tops of the
flasks were taken off, just above the surface of the liquid, and the
eighteen flasks thus became transformed into eighteen basins, each
containing about 100 c.c. (about 3-½ fluid ounces) of grape must. These
eighteen basins were placed on a table in the room, and left there for
five days, precautions being taken to prevent any one from entering the
room.

The basins were examined on June 2nd: they all contained little flakes
of floating _mycelium_, but none of them had any white streaks on their
sides—a proof that they were destitute of _torulæ_—the liquid had
remained very bright. With the contents of nine of the basins we filled
two long-necked flasks that we had prepared for our purpose—that is to
say, had heated to a certain point, just before using them, with the
object of removing from their sides any foreign germs which they might
have picked up in the laboratory. Up to July 10th, when we deemed it
useless to carry our observations further, these flasks presented no
signs of fermentation whatever. With the contents of the remaining nine
basins, we filled two other long-necked flasks, but before doing so, we
kept the basins for twenty-four hours (June 2nd to 3rd) in the basement
of our laboratory. These two flasks soon set up an active fermentation,
and deposited an abundance of yeast—an additional proof of the great
difference in character of the germs floating about the room and those
floating about the laboratory.

On June 3rd we exposed, simultaneously, in the aforementioned room and
also in our laboratory, seven basins prepared as just described. On June
8th all the basins in the room showed signs of fungoid growths, without
any trace of _mycoderma vini_, or lines indicative of the presence of
_torulæ_, whilst six of the seven in our laboratory had their sides
covered with a white precipitate, and on the surface of the liquids a
layer of isolated patches of fungoid growth. The liquid in these latter
basins was poured into a long-necked flask, which it nearly filled, and
in the course of forty-eight hours it began to show signs of alcoholic
fermentation.[45] This is another striking proof of the difference
between the number of germs of ferment and _torulaceæ_ in the air of our
laboratory and that of an ordinary room.

[Illustration: Fig. 12.]

We append drawings of the _torulæ_ found in the six laboratory basins
(see Sketches I., II., III., IV., V., VI. of Fig. 12). The abundance of
the germs of these organisms in our laboratory is very striking, and is
doubtless due to the nature of the work carried on there, as well as to
the power of endurance peculiar to the germs, or the minute vegetative
cells of these microscopic plants—a tenacity of life which prevents them
from losing their reproductive powers, even after being dried up into
dust. But varied as are the formations represented in Fig. 12—and it
will be observed that in IV. and VI. we have shown four distinct forms,
marked _a_, _a_; _b_, _b_; _m_, _m_; _n_, _n_ respectively—it must not
be supposed that they correspond necessarily to distinct species. From
the ends of a compound organism like those in No. III, a little
spherical cell may detach itself and then, by a process of budding, give
rise to a series of other minute spherical cells reproducing the form
shown in Nos. I. and II.

The forms figured in No. III. represent one of the types of _mycoderma
vini_, which is often found in this branching arborescent state; but it
frequently also occurs in short forms, and it is in this shape that it
is generally met with on the surface of wines.

It is true that the nature of the substratum has a great influence on
the changes of aspect in the organisms which we are describing, but this
is not the sole cause of their morphological modifications. We are
strongly inclined to believe that each of the cells, or vegetating forms
so represented, differing so greatly as they do in aspect, and begotten,
all of them, spontaneously in certain appropriate liquids, in a
laboratory where researches on fermentation are pursued, is capable of
furnishing a distinct variety. In fact, there is not a single one of the
cells in the six varieties in Fig. 12, which, taken alone, has not its
own peculiar characteristics, which, by hereditary transmission, it can
impart, in a greater or less degree, to all the individuals of the
generations that succeed it.

We may remark, on the other hand, that nothing can be more favourable to
the isolation of different varieties of _torulæ_ or _mycoderma vini_
than the spontaneous impregnations to which we submit our liquids. For
when suitable liquids contained in flasks exhausted of air are
impregnated with the particles of atmospheric dust, by opening the
flasks for a moment and then immediately re-sealing them, it must
generally happen that we admit only one species of reproductive
organism, so that we shall have a vegetation exclusively of one kind, as
being derived from the same mother-cell. If we could take from a crowd
composed of men and women separate couples, and forthwith transport each
couple to a separate isolated and unpeopled island, they would, in the
course of time, beyond doubt form so many distinct tribes.

It is very remarkable that some of the _torulæ_ in Fig. 12 are not
ferments; they do not cause sugar to decompose into alcohol and carbonic
acid, any more than the _mycoderma vini_ does; but, nevertheless, there
may be an absolute similarity in aspect, development, shape, and size
between the alcoholic ferment, properly so called, and these
_torulaceæ_.

We must here cite a case in proof. On May 28th, 1872, in one of the
rooms of our laboratory, we broke the fine points of a series of flasks,
similar to those used in our previous experiments, containing must of
grapes and deprived of air. We then closed up the ends immediately after
the sudden entrance of the exterior air. One of these flasks developed
only one kind of organism, which belonged to the _torulaceæ_. On June
7th this was sufficiently abundant to cover all the sides with a white
deposit, and the surface of the liquid appeared quite free of any
_mycoderma vini_. To assure ourselves that we were actually dealing with
one kind of _torulæ_, unattended by fungoid growths, we waited until
June 14th, but the aspect of things remained unchanged. On that day we
opened the flask; there was no escape of gas to indicate that the
interior pressure was greater than the exterior. We then subjected the
plant to microscopic examination. It was quite homogeneous, and formed
of a mass of cells, absolutely identical in aspect and size with old
cells of ordinary yeast (Fig. 13).

[Illustration: Fig. 13.]

We distilled all the liquid, of which there was about 100 c.c., (3-½ fl.
oz.) without obtaining any trace of alcohol in our distillate; we
collected 33 c.c. (about an ounce) of the distillate, which we distilled
again, and we submitted the distillate a third time to distillation; but
even then there were no more signs of the presence of alcohol than there
had been in our first distillate.[46]

We may safely conclude that our _torula_, in the course of its
development in must, with a weight that would have been very
appreciable, did not produce, by its action of multiplication 1/10,000
of 1 c.c. of alcohol.

Under the following conditions we obtained a slightly different result,
which, nevertheless, confirmed the preceding one.

On July 5th, 1872, we opened and closed twelve flasks similar to those
we had used before, the sole difference being that they contained yeast
water[47] sweetened with ten per cent. of sugar. One of these flasks
furnished us similarly with one kind of _torula_, which bore the
greatest resemblance to the ferment of beer. When this _torula_ was
beginning to spread all over the bottom of our flask, we shook up the
liquid, and turned the flask upside down, with the object of submerging
the torula and depriving it of air, at least at the bottom of the neck.
For some days, and even months, there were no signs of the liberation of
gas. On July 22nd, 1873, after the interval of a year, we opened the
flask (which gave no indication of the existence of an interior
pressure) and endeavoured to discover the presence of alcohol, by means
of successive distillations, as just described. In the two first
distillates there seemed to be no alcohol, but in the third we detected
its presence in very small quantity. We shall see, later on, that the
formation of such a small quantity of alcohol may be attributed to the
fact of the plant having been submerged when in full growth, and to its
having continued to live for some time after its submersion quite
independently of the oxygen contained in the air of the flask.

From the previous facts it is obvious that there exist certain
productions of various aspects, the germs of which are particularly
abundant in the dust of a laboratory where the phenomena of fermentation
are studied, productions essentially aerial, and incapable of giving
rise to fermentation, although it may be impossible for the microscope
to distinguish their forms from those of true alcoholic ferments.

The idea of some physiological bond between these plants and the
ferments which resemble them in so remarkable a manner, is one that
impresses itself forcibly and, so to say, instinctively upon the mind.
This remark holds good also in the case of _mycoderma vini_, properly so
called, when compared with alcoholic ferments. There appears to be no
other difference between the _mycoderma vini_ and the _torulæ_ of which
we are speaking, than that afforded by peculiarities of physical
structure and a certain greasiness in the cells of the former which
permits it to exist, in the form of a scum, upon the surface of liquids,
that is to say unsubmerged.[48]

We have frequently, but without success, endeavoured to bring about the
conversion of these unsubmerged _torulæ_ and _mycoderma vini_ into
alcoholic ferments; in other words, we have never succeeded in imparting
to these _torulæ_ or to _mycoderma vini_, which bear so striking a
resemblance to alcoholic ferments, the permanent fermentative character
peculiar to the latter. At one period of our researches, in 1862, and
more recently, in 1872, we thought that we had discovered the conditions
under which such conversion might be possible, but, as we shall explain
in a subsequent chapter, our experiments were affected by certain errors
that had escaped our notice.


  § VI.—Yeast may become Dry and be Reduced to Dust without losing its
                        Faculty of Reproduction.


In the preceding paragraphs we have given examples of liquids becoming
impregnated with self-sown alcoholic ferments. We shall proceed to show
that this little cellular plant may actually exist in the form of dust,
floating in the air, after the manner of spores of fungoid growth and
the encysted forms of certain infusoria, without losing its powers of
reproduction.

On December 16th, 1872, we collected and pressed all the yeast resulting
from a brewing of about one hectolitre (about 22 gallons). From the
centre of the cake we took a few grammes (50 or 60 grains) of yeast,
which we mixed in a porcelain mortar with five times its weight of
plaster—both mortar and plaster having been heated, just before, in an
oil bath, to a temperature of about 200° C. (392° F.), and then cooled
rapidly. The powder thus prepared was immediately done up in a twist of
paper, which had been passed through the flame of a spirit lamp, and the
twist and its contents were then placed in an oven at a temperature of
from 20° C. to 25° C. (about 75° F.). The object of these several
precautions was to free the powder composed of the yeast and plaster, if
not from the germs contained in floating particles of dust, at all
events from those contained in the dust existing on the surface of the
articles we used—the mortar, plaster, and paper.

[Illustration: Fig. 14.]

On December 18th, we took up with a platinum spatula, previously passed
through the flame, a pinch of the yeast-and-plaster powder, and sowed it
in a two-necked flask (Fig. 14) containing some pure wort. We then
placed the flask in an oven, at 20° C.

On December 21st, three days after we had sown the powder, fermentation
began to manifest itself by the appearance of patches of froth on the
surface of the wort. On December 19th and 20th the yeast was sensibly
developing, although there was no liberation of gas to denote the
presence of actual fermentation. The yeast, examined under the
microscope, appeared very pure.

On March 5th, 1873, we took another pinch of the yeast-and-plaster
powder from the twist of paper, and placed it in a flask of pure wort,
as in the foregoing experiment.

On March 9th, that is, after having been subjected to a heat of 20° C.
(68° F.) in the oven for four days, fermentation began to manifest
itself by the appearance of patches of froth on the surface of the wort.
From this it was evident that the yeast had not been destroyed, but only
retarded in its revival.

On July 25th, 1873—that is, after a lapse of seven months and a half—we
resumed our experiments, and sowed some more of the yeast-and-plaster
powder in another flask of wort. On August 2nd, eight days from the time
of our sowing, the little islets of froth appeared on the surface of the
liquid. Observed under the microscope, the yeast still seemed pure, and
resembled the original yeast; we append a sketch, which will give an
idea of its shape (Fig. 15).

[Illustration: Fig. 15.]

On November 7th, 1873, we once more sowed some of the powder. This time
the yeast was dead; we observed the flask which contained it, day by
day, until February 1st, 1874, without detecting the slightest sign of
fermentation or development of the yeast that we had sown. On February
1st, we made a microscopical examination of the yeast, and found it
mixed with the plaster and absolutely inert at the bottom of the
saccharine liquid; its cells were isolated, very old-looking and
granulated, without any appearance that might denote the possibility of
their ever budding again.

Thus we determined that alcoholic ferment may be dried at the ordinary
temperature of the atmosphere, and preserved, in the form of dust, for a
period of seven months or longer, without losing its faculty of
reproduction. This faculty evidently diminishes in the course of time,
for our dried yeast, after having been kept for seven months and a
half—all the other conditions of the two experiments having been
precisely the same—required about eight days to develop sufficiently to
reveal fermentation, whilst, immediately after the drying, it only
required three or four days to accomplish the same thing.

[Illustration: Fig. 16.]

Side by side with these experiments on alcoholic ferment, we carried on
exactly similar ones with yeast obtained from “high fermentation”
breweries. On December 16th, 1872, we prepared a powder of this yeast
and plaster as before. Our last sowing took place on July 25th, 1873, in
a flask of pure wort, which showed signs of fermentation on July 27th.
We append a sketch (Fig. 16) which gives the general aspect of this
“high ferment,” when revived after such a lapse of time; it had
preserved the distinctive features of the cells of “high ferment.”

These facts can leave no doubt whatever as to the possibility of cells
of yeast existing, in a state of suspension in the air, in the form of
fine dust, particularly in a laboratory where researches on alcoholic
fermentation are pursued.

Footnote 21:

  We have heard of liquids even less sensitive than these, which
  required a temperature of 120° C. (248° F.) or more, but we have had
  no opportunity of studying them.

Footnote 22:

  See Pasteur, _Mémoire sur les Générations dites Spontanées_ (_Annales
  de Chimie et de Physique_, t. lxiv. 3^e série, année 1862).

Footnote 23:

  See my _Mémoire sur les Générations dites Spontanées_, already cited.

Footnote 24:

  Jules Duval (of Versailles), _Nouveaux faits concernant la mutabilité
  des germes microscopiques. Rôle passif des êtres classés sous le nom
  de ferments._ (See the _Journal d’Anatomie et de Physiologie_, edited
  by C. Robin, Sept. and Oct. 1874, and _Compte-rendus de l’Académie des
  Sciences_, Nov. 1874). M. Béchamps had previously fallen into similar
  errors.

Footnote 25:

  On this subject see the observations of M. Coste (_Compte-rendus de
  l’Académie_, t. lix. pp. 149 and 358, 1864).

Footnote 26:

  Chauveau’s experiments were directed to show that the operation
  _bistournage_, employed by veterinary surgeons for castrating animals
  by twisting and subcutaneous rupture of the spermatic cord, an
  operation which, though leading to the mortification and subsequent
  absorption of the testicles, is commonly attended with no other
  mischief to the animal, does, nevertheless, lead to septic effects of
  a serious character, provided that septic germs—decomposing serum
  containing _vibrios_, for example—be introduced into the blood
  current. From the fact that the operation is ordinarily harmless, M.
  Chauveau concludes that septic organisms are not produced by the
  action of the constituent gases of the atmosphere—always present in
  the blood—upon albuminous matter when outside vital influences;
  whilst, from the success of the direct experiment of introducing
  septic germs, he concludes that the phenomena always arise from the
  actual presence of such germs.—D. C. R.

Footnote 27:

  See Pasteur, _Mémoire sur les Générations dites Spontanées_, pp. 51
  and 52, 1862.

Footnote 28:

  DAVAINNE, _Compte-rendus de l’Académie des Sciences_, t. lvii. p. 220,
  1863.

  COZE and FELTZ, _Recherches cliniques et expérimentales sur les
  maladies infectieuses_, Paris, J. B. Baillière, 1872. Summary of all
  their works published before 1865.

  Dr. LISTER, Medical and surgical journals, particularly the _Lancet_,
  1865-67.

  Dr. GUÉRIN, _Compte-rendus de l’Académie des Sciences_, March 23,
  1874, and May 28, 1874, also the Report of M. Gosselin, December,
  1854.

  Dr. SÉDILLOT, _Compte-rendus de l’Académie des Sciences_, November,
  1874, t. lxxix. p. 1108.

  PASTEUR, _Mémoire sur la fermentation appelée lactique_. (_Annales de
  Chimie et de Physique_, t. lii. 3^e _série_, 1875.)—_Animalcules
  infusoires, vivant sans gaz oxygène libre et déterminant des
  fermentations._ (_Compte-rendus de l’Académie des Sciences_, t. lii.
  1861.)—_Recherches sur la putréfaction._ (_Compte-rendus de l’Académie
  des Sciences_, t. lvi. 1863.)

  GOSSELIN, ROBIN, and PASTEUR, _Compte-rendus de l’Académie des
  Sciences_, January 5, 1874. _Urines ammoniacales._

  TRAUBE, _Gazette hebdomadaire de médecine et de chirurgie. Sur la
  fermentation alcaline de l’urine_, April 8, 1864.

  CHAUVEAU, _Putréfaction dans l’animal vivant_. (_Compte-rendus de
  l’Académie des Sciences_, April 28th, 1873.)

Footnote 29:

  We must mention one curious result, which relates to what have been
  called the _crystals of the blood_. We could hardly have recourse to a
  better method of preparing these crystals, at least in the case of
  dog’s blood, which seems to yield them with the greatest facility in
  any quantity we might desire to procure. Under the circumstances just
  recounted, in which dog’s blood exposed to contact with pure air
  underwent no putrefactive change whatever, the crystals of that blood
  formed with a remarkable rapidity. From the first day that it was
  placed in the oven and exposed to an ordinary temperature, the serum
  began gradually to assume a dark brown hue. In proportion as this
  effect was produced, the globules of blood disappeared, and the serum
  and the coagulum became filled with very distinct crystals, of a brown
  or red colour. In the course of a few weeks, not a single globule of
  blood remained, either in the serum or coagulum; every drop of serum
  contained thousands of these crystals, and the smallest particle of
  coagulum, when bruised under a piece of glass, presented to view
  colourless and very elastic fibrine, associated with masses of
  crystals, without the slightest trace of blood-globules. Where our
  observations were protracted, it sometimes happened that all the
  fibrine collected into one hyaline mass, which gradually expelled
  every crystal from its interior.

Footnote 30:

  PASTEUR, _Comptes rendus de l’Académie des Sciences_, t. lvi. p. 738,
  1863.

Footnote 31:

  GAYON, _Comptes rendus de l’Académie des Sciences_, and _Annales
  Scientifiques de l’École Normale Supérieure_, 1874-75.

Footnote 32:

  Amongst these influences one of the most important, according to M.
  Fremy, is “_organic impulse_,”—another gratuitous assumption.

Footnote 33:

  FREMY, _Comptes rendus de l’Académie des Sciences_, t. lviii. p. 1167,
  1864.

Footnote 34:

  FREMY, _Comptes rendus de l’Académie des Sciences_, t. lxxiii. p.
  1425, 1871. M. Trécul shares M. Fremy’s opinions, and extends them to
  the development of different fungoid growths.

Footnote 35:

  This observation had already been made by Anthon and H. Hoffmann. “If
  we scrape the surface of a gooseberry with a blunt knife,” says H.
  Hoffmann, “and put under the microscope the scrapings, which are of a
  whitish colour, we shall recognize amongst many varieties of shapeless
  dirt, earthy particles and other things, the same fungoid spores that
  we find in the expressed juice, but we shall see them there in
  infinitely larger quantities. Some of them will be of a dusky colour
  (_Stemphylium_, _Cladosporium_), and others will be colourless; the
  shape of these latter will be round or oval, and cylindrical. Most of
  them will bear resemblance to beads of the _chaplets_ of _Oidium_,
  _Monilia_, _Torula_ (that is to say, to spores of certain
  _Hyphomycetes_), which have been detached and carried off by the wind,
  and have attached themselves to the fruit. Some of these spores will
  be already provided with short germinating filaments.” (_Annales des
  Sciences Naturelles, Botanique_, t. xiii. p. 21, 1860).

Footnote 36:

  The experiments that we have described give rise to a useful remark.
  All the organic liquids, boiled or not, in the course of time must
  take up oxygen from the air. At the same time, and certainly under
  this influence, they assume an amber or brownish colour, but this
  effect is only produced when the liquids are placed under conditions
  of unalterability. Should fermentation or the development of fungoid
  growths be possible, scarcely any change of colour will take place.
  Doubtless this non-coloration may be attributed to the fact that these
  organisms consume the oxygen necessary for coloration. In these
  experiments on must, all the unchanged flasks assumed a pale yellowish
  brown colour; those which fermented or contained fungoid growths
  remained colourless, or nearly so.

Footnote 37:

  _Comptes rendus de l’Académie, séance du 28 Octobre, 1872._

Footnote 38:

  GAY-LUSSAC, _Annales de Chimie_, t. lxxvi. p. 245; read at the
  Institute, December 3rd, 1810. Long before Gay-Lussac, it had been
  remarked that atmospheric air had a great influence on fermentation.
  See M. Chevreul’s articles on the history of chemistry in the _Journal
  des Savants_.

Footnote 39:

  GAY-LUSSAC, _Annales de Chimie_, t. lxxvi. p. 247, _Mémoire cité_,
  1810.

Footnote 40:

  It is well to notice that under the influence of fungoid growths,
  properly so called, the wort of beer speedily becomes bright. We may
  say that fungoid growths, by their rapid development, clarify the
  must, which serves to nourish them.

Footnote 41:

  It has already been observed in our Memoir on spontaneous generation,
  that alcoholic fermentation is not always to be obtained by sowing
  wads of cotton or asbestos, charged with the particles of dust which
  float through the air, in saccharine musts that are in contact with
  much air. The air which furnished the particles of dust, in the
  experiments to which we are alluding, was taken outside the
  laboratory, in a neighbouring street.

Footnote 42:

  The decanting into the flasks is necessary, because of the possibility
  of the fermentation in the basins being masked. See further on the
  note on p. 75.

Footnote 43:

  This small ferment is very curious, although it scarcely affects
  industrial fermentation. It was first described in 1862. (PASTEUR,
  _Bulletin de la Société chimique_, 1862, page 67, and following:
  _Quelques faits nouveaux au sujet des levûres alcooliques_.) It has
  since been described by Dr. Rees under the name _Saccharomyces
  apiculatus_. (Dr. Rees, Leipzig, 1870: _Sur les champignons de
  fermentation alcooliques_. See also Dr. Engel, _Thèse pour le
  Doctorat_, Paris, 1872.) If we carefully filter some grape must at the
  time of vintage, we may be sure that we shall see it appear in the
  clear liquid at the bottom of our vessel, without intermixture with
  any other ferment.

  Should we not filter the must this ferment will appear all the same,
  but it will soon become associated with another, thicker in appearance
  and more elongated, which also is one of the ferments peculiar to the
  fermentation of grape must.

Footnote 44:

  We may remind the reader that in 1862, in our _Mémoire sur les
  Générations dites spontanées_, we applied the expression of _torula_
  to all the little cellular plants of spontaneous growth, excepting
  _mycelium_, propagated by budding, after the manner of the ferment of
  beer. At the same time, stress was laid upon the frequent occurrence
  of their germs, especially in our laboratory, where studies on
  fermentation were, even then, carried on. Plate III. represents two of
  these ferments.

Footnote 45:

  It is to be remarked that in this case, as in the case recorded § IV.
  p. 70, in order to detect with certainty any alcoholic ferment, the
  contents of the basins were transferred to a long-necked flask; since
  where, as in the basins, a liquid has a large surface exposed to
  spontaneous impregnation, the strictly alcoholic fermentation may
  escape observation. The reason of this is that, when a liquid of large
  surface but small depth is exposed to the air it affords a suitable
  medium for the active development of moulds, which, by absorbing the
  oxygen which would dissolve in the liquid, checks the growth of the
  ferment, or even prevents its germination altogether. As a matter of
  fact we shall see that for their growth and multiplication ferment
  _cells_, and still more ferment _germs_ (the difference between the
  two will appear in Chap. V.), require a larger supply of oxygen just
  in proportion to their age, state of desiccation, and distance from
  the budding condition. Now, if the spores of moulds be present and
  effect a settlement in the liquid, the increase of the ferment, or
  even its actual germination, is prevented. But by collecting the
  liquid in a deep and narrow vessel, such as a long-necked flask, after
  it has been exposed to spontaneous impregnation, we deprive the moulds
  almost completely of oxygen, and so allow the ferment to exert its
  peculiar energies. The mere act of transferring enables the liquid to
  take up a sufficiency of oxygen, and a liberation of gas very speedily
  shows that fermentative action is going on. We must add further that
  sometimes in a liquid of large surface and shallow depth, in which but
  little ferment is formed, the evolution of carbonic acid gas may fail
  to be detected, by reason of its diffusing itself into the air slowly
  as it is formed.

Footnote 46:

  Here we had to seek for a most minute quantity of alcohol, that no
  alcoholometer could have indicated. A certain sign of the presence of
  alcohol is contained in the first few drops distilled; these always
  assume the form of little drops or _striæ_ or, better still, oily
  tears, when alcohol is present in the distillate. The distillation
  should be effected with a small long-necked retort and a Liebig’s
  condenser. We must carefully watch the neck of the retort at the
  moment of boiling; should the liquid contain 1/1000 part of its volume
  of alcohol, we shall observe the indications given above for a short,
  but appreciable time. 1/10,000 of alcohol is difficult to judge, but
  with care and practice we may do it without failing. Collecting a
  third of each distillate, and supposing the limit of appreciation to
  stop at thousandths, in three distillations we may easily detect the
  presence of 1/10,000 of 1 c.c. of alcohol in a total volume of 100
  c.c.

Footnote 47:

  Yeast when macerated in water imparts to it certain soluble
  nitrogenous materials. The solution so obtained, filtered from yeast
  globules, is known as _yeast water_.—D. C. R.

Footnote 48:

  It is possible that this greasiness in the cells of the common
  _mycoderma vini_ arises simply from the composition of the liquid in
  which it vegetates. It is in saccharine liquids that the submerged
  _torulæ_ are found; fermented liquids more readily give birth to the
  forms of _torulæ_ and _mycoderma vini_ which exist as a scum. In all
  probability, however, there is no radical difference between these two
  kinds of little cellular plants of aerial growth, the floating
  _torulæ_ and _mycoderma vini_.




                              CHAPTER IV.
     The Growth of Different Organisms in a State of Purity: Their
                            _Autonomy_.[49]


Our observations in the preceding chapter will have shown that organic
liquids, natural or artificial—the wort of beer amongst others—if
exposed to contact with the air, rapidly develop various forms of life.
This is a natural consequence of the mode of impregnation. The fertility
of the liquid depends on the various microscopic germs which are
deposited in it by the common air, and these, again, as regards their
nature and number, are dependent upon the situation of the vessel
containing them, its height above the ground, the time of year, the
disturbance of the atmosphere, and other causes.

The fortuitous association of other forms, in growths which we believe
to be uniform and independent, constitutes one of the principal
difficulties that occur in the study of the lower organisms,
particularly that of fungoid growths. The fact that the germs of many of
these little beings exist in the atmosphere in the form of dust,
invisible to the naked eye, or, as such, spread over the surface of the
different materials and objects used in experiments, exposes the student
to constant risk of wrongly interpreting the results which come under
his notice. He has sown a plant, and is observing the course of its
development. Without his knowledge, spores of another plant have got
mixed with his growth, and germinated. In his ignorance, he will
attribute all that he sees, all the changes which he describes and which
he sketches, and all the conclusions which he draws, to the one plant
which engages his attention. If he is dealing with _bacteria_,
_vibrios_, and, generally speaking, the infinite variety of mobile
microscopic organisms, his embarrassment will be greater still. Again,
inasmuch as the medium which serves as substratum for growths has a
considerable influence on the fertility of the germs in contact with it,
as well as on their ulterior development, it often happens that germs
deposited fortuitously by the particles of dust which fall from the
atmosphere or collect on objects are fertile and multiply with rapidity,
whilst those which have been directly sown, no matter in what number,
remain sterile, or multiply very slowly. If we place in a young wine
some _mycoderma aceti_, we shall obtain _mycoderma vini_; by placing
some _mycoderma vini_ in an old wine, especially if it is a little acid,
we shall obtain _mycoderma aceti_.[50] It is from facts of this kind,
wrongly interpreted, that many errors have crept into our knowledge of
the lower organisms, and that we are constantly seeing old discussions
crop up, both on the subject of so-called _spontaneous_ generation and
on that of the theory of fermentation. At every step in the course of
this work we shall see the trace of these complications, as well as the
influence they have had on the progress of our knowledge.

In opposition to these results, we will study the case of wort sown
directly with germs distinctly of one kind, unmixed with any other.


§ I.—Growth of Penicillium Glaucum and Aspergillus Glaucus in a State of
Purity—Proofs that these Fungoid Growths do not become Transformed into
  the Alcoholic Ferments of Beer or Wine.—Preliminary Enquiry into the
                         Cause of Fermentation.


Let us again take one of the flasks furnished with two necks such as we
have already described, and let it be supposed that this flask contains
a quantity of saccharine wort, brewed some considerable time ago, which
has undergone no change whatever, except in colour, the slow process of
oxidation having gradually darkened the original colour of the liquid.
What we have to do is to drop into this unchanged and fertile liquid
some grains or spores of _penicillium_ which are free from the slightest
trace of the spores or germs of other microscopic organisms.

One means of effecting this consists in taking up with a pair of
metallic forceps, previously heated, a piece of platinum wire, one or
two centimetres (about 3/4 in.) in length, which we also pass through
the flame of a spirit lamp, and with which, as soon as it is cold, we
touch a mass of sporanges of a growth of _penicillium_. No matter how
few spores may be taken up on the end of the platinum wire, we shall
have far more than we require for the impregnation of the liquid. At the
moment of charging the point of the wire we withdraw the glass stopper
which closes the india-rubber tube on the right-hand neck of the flask
(Fig. 14) and drop the wire through that tube; we then replace the glass
stopper, after having, by way of additional precaution, passed it
rapidly through the flame of the lamp. There is no doubt that we expose
ourselves to error in consequence of having to convey the wire through
the surrounding air, and also, in consequence of having previously to
open the flask; but, as we have already remarked, this double cause of
error has never, we may say, interfered with the exactness of our
experiments, the volume of air with which we are concerned being
exceedingly limited. Moreover, our flask being in free communication
with the exterior air, by means of the opening in the curved, slender
tube, there is no inrush of air when we withdraw the stopper. The chance
of encountering a spore or fecund germ and introducing it into the flask
on the wire that is charged with the others, is so remote that we have
considered it unnecessary to adopt a more perfect apparatus, which might
easily have been devised had we felt that it was necessary.

A more serious cause of error may occur in the preceding method;
resulting from the possible impurity of the spores taken from a field of
_penicillium_ which has developed in contact with common air. This field
receives, every instant, and has received throughout its growth,
particles of dust which have fallen from the atmosphere; thus, it may
not be, and, as a matter of fact, is not, free from the germs of other
fungoid growths.[51]

The operator, without knowing it, may frequently sow, besides the
_penicillium_, which is all he can see, spores of _mucor mucedo_ and
_mycoderma vini_, in short, of all the most common fungoid growths.

[Illustration: Fig. 17.]

This process of impregnation, therefore, does not afford us sufficient
safeguards, but by means of the following device we shall render it more
satisfactory. Let us take a series of flasks shaped as in Fig. 17,
containing an organic liquid suitable to the development of fungoid
growths, that is to say, slightly acid—yeast-water, plain or sugared,
the wort of beer, or Raulin’s fluid[52] will answer our purpose; let us
boil the liquid, and having previously drawn out the necks, let us close
the ends in the flame of a lamp whilst the steam is escaping, as soon as
we judge that the air has been nearly all expelled. Having prepared ten
or twenty of these flasks in this manner, when they are cold we may
break their points in any place we may be in. The air will rush into the
flasks, and we must then seal them up again in the flame of the lamp,
and put them aside for future observations. In a certain number of these
flasks, as we have already explained in our experiments carried on after
this fashion, we shall see some fungoid growths appear, first in the
shape of flakes of _mycelium_ floating in the liquid, and afterwards
coming to the surface to fructify. Now, it often happens that
_penicillium glaucum_ appears alone, so numerous are the spores of this
fungus floating in the air. Under such conditions, we shall evidently
obtain a field of sporanges quite free from the presence of other
organisms. If we now take off the neck of one of the flasks containing
the pure _penicillium_, and take out some of the germs with our platinum
wire,[53] we shall thus obtain with most certainty spores of
_penicillium_ free from impurities.

Our readers will excuse the length of these details and the minutiæ of
our precautions, but we shall again and again see that to neglect them,
or any part of them, is to expose ourselves to hazard in drawing sure
conclusions from facts which come under our observation.

[Illustration: Fig. 18.]

On June 17th, 1872, we placed some pure spores of _penicillium_ in a
series of three flasks containing wort (Fig. 18), observing all the
precautions that we have indicated. We shall designate these flasks by
the letters A, B, C. On the following day the spores germinated, and the
liquid became full of flakes of mycelium, some of which came to the
surface to fructify. The temperature varied between 25° C. and 30° C.
(77° to 86° F.).

On June 22nd, small patches, with whitish borders and green centres,
developed on the liquids. We then shook up the flask A, in order to
submerge the plant and the spores.[54] We also shook the flask B, after
observing the precaution of sealing up the slender bent tube.[55] The
flask C was attached on one side to an aspirator, on the other to a tube
filled with cotton, and every day we renewed the air in it.

During the weeks and months over which our observations extended, there
was not the least formation of yeast in these flasks; moreover, we have
frequently repeated this and other experiments of a similar kind,
without ever detecting the appearance of either ordinary yeast or any
other true alcoholic ferment. The experiments may be made with
saccharine juices that are highly favourable to the development of
_bacteria_ and lactic ferment. These latter appear equally incapable of
transformation into yeast, which has never been seen to develop in
experiments where they were used, if proper precautions have been taken
to secure a pure growth. Should we neglect any of the precautions that
are necessary to secure the purity of our spores, we may of course
obtain different results. If, for instance, we sow spores of
_penicillium_ grown in free contact with the atmosphere, and
consequently exposed to the particles of dust floating therein, we shall
frequently observe, mixed with the fruiting hyphæ of the fungoid growth,
yeast and _mycoderma vini_ and _torulæ_, or even _bacteria_ and lactic
ferment. Thus we shall be led to believe, in all good faith, that we
have under our eyes examples of the changes of spores of fungoid growth
into cells of ferment, or proofs of the conversion of _bacteria_ or
lactic ferment into the same cells.

Causes of error of this nature have induced some German naturalists to
believe that they have succeeded in proving, beyond the possibility of
doubt, that a number of fungoid growths may produce alcoholic ferment,
and that they have clearly demonstrated that spores of these fungi may
become transformed into yeast. In 1856 M. Bail, and, about the same
time, Berkeley, and, later on, H. Hoffmann and Hallier, have
successively entertained these views, which were introduced into science
by M. Turpin. We have combated them since the year 1861.[56] Since that
period they have lost rather than gained ground abroad, in spite of the
growing favour bestowed on the Darwinian system. One of the mycologists
who enjoy the most legitimate authority beyond the Rhine, M. de Bary,
has arrived, as we have, at absolutely negative results.

A simple perusal of what has been written in favour of the
transformations which we are discussing causes us to entertain the
gravest doubts as to the correctness of results which are quoted as
decisive. We need only give one example, which we extract from a paper
by M. H. Hoffmann.

“In some cases,” this author writes, “and under favourable
circumstances, I have been able to see the ferment produce filaments,
both small specimens that could be examined immediately under the
microscope, and also large specimens, and I have recognized, amongst
other varieties, _penicillium glaucum_, _ascophora mucedo_, _ascophora
elegans_, and _periconia hyalina_, sometimes isolated, sometimes
intermixed. This result is most easily to be obtained by the following
method:—Pour a small quantity of water into a test-tube, which should
then be placed slantingly: introduce some fresh yeast into the middle
part of the tube, and close the tube with a wad of cotton, to prevent
the entrance of exterior particles of dust. In this vapour-filled
receptacle we shall sometimes see flakes develop. It seems that Messrs.
Berkeley and G. H. Hoffmann have also obtained _penicillium_ from
ferment, by a similar process.”[57]

Why, however, should it not be admitted that _penicillium_ found under
such conditions may be derived from spores of that growth, adhering to
the sides of the tube before it is closed with the wad, or mixed with
the yeast that is put into the tube?

The facts alleged during the last few years by M. Bail, who persists in
his views, in spite of their apparently greater exactness, are also far
from being satisfactory.[58]

M. Trécul, who is almost the only one in France, besides Messrs. Ch.
Robin and Fremy, to participate in these errors, does not confine
himself to affirming the change of the spores of _penicillium_ into
yeast, and _vice versâ_; his system is a far more extensive one.
“According to my observations,” he says, “there would be the following
series of changes; albuminous matter changed into _bacteria_, or
directly into alcoholic ferment, or into _mycoderma_; _bacteria_ into
lactic ferment, there becoming immoveable; lactic ferment into alcoholic
ferment; alcoholic ferment into _mycoderma cerevisiæ_; finally,
_mycoderma cerevisiæ_ into _penicillium_.[59]”

M. Trécul does not stop here. He goes on to explain the principal of
these changes, as though his testimony were quite beyond refutation:

“If we use a perfectly filtered wort, containing no granulations, and
prepared at a temperature between 60° C. and 70° C. (140° F. and 160°
F.), there will first of all appear a multitude of fine granules that
will develop into active bacteria, which, losing the faculty of motion,
will constitute lactic ferment, as I have repeatedly pointed out. A few
days after the appearance of the first granules, we shall perceive
others rather larger in size and isolated. These will increase in size,
and, in the course of time, assume the form of little globuloid, or
elliptic cells; they will not commence to bud before they have attained
a comparatively large size, approaching that of ordinary yeast,
consequently, there will be a considerable interval of time, during
which the young cells will present no buds, especially if we work at a
low temperature, as from 20° C. to 35° C. (68° F. to 95° F.)

“As for the transformation of the spores of _penicillium_ into alcoholic
ferment, the possibility of which M. Pasteur also denies, I have very
often obtained it by using liquids, such as boiled wort and sugared
barley water, which had stood for a month or six weeks without setting
up an alcoholic fermentation. These liquids, sown with spores of
different forms of _penicillium_, chosen when young and in full growth,
fermented after a varying number of days, even at a temperature of 12°
C. (54° F.), the condition of fermentation being that the flasks were
closed with very elastic corks, which had been boiled for a quarter or
half an hour; these corks, as I have already pointed out, it is best to
keep for a month after the boiling, to make sure, by drying them
thoroughly again, of destroying any _mycelia_ adhering to them. It is
necessary to keep the flasks stoppered that the corks may be always
moist, and it is also advisable to shake the flasks once or twice a day,
to secure the submersion of the spores. If these conditions are carried
out, we shall soon see the spores increase in size, gradually lose their
green colour and then bud, and often a very active fermentation will
manifest itself. All the spores will be transformed, if the flasks are
perfectly air-tight.”[60]

Such is the manner in which M. Trécul regards these changes. His is
entirely a system of spontaneous generation, worked out into minutest
detail, from the transformation of the albuminous substances to the
formation of cells of the higher organisms, passing from the
disintegration of the original substances to the formation of very fine
granules, from these to the creation of active _bacteria_, which last,
in their turn, become lactic ferment through the simple cessation of
their faculty of moving and so on. We regard all this as purely
imaginary. As a matter of fact, M. Trécul’s argument is based on the
successive phenomena which manifest themselves in filtered wort
“containing no granulations.” As M. Trécul reasons, this condition is a
necessity, for he starts with the assertion that the albuminous
substances in the wort become changed into granulations “that will
develop into active bacteria.” This is another of M. Trécul’s illusions.
No doubt we may filter hopped wort to almost perfect clearness, but we
can only do this when it is cold. If we filter it warm, it will be
bright as long as it remains warm, but as soon as cold it will appear
turbid, in consequence of the great number of minute granules floating
in it. Again, cold wort, however little it may be or has been in contact
with air, undergoes a process of oxidation, and this oxidation, which
acts principally on the colouring or resinous matter, causes a deposit
of fine granules, the number of which is constantly increasing as
oxidation goes on. These granules form an absolutely inert precipitate
which, under no possible circumstances, can become transformed into
active bacteria. Nothing can be easier than to prove this fact, by
taking some of our two-necked flasks (Fig. 4) and preparing pure wort in
them, by boiling, and then leaving it to cool and undergo the process of
oxidation. In wort thus exposed to the air—the air being pure and free
from germs—there will be formed a granular deposit, which will never
become active or transform itself into any kind of organism whatever.

We may also remark that whilst M. Trécul used wort in his experiments,
he does not tell us if it was hopped or not. Had M. Trécul informed us
that the facts which he described applied to unhopped wort, we should
reply that a temperature of 60° C. or 70° C. (140° F. or 160° F.) is
quite insufficient to kill the germs of bacteria existing in such a
wort. Hopped wort should be heated to 70° C. or 75° C. (160° F. to 170°
F.), that it may remain inert after having cooled down in contact with
pure air; unhopped wort must be heated to about 90° C. (194° F.).

In short, whatever may be the case, it must be evident to our readers
that the active bacteria observed by M. Trécul existed in the form of
germs in the wort that he used, and that what he observed was nothing
more than the development of these germs when brought into contact with
the air held in solution in the liquid.

As for the success of M. Trécul’s experiments on the _penicillium_, we
have no doubt that that gentleman has sown germs of yeast or _torulæ_,
which bear so striking a resemblance to yeast, at the same time that he
sowed spores, since he took his spores from sporangia of _penicillium_
that had been exposed to contact with ordinary air. The conditions under
which M. Trécul conducted his experiment rendered it difficult for the
spores—although, relatively, much more numerous than the contaminating
germs of ferment with which they happened to be associated—to make way
against these latter, since the spores were unable to continue their
development in a medium that was deprived of oxygen. On the other hand,
the cells of ferment might easily have multiplied to such an extent as
to make the discovery of the spores that had been sown a matter of some
difficulty, since those spores would have been lost amongst the great
number of cells of ferment. This was, probably, one of the causes which
led to the mistaken notion that such spores underwent conversion into
cells of ferment. Now although botanists describe several varieties of
_penicillium glaucum_, we do not suppose that the cause of the
difference between our results and those obtained by M. Trécul can be
attributed to our having operated upon a different variety of
_penicillium_ from that which he used. Supposing that there had been a
great difference between our two varieties, still M. Trécul declares
that he has realized the phenomenon with numerous varieties of this
fungus.

M. Trécul expresses himself as follows: “I have used spores of
_penicillium_ of several varieties in these experiments: Firstly, thick,
green, elliptic spores of a variety of _penicillium_ that grows on
lemons; secondly, elliptic spores of a bluish colour and smaller than
the preceding, of another variety of _penicillium_ found on lemons;
thirdly, spherical spores of the variety termed _penicillium
crustaceum_; fourthly, spores of the _penicillium_ that develops on the
yeast of beer.[61]”

This criticism of M. Trécul’s opinions was written on the occasion of a
discussion at the Academy, after we had been induced to read over again
the remarks which he had published on the subject. We were so impressed
by the positive manner in which his conclusions were stated, that we
asked ourselves, once more, which of us could be mistaken, and once
more, also, we applied ourselves to fresh experiments, which we
conducted with every possible precaution, following, as far as we could
without falling into the errors of which we accuse the learned botanist,
the mode of procedure that he adopted. As his descriptions struck us as
being at times insufficient, we resolved to ask him for certain
explanations _vivâ voce_ (November 3rd, 1873), which he gave us with the
greatest willingness.

“Every variety of _penicillium_,” said M. Trécul, “especially when young
and vigorous, is amenable to transformation into ferment. This is the
way in which I operated; I had some little flasks of 30 c.c. to 40 c.c.
(about 1-½ fluid ounces) in capacity, filled quite full with wort, or,
at least, containing very little air, closed perfectly air-tight with
corks which I had kept for a quarter of an hour in boiling water. These
flasks when corked were heated to 60° C. or 70° C. (140° to 158° F.).
After they had cooled I uncorked them, and introduced into them the
spores which had been prepared as follows: I placed on a piece of glass
some spores of the variety of _penicillium_ that I wished to study,
taken with a pair of forceps from a mouldy lemon, and I mixed these
spores with a drop of wort and observed them under the microscope to
assure myself that they contained nothing of a foreign nature; then I
poured my drop of wort from the piece of glass into one of the flasks,
which I recorked and laid down. The transformation into ferment took
place next day.”

Provided with these new data we set to work again and prepared a series
of little flasks which were filled quite full with hopped wort, or
contained but very little air, as M. Trécul had recommended. These we
heated in a hot water bath to 70° C. (158° F.) at least; we then
impregnated them, observing the necessary precautions, which we
described at the commencement of this paragraph—not working in the
evidently defective manner in which M. Trécul had done. Taking his
spore-seeds from a field of sporanges exposed to the air, and afterwards
manipulating them, in contact with the air, in water on a piece of
glass, before he made his microscopical examination, his experiments
were conducted under circumstances in every way conducive to the
introduction of causes of error. One of the most serious of those causes
is that which results from the substratum of the spores as taken from a
mouldy lemon. If M. Trécul will examine under the microscope the water
in which any lemon has been washed—even a sound lemon, unattacked by any
fungoid growth—he will immediately see the cause of error to which his
method of working exposes him. Germs of microscopic organisms exist
abundantly on the surface of all fruits.

Having impregnated the liquids in our flasks with spores from a quantity
of pure sporangia grown in a closed vessel, gathered on the point of a
platinum wire, which had first been heated and then allowed to cool, we
found that in each case, without exception, germination took place, then
a _mycelium_ was developed, which soon, however, ceased to grow from
want of a proper supply of air; but not in any single case was there the
faintest trace of fermentation, formation of yeast, or appearance of
_bacteria_ or lactic ferment.

We repeated these experiments, using unhopped wort instead and obtained
similar negative results. We had previously determined that it was
necessary to heat flasks of hopped wort to 70° C. (158° F.), at least,
and those of unhopped wort to 90° C. (194° F.) to secure them from
further change.

In short, contrary to the assertions of M. Trécul, M. H. Hoffmann, and
other naturalists, it is not true that the spores of _penicillium_ can
change into alcoholic ferment.

Regarded from another point of view, growths of pure _penicillium_ will
give us some remarkable results, the interpretation of which seems to us
to be intimately connected with the physiological theory of fermentation
that we shall discuss in a subsequent chapter. It is a question as to
the production of alcohol whilst the life of the plant is carried on
under certain conditions of growth.

If we distil saccharine liquids on the surface or in the body of which
we have grown _penicillium_, and repeat the distillation in the manner
that we have already described for the detection of the minutest
quantities of alcohol, we shall readily find that those liquids
frequently do contain a little ordinary alcohol. Moreover, if we regard
the quantities of alcohol produced, which are always very minute, seldom
exceeding 1 or 1·5 thousandth of the total volume of the liquids, we
shall find that there is no fixed proportion between this alcohol and
the weights of the plants formed. It is possible, for instance, that we
may obtain more alcohol from one plant than from another weighing a
hundred times as much. Often, however, when the vegetation is abundant
we cannot make out the occurrence of alcohol in spite of the
sensitiveness of the process described (p. 78).

What can be the cause of these varying results relating to the
production or non-production of alcohol in the vegetation of the little
plant? The numerous experiments that we have made seem to demonstrate
positively that they are dependent upon variations in the amount of air
or oxygen that is supplied to the fungoid growths, whether, that is, the
vegetating mycelium alone be submerged, or the whole plant with its
organs of fructification. When the plant has at its disposal an excess
of oxygen, as much as its vitality can dispose of, there is no alcohol,
or very little, formed. If, on the other hand, the plant vegetates with
difficulty, in presence of an insufficiency of oxygen, the proportion of
alcohol increases; in other words, the plant shows a certain tendency to
behave after the manner of ferments.

Some time ago, wishing to assure ourselves that the spores of
_penicillium_ could not become transformed into ferment, we sowed some
pure spores in small flasks, holding from 50 c.c. to 100 c.c. (from 2 to
4 fl. oz.), which contained very little air, and which were sealed
hermetically after the sowing. Under these conditions, the germination
and growth of the spores proceeded with great difficulty, and soon
ceased through want of air. The total weight of the little plant was too
small to be determined. In cases of this kind, if we distil the whole of
the liquid we shall often see the alcohol appear in the second
distillation, even though the weight of the plant may have been scarcely
appreciable. If, on the other hand, side by side with experiments of
this kind, we grow pure _penicillium_ in flasks containing air and
having quantities of saccharine liquids equal to the quantities in the
small flasks of which we have been speaking, the plant, in consequence
of the large volume of air at its disposal, will develop vigorously, and
in the course of even a few days will have become perceptibly heavier.
In distilling the subjacent liquid, however, we shall generally find
that it contains no alcohol at all, even though the weight of the plant
be half a gramme, or more (6 or 7 grains).

These results apply to all the fungoid growths that we have studied, but
they vary considerably with the nature of the organisms. _Aspergillus
glaucus_ is, in this respect, one of the most curious.

On June 15th, 1873, we impregnated three flasks of wort, A, B, C, with
pure spores of _aspergillus glaucus_. The development was rapid and the
fructification abundant. On June 20th, we shook up the liquid and the
supernatant fungoid growths in the three flasks; the flasks A and B were
then treated as follows:—

We distilled the Liquid in A to discover the presence of alcohol; but
could find none.

[Illustration: Fig. 19.]

The flask B was connected with a test flask (Fig. 19), into which the
liquid, together with its fungoid growth, was transferred from B. The
next day, June 21st, the mycelium which was on the surface of the
liquid, in the neck of the flask, was studded with bubbles of gas; these
we dispersed by shaking. On June 22nd, many others had formed again, and
a large flake of mycelium that had risen from the bottom of the test
flask had been stopped at the bottom of the neck, quite distended by gas
bubbles. We liberated the gas by shaking, but the bubbles formed again
by the next day, and this effect continued for several days;
nevertheless the liberation of gas was not continuous, as is the case in
an ordinary fermentation.

On July 20th we drew off the liquid and distilled it; it was still very
sweet, but though it contained a sensible quantity of alcohol, the
microscope failed to detect a single cell of ordinary alcoholic ferment.

These results show that the _aspergillus_ when in full growth, with
plenty of air at its disposal, does not yield alcohol, and that if we
submerge it, so as to prevent the oxygen of the air from readily coming
into contact with its various parts, it decomposes sugar, after the
manner of yeast, forming carbonic acid gas and alcohol.

These effects were still more marked in the case of the flask C, the
liquid in which, after having been shaken up, was not decanted to any
great depth in our test flask, as had been the case with B. From June
21st, there was _mycelium_ on the surface of the liquid, studded with
large bubbles of gas, which formed again after having been liberated by
shaking. This last flask was examined on November 1st, 1873. Its aspect
was unchanged; the liquid was covered with _mycelium_ loaded with
sporanges and borne up by large old bubbles that had not disappeared.
The following was the analysis of the liquid:—

                         Alcohol           1·2
                         Glucose          84·0
                         Dextrine (?)     32·0

The liquid was very bright, and contained an amorphous granular deposit,
formed by the wort after it had been boiled, at the time when we
prepared our flasks. We crushed a small quantity of mycelium that had
risen to the surface of the liquid, and obtained a field such as is
represented in Fig. 20. Amongst the ordinary filaments of mycelium
belonging to the plant, which are not represented in our engraving, and
which were not more than 1/300 of a millimetre (nearly 1/7500 in.) in
diameter, we perceived much larger ones, swollen and contorted in the
most singular manner, and measuring as much as 1/50 of a millimetre
across their broadest parts. There was also a multitude of the ordinary
spores of _aspergillus_ mixed with others of larger size, and big,
inflated cells, with irregular or spherical protuberances, full of
granular matter. As there are all the stages between the normal spores
of the plant and the big cells, and between these latter and the
filaments, it must be admitted that the whole of this strange vegetation
results from spores which change their structure under the influence of
special conditions to which they are exposed.[62] Beyond all doubt these
cells and irregularly shaped segments, in vegetating with difficulty,
gave rise to the fermentation, which, although insignificant, was
sufficiently marked to produce more than a gramme (15 grains) of
alcohol. The oxygen of the air failing, or existing in insufficient
quantity for the regular development of the filaments of mycelium
belonging to the plant, and for the germination of its submerged spores,
filaments and spores vegetated as the yeast of beer might have done if
deprived of oxygen.

[Illustration: Fig. 20.]

If we study the vegetation of _aspergillus glaucus_ with this
preconceived idea, we shall soon recognize the fact that these spherical
forms of mycelium are the result of a greater or less deprivation of
air. The filaments of this mycelium which develop freely in the aerated
liquid are young and transparent, small in diameter, and exhibit the
ordinary ramifications. Those which are situated about the centre, in
the denser or more complicated parts, to which the oxygen cannot
penetrate in consequence of its absorption by the surrounding parts, are
more granular in appearance as well as larger, and inclined to develop
swellings. We can observe no _conidia_[63] on these filaments, but we
may say that they are on the point of appearing, for the spherical
segments often tend to assume an appearance of close jointing, as when
they take the form of those rows of swelling, or cells, which has given
rise to the idea of the _chaplets of the conidia-cellules_. This is
represented in the accompanying sketches (Fig. 21), which we have
purposely contrasted with two similar ones which relate to the _mucor_,
of which we shall soon speak. The _conidia_ of these latter are very
remarkable, and their fermentative character becomes apparent as soon as
their growths are deprived of air.

[Illustration: Fig. 21 A. Growth with abundant air-supply at the edge of
the mycelium crust.]

[Illustration: Fig. 21 B. Growth with reduced air-supply in the central
and deeper parts of the mycelium.]

[Illustration: Fig. 21 C. Growth with abundant air-supply at the edge of
the mycelium tuft.]

[Illustration: Fig. 21 D. Growth with reduced air-supply in the central
and deeper parts.]

It is scarcely necessary to add that in these vegetations of
aspergillus, which were accompanied by a corresponding alcoholic
fermentation, it was impossible to find cells of yeast; and that,
notwithstanding this, the liquid was so adapted to ordinary alcoholic
fermentation, that, when we added a small quantity of yeast to it, in
the course of a few hours, a most active alcoholic fermentation declared
itself.

We may give some other facts relating to a crop of _aspergillus glaucus_
which was also grown in ordinary hopped wort, and which was left to
itself for a year.

A two-necked flask, holding 300 c.c. (rather more than 10 fl. oz.) was
prepared and impregnated on December 21st, 1873, and was then placed in
an oven at a temperature of 25° C. (77° F.). The fungoid growth
developed in isolated tufts, which subsequently united, but without
entirely covering the surface of the wort. A few tufts also vegetated at
the bottom of the liquid; those on the surface soon became surrounded by
large bubbles of gas.

On December 12th, 1874, we examined the liquid and the plant, which for
a long time had appeared dead. Its mycelium was formed of aged,
granulated filaments, with few swellings. The weight of the dry fungoid
growth was 0·50 gramme (about 8 grains) for a total volume of liquid of
122 c.c. (4-1/4 fluid ounces). We obtained 4·4 c.c. of alcohol of 15°,
which was about seven times the weight of the plant. Finally, we
determined the acidity of the liquid, and found 2·8 grammes, in
equivalents of sulphuric acid, a quantity greatly in excess of the total
acidity of an equal volume of wort, a fact which shows us that
fermentation caused by _aspergillus glaucus_ is accompanied by the
formation of an organic acid, the nature of which it would be
interesting to determine. M. Gayon has commenced the study of this
subject in our laboratory.

In concluding our observations on the _aspergillus glaucus_, we may give
the comparative results of two growths that were obtained under
precisely similar conditions, in flasks of exactly the same size, but
differing in this respect—that one of them was constantly subjected to a
current of pure air that played on the liquid. In the course of a few
days, when the fungoid growth in the flask that had been aerated had
attained a considerable size, in comparison with the other, we broke the
flasks in order that we might take out the two growths and compare their
weights. After drying them at 100° C. (212° F.) we found:—

             Growth in the aerated flask              0·92
             Growth in the closed flask               0·16
             Ratio of weights, 92/16 =  5·75.

Again, although we had taken the precaution of condensing in a U tube,
over which cold water played, the vapours carried away by the current of
air, the liquid in the aerated flask gave no evidence of alcohol. That
in the other flask contained a very appreciable quantity, although the
weight of fungoid growth in that flask was scarcely a sixth part of what
it was in the other.

The preceding facts taken altogether, seem to us to demonstrate once
more, in the most conclusive manner:—

Firstly, That neither _penicillium_ nor _aspergillus glaucus_ can change
into yeast, even under conditions that are most favourable to the life
of that ferment.

Secondly, That a fungoid growth which vegetates by using the oxygen of
the air, and which derives from the oxidating action of that gas, the
heat that it requires to enable it to perform the acts necessary to its
nutrition, may continue to live, although with difficulty, in the
absence of oxygen; that, in such a case, the forms of its mycelian or
sporic vegetation undergo a change, the plant, at the same time,
evincing a great tendency to act as alcoholic ferment, that is to say,
decomposing sugar and forming carbonic acid gas, alcohol, and other
substances which we have not determined, and which probably vary with
different growths.

Such, at least, is one interpretation of the facts that we have
reviewed. The observations in the following paragraphs and chapters may
the more incline our readers to accept it as the true one.


§ II.—Growth of Mycoderma Vini in a state of Purity—Confirmation of our
original Conjectures as to the cause of Fermentation—Mycoderma Vini does
   not Change into Yeast, although it may give rise to Fermentation.


The efflorescence of wine, cider, and beer is pretty generally
known.[64] Fermented liquors cannot be exposed to the air without soon
becoming covered with a white film, which grows thick and becomes
wrinkled in a marked manner in proportion as it is deprived of room
wherein to spread horizontally, in accord with the extraordinary
multiplication of the cellules that compose it. The rapidity of this
multiplication is sometimes astounding. During the heat of summer, when
the medium is well adapted to the life of the plant, we may count the
number of cells which grow in the course of a few hours by millions. The
absorption of the oxygen necessary to the activity of this growth, and
the heat developed in the film, as well as the liberation of carbonic
acid gas, that result from it are considerable. A piece of glass
covering the mycoderma, at some distance above it, becomes wet with
moisture, that soon accumulates to form large drops of water. The
quantity of oxygen absorbed is so great that we never see any other
fungoid growth on the surface of this film, although the air is
constantly depositing on it, as dust, spores of an entirely different
character; for, notwithstanding that the warm and moist surface is in
contact with an atmosphere that is being continually renewed, yet the
_mycoderma_ appropriates to itself all the oxygen contained in the air.
When, however, the vegetation begins to languish, we often find, on the
other hand, that the plant becomes associated with other species of
mycoderma, notably _mycoderma aceti_, as well as other fungi, amongst
which _penicillium glaucum_ generally appears. This is one of the facts
which, wrongly interpreted, have led to the belief that _mycoderma vini_
or _cerevisiæ_ may possibly, or even readily, become transformed into
_penicillium_, and _vice versâ_.[65] As the study of the growth of
_mycoderma vini_ on the surface of saccharine liquids and in their
depths, unaccompanied by any other species, has the most important
bearing on the theory of alcoholic fermentation, we may pursue it
through a few examples with all the detail that it allows of.

[Illustration: Fig. 22.]

On June 21st, 1872, we sowed some _mycoderma vini_ in three flasks, with
double necks, A, B, C (Fig. 22), containing some wort. The spores
employed for the purpose were obtained from plants growing on sweetened
yeast-water in an ordinary closed flask. This had been impregnated with
spores from plants grown on wort, which in turn had sprung from spores
taken directly from _mycoderma vini_ growing on wine.

The several impregnations were effected by means of a platinum wire,
held by forceps, both having been first cleaned by passing through
flame, and then smeared with the fungoid films.

By this series of growings in closed vessels, which were but momentarily
open at the time when we dropped the spores into them, we secured the
separation of the mycoderma from all foreign organisms; and more
particularly from germs of _mycoderma aceti_, which is generally found
along with it, but which propagates with difficulty in neutral
saccharine liquids.

On the following days films of _mycoderma vini_ had spread over the
surface of the liquid in the three flasks. To all appearance they were
very pure; and the microscope showed the complete absence of any mixture
of _mycoderma aceti_, lactic ferment, or other foreign growths.[66]

On June 26th we decanted and distilled the liquid in A without finding
any trace of alcohol. We shook up the liquids in B and C, with all due
precautions, so as to submerge their films as much as possible, and then
we raised the temperature of the flasks to 26° C. or 28° C. (82° F.).
For some days afterwards we saw a constant succession of minute bubbles
of carbonic acid gas rising through the liquid, which remained bright
under the part of the film that had not fallen in. It had all the
appearance of a slow but continuous fermentation.

On June 29th we decanted and distilled the liquid in B, and found in it
an appreciable quantity of alcohol, which showed itself in the first
distillation. The flask C, which was shaken afresh, continued to give
signs of fermentation, but, some days later, the evolution of the
bubbles ceased.

On July 15th, 1873, we examined the flask with its film and its deposit
of _mycoderma vini_, without finding a trace of any foreign growths,
either in the shape of _penicillium glaucum_, or _mucor mucedo_, or
_rhyzopus nigrans_, or _mycoderma aceti_, or, in short, any of the
organisms which could not have failed to appear on the surface of a
substratum so peculiarly adapted to their development, had it been in
the nature of _mycoderma vini_ to transform itself into one or other of
those common fungoid growths. The liquid, moreover, still remained
sweet, and did not contain any cells of actual yeast. We may conclude
then that when one or more of these fungi occur, after an interval of
some days, in a growth of _mycoderma vini_ conducted in contact with
common air, it does so in consequence of that air having, without the
knowledge of the observer, impregnated the liquid spontaneously with
germs of these foreign organisms.

There might perhaps be room for some fear that the conditions of growth
in our flasks were not favourable to the simultaneous appearance of
these common fungoid growths along with the _mycoderma vini_. On June
24th, 1872, we sowed, in three flasks of sugared yeast-water, prepared
as before—in the first, _mycoderma vini_, together with _penicillium
glaucum_; in the second, _mycoderma vini_, together with _mucor mucedo_;
in the third, _mycoderma vini_ alone.

We effected this by plunging the platinum wire, which we used for
impregnating the liquids, into the pure film of _mycoderma vini_, and
then touching with the wire the sporanges of the other fungus. On June
29th, we saw on the surface of our first flask some green patches of
_penicillium_, along with some spots of _mycoderma vini_; in the second
flask a voluminous mycelium of _mucor mucedo_, distended by large
bubbles, had risen to the surface of the liquid, and was entirely
covered by a film of _mycoderma vini_. As for the liquid in the third
flask, there were only a few spots of very pure _mycoderma vini_. This
last flask, after being kept in an oven at 25° C. (77° F.) for several
months, still contained nothing but _mycoderma vini_, unmixed with any
other fungoid growth whatever.

We may therefore be sure that _mycoderma vini_, vegetating on the
surface of liquids adapted to its nutrition, in contact with air
deprived of its germinating dust, will not present the least sign of a
transformation into any of these other common fungi, or into yeast,
however long may be the duration of its exposure to contact with that
pure air.

We may now return to that feeble and limited production of carbonic acid
gas and alcohol, the formation of which we have shown experimentally to
take place at a high temperature, after submerging the film of
_mycoderma vini_.[67] There can be no doubt that we have here a
phenomenon similar in every point to that presented by _penicillium_ and
_aspergillus_, which we studied in the preceding paragraph. When the
germs or jointed filaments of _mycoderma vini_, growing on a saccharine
substratum in contact with the air, are in the full activity of life,
this activity is carried on at the expense of the sugar and other
materials in the liquid, in the same way that animals consume the oxygen
of the air and evolve carbonic acid.

The consumption of the different materials is attended with a
proportionate formation of new materials, development of structure, and
reproduction of organisms.

Under these conditions, not only does the _mycoderma vini_ not form a
sufficiency of alcohol for analytical determination, but, if any alcohol
exists in the subjacent liquid, the _mycoderma_ consumes it, converting
it into water and carbonic acid gas, by fixation of the oxygen of the
air.[68] If, however, we suddenly submerge the _mycoderma_, we shall
obtain a different result. If, on the one hand, the conditions of life
of this fungus are incompatible with the altered circumstances in which
it is placed, the plant must perish, just as an animal does when
deprived of oxygen. But if, in spite of these changed conditions of
nutrition, it can still continue in life, we should expect to see marked
changes in its organic structure, or chemical metamorphoses. The result
of our observations points to the continuance of life, in a distinct
though sluggish and fugacious activity, accompanied by the phenomena of
alcoholic fermentation, that is, the evolution of carbonic acid gas, and
the production of alcohol.

If we take a drop of liquid charged with disjointed cells of
_mycoderma_, a day or two immediately after the submersion of the film,
we shall observe changes, small but appreciable, in the aspect of a
great number of these cells; they will show increase in size, their
protoplasm will be in process of modification, and many of them will
have put forth little buds. It will be quite evident, however, that
these acts of interior nutrition and the changes of tissue resulting
from them, proceed with difficulty; the buds when they form will soon
wither, and there will be no multiplication of new cells. These changes
will, nevertheless, be accompanied by the decomposition of sugar into
alcohol and carbonic acid.

In comparing these facts with those which we have pointed out in
connection with the cultivation of _penicillium_ and _aspergillus_, we
are compelled to admit that the production of alcohol and carbonic acid
gas from sugar—in one word, alcoholic fermentation—is a chemical action,
connected with the vegetable life of cells which may differ greatly in
their nature, and that it takes place at the moment when these cells,
ceasing to have the power of freely consuming the materials of their
nutrition by respiratory processes—that is, by the absorption of free
oxygen—continue to live by utilizing oxygenated matters which, like
sugar or such unstable substances, produce heat by their decomposition.
The character of ferment thus presents itself to us, not as being
peculiar to any particular being or to any particular organ, but as a
general property of the living cell. This character is always ready to
manifest itself, and, in reality, does manifest itself as soon as life
ceases to perform its functions under the influence of free oxygen, or
without a quantity of that gas sufficient for all the acts of nutrition.
Thus we should see it appear and disappear concomitantly with that mode
of life; feeble and fugacious in its action when the conditions of this
vitality are of a similarly restricted character; intense, on the other
hand, and of long duration and productive of large quantities of
carbonic acid gas and alcohol, when the conditions are such that the
plant or cell can multiply with facility in this novel manner. To this
we may attribute all possible degrees of activity in fermentation, as
well as the existence of ferments of every variety of form and of very
different species. It may readily be imagined that sugar may undergo
decomposition in a quite different manner from that of which we have
spoken, that instead of alcohol, carbonic acid gas, glycerine, and
similar substances, it may yield lactic, butyric, acetic, and other
acids. It would be only one definite class of cellular organisms, the
members of which resembled each other more or less, that decomposed
sugar into alcohol and carbonic acid; others, specifically different,
would act in a different manner. In short, we may say that the number of
these living organisms is a measure of the number of different ferments.

Plate IV. represents in its two halves the condition of the _mycoderma
vini_ at two different and unequal periods after its submersion. In the
left-hand semi-circle, it is evident that many of the figures are
swollen, that modification of their protoplasm has taken place, and
incipient budding is going on in several of them. A budding of this kind
would not wither; the buds would grow and, detaching themselves, would
form new cells capable of budding in their turn. We should have under
our eyes all the characteristics of a yeast, which, beyond doubt, would
give rise to a very active fermentation, inasmuch as it would belong to
the order of phenomena of nutrition and vital energy of which we are
speaking. Instead, however, of insisting upon the acceptance of our
interpretations, based on a few facts merely, let us go on to accumulate
facts, varying the conditions as much as possible. Our examples, taken
singly, may seem insufficient to establish the theory that it will be
our endeavour to substantiate, but taken together we trust that they
will secure our readers’ confidence.

We may now, perhaps with advantage, introduce two new expressions to
embody the preceding facts, by the help of which we may often shorten
our subsequent explanations. Since life can continue, under certain
conditions, away from contact with the oxygen of the air, and since the
altered nutrition is accompanied by a phenomenon which is of great
scientific as well as industrial importance, we may divide living beings
into two classes, _aërobian_, that is those which cannot live without
air, and _anaërobian_, which, strictly speaking, and for a time, can do
without it; these latter would be ferments, properly so called. Again,
since we can conceive, in an entire organism, some organ or even a cell
capable of existing, at least momentarily, apart from the influence of
the air, and endowed at a given moment with the character of a ferment,
we may, in like manner, make use of the expression _anaërobian_ cell, in
opposition to a cell that is _aërobian_.

As long ago as 1863, in our work on putrefaction, we proposed to adopt
the preceding expressions, and since then we have had the satisfaction
of seeing them used by different authors in France and other countries.

One of the principal assertions in this paragraph relates to the
non-transformation of _mycoderma vini_ into other moulds or into
yeast.[69]

[Illustration: Plate 4. Mycoderma Vini functioning as an Alcoholic
Ferment.—Right Half, showing Appearance of Spores just Sown; Left Half,
their Appearance after an Interval of Submerged Life.]

For a long time, like Turpin and many other observers, although we had
no belief in the transformation of _mycoderma vini_ into any one of the
common moulds, yet we did believe in its transformation into alcoholic
ferment. In the course of more elaborate researches, however, we at last
discovered that our previous experiments had been vitiated from the same
source of error which we have so often had occasion to point out as
affecting the observations of our opponents, namely, the fortuitous and
spontaneous introduction, unknown to the experimentalist, of germs of
the very plant for whose appearance by way of transformation he is
seeking.

When we consider that every fermented vinous liquor, when put on
draught, is liable to efflorescence, it is difficult to avoid the
supposition that this efflorescence is primarily due to cells of the
yeast that has caused the liquid to ferment, from which cells the
liquid could not be completely freed, no matter how bright it might
have been, and which come to the surface of the liquid to live after
the manner of fungoid growths. We wished to test this supposition by
means of experiments. So great, however, was the resemblance between
the forms possible to yeast and mycoderma, of which latter
efflorescence is really composed, that we quite despaired of being
able to solve the question by microscopical examination, that is, by
observing the actual conversion of a cell of yeast into a cell of
mycoderma. In order, then, to overcome that difficulty, we endeavoured
to produce an inverse transformation—that of mycoderma into yeast. We
imagined that we should doubtless obtain this result by submerging
some of the efflorescence of wine or beer in a saccharine liquid well
adapted to alcoholic fermentation. By submerging the mycoderma we
would do away with the ordinary conditions of life in this kind of
fungoid growth; for we would thus prevent the supply of oxygen from
the air, since that oxygen would always be excluded, in the most
effectual manner possible, by the portion of mycoderma that would
remain on the surface of the liquid, even after the submersion
process; and on the other hand, we would be subjecting our growth to
the ordinary conditions of ferment life, which acts at the bottom or
in the bulk of liquids fermenting.

Our experiments were conducted in the following manner:—In some flat
porcelain basins, we grew some pure _mycoderma vini_[70] on fermented
liquids, such as wine or beer, or on artificially vinous liquids, such
as alcoholized yeast-water, taking care to boil these liquids previously
to kill any germs of yeast or other organism that they might contain.
The basins themselves, as well as the plates of glass with which they
were to be covered, were plunged into boiling water just before they
were wanted for use. As soon as the film of mycoderma had become well
developed and thick, and even wrinkled—a process requiring not more than
two or three days during summer heat—we decanted the subjacent liquid,
by means of a siphon, so as to leave the film on the bottom of the
basin. We then diffused the whole mass of efflorescence in a saccharine
liquid that had been boiled and afterwards cooled down in a closed
vessel; generally, we used wort or must preserved by Appert’s process.
After that, we emptied the mixture of saccharine liquid and
efflorescence into long-necked flasks that had likewise been previously
heated, as also had the funnels used in the process of transference.

It seemed to us that experiments conducted with all these precautions
must be free from causes of error. It was true that we were working more
or less in contact with atmospheric air, but all that we had to fear for
the soundness of the conclusions which we might draw was the presence of
germs of alcoholic ferment, and we considered how few of these there are
amongst floating particles of dust. Consequently, if we succeeded in
observing the advent of yeast in each of the long-necked flasks,
accompanied by an active alcoholic fermentation, we thought that we
might, without danger of error, admit as a fact the transformation of
cells of mycoderma into cells of yeast. Again, we thought that we should
probably find in the forms of the cells of yeast which were directly
derived from the cells of mycoderma, a more or less elongated structure,
which would be a convincing proof of the transformation that we were
seeking, if, indeed, such transformation were possible.

Strange to say, everything happened in a manner that seemed to realize
our expectations. The saccharine worts in the flasks in which we had
mixed and submerged the mycoderma, fermented in the course of a few
days; the yeast first appeared in elongated shapes; lastly, we could see
under the microscope that many of the cells or jointed filaments of
mycoderma were inflated and presented the appearance of undoubted
gradations between their natural state and that of the cells of yeast
which soon formed part of the deposit in the vessels. In spite of all
this, however, we were the victims of an illusion.

In experiments conducted as we have just described, the yeast which
appears, and which soon sets up an active alcoholic fermentation, is
introduced in the first place by atmospheric air, from which germs are
constantly falling either upon the film of mycoderma or upon the objects
that are employed in the successive manipulations. Two peculiarities in
these experiments first opened our eyes to the existence of this cause
of error. We sometimes found at the bottom of the flasks in which we had
submerged the efflorescence, along with the cells of mycoderma, large,
spherical cells of _mucor mucedo_ or _racemosus_, ferment-cells that we
shall soon learn to recognize in studying this curious fungoid growth.
The existence of _mucor mucedo_ or _racemosus_, where we had only sown
_mycoderma vini_, was to us a proof that one or more spores of that
mucor had been introduced by the surrounding air. If then, we reasoned,
the air can introduce spores of mucor into our field of operations, why
should it not introduce cells of yeast, especially in our laboratory?
Again, it sometimes happened that a negative result was obtained.
Harassed by doubts about the reality of this transformation, which
accorded so well with, the physiological theory of fermentation we had
been led to adopt, we repeated the experiments many times, and in some
cases we failed to detect any appearance whatever of a transformation of
mycoderma into yeast cells, although the conditions under which each of
the experiments was conducted had been as similar as could be.

We were at a loss to account for this inactivity in the cells of the
mycoderma. Even in the most favourable cases of the supposed
fermentation, it was evident that a host of cells of _mycoderma vini_
did not become cells of yeast; but how could it possibly be admitted
that amongst the millions of submerged cells, none were adapted for
transformation, if that transformation were at all possible?

Thereupon, to find a way out of the difficulty, we resolved to modify
completely the conditions of our experiments, and to apply to the
research that we had in view a mode of cultivation that might
completely, or nearly so, obviate the sole cause of error that we
suspected, namely, the possible fall of cells or germs of yeast during
the manipulations. We secured this by the use of flasks with two tubes,
the right hand one of which was closed by means of a piece of
india-rubber tubing with a glass stopper, the other one being drawn out
in the shape of a swan’s neck. The use of these flasks, which was then
new to us, permitted us to grow mycoderma and to study it under the
microscope without fear of disturbance from exterior particles of dust.
This time we obtained the results given in the first part of this
paragraph. We no longer observed yeast or alcoholic fermentation
following the submersion of the efflorescence, either in the flasks
themselves, or in the test-flasks attached to them, as represented in
Fig. 19. We observed, however, that kind of alcoholic fermentation of
which we have already spoken and which is due to the mycoderma itself, a
fermentative action that is still more instructive than the one which we
thought we had determined, and certainly not less calculated to support
the theory of fermentation which we have already briefly sketched.

In an age when ideas involving transformation of species are so readily
accepted, perhaps in consequence of their requiring no rigorous
experimental work, it is not without interest to consider that, in the
course of our researches upon the growths of microscopic plants in a
state of purity, we once were inclined to believe in the transformation
of one organism into another—the transformation of _mycoderma vini_ or
_cerevisiæ_ into yeast, and that, on that occasion, we were altogether
wrong, through having ourselves fallen a victim to the identical source
of error which confidence in our theory of germs had led us so
frequently to detect as affecting the observations of others.


         § III.—Growth of Mycoderma Aceti in a State of Purity.


The study of _mycoderma aceti_ has not escaped the numerous causes of
error which are apt to attend all observations made on microscopic
organisms. This little fungus is still believed by many authors to be
one of those polymorphous species capable of great modifications,
according to the conditions of their cultivation—it could be, in turns,
bacterium, vibrio, yeast, &c. Respecting it, we have seen resuscitated
under a modern name, in the course of the last few years, the old
hypothesis of Buffon concerning _organic molecules_, that of Turpin
concerning the _punctiform globulines_ of barley, milk, and albumen, and
the theory maintained by Dr. Pineau, of Nancy, and by Pouchet concerning
_proliferous pellicles_.[71]

M. Béchamp, Professor in the Faculty of Medicine at Montpellier,
disdaining to adopt the expressions which we have just used, has
substituted for them that of _microzyma_, whilst adhering to the
opinions and errors represented by the other expressions. This savant
designates under the name of _microzyma_ all those punctiform globulines
that are met with in most organic liquids when submitted to the
microscope; and attributes to them, with Turpin, the faculty of playing
the part of ferments, as well as of transforming themselves into yeast
and various other organisms. They are contained in milk, blood, eggs,
the infusion of barley, and such like; nay, we may even find them in
chalk, and so we have the fine discovery of _Microzyma cretae_ as a
distinct species!

Those who, like ourselves, cannot see in these granulations of organic
liquids ought besides things whose nature is still undetermined, term
them _molecular granules_, or, in reference to their Brownian movements,
_mobile granules_. Indefinite expression is the best exponent of
imperfect knowledge; when a precise terminology is invented, without any
basis of precise ideas derived from a rigid observation of facts, sooner
or later the hypothetical facts disappear, but the terminology
prematurely created to explain them, hangs about the Science, and,
bearing an erroneous interpretation, retards rather than promotes real
progress.

We may here introduce a summary of Turpin’s system, as given by himself.
It forms a complete biogenesis, which leaves far behind it M. Béchamp’s
theory of _microzymata_, M. Fremy’s descriptions of _hemi-organism_, and
M. Trécul’s account of the genesis of bacteria and lactic ferment:—

“When a mucous substance presents nothing visible through the
microscope, as, for example, gelatinous matter, dissolved gum, the white
of eggs, or plant-sap, simply thickened on its way to _cambium_, we call
it _organic matter_ or _organizable matter_. We attribute to it the
fecundating power of organic life in the simplest degree; we consider it
as material still isolated from organization. We suppose that the
invisible molecules, of which this organizable matter is composed, come
together, combine and serve through this association in the construction
of the different elementary forms of future tissues.

“May we not with greater truth believe that organizable matter is of
varied origin, formed of innumerable globulines, too minute and
transparent as yet to be observed by our present microscopical means,
and that these globulines which are always endowed with motion and a
special vital centre, are all capable, although many of them _do_ abort,
of separate development either into a formative element of tissue or
into a mucedinous plant?

“Organizable matter may, according to its successive states of
development or age, and according to the different forms it takes in the
tissues, be distinguished by special names:—

“1.—We may term matter _organizable_ as long as the globulines composing
it are not yet visible to microscopes of existing power.

“2.—We may speak of _amorphous_ or _globuline tissue_ when even the
globulines, previously invisible, have increased so as to be seen under
the microscope, the term amorphous, or shapeless, being here applied to
the association of globulines, and not to the globulines themselves.

“3.—Then we have _vesicular tissue_, when the globulines, continuing to
increase, have developed in such a manner as to present a mass of
continuous vesicles, still empty or already containing a new generation
of globulines.

“4.—Lastly we have _filamentous_ or _tubular tissue_, when the
globulines, instead of vesiculating, form threads or tubes.”[72]

Such are the purely hypothetical and exploded ideas which MM. Fremy,
Trécul, Béchamp, H. Hoffmann, Hallier, and others would revive in our
own day, in opposition to a theory so clear and so well supported by
facts as that of germs floating in the air, or spread over the surface
of objects, as fruits, dry or green wood, and so on.

M. Béchamp believes that he has discovered that _mother of vinegar_,
introduced into various saccharine liquids, in the presence of carbonate
of lime, generates bacteria, which, with the sugar or dregs, produce
butyric, lactic, and acetic acids, and that this same mother of vinegar,
without the addition of the carbonate of lime, “generates, on the other
hand, the fine cells, which produce the normal alcoholic fermentation of
cane sugar.” Further, M. Béchamp advances the hypothesis that mother of
vinegar is a conglomeration of _microzymata_, and, as he fails to see in
the experiments on which he bases the conclusions which we have just
given, that bacteria and ferment cells are the result of spontaneous
impregnation, having no connection with the presence of mother of
vinegar, on which he experimented, he arrives at this conclusion: “In
the experiments which I have just described, things happened as though
the microzyma, under some peculiarly favourable conditions, had been the
parent both of the bacteria and the cells.”[73] ...

The object of the following experiments was the study of these assumed
transformations of the _mycoderma aceti_ in saccharine liquids, in the
presence and in the absence of carbonate of lime.

We prepared some two-necked flasks, containing as a growing medium a
liquid composed of one-third of Orleans vinegar, and two-thirds of a
white wine used by vinegar-makers in Orleans. This liquid is peculiarly
adapted to the development of _mycoderma aceti_.

On December 13th, 1872, we sowed the little plant in a state of purity,
by means of a piece of platinum wire, in the manner already explained in
connection with propagation of other fungoid growths. On December 19th a
young and thin film of _mycoderma aceti_ covered the surface of the
liquid. We then poured out the liquid through the right-hand tube, at
the same time heating the end of the bent tube, to purify the air that
passed into the flask. The whole film of _mycoderma aceti_ remained
adhering to the interior sides of the flask during this decanting. The
question then was how to convey this film of the little plant into a
saccharine liquid of a particular kind. We effected this easily by the
following means: After having emptied the flask, as just described,
instead of re-closing the india-rubber nozzle on the end of the
right-hand tube, we attached it to a test-flask containing the
saccharine liquid on which we wished to operate. This had been
previously boiled in the test-flask, and when we attached the neck of
the test-flask, previously slightly drawn out and curved, to the
india-rubber tube, the liquid was still very warm. We permitted the
liquid in the test-flask to cool down, and, then, taking up the
test-flask, we decanted its contents into the other flask, in which, as
we have already said, the film of _mycoderma aceti_ had been left. In
this way the film became partly submerged, partly spread over the
surface of the new liquid. Experiments were made with two saccharine
liquids, must and wort. In the case of the latter, from December 22nd
the whole surface of the liquid was covered by a film of _mycoderma
aceti_, which even spread up the moist sides of the flask above the
level of the liquid. In the case of the must, on the other hand, the
plant for some time did not seem to be developing; on December 24th,
however, it was visibly spreading over the surface of the must. The
following days we frequently shook up the films to separate them, and
spread them over the subjacent liquid. There were no signs of alcoholic
fermentation.

On December 30th we introduced several grammes (50 or 60 grains) of
carbonate of lime into each of the flasks, an operation of little
difficulty, which we effected in a manner similar to that just
described. We substituted for the test-flask another flask—or, better
still, a simple glass tube—containing carbonate of lime that had been
subjected to great heat in the flask or tube, and there left to cool
down. When cold, we poured the powdered carbonate of lime into the
liquid in the flask, in this way avoiding the possibility of any error
from the introduction with the carbonate of lime of any foreign germ.

In neither case did we obtain alcoholic fermentation, nor was there any
appearance of lactic fermentation, or bacteria, or _vibrios_, properly
so called. The flasks remained in the oven, at a temperature of about
25° C. (77° F.), until the end of January, 1873, when we made a
microscopical examination of their deposits, exercising greater care and
precaution than we had adopted in the case of those examinations which
we had made from time to time in the course of the experiment to assure
ourselves of the nature of the organisms present.[74] The result was
that we never found anything besides the _mycoderma aceti_, which had
developed, although with great difficulty, on the surface of the liquids
neutralized with carbonate of lime. The beaded filaments had, under
these circumstances, only become a little larger than they had been in
the unsweetened acid liquids.

_Mycoderma aceti_, then, grown on sweetened acid or neutral liquids,
grown in the absence or in the presence of carbonate of lime, undergoes
no transformation into bacteria or vibrios or yeast, if only we operate
with pure germs, free from the dust floating in the air, and from that
which, unknown to the operator, may be introduced by means of the
vessels and materials employed. It may be asked, do we, therefore,
absolutely, reject the theory of the polymorphism of _mycoderma aceti_?
On the contrary, we have endeavoured to prove the existence of this
polymorphism again and again in a variety of ways. We have been mostly
concerned with physiological polymorphism; that is, our efforts have
been directed to ascertain if _mycoderma aceti_ might be, for example,
the _aërobian_ form of a ferment from which it differed physiologically,
as, for instance, lactic ferment, which, in shape, sometimes bears a
striking resemblance to _mycoderma aceti_. We have not succeeded in
discovering anything of the kind up to the present time.

What, in view of the positive proofs to the contrary, we do absolutely
reject in the matter of this mycoderma, is the theory of polymorphisms,
advocated by M. Béchamp and other authors, which, in our judgment, can
only be founded on incomplete and erroneous observations.


  § IV.—Growth of Mucor Racemosus in a state of Purity—Example of Life
    more active and lasting when removed from the influence of Air.


Side by side with the facts explained in the last paragraph, the study
of varieties of the genus _mucor_, grown in natural or artificial
saccharine liquids, is of great importance to the establishment of the
physiological theory of fermentation, which we shall explain later on.
There is a very remarkable work on the subject of this mucedinous fungus
by a German botanist, M. Bail, who, in 1857, declared that _mucor
mucedo_ caused alcoholic fermentation, and could change into ordinary
yeast. The first assertion, relating to the alcoholic fermentation that
this fungoid growth which is everywhere so abundant may cause, is quite
correct; the second which relates to its faculty of changing into yeast
is erroneous.[75]

On June 13th, 1872, we sowed by the help of a platinum wire in some
wort, contained in two-necked flasks, A, B, and C, several of the minute
sporange-bearing filaments of _mucor_ along with the heads containing
the spores.

On June 14th, there was no mycelium visible to the naked eye in the
liquids.

On June 15th mycelium was very abundant, and was borne up by bubbles of
gas. In addition to this there were a few scattered patches of bubbles
on the surface of the liquid, showing that fermentation had commenced.

On June 16th fermentation continued to show itself by the frothy state
of the crusts of mycelium buoyed up by the bubbles of gas.

On June 17th we attached B and C separately, as indicated in Fig. 19 (p.
101) to test-flasks, into which we transferred nearly all their
contents. Some clusters of entangled filaments of mycelium remained on
the surface of the liquids in the test-flasks.

On June 18th a very slow fermentation commenced in the test-flasks; it
continued for some days without becoming more active. A little bubble
would slowly rise from the bottom of the vessel, succeeded after a short
interval by another, and so on. The temperature of the oven was 24° C.
(75° F.). On June 22nd we raised it to 28° C. (82° F.). The fermentation
became more rapid, a constant succession of bubbles rose quickly from
the bottom of the test-flasks; still there was none of the vivacity of
an alcoholic fermentation produced by yeast.

On June 25th the fermentation was in much the same condition, if
anything rather less active.

On June 28th temperature 25° C. (77° F); fermentation had stopped.

On June 29th we raised the temperature to 27° C. (81° F.) again, and
some slight revival of fermentation manifested itself.

The increase in temperature, therefore, as might have been expected,
exercises a considerable influence on this kind of fermentation.

The vessels were then left to themselves, and during the course of three
months they did not show the least sign of fermentation; moreover, we
did not observe, either on the interior walls of the empty flasks, or on
the surface or throughout the body of the liquid in the test-glasses,
any fungoid production or organism whatever different from _mucor_
itself.

The same observations apply to the vessel A; in this case the liquid
that remained in the flask was covered with a gelatinous and frothy
mycelium.

On October 20th, 1872, after a lapse of three months and a half, we
poured the liquid from the test-flask attached to flask C back again to
that flask. The test-flask connected with flask B we left untouched
alongside the other flasks to serve as a means of comparison.

On October 21st, 22nd, 23rd, we observed nothing; on succeeding days,
however, some patches of bubbles appeared on the surface of the liquid
in flask C, and clusters of mycelium buoyed up by the bubbles of gas
which they imprisoned. Life had resumed its course, and with life
fermentation had recommenced. What had been the cause of this change in
the condition of the liquid, after an absolute quiescence of three
months? There can be but one answer to this question: for in the other
vessels there was no corresponding movement, or sign of life to be
detected. In this vessel, however, an aeration of the plant had
evidently taken place, consequent on the decantation and contact with
the atmosphere of the flask, which communicated with the exterior air
through the curved tube. This aeration had been absent or ineffective
before decantation, in consequence of the great depth of liquid in the
test-flask, the surface of which, too, was covered by a mass of mycelium
filaments, itself effectually opposing any aeration of the liquid.
Moreover, the surface of the liquid in the narrow neck of the test-flask
had necessarily been covered by a layer of carbonic acid gas. We may
investigate more thoroughly the influence of aeration, and its relation
to the resumption of life in the mycelium of _mucor_, by restoring the
liquid to its previous condition of depth and so cutting off again
contact with the air.

For this purpose, on October 31st we decanted once more the liquid and
its deposit from the flask into the test-glass. The same evening a
slight but continuous fermentation, with formation of froth, appeared on
the surface of the liquid in the neck of the test-glass. Fermentation
although never vigorous, continued the following days, and until
December 20th.

Between December 20th and 23rd, it ceased altogether to manifest itself
by liberation of gas. As for the flask B, during all this time it had
remained quite inactive and in the same state in which it had existed
since June 29th, although the oven had on several days been heated to
28° C. (82° F.).

On December 23rd, 1872, wishing to assure ourselves of the state of the
plant in flask B, we subjected it to the same operation to which the
flask C had been subjected on October 20th: that is to say, we poured
the contents of the test-glass back into the connected flask, with the
object of supplying the plant with oxygen.

On December 24th, 25th, 26th, 27th, there was no apparent change.

On December 28th bubbles of gas began to be evolved carrying up clusters
of mycelium to the surface of the liquid. It was evident, therefore,
that the quiescence in the test-glass attached to flask B, was solely
due to deprivation of air, as had happened in the case of the test-glass
attached to flask C, up to the date of October 31st.

On this day, December 28th, we re-decanted the contents of the flask
into the test-glass, and the following day a continuous but feeble
fermentation proceeded. This lasted until January 22nd, although very
sluggish in character; it is evident that these effects were exactly the
same as those which took place in flask C.[76]

We should observe before we proceed further, that we took specimens from
the flasks A, B, C, at different times between June and January, and
that the microscope never revealed the least trace of yeast in them. We
may note besides that, during this interval, we impregnated fresh flasks
of wort with specimens taken from the deposits in the flasks A, B, C,
and that we always obtained reproduction of the _mucor_ and its peculiar
fermentation without the least appearance of ordinary ferment.

The inferences from the results that we have just detailed follow
readily, and are besides of great interest. In the first place, it is
evident that even if the _mucor mucedo_ may be able to produce alcoholic
fermentation, it is totally incapable of changing into yeast. The two
plants are necessarily and radically distinct, and, if different authors
have succeeded in obtaining them mixed one with another in growths of
_mucor_, this intermixture was doubtless the result of a spontaneous
sowing of the yeast, the germs of which abound, particularly in the
particles of dust existing in the atmosphere of any laboratory in which
studies relating to fermentation are pursued.

This, however, is not the most striking inference from the facts which
the cultivation of these organisms revealed. The _mucor_ is evidently a
plant, at the same time _aërobian_ and _anaërobian_. If we had sown the
spore-bearing filaments of _mucor_ on slices of pear, lemon, or similar
fruit, we should have seen the spores germinate, tubes of mycelium
ramifying on the surface of the substratum, and reproducing sporiferous
aerial _hyphae_. In this case the plant would have effected all its
phenomena of nutrition by absorbing oxygen and emitting carbonic acid,
after the manner of animals, as, in our essay on the organic corpuscles
which exist in a state of suspension in the atmosphere, we have shown to
be the case generally with fungoid growths. Under these circumstances,
the only sugar decomposed would have been a quantity equivalent to that
assimilated in forming the cellulose of the young tissues of the fungus,
or in entering into combination, either with the elements of ammonia or
with the sulphur of the sulphates, or the phosphorus of the phosphates,
to form the albuminous substances of the interior of the cells.[77] In
this case the sugar used up would furnish no alcohol, or at least, if
alcohol were formed, it would be decomposed immediately. All _aerial_
growths take place in the same manner; and such is the nature of
nutrition and life in all the larger forms.

In our flasks, on the other hand, the life of the little plant functions
quite differently. Deprived of oxygen, or having at its disposal but an
insufficient quantity of that gas, after a life of activity in contact
with air, it can, nevertheless, live apart from the direct action of
that element, and the combinations to which it gives rise. On the other
hand, we see all the signs of alcoholic fermentation appear; that is, a
notable proportion of sugar, in comparison with the weight of solid
matter assimilated and fixed by the plant, is decomposed into alcohol
and carbonic acid gas; and this decomposition continues as long as life
itself continues in the cells, and they remain submerged, this last
condition being effected by the decantation of the liquid and its
deposit into the test-glass. Along with the disappearance of the
phenomena of vital activity in the cells, the fermentation ceases
absolutely, or at least is no longer visible externally, by reason of
its extreme feebleness. The cells then assume an old, shrivelled,
worn-out appearance, with irregular outlines and granular markings.
Their life is merely suspended, however, not extinct; for if they be
supplied once more with oxygen, and suffered to exist under the
influence of that gas, they will vegetate again, and become capable of
producing fermentation afresh, even after having been excluded from the
air for a considerable time.

Oxygen then presents itself to us as being endowed with a certain
determining stimulus in the matter of nutritive action enabling this
action to be prolonged beyond the point where the direct influence of
oxygen ceases. In time the energy that has been imparted to the cells
will die away, and then also fermentation will cease, to be resumed,
however, when the plant is once more submitted to the revivifying action
of the gas. It seems as though the vital energy derived from the
influence of gaseous oxygen were capable of effecting an assimilation of
oxygen, not in the gaseous state, but existing in some state of
combination, and hence its power of causing the decomposition of sugar.
Looking at the matter in this light, it seems to us that we may discover
in it a fact of general occurrence, that this peculiar action of the
oxygen and the cells is to be seen in all living beings. For indeed is
there any cell which, if suddenly and completely deprived of air, would
perish forthwith, and absolutely? Probably there is not a single one
that would do so. With certain modifications of greater or less amount
the assimilative and excretive acts which have taken place during life
must be carried on after the suppression of oxygen, resulting in
fermentations ordinarily obscure and feeble, but in the case of the
cells of ferments, properly so called, manifesting an activity both
greater in amount and more enduring.

Let us now proceed to compare the weight of alcohol formed by the
_mucor_ during fermentation with the weight of the plant itself.

_First experiment._—One of the double-necked flasks contained at
starting 120 c.c. (about 4 fl. oz.) of wort.

On January 2nd, 1873, we attached this flask to a test-glass, containing
a deposit of _mucor_ ferment (Fig. 19, p. 101), a few drops of which we
poured into the wort in the flask, to impregnate it. On January 3rd we
decanted the wort from the flask into the test-glass; under these
conditions we have seen that the wort must ferment.

On January 18th the fermentation in the test-glass ceased. On July 31st,
1873, we transferred the liquid from the test-glass back to the flask.
On August 4th, 1873, we again decanted this same liquid from the flask
into the test-glass. On December 25th, 1873, we once more removed the
liquid from the test-glass to the flask, and allowed it to remain so
until December 23rd, 1874, on which day we submitted it to examination.
It was found to contain per 100 c.c. (3-½ fl. oz.)

                                             Grains.   Grammes.[78]

    Total weight of the fungus                   5·7           0·37

    Absolute alcohol                            50·9            3·3

    Acidity, estimated in its equivalent of      1·7           0·11
    sulphuric acid

    Sugar, determined by cupric solution        82·2            5·2

    Dextrine (?)                                24·6            1·6

The total weight of fungoid growth being 0·37 gramme, and the total
weight of absolute alcohol for the 120 c.c. of fermented liquid being 4
grammes, we had, consequently, from ten to eleven times by weight more
alcohol than fungus.

_Second experiment._—On June 13th, 1872, we sowed two or three
sporiferous heads of _mucor_ in some wort contained in one of the
double-necked flasks. The temperature of our oven varied between 23° C.
and 25° C. (73° F. to 77° F.) The total volume of liquid was 120 c.c.,
as before.

June 15th, mycelium had developed, buoyed up on bubbles of gas.

June 16th, patches of bubbles, due to fermentation, covered the surface
of the liquid.

June 17th, we transferred the liquid to the test-glass.

June 28th, fermentation in the test-flask had ceased.

June 28th, fermentation recommenced, the temperature of the oven being
raised to 27° C. (80° F.).

October 20th, the liquid was transferred back from the test-glass to the
flask.

October 24th, mycelium had developed, supported by big bubbles on the
surface of the liquid in the flask.

October 31st, we retransferred the liquid to the test-glass.

November 1st, a feeble, but continuous fermentation commenced. This was
kept up until January 2nd, 1873, on which day we transferred the liquid,
with its deposit from the test-glass to the flask, when it now seemed to
be quite inert. We left it in this flask until December 24th, 1874,
without its manifesting during this long interval any sign of
fermentation; nor did the fungus appear to grow at all.

We then submitted the liquid to analysis, and found in it, per 100 c.c.—

                                                       Grammes.[79]

    Total weight of fungoid growth                             0·25

    Absolute alcohol                                            3·4

    Acidity, estimated in its equivalent of sulphuric          0·12
    acid

    Sugar, determined by copper solution                        6·2

    Sugar, determined after treatment by boiling with           1·0
    sulphuric acid, and deduction of amount of sugar
    already obtained (dextrine)?

The total weight of absolute alcohol for the 120 c.c. of fermented
liquid was 4·1 grammes—that is, the weight of the alcohol was sixteen or
seventeen times that of the plant.

[Illustration: Plate 5. Mucor, Vegetating Submerged, in Deficit of Air.]

The structure of the plant differs considerably when it lives surrounded
by air, and when it is more or less completely deprived of that fluid.
If it has an abundance of air at its disposal, if it vegetates on the
surface of a moist substance or in a liquid in which the air held in
solution may be renewed without being incessantly displaced by carbonic
acid gas, we shall see it develop as an ordinary fungoid growth, with a
mycelium consisting of filaments more or less slender, branching, and
entangled, sending up from the surface of the liquid aerial organs of
fructification. This is the well-known form of vegetation of the common
_mucor_. On the other hand, if we compel the _mucor_ to live in a
saccharine liquid with insufficiency of air, at least for some of its
parts, the mode of vegetation will change completely, as we have seen in
the case of _penicillium_, _aspergillus_, and _mycoderma vini_ when
submerged, but with this difference, that in the case of the _mucor_ the
changes in question, and the activity of nutrition under these new
conditions, are much more marked than in the case of those other
organisms. The spores grow larger and the filaments of mycelium which do
develop are much stronger than those in the normal plant. These
filaments put forth, here and there, other filaments which detach
themselves and vegetate at the side of the others, being terminated or
interrupted by chains of large cells, species of spores which can live
by budding and reproducing cells similar to themselves or by elongating
into filaments.

Plate V. represents the living plant submerged at a little depth, and
having, consequently, still at its disposal a certain quantity of air,
insufficient, however, to supply the oxygen needed for all the acts of
nutrition. In this case the _mucor_ appears very different,
morphologically, from what it is when in free contact with air. Here it
forms short filaments, having a diameter double or triple that of the
filaments of the ordinary mycelium with branches and buds all over, and
what is especially characteristic, forming a network of chains of cells,
sometimes spherical, sometimes oval or pear-shaped, which are the actual
spores. These, as soon as they are detached, bud in their turn, and
reproduce either cells or branching tubes; these cells or the chaplets
which they form being known under the name _mycelian spores_ or
_conidia_. Our plate gives these different aspects very correctly, and
affords us a good idea of the luxuriant state of this remarkable
vegetation.

Plate VI. represents the plant living at a greater depth with less air,
expending, by means of sugar as source of heat, the energy which it
acquired in vegetating under the influence of the oxygen of the air. The
filaments are fewer and older in aspect, and the number of cellular
forms is proportionately larger than in the former case, the budding
giving rise by preference to spherical or oval cells. On a single cell
we often see two, three, four, five, six, and even more buds.

When the buds of the oval or spherical cells detach themselves whilst
young, they often resemble in form and size cells of ordinary yeast, nor
can even considerable experience in this kind of observation always
enable us to distinguish them. Hence we may easily understand how many
have come to believe, with so skilful a botanist as Dr. Bail, in the
transformation of _mucor_ into yeast.

With the forms represented in Plates V. and VI., the plant is more of a
ferment than of a fungoid growth. In such cases the weight of sugar
decomposed in comparison with the weight of new cell-globules formed is
very considerable, an effect which is more marked the less air the plant
has at its disposal. Under the latter conditions, however, vegetation is
slow and laborious, and the ferment very soon assumes an aspect of age,
and we must constantly rejuvenate the cells by bringing them into
contact with oxygen, and subjecting them to the action of limited
quantities of that gas, and so promote their vegetation and prolong
their fermentative activity. This effect we brought about when we
retransferred the liquid and its deposit of _mucor_ from the test-glass
to the flask, thus bringing them into contact with fresh air. We saw
cells that appeared old, dark, and highly granulated, become inflated,
grow more transparent, and fill with a gelatinous protoplasm, the few
granulations which they still exhibited assuming a brilliant appearance
when we succeeded in distinguishing them; and finally, a very active
budding was set up. Under this reviving influence life could continue
once more away from the air, although with difficulty, so that
fermentation would be most intense if the large filaments and their
conidia were constantly being removed from and to the action of air.

The preceding plates show several instances of this rejuvenescence of
the old cells of _mucor_ ferment.

[Illustration: Plate 6. Ferment of Mucor.]

We have omitted to represent amongst the old cells some cells which have
their granulations collected about the centre, with an empty space
between the granulations and the exterior borders.[80] In this state,
cells are generally dead and incapable of any revival. It is impossible
to avoid being impressed by the striking analogies which exist between
all these facts and those presented by cells of yeast.

In concluding our study of the vegetation of _mucor_ as a mould and
_mucor_ as a ferment, we may again remark that the most striking
analogies also exist between the preceding observations and those we
have seen in the case of _penicillium_, _aspergillus_, and _mycoderma
vini_. These latter plants do not furnish alcohol or carbonic acid gas
by direct fermentation of the sugar, as long as we let them vegetate
with plenty of air at their disposal. Once submerged, however, their
vital aspect changes; on the one hand the cells or filaments of the
mycelia evince a tendency to become larger; on the other hand there is a
tendency to greater closeness in these latter, and, consequently, a
transition to the state of _conidia_. Lastly, there is a correlative
budding of cells, accompanied by a formation of alcohol and liberation
of carbonic acid gas; in short, all the ordinary signs of alcoholic
fermentation.

The principal difference in the case of _mucor_ consists in this, that
the vegetation of this latter, under the conditions of insufficient
aeration or none at all, is more decided, both as to extent and
duration.

It may be thought that all the varieties of _mucor_ are capable of
yielding the kind of ferment that we have just mentioned. But this is
not the case; and here we have another striking proof of the great
physiological differences presented by forms of vegetation so intimately
connected with each other that, in botanical classifications, they must
be put as closely as possible together. Of this fact we have the most
striking example in _mycoderma vini_ and the alcoholic ferments,
properly so called, which so closely resemble each other in form and
development that they might be supposed to be identical, at least,
according to our present knowledge, but which differ so widely in their
physiological aspects.

On November 17th, 1873, we found a very beautiful specimen of _mucor
mucedo_ on a pear, under a glass bell jar. It was a mass of perfectly
straight filaments, simple and isolated, very large in comparison with
those ordinarily met with, each terminating in a sporange, identical to
that of _mucor mucedo_, and proportionately well developed. We are able
to distinguish _mucor racemosus_ from _mucor mucedo_ only by the
circumstance of its having on its sporange-bearing hyphæ lateral
branches which also terminate in sporanges.

We sowed only one of the terminal heads of the large erect hyphae in
some wort, in which it soon produced an abundant mycelium, but without
the least appearance of gas. For a very long time, up to January 7th,
1875, we studied the developments of this organism, which remained all
the time perfectly pure, in consequence of our having cultivated it in
one of our two-necked flasks on pure wort.

The total volume of liquid, which was 130 c.c., (4·57 fl. oz.) contained
2·3 grammes (35·3 grains) of alcohol. In spite of this rather large
proportion of alcohol, a clear sign of undoubted fermentation, the plant
had yielded no _conidia_ at all, nor any cell-globules of ferment. Some
of the filaments, however, were larger than the rest, and exhibited
irregularly-shaped swellings, which in some cases were of enormous size.
Whilst the natural mycelial filaments, by which we mean the vegetating
part of mucor, which were supplied with abundance of air, only measured
3/450 of a millimetre[81] in diameter, the filaments that had grown
probably with an insufficiency of oxygen, and performed the functions of
ferment, measured 8/450, and the swellings as much as 30/450 of a
millimetre in diameter, as represented in Fig. 24.

[Illustration: Fig. 24.]

In concluding this paragraph, we may mention a very able research on
fermentation which we have lately studied, the author of which, Dr.
Fitz, communicated it to the Chemical Society of Berlin in 1873. In
section II., page 48, this author explains his observations in a manner
conformable to our own views, as may be seen from the following passage
of the memoir:—

“In the presence of oxygen, the ferment of _mucor_ develops into a
mycelium and consumes the sugar; in the absence of oxygen, on the other
hand, the spores develop into ferment of _mucor_, that buds and
decomposes the sugar into the products of fermentation.

“The properties of _mucor mucedo_ in a fermentable liquid, in the
presence or in the absence of oxygen, accord perfectly with the theory
of fermentation established by Pasteur in 1861 (_Comptes rendus de
l’Académie des Sciences_, t. lii., p. 1260). According to this theory a
fermentative fungus needs oxygen for its development; if it finds any
free oxygen it utilizes the whole of it, assimilating one part of the
sugar and burning the other; whilst in the absence of free oxygen, the
fungus appropriates what it requires from the sugar.”

Footnote 49:

  In the course of this work we shall combat, by means of experimental
  proofs which appear to us irrefragable, the opinions which many
  writers entertain on the subject of certain transformations of
  organisms—that of _penicillium glaucum_ into ferment, or _mycoderma_;
  of _bacteria_ into lactic ferment; of ferment into _vibrios_; of
  _mycoderma aceti_ into ferment, and so on. Nevertheless, we shall
  pronounce no _a priori_ opinion on the question whether the inferior
  organisms, which will be the subject of this chapter, and which
  include yeast and the ferments properly so called, are perfect beings
  in their habitual form, or whether they are susceptible of
  polymorphism. It is with this reservation that we employ the word
  _autonomy_. If we claim polymorphism for any species, we shall not do
  so without furnishing proofs. Some organs detached from higher
  organisms, and some beings in a certain phase of their existence, may
  reproduce themselves under a special form, with special properties,
  when brought into media and under conditions that are unfit for the
  production of the plant or animal under its other shape or ordinary
  mode of reproduction. Modern Science affords many examples of this,
  and certain alcoholic ferments present us with analogous facts; but to
  wish to stretch these facts beyond their due significance, and to
  admit a polymorphism that cannot be proved, in consequence of a belief
  that it is possible, or on the faith of confused observations, is to
  indulge in gratuitous assertion from a mere spirit of system.

Footnote 50:

  See, on this subject, the author’s _Études sur le Vinaigre_, Paris,
  1868, p. 76, note; and especially _Études sur le Vin_, 2nd Edition,
  1873, p. 19.

Footnote 51:

  Some observations in the preceding chapter enable us to account for
  the vast number of germs which are constantly falling on the surface
  of everything. We may here allude to the use we have made of flasks,
  shaped as in Fig. 17, and holding from 250 c.c. to 300 c.c., which are
  a third part filled with an organic liquid, and are closed up when
  boiling. They contain no air when cool, and are opened in series of
  10, 20, &c., out of doors, and closed up again immediately. The air
  rushes violently into the vacuum, and thus we introduce about 200 c.c.
  of air, with all the particles of dust contained in that air, into
  each flask. It has been proved that a certain number of these flasks
  undergo change in the course of time, the number of those changing and
  the nature of their changes being in close proportion to the probable
  number and nature of the floating germs able to develop in the
  particular nutritive liquid used. If we work at great elevations, far
  from houses and the dirt of towns and inhabited plains, as we did at
  Montanvert, near the _Mer de Glace_, change will seldom occur. The
  opposite will be the case if we work in a place like the living-room
  of the little, dirty, ill-kept inn at Montanvert. In a laboratory
  where fermentation is studied we obtain certain kinds of germs which
  often differ from those found in the air of the open country. If we
  desire to have organisms in all our flasks, we have only to stir up
  the dust on the ground or on surrounding objects at the moment when we
  open the flasks. This simple and easy experiment clearly shows us that
  it is impossible for a field of sporanges of fungoid growth, existing
  in an uncovered vessel or on the surface of a fruit, to escape
  becoming mixed with germs that are foreign to the little plant; in
  other words, the student who sows spores of _penicillium_, which he
  has collected from one place or another on a brush, exposes himself to
  serious causes of error.

Footnote 52:

  M. Jules Raulin has published a well-known and remarkable work on the
  discovery of the mineral medium best adapted by its composition to the
  life of certain ordinary fungoid growths; he has given a formula for
  the composition of such a medium. It is this that we call here
  “Raulin’s fluid” for abbreviation.

                    Water                     1,500
                    Sugar Candy                  70
                    Tartaric Acid                 4
                    Nitrate of Ammonia            4
                    Phosphate of Ammonia        0·6
                    Carbonate of Potassium      0·6
                    Carbonate of Magnesia       0·4
                    Sulphate of Ammonia        0·25
                    Sulphate of Zinc           0·07
                    Sulphate of Iron           0·07
                    Silicate of Potassium      0·07

  J. RAULIN. Paris, Victor Masson, 1870. _Thèse pour le doctorat._

Footnote 53:

  If we do not wish to take the chance of procuring the pure
  _penicillium_ by means of these spontaneous sowings, effected by
  opening and then closing in the flame a certain number of flasks with
  drawn-out points, we may utilize one of the flasks, which, having been
  opened and closed again, has notwithstanding developed no organized
  forms, as follows:—We impregnate the contained liquid directly, by
  dropping into it from a metallic wire spores taken from any growth of
  _penicillium_ exposed to the common air; and then from the new field
  of sporanges formed by this sowing in the flask that has been
  re-closed, we must, later on, take the pure spores that we require.
  This method is quicker and almost as safe.

  We should add that, if we wish to use for our purpose spores of
  _penicillium_ from a closed flask, in which the plant has fructified,
  we must be careful not to leave the plant too long closed up. A few
  days after the sowing the growth of the fungus is arrested, in
  consequence of all the oxygen being absorbed, and its place being
  supplied by a mixture of carbonic acid and nitrogen; and the spores,
  if kept too long in this atmosphere, will all perish.

Footnote 54:

  To shake the liquid without danger of introducing exterior particles
  of dust, we apply the flame of the spirit lamp to the drawn-out neck
  of the flask, and close up the open end; we may then shake our flask
  without risk. We must afterwards reopen the end of the drawn-out neck
  for the purpose of re-establishing communication with the exterior
  air.

Footnote 55:

  The flask B was closed with the lamp in consequence of one of the
  objects of these experiments being to test M. Trécul’s experiments on
  the transformation of _penicillium_ into ferment. Strangely enough,
  according to M. Trécul, as we shall see later on, the spores of
  _penicillium_ refuse to change into ferment, if the vessels in which
  they are sown are not “perfectly air-tight.”

Footnote 56:

  _Bulletin de la Société Philomathique._

Footnote 57:

  HERMANN HOFFMANN, _Études Mycologiques sur la Fermentation_.
  _Botanische Zeitung_ and _Annales des Sciences Naturelles_, 4^e série,
  t. xiii. p. 24, 1860.

Footnote 58:

  _Communication sur l’Origine et le Développement de quelques
  Champignons._ Dantzig, 1867.

Footnote 59:

  TRÉCUL, _Comptes rendus de l’Académie_, t. lxxiii. p. 1454; December
  28, 1871.

Footnote 60:

  TRÉCUL, _Comptes rendus de l’Académie_, t. lxxv. p. 1169, November 11,
  1872. A proof of M. Trécul’s carelessness in experiments of this kind
  is the fact that in studying the fertility of an impregnated wort, he
  often obtains different productions. Our experiments give opposite
  results. If we sow nothing, we obtain nothing. If we sow a plant, we
  obtain a similar plant; or, should there be any difference, the change
  may be traced, beyond question, to its origin in the plant sown, and
  is the consequence of some alteration in the conditions of our
  experiment.

Footnote 61:

  _Comptes rendus des Séances de l’Académie des Sciences_, t. lxxv. p.
  1220; Nov. 18, 1872.

Footnote 62:

  Since writing the above we have experienced some doubt as to whether
  the forms of development represented in Fig. 20 are actually those of
  the _aspergillus glaucus_, which we supposed our fungoid growth to be.
  In some of the later sketches of our observations we find similar
  forms, which belong to a bluish kind of _penicillium_, with rather
  large spores. Fortunately, this doubt affects our argument in no
  essential particular. It matters very little what variety of fungoid
  growth it is that gives rise to alcoholic fermentation attended by
  peculiarities of shape that only occur in the development of its
  spores when air fails it.

Footnote 63:

  By the term _conidia_ is meant certain chains of cells, which are in
  reality mycelial spores.

Footnote 64:

  See PASTEUR, _Études sur le Vin_, 1st Edition, pp. 20 and following.

Footnote 65:

  Since writing this paragraph, we have found in M. Ch. Robin’s _Journal
  d’Anatomie et de Physiologie_, an article signed by that gentleman,
  and entitled _Sur la Nature des Fermentations_, &c. (July-August,
  1875), in which the learned microscopist says:—“The _torula cerevisiæ_
  is derived from the _mycoderma cerevisiæ_. My observations leave no
  doubt on my mind that _penicillium glaucum_ is one of the forms
  evolved from spores or ferments that have preceded it, as M. Trécul
  showed a long time ago, and that, moreover, the spores of
  _penicillium_, germinating in suitable media, give us the sporical
  form termed _mycoderma_.”

  We take the liberty to observe that these assertions of M. Robin’s are
  purely gratuitous. Up to the present time it has been impossible to
  discover a suitable medium for the proof of these different
  transformations or polymorphisms. From the time of Turpin, who firmly
  believed that he had observed these changes, to our own, none of the
  microscopists who have affirmed these transformations have succeeded
  in adducing any convincing proof of them, and M. Trécul’s latest
  observations, especially as regards _penicillium_ and its
  transformation into ferment or into the _mycoderma_ of beer, have been
  positively disproved by ours, supported, as they are, by proofs that
  we consider irrefutable.

Footnote 66:

  It is a very easy matter to study the liquids and growths in our
  flasks during the course of a single experiment. We take out the glass
  stopper that closes the india-rubber tube on the straight-neck, and,
  by means of a long rod or a glass tube previously passed through the
  flame, take up a quantity, which we draw out immediately for
  microscopical examination. We then replace the glass stopper, taking
  care to pass it through the flame before doing so, to burn up any
  organic particles of dust that it may have picked up from the table on
  which we laid it.

Footnote 67:

  We may prove the occurrence of alcoholic fermentation by the cells of
  submerged _mycoderma vini_ in a different manner. To do this, after
  having made all our preparations as before and shaken up the film of
  _mycoderma vini_ in its liquid, we must attach our flask to a test
  flask (Fig. 19), and pass the turbid liquid into the latter. On
  succeeding days we shall detect a very protracted fermentation in the
  test flask; there will be a succession of minute bubbles rising from
  the bottom, but in small number at a time. The fermentation is very
  evident whilst it lasts, but is rather sluggish, and, although of very
  long duration, ceases long before the sugar is exhausted.

  This experiment proves better than any other the non-transformation of
  _mycoderma vini_ into other ordinary fungoid growths. For after
  decanting the liquid into the test flasks, the sides of the
  experimental flask remain covered with streaks of _mycoderma vini_
  along with some of the liquid. Moreover, the flask is refilled with
  air, and this air is being constantly renewed, in part, by variations
  of the temperature of the oven, so that the _mycoderma_ remaining on
  the sides is thus placed under the most favourable conditions for
  transformation into other fungoid growths, if that were possible. It
  is still more easy to detach the experimental from the test flask, and
  to pass pure air into it, once or twice a day, or constantly. In any
  case, we shall never see anything besides the _mycoderma vini_ spring
  up within it.

Footnote 68:

  See PASTEUR, _Comptes rendus des Séances de l’Académie des Sciences_,
  t. liv., 1862, and t. lv., 1862. _Études sur les Mycodermes, &c._

Footnote 69:

  In a subsequent chapter we shall prove that yeast is likewise
  incapable of transformation into _mycoderma vini_.

Footnote 70:

  We secured the purity of our mycoderma by the same means that we have
  already described for the procuring of spores of _penicillium_ or
  other fungoid growths in a state of purity.

Footnote 71:

  BUFFON, _Histoire de l’Homme_, t. viii., edition 12mo, 1778; TURPIN,
  _Mémoires de l’Académie des Sciences_, t. xvii.; Dr. PINEAU, _Annales
  des Sciences Naturelles_, t. iii., 1845; POUCHET, _Traité de la
  Génération dite Spontanée_, p. 335, 1859. See also our _Mémoire sur
  les Générations dites Spontanées_, 1862, pp. 100 and following, in
  which we give a _resumé_ of some of these theories.

Footnote 72:

  The following is Turpin’s application of his theory to the formation
  of the ferments of fruits (_Mémoires de l’Académie_, t. xvii., 1840,
  p. 155), where also, on p. 171 the above quotation will be
  found:—_Ferments Produced by the Filtered Juice of the Pulp of
  Different Fruits_—“By the word pulp we mean the soft and juicy
  cellular tissue of the fleshy part, mesocarp or middle layer of the
  pericarp of certain ripe fruits. This cellular tissue, which is very
  abundant in the peach and all stone-fruit, in the apple and pear, in
  the orange and grape, and similar fruits, is the same as that which
  forms the body of a leaf. Being in every case composed of a simple
  agglomeration of contiguous mother-vesicles, which are always filled
  with globulines that are more or less developed, more or less
  coloured, and individually endowed with a special vital centre, it is
  not surprising that its globulines when free and detached from the
  compound organisms to which they belong, and from association with its
  vegetable life, should, when placed in a suitable medium, themselves
  vegetate and become transformed, under these new influences, into a
  mucedine, with filaments and articulations. Such are the very fine,
  and, consequently, very transparent globulines, which, when left to
  themselves in sweetened water, grow and become vesicular, producing
  other globulines in their interior, then bud, vegetate into mucedinous
  filaments, decompose sugar, and produce all the effects that
  constitute what we term _alcoholic fermentation_.”

Footnote 73:

  BÉCHAMP, _Recherches sur la Nature et l’Origine des Ferments (Annales
  de Chimie et de Physique, 4^e série_, t. xxiii., and _Comptes rendus
  de l’Académie des Sciences_, Oct. 23, 1871).

Footnote 74:

  We need scarcely here observe, having done so on previous occasions,
  that whenever we opened our flasks to obtain specimens, we made use of
  a fine tube, previously passed through the flame of a spirit lamp, and
  that we also passed this flame over the surface of the india-rubber,
  glass stopper, &c., to consume the organic particles of dust which
  floating about might introduce themselves at the moment when we opened
  the right-hand tube of the flask.

Footnote 75:

  Ever since the year 1861 (see p. 92), this question of the possible
  transformation of the ordinary fungi, especially _penicillium_ and
  _mucor mucedo_, into yeast has engaged our attention. The results
  attained have been entirely negative; but hitherto only the
  conclusions of our work have been published, some account of which was
  given at the meeting of the _Société Philomathique_ of March 30th,
  1861. The following extract is from the _Bulletin_ of the
  society:—“Meeting of March 30th, 1861. At this meeting a paper was
  read by M. Pasteur ‘On the supposed changes in the form and vegetation
  of yeast-cells, depending on the external condition of their
  development.’ It is well-known that Leuwenhoeck was the first to
  describe the globules of yeast, and that M. Cagnard-Latour discovered
  their faculty of multiplying by budding. This interesting vegetable
  organism has been the subject of a host of researches by chemists and
  botanists. The latter, from the days of Turpin and Kutzing, have
  almost unanimously regarded yeast as a form of development of various
  inferior vegetable types, especially _penicillium_. The studies of
  this subject which seem to have won most favour during the last few
  years are those of MM. Wagner, Bail, Berkeley, and H. Hoffmann. The
  researches of these botanists seem to strengthen and confirm the
  original observations of Turpin and Kutzing. M. Pouchet has, quite
  recently, expressed the same ideas, and has determined certain points
  in connection with them with much precision of detail. M. Pasteur has
  long studied this important question, which is so intimately connected
  with the essential nature of yeast and with those phenomena of the
  polymorphism of the inferior types of vegetable life, to which most of
  the remarkable works of M. Tulasne relate; he has, however, arrived at
  results that are altogether negative, and he declares that he was
  unable to detect the transformation of yeast into any of the
  _mucedines_ whatsoever, and, inversely, that he could never succeed in
  producing the smallest quantity of yeast from ordinary _mucedines_.”
  These same results we communicated to the _Société Chimique_ of Paris,
  at a meeting held April 12th, 1861. Throughout the investigation of
  which we have just indicated the conclusions, we insisted on the
  necessity of cultivating the separate organisms in a state of purity
  in all researches relating to these inferior forms of life, if we
  desire to attain to sure inferences about them; and the method of
  working, which we recommended, did not differ essentially from that
  adopted in the present work. Since then the study of these growths has
  been conducted with the utmost precautions; and other apparatus,
  perhaps as safe as those which we employ and better adapted than ours
  for the study of polymorphism of species, have been invented by
  botanists of great skill—M. de Bary, in Germany, and M. Van Tieghem,
  in France.

Footnote 76:

  We found, after the lapse of another year, in December, 1873, that the
  ferment of the _mucor_ in the test glass might still be easily
  revived; that it was able to propagate, both in the mycelium and in
  the cellular form, in wort, and that it might produce a fermentation,
  more or less active, according to the condition of aeration; in short,
  that it was capable of producing all the characteristic phenomena
  described. By means of the method of cultivation that we employ, our
  study, which was continued for years, was pursued without the least
  fear of any foreign fungoid growths being introduced into the vessels,
  although they remained constantly open, and the air in them was being
  perpetually renewed by the action of diffusion and variations of
  temperature. In 1875 nothing remained alive in our flask, and further
  revival became impossible.

Footnote 77:

  We do not here take into account certain phenomena of oxidation of
  which the fungoid growths are the seat, and which remind us of those
  that are presented in so remarkable a degree by _mycoderma vini_ and
  _mycoderma aceti_.

Footnote 78:

  [There are 15·43 grains in the gramme.]

Footnote 79:

  [For English equivalent see Experiment 1, p. 135.]

Footnote 80:

  The figure given below supplies this omission. The cells that are
  isolated or are in chains, _b.b.b._, show this state of the old cells.
  The cells _a.a.a._ are younger, and may be more easily revived. We may
  see by the dimensions of some of these how greatly, in certain cases,
  the cells of _mucor_ resemble cells of yeast; nevertheless, in the
  state of the contents and the aspect of the outlines, there are always
  some differences sufficiently appreciable to strike the practised
  observer.

  [Illustration: Fig. 23.]

  The figures adjoining the cells indicate fractions of a millimetre. (A
  millimetre may be taken as ½5-in.)

Footnote 81:

  [0·000089 in., 0·00026 in. and O·00089 in. respectively.]




                               CHAPTER V.
                        The Alcoholic Ferments.


                     § I.—On the Origin of Ferment.


Amongst the productions that appear spontaneously, or, we should rather
say, without direct impregnation, in organic liquids exposed to contact
with the air, there is one that more particularly claims our study. It
is that one which, by reason of its active energy as an agent of
decomposition, has been distinguished and utilized from the earliest
times, and is considered as the type of ferments in general; we mean the
ferment of wine, beer, and more generally, of all fermented beverages.

Yeast is that viscous sort of deposit which takes place in the vats or
barrels of must or wort that is undergoing fermentation. This kind of
ferment presents for consideration a physical fact of the most
extraordinary character. Take a morsel of the substance and put it in
sweetened water, in must, or in dough, which always contains a little
sugar; after a time, the length of which varies, a few minutes often
sufficing, we see these liquids or the dough rise, so to speak. This
inflation of the mass, which is due to a liberation of carbonic acid
gas, may cause it to overflow the vessels containing it, if their
capacity is not considerably greater than the volume of the matters
fermenting. It is equally remarkable that these phenomena are natural
and spontaneous; that is to say that the must, the wort, and the dough
are able to rise, as we have termed it, when left to themselves, without
the least addition of foreign substances. The only difference that may
occur in these phenomena is a certain amount of retardation, in cases
where the yeast does not reach the saccharine matters in a perfectly
natural form, inasmuch as it then requires a certain time to get itself
together before it can begin to act.

It is necessary, indeed, that sugar should be present; for if we
abstracted by some means or other from the must or dough all the sugar
contained in it, without touching the other constituents, the addition
of yeast would produce no gas. Everything would remain quiet until the
moment when signs of a more or less advanced putrefaction showed
themselves. Yeast is one of the most putrescible of substances, and it
is worthy of notice that its alteration is also the consequence of the
formation of one or more ferments, very different, however, from that of
which we are speaking. As for the nature of yeast, the microscope has
taught us what it is. That marvellous instrument, although still in its
infancy, enabled Leuwenhoeck, towards the close of the 17th century, to
discover that yeast is composed of a mass of cells. In 1835
Cagnard-Latour and Schwann took up Leuwenhoeck’s observations, and by
employing a more perfect microscope, discovered that these same cells
vegetate and multiply by a process of gemmation. Since then the physical
and chemical phenomena already mentioned, such as the raising of the
mass, the liberation of carbonic acid gas, and the formation of alcohol,
have been announced as acts probably connected with the living processes
of a little cellular plant, and subsequent researches have confirmed
these views.

In introducing a quantity of yeast into a saccharine wort, it must be
borne in mind that we are sowing a multitude of minute living cells,
representing so many centres of life, capable of vegetating with
extraordinary rapidity in a medium adapted to their nutrition. This
phenomenon can occur at any temperature between zero and 55° C. (131°
F.), although a temperature between 15° C. and 30° C. (59° F. and 86°
F.) is the most favourable to its occurrence.

As regards the rapidity of the budding, the following observations will
give some idea of what it is in the case of one of the ferments of
natural must. The temperature was between 12° C. and 13° C. (55° F.).

“On October 12, 1861, at ten o’clock in the morning, we crushed some
grapes, without filtering the juice that ran from them; afterwards, at
different times during the day, we examined the juice under the
microscope, until at last, although not before seven o’clock in the
evening, we detected a couple of cells, as represented in Fig. 25, _a_.

[Illustration: Fig. 25.]

From that time we kept these contiguous cells constantly in view. At
7.10 we saw them separate and remove to some little distance from each
other (Fig. 25, _b_). Between 7 and 7.30 we saw, on each of these cells,
a very minute bud originate and grow little by little. These buds
developed very near the point of contact, where the disjunction had just
taken place. By 7.45 the buds had increased greatly in size (Fig. 25,
_c_). By 8 they had attained the size of the mother-cells. By 9 each
cell of each couple had put forth a new bud (Fig. 25, _d_). We did not
follow the multiplication of the cells any farther, having seen that in
the course of two hours two cellules had furnished eight, including the
two mother-cells.”[82]

An increase like this, which would have been more rapid at a temperature
between 15° and 25° (59° and 77° F.), and still more so between 25° and
30° (77° and 86° F.), may indeed seem surprising. It is really, however,
nothing to what sometimes occurs. In choosing proper conditions of
temperature and medium, of state and nature of yeast, it has sometimes
happened that the bottom of a vessel has become covered with a white
deposit of yeast cells, in the course of not more than five or six hours
after we had sown a quantity of yeast so small as to effect no change at
all in the transparency of the liquid contained in the vessel after it
had been shaken up. Such a rapidity of vegetation reminded us of those
exotic plants which are said to grow several feet in height in the
course of twenty-four hours.

[Illustration: Fig. 26.]

Budding commences in the form of a simple protuberance on the cell—a
kind of little boss, as represented in Fig. 26, No. 1. This protuberance
goes on increasing, and assumes a spherical or oval form. At the same
time, there is a tendency in the points of attachment in the young cell
to meet—a kind of strangulation occurs (Fig. 26, No. 2). The junction
takes place a little sooner or later, according to the species (Fig. 26,
No. 3); the two individuals then separate (Fig. 26, No. 4). In certain
cases a single cell may give rise to several protuberances, and,
consequently, to several daughter-cells. Where there is only one
protuberance or bud we generally see it originate at the thick end and a
little on one side of the apex of the oval outline, which, in a greater
or less degree, characterizes the cells of the majority of ferments.

Certain authors have maintained that the method of budding which we have
just described, and which we think was first promulgated by
Mitscherlich, is merely an illusion, and that the cells of yeast break
up and scatter their granular contents, and that these scattered
granules eventually attach themselves to the cells, growing there, and
so giving the appearance of buds or daughter-cells. This error has been
revived quite recently.[83] Nothing can be less admissible. We could
count the number of yeast cells which we have seen undergo this process
of rupture in the course of some ten years of observation, every day of
which, we may say, thousands of these cells have passed under our eyes.
This breaking up of the cells is really of the most rare occurrence, and
may always be explained by some abnormal circumstance affecting the
yeast; being indeed a mechanical accident, not a physiological fact. We
may easily convince ourselves of this by growing some yeast in a
saccharine wort, filtered perfectly clear, and, consequently, deprived
of all granular amorphous deposit that might deceive the observer. The
cells will be observed to bud and multiply without exhibiting the most
minute appearance of granulation, or disruption; moreover, there will
always be cells of all sizes, ranging from the smallest visible up to
the largest. This very simple piece of observation may be made in all
the alcoholic ferments, and with any wort capable of fermenting, and in
its presence the hypothesis, which we have been repudiating, cannot hold
its own.

In Plate VII. (left side) there is represented a field of yeast,
magnified 400 times. We see a mass of disjointed cells, such as appear
after fermentations without sufficient aliment; of the kind represented
some are nearly spherical, others oval or cylindrical, more or less
elongated. If we mix a little of this yeast, of about the size of a
pin’s head, with wort, and put the wort into a small, shallow,
flat-bottomed basin, having a surface of about 1 square decimetre (10
sq. ins.) exposing it to the surrounding temperature, we shall find next
day the bottom of the basin covered with a fine white deposit, of the
forms of which we give a sketch in the right half of the plate. In this
it will at once be observed that the cells sown have lost their interior
granulations, having become more transparent and filled with a
gelatinous protoplasm. The principal difference between the two halves
of the plate consists in this, that whereas the cells in the left half
are isolated and granular, those in the right half are more inflated,
more transparent, and provided with buds, which may be seen in every
stage of development, from their first appearance till they become as
large as the parent cells. They continue to grow until they detach
themselves; then they bud in their turn, so that the same figure may
furnish examples of cells of the first, second, and third generations.
In the right half, the protoplasm contained in the cells exhibits
circular spots or vacuoles, which may be made to appear lighter or
darker than the rest of the cell by slight movements of the object-glass
of the microscope. These spots are due to a migration of the protoplasm
towards the sides; they commonly occur in yeast cells the vitality of
which, from deficient nourishment, has become suppressed—the shrivelled
appearance which they then assume being due to their being forced to
live upon themselves, so to speak. However, by introducing such cells
into a nutritive and aerated liquid the vacuoles quickly disappear.

[Illustration: Plate 7. Yeast-cells—Worn out and Dissociated (left),
after Revival in a Sweet Wort (right).]

In the ordinary yeast, as met with in breweries, the majority of cells
show one or more of these vacuoles; if, however, we place a little of
this yeast in an aerated wort, and watch under the microscope the
changes that occur in the cells, we shall witness, often in the course
of a few seconds, a kind of turgescence, a greater tension of the
cell-walls, which seem to grow thinner, and a complete disappearance of
the vacuoles. At the same time the interior gelatinous matter will
become filled with fine granulations that are scarcely visible, but
which at a certain distance appear brilliant. At the same time
protuberances begin to show themselves, and next day the budding will
have already become very active. The newly-formed cells will have such a
delicacy of aspect and contour as to be scarcely discernible in the
field of the microscope. There will also be a tendency to ramification
in the budding, which appearance will be more or less marked according
to the kind of alcoholic ferments present, as we shall see presently,
attaining its maximum in each case when the cells have been revived
after exhaustion by rest and want of food. In the latter case, the
process of rejuvenescence may be protracted; but this is not the case
with cells of commercial yeast, which is always used within a few days
of its formation. And thus, as I said a little ago, speaking of these
cells, they often manifest the first signs of their budding in a few
seconds.

In our preceding remarks we have expressly assumed that there are many
kinds of alcoholic ferment. This is, beyond doubt, the case, as we have
given incontestable proofs, first in 1862, in the _Bulletin de la
Société chimique_ of Paris, and later on, in 1864 and 1866, in a Note in
the _Comptes rendus_, on the diseases of wines, as well as in our
“Studies on Wine.” Moreover, we know that brewers have long recognized
two distinct methods of fermentation—“high” fermentation and “low”
fermentation—and two corresponding yeasts. It is true that the
differences presented by these fermentations were believed to be caused
by the different conditions under which they took place, and that it was
supposed that we might change “high” yeast into “low” yeast, or
inversely, by subjecting the first to a low temperature, or the second
to a high one. In our observations of 1862, which we have just
mentioned, we discovered that must gives rise to several yeasts; that
the ferment of “high” beer cannot develop except with great difficulty
in must, whilst one of the ferments of the grape grows rapidly and
luxuriantly in wort; that it is easy to isolate the smallest of the
ferments of the grape from its congeners, by subjecting filtered must to
fermentation; and finally, that the secondary fermentations of wines
which remain sweet furnish a remarkable ferment, very different in
aspect to the ferment of beer.

We have not given specific names to these different ferments, any more
than we have to the other microscopic organisms which we have had
occasion to study. This was not from any disregard for names, but from a
constant fear that, since the physiological functions of these minute
forms was the exclusive object of our study, we might be led to attach
too much importance to exterior characters. We have often found that
forms, having nothing apparently in common, belong to one and the same
species, whilst similarity of form may associate species far apart. We
shall give some fresh examples of this fact in the present paragraph. A
German naturalist, Dr. Rees, who has discovered new proofs of the
diversity of alcoholic ferments, putting aside, perhaps rightly, such
scruples, has attached specific names to the different kinds of
ferments, in his _brochure_ published in 1870, which we have already
cited (p. 71). Indeed, we have often ourselves, for brevity’s sake, made
use of the names proposed by Dr. Rees.[84]

[Illustration: Fig. 27.]

In a Note inserted in the _Bulletin de la Société chimique de Paris_, in
1862, we figured a ferment of small dimensions, which develops
spontaneously in must, filtered or unfiltered, and which is very
different from the ordinary ferment of wine. It is the first to make its
appearance in the fermentation of the grape, and may even appear alone
if the must has been previously well filtered, doubtless because its
germs, being smaller than those of other ferments, pass through the
filter more easily and in greater number. Fig. 27, extracted from our
Note of 1862, represents this ferment, together with some spherical
cells of _high_ yeast, with the object of giving a more exact idea of
the relative dimensions of these two ferments and their dissimilarities.
Dr. Rees has named it _saccharomyces apiculatus_.

The same savant has given the name of _saccharomyces pastorianus_ to the
yeast of the secondary fermentations of sweet liquids, such as wine that
has remained sweet after its principal fermentation. We have described
this yeast in a Note published in 1864, on the diseases of wine, from
which we give the following extract:—[85]

[Illustration: Fig. 28.]

“Fig. 6 (Fig. 28 in this work) represents a very interesting variety of
alcoholic ferment. It happens pretty often, especially in the Jura,
where the vintage takes place about October 15th, when the season is
already cold and little favourable to fermentation, that the wine is
still sweet at the moment when it is put into casks. This is especially
the case in good years, when the sugar is abundant and the proportion of
alcohol high, a circumstance which prevents the completion of
fermentation when effected at a low temperature. The wine remains sweet
in cask sometimes for several years, undergoing a continuous but feeble
alcoholic fermentation. In such wines we have always observed the
presence of this peculiar ferment. In form it consists of a principal
stem, forming nodes at various points, from which short branches arise,
ending in spherical or ovoid cells. These cells readily detach
themselves, and act as spores of the plant. It is rarely, however, that
we see so perfect a vegetation as we have represented, because the
different parts fall to pieces, as we have shown in the left half of the
figure.”

What is the origin of cellular plants of this remarkable type? Where and
how are the ferments of the grape generated?

In Chapter III. § 3 we were on our way to a solution of this question.
It has been shown that fermentation cannot take place in the juice of
crushed grapes if the must has not come into contact and been mixed with
particles of dust on the surface of the grapes, or of the woody part of
the bunch. It would, however, be sufficient that a vintage vat, of any
capacity whatsoever, should receive the particles of dust existing on a
single bunch in some cases, on even a single grape, for the whole mass
to enter into fermentation.

[Illustration: Plate 8. Fertile Mould-cells from the Outer Surface of
Grapes.]

What, then, we must ask ourselves, is the nature of these particles of
dust? On September 27th, 1872, we picked from a vine, in the
neighbourhood of Arbois, a bunch of grapes, of the variety called _le
noirin_. The bunch selected, without any injury to a single grape, was
brought to our laboratory in a sheet of paper that had been previously
scorched in the flame of a spirit lamp, and the grapes were cut off with
a pair of fine scissors, which had also been passed through the flame.
By means of a badger-hair brush, thoroughly purified in water, each
grape to which a portion of its peduncle remained attached, was washed
in a little pure water. The successive washing of a dozen grapes in 3
c.c. of water was sufficient to make the water turbid; we then examined
it under the microscope. Each field contained many little organized
bodies, accidentally associated, now and again, with some very scarce
crystalline spicules. As a rule, the organisms consisted of simple,
transparent, colourless cells; some, indeed, of larger size had a
yellowish brown colour, and were detached or united in irregular masses;
and, lastly, there were club-shaped or bottle-shaped vessels, full of
spores ready to germinate. We repeated this experiment with bunches of
other varieties of grape, and also submitted to examination water in
which the outer surfaces of gooseberries, plums, and pears had been
washed; the result was in each case the same, that is, we found a great
number of the same colourless cells, and the same irregular masses of
darker cells, which latter, however, we must not confound with the
masses of dead cells sometimes found covering parts of the epidermis of
certain fruits.

As we had purposely left each fruit attached to part of its peduncle, we
wished to ascertain if these corpuscles proceeded from the grapes or
from the wood of the peduncle. For this purpose we washed separately the
surface of the grapes and the woody part of the bunch. The water in
which the latter was washed was visibly more charged with the minute
organisms than that in which the grapes was washed, although the latter
was by no means free from them.

Plate VIII. represents these corpuscles as they exist on the surface of
fruits, magnified 500 times. The groups, _b_, _b_, _b_, ..., _c_, _c_,
... are of a brown colour, more or less dark, or of a reddish yellow;
the cells _a_, _a_, ... are transparent. Amongst them are some spores of
ordinary fungoid growths, and several cells which are probably the issue
of a germination that had commenced in certain groups which have a hard,
yellowish appearance, and which are provided with what seems to be a
double case—_b_, _b_, _b_, ..., _c_, _c_, ..., a result of the moisture
of the woody part of the bunch, or of rain that fell just before the
commencement of our observations.

It is an easy matter to trace the germination of these different
varieties of cells with the microscope. We put a drop of the water in
which the woody part of a bunch of grapes has been washed into a small
quantity of wort, previously boiled and filtered bright. Plate IX.
presents a series of developments observed in the case of simple or
grouped cells, A, D, G, and J. The process is as follows: The
yellowish-brown cells soften and grow larger in the nutritive medium,
and gradually become almost transparent and colourless. At the same time
we see some very young buds appear on their margins; these rapidly
increase in size, and detaching themselves to make room for others, move
off as young cells that after a time bud in their turn. The rapidity
with which these cells bud and multiply is often extraordinary. The
group A and the cell D produced the groups C and F within twenty-four
hours, passing through the intermediate stages represented in groups B,
E. The cells A and D did not give rise to any filamentous growths, at
least whilst under our observation. Some groups of cells, however, put
forth, from the first, long filaments, having cross-partitions and
resembling the mycelium in ordinary fungoid growths. Together with
these, and along their whole length, was an abundance of cells, often in
clusters, as represented by Fig. G, the whole of which growth took place
in less than twenty-four hours.[86] But apart from contact with the air,
there was a complete absence of life.

[Illustration: Plate 9. Various Examples of the Mode of Growth of
Mould-cells from the Outer Surface of Grapes.]

The figures H, I, J, K, represent other aspects of developing cells and
filaments. The cells H are spherical; the cells I have numerous buds, as
also have those marked K. These different forms were all produced in the
course of twenty-four hours by the cell which may be observed in the
centre of the group J. In connection with this same group, J, we may
remark that on September 30, 1872, at 10 A.M., we witnessed the
detachment of three oval cells at the points _a_, _b_, _c_; by 10.45
other buds of the size represented in our engraving had formed in their
place; by about five o’clock that same afternoon these buds, _a_, _b_,
_c_, having become transformed into cells, fell off in their turn.[87]

It may be asked, what proof have we that amongst the filamentous and
cellular growths which spring from the small, dark bodies existing in
the particles of dust adhering to the surface of fruits, and which we
here see bud and multiply with such marvellous rapidity, the ferment or
ferments of vintage do actually exist? A very simple experiment will
prove conclusively that this is the case. When in the course of
twenty-four or forty-eight hours, by contact with saccharine must, and
in presence of excess of air, the revival and development of the cells
has taken place on the bottom of the little troughs employed in our
observations; if then we fill up the trough with the same must, so that
there remains no free air under the cover-glass, within a very short
time—an hour, half-an-hour, or often less—we shall see bubbles of gas
rise from the bottom, accompanied by an increase in the deposit of
cells. This will be the must fermenting after the submersion of the
cellular plants. It follows that the cells, or groups of cells, of a
dark colour which cover the grapes, or the woody part of the clusters,
are actual germs of the cells of yeast; more correctly speaking, that
germs of yeast-cells exist amongst these groups, for it would not be
consistent with truth to say that the various germinating forms present
in the dust on the surface of grapes must all of them give rise to
actual corresponding ferments. Thus the flask-shaped spores _c_, _c_,
... in Plate VIII., are reproductive organs of _alternaria tenuis_,
which have probably nothing in common with alcoholic ferment or
ferments, properly so called, except their outward form. We may repeat,
however, and it is a point of great importance to bear in mind, that the
cells of yeast originate from some or other of the little, brownish,
organized bodies, which the microscope reveals in such numbers amongst
the particles of dust existing on the surface of fruits.

The impossibility, which we have already demonstrated (Chapter III. §
3), of making grape juice ferment apart from the action of external
particles of dust, and the knowledge which we have just acquired, that
the particles of dust on the surface of the grapes and woody peduncles,
at the moment when the grapes have attained maturity, contain certain
reproductive cells which give rise to certain ferments, naturally lead
us to the investigation of another point, which concerns the period at
which these germs make their appearance on the different parts of the
vine plant. The two following experiments tend to prove that the ferment
can only appear about the time when the grapes attain maturity, and that
it disappears during the winter, not to reappear before the end of the
following summer.

I. In the month of October, 1873, we procured from a vineyard in the
canton of Arbois some of the woody parts of very ripe clusters of
grapes, taking the precaution to cut off all the grapes, one by one,
with a very clean pair of scissors, whilst still on the vine; we then
wrapped up the woody parts of the clusters, thus deprived of their
grapes, in thin paper, to convey them to Paris. Our only object at that
time was to secure for use in our subsequent studies the ferment-bearing
dust found in October on the woody part of the vine, and, more
particularly, on the clusters themselves, as already stated. After our
return to Paris, and during the course of our experiments in October and
November, it sufficed to wash a few scraps of the bunches in a little
pure water, in order to obtain the grape-ferment in abundance; but later
on in the winter we were astonished to find that the same procedure
yielded no ferment, only some moulds. The bunches which, when put into
boiled and filtered must, in October, very readily caused that must to
ferment, at the end of winter could no longer produce the same effect,
however favourable might be the temperature to which we raised the must.
The particles of dust on the bunches had, therefore, become sterile, as
sources of alcoholic ferments.

II. On February 17th, 1875, we purchased of Chevet, a dealer in
provisions, two bunches of white grapes, which were perfectly sound,
presenting not the slightest trace of injury or bruise. We took an iron
pot full of mercury, which had been heated to 200° C. (392° F.), and
then covered over its surface with a sheet of paper that had also been
subjected to flame. When the mercury had cooled down we placed several
of Chevet’s grapes, singly and in small bunches, on the surface of the
metal, and, after having enclosed them in a glass cylinder that had been
previously heated with and by means of the mercury, we crushed them in
this vessel, in contact with air, by means of a strong, crooked iron
wire that had been passed through the flame of a spirit lamp. The object
of all these precautions was to prevent any cause of error, such as
might have resulted from the accession of particles of dust associated
with the mercury, or floating about our laboratory. We then placed our
cylindrical jar in an oven, at a temperature of 25° C. (77° F.); but
though the experiment was continued for several days following, no
fermentation manifested itself. At last, to assure ourselves that the
pulp and liquid were, notwithstanding this, well adapted to
fermentation, we introduced into the test-flask an almost imperceptible
quantity of yeast. This readily developed, and promptly produced
fermentation.[91]

It seems possible, therefore, that the germs of ferment may not exist on
bunches of sound grapes during winter, and that the well-known
experiment of Gay-Lussac on the influence of air on the fermentation of
the must of crushed grapes cannot succeed at all times.

The following observations will afford more than sufficient proof of
this statement, being, after all, but an easy method of carrying out
Gay-Lussac’s experiment, without having recourse to the use of mercury.

It may already be inferred from the preceding facts that there must be,
in the course of the year, between the end of winter and autumn, a
period when the vegetation of the cellules from which yeast proceeds
undergoes a revival. When does this period occur? In other words, how
long after winter does sterility of the plant continue, until it is
again capable of yielding ferment? To ascertain this, we conducted
numerous experiments during the summer and autumn of 1875 and the winter
of 1876. Having to conduct them in a vine-growing country—in the
vineyards of Arbois, Franche-Comté—at a distance from our laboratory, we
were compelled to adopt a simple form of apparatus for our experiments,
which, besides being very convenient, was at the same time sufficiently
exact for the object we had in view.

[Illustration: Fig. 32.]

Into common test-tubes we poured some preserved must; we then boiled it,
with the object of destroying all the germs that it might contain, and
then, having passed the flame of a spirit lamp over the upper sides of
the tubes, we closed them with corks which had been held in the flame
until they began to carbonize (Fig. 32). Having provided ourselves with
a series of tubes prepared in this manner, we carried them to a vine,
and there dropped into some of them grapes, into others bunches, from
which we had taken all the grapes, by cutting their peduncles; into
others, fragments of leaves or the wood of the branches. The corks were
again passed through the flame and replaced successively in each tube.
Some of the grapes we dropped in whole, some we crushed at the bottom of
the tubes with an iron rod that had previously been passed through the
flame; others, again, at the same moment that we introduced them into
the tubes, were cut open with scissors, likewise passed previously
through the flame, so that a portion of their interior juice might mix
with the must in the tube.

Our experiments gave the following results:—As long as the grapes were
green, about the end of July and during the first fortnight of August,
we obtained no fermentation in our must. Between the 20th and 25th of
August a few tubes underwent fermentation, by the action of the little
apiculated ferment; and in the course of September the number of tubes
that fermented increased progressively. In each series of tubes,
however, we always found a few in which there was a complete absence of
fermentation.

Here are a few actual examples. In the beginning of September we placed
grapes in thirteen tubes, into some whole, into others crushed ones,
taken from bunches of the variety known as the _ploussard_, the fruit
being already sufficiently ripe to be very pleasant to the taste. All
the tubes of this series failing to give us any trace of fermentation,
or anything besides ordinary moulds—which indeed appeared in all our
experiments, whether there was or was not fermentation—we began a new
series of experiments, under similar conditions, on September 28th, as
follows:—

Nos. 1, 2, 3 and 4 tubes containing one uncrushed grape.

No. 5 tube containing two uncrushed grapes.

No. 6 tube containing two crushed grapes.

No. 7 tube containing two crushed grapes, in 2 c.c. of water previously
boiled.

No. 8 tube with a fragment of a bunch from which grapes had been cut,
and occupying the entire depth of liquid.

No. 9 tube with a fragment of wood from a branch.

Nos. 10, 11, and 12 tubes with a fragment of leaf.

On September 29th and 30th there was no appearance of fermentation in
any of the flasks, but all contained flakes of fungoid mycelium. On the
1st of October fermentation more or less marked and active occurred in
2, 3, 4, and 5, in which uncrushed grapes were, accompanied by a general
turbidity of the liquid, and a suspension of the development of the
fungoid growths. It was still absent in 1, 6, and 7, of which the first
contained an entire, the latter crushed grapes. No. 8, containing the
woody part of the bunch, was in active fermentation. Nos. 9, 10, 11, and
12, with fragments of branch or leaves, showed no signs of fermentation.
The following day No. 1 was fermenting; but from October 5th onwards
there was no alteration in the number of fermenting tubes.

In this series we determined the presence of the small apiculated form
of yeast (_S. apiculatus_) in the tubes that fermented, only once
finding it associated with _saccharomyces pastorianus_.

We need hardly say that the grapes which we employed were perfectly
ripe, the vintage having already commenced in some of the Jura cantons.

This experiment shows that, even when the grapes are perfectly matured,
it by no means follows that each individual grape must carry germs of
ferment, and that some grapes may be crushed, in some instances several
together may be crushed, without being able to set up a fermentation. In
the presence of these novel facts, those who support the hypothesis of
the transformation of the albuminous matter contained in the juice of
grapes into yeast will no doubt admit the untenability of their
opinions, since their hypothesis requires that every grape or number of
grapes, when crushed, should ferment, in contact with air.

On the same day we prepared another series of tubes, using grapes of a
variety called the _trousseau_.

Nos. 1, 2, 3, and 4 tubes containing one whole grape.

Nos. 5 and 6 tubes containing some of the wood of a branch.

No. 7 tube containing some of the wood of a branch from which the grapes
had been detached.

In the course of the following days fermentation took place in 4, 5, and
7.

In this case three out of four of the uncrushed grapes did not cause the
must in which they were placed to ferment; whilst the same must
fermented in one of the two tubes containing wood of the branch, and in
the other remained unchanged; and, lastly, the tube containing the woody
peduncles of the bunch fermented.

We have already remarked that it was more particularly the wood of the
bunch that was charged with germs of ferment. The truth of this
assertion was proved by the following series of experiments.

On October 2nd, 1875, we charged at the vineyard twenty-four tubes, all
of which were about a third filled with pure must that had been
previously boiled.

Nos. 1, 2, 3, 4, 5, and 6 tubes containing one crushed grape.

No. 7 tube containing two crushed grapes.

No. 8 tube containing one crushed grape.

Nos. 9, 10, 11, and 12 tubes containing some wood of a branch of the
vine.

Nos. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 tubes containing
a fragment of the wood of a bunch from which the grapes had all been
detached and removed.

In the course of the following days some of these tubes began to
ferment; but in others only fungoid mycelia were visible.

On October 7th the following tubes were fermenting: the second of the
first eight containing whole or crushed grapes; not one of the four that
contained wood of the branches; whilst, on the other hand, of the tubes
containing wood of the bunches, 15, 17, 20, 21, 22, 23, and 24 were all
in full fermentation. In short, fermentation, and therefore germs of
yeast, were present in one single tube out of eight containing grapes;
in none of the four tubes containing wood of the vine branch; but in
seven out of the dozen containing wood of the bunches. There was no
subsequent change in the number of tubes that fermented.

The same day that we arranged this series of tubes we prepared
twenty-four other similar ones, using the _wood of bunches preserved
from the vintage of the preceding year_. Not one of these twenty-four
tubes showed the least sign of fermentation, although they contained
grape juice in presence of the wood of the bunches; this was a further
confirmation of the sterility of the germs of ferment in the case of
bunches of grapes preserved for a sufficient time.

The next question to be considered was, what length of time after the
vintage do the germs on the surface of the woody part of bunches of
grapes preserve the faculty of producing yeast? The following
experiments were undertaken to determine this point.

We have just seen that on October 2 fragments of the woody peduncles
introduced with must, on the spot, caused that must to ferment in seven
cases out of twelve. In order that we might test the wood of these same
bunches during winter we took care to wrap some fragments up in paper
previously passed through the flame. We afterwards took occasion to test
portions of these fragments as follows:—

On December 21st, 1875, we conducted an experiment with twelve. On the
following days all began to show flakes of mycelium, or numerous
multiplying cells of _mycoderms_, _torulæ_, and _dematium_; and only
four subsequently produced yeast and alcoholic fermentation. From this
we may conclude that, three months after the vintage, a large number of
the germs of yeast spread over the woody part of the bunches lose their
vitality, through desiccation by the surrounding air, since two-thirds
of the examples taken had become sterile by that time.

On January 21st we conducted a similar experiment with twelve other
tubes. At a temperature between 20° and 25° C. (68° to 77° F.)
fermentation occurred in only two of them. On March 2nd we undertook
another experiment, again using twelve tubes, and again fermentation
occurred in two tubes.

By the beginning of April the sterility was absolute. At that period of
the year (April and May) we made numerous experiments of this kind,
using the woody parts of fresh bunches of grapes and white grapes
preserved from the last vintage, plenty of which were still to be had in
a state of freshness at the provision store. We also operated on some
wood obtained from a vineyard at Meudon. In a great number of cases no
fermentation occurred; it even happened that a whole bunch of fresh
black grapes, very ripe, which were bought at Chevet’s on April 16th,
and which had been grown in a hot-house, after having been crushed, did
not ferment at all.

Up to March not one of the tubes containing the wood of dry bunches
brought from the Jura, which had fermented, showed any signs of
_apiculatus_ or of _pastorianus_, or anything besides the ordinary low
yeast of wine, _saccharomyces ellipsoideus_.[93]

It would be a study of much interest to determine if yeast exists on
other species of plants besides the vine. During the winter we could
discover it on no others. Once during the winter, experimenting on box,
we obtained fermentation in one of our tubes which contained must. In a
great number of other experiments we obtained nothing besides moulds and
growths of _dematium_, _alternaria_, and _torulaceæ_.

Our observations in Chap. III § 6, taken in connection with those which
we have just made, prove that the yeasts of fermentation, after being
dried, preserve the faculty of germination longer than the germ-cells
which are scattered over the dead wood of the vine.

As might be expected, a microscopical examination of the particles of
dust scattered over the surface of the fruit and woody peduncles of
grapes, reveals great differences in the number of these fertile
particles at different periods of the vegetation of the grapes. As long
as the grapes are green and the vine in full activity we find scarcely
any, or, at all events, very few spores which seem to belong to ordinary
fungoid growths. Towards autumn, however, when the grape is ripening and
the leaves becoming yellow, fungoid growths and numerous productions of
great fertility accumulate on the vine, on the leaves, the branches, and
the bunches. At this period we find the water in which the grapes and
the woody parts of the bunches are washed swarming with different kinds
of organized corpuscles; it is at this period, too, that the
ferment-yielding moulds attain that phase of their vegetation in which,
when mixed with the juice of grapes, they produce fermentation.

In the Jura district a peculiar kind of wine, called straw wine (_vin de
paille_), is manufactured, which seems to contradict what we have said
as to the advent of sterility towards the end of winter in the
yeast-germs formed on the surface of preserved bunches of grapes. This
straw wine is made of grapes preserved for long after the vintage on
straw. From what we have said it might be supposed that fermentation
could not occur under these circumstances. We have, in fact, no doubt
that it is often really produced by quite different yeast-germs from
those which cause fermentation in the vintage gathered in autumn.
Fermentation as effected in the manufacture of straw wine is probably
due to yeast-dust spread over the utensils of the vine-grower, and
derived from the preceding vintage. We have seen (Chap. III. § 6) that
yeast may be dried and reduced to powder, and yet preserve its faculty
of germination for several months. It would be useful, however, to
submit this surmise to the test of experiment, and it would be easy to
do so provided we took care to crush the grapes so preserved in very
clean vessels, previously heated to a temperature of 100° C. (212° F.),
having first rejected every bunch containing injured grapes, which might
have fermented or given occasion to the development of yeast.
Fermentation, we believe, would not then take place.

[Illustration: Plate 10. One of the Ferments of Acid Fruits at the
Commencement of Fermentation in its Natural Medium.]

Another consequence results from the various facts that we have brought
out in relation to the origin of the wine-ferments, which is, that it
would be easy to cultivate one or more vine-stocks so that the grapes
gathered from them, and crushed to extract their juice, would be unable
to ferment spontaneously _even in autumn_. For this purpose it would be
sufficient to keep the bunches out of contact with particles of dust
during the vegetation of the bunches and the ripening of the grapes, and
then to effect the crushing in vessels thoroughly freed from germs of
alcoholic ferments. Moreover, every fruit and every vegetable might be
submitted to important investigations of this kind, the results of
which, in our opinion, could hardly be doubtful.

The following observations, which relate to the polymorphism of
_saccharomyces pastorianus_, seem to me to have an important bearing on
the history of alcoholic ferments, as presenting a close analogy between
the species of ferment and fungoid growths of a higher order, for
example, such fungi as _dematium_, which are generally found on dead
wood; and we would say that between the vine and other shrubs there is
only this difference, that amongst the _dematium_ forms of the vine
there occur one or more which are anaërobian, at a certain period of the
year, whilst, on the other hand, the _dematia_, _alternaria_, &c., of
other shrubs are more generally aërobian. There would be nothing
surprising in this result, considering that amongst the mucors, for
instance, we find both aërobian and anaërobian forms, and that there are
likewise torulæ-ferments or anaërobian forms, as well as torulæ-forms
exclusively aërobian.

When _saccharomyces pastorianus_ begins to develop from its natural
germs, such as are scattered over the surface of acid fruits, it takes
the form of elongated jointed filaments, branching, often pear-shaped,
and more or less voluminous. In proportion as the oxygen held in
solution in the liquid disappears and the buddings are repeated, the
length and diameter of the filaments and cells diminish, and such is the
transformation that we might, at last, suppose that we were dealing with
a different ferment of smaller dimensions.

Plate X. represents this ferment, at the commencement of fermentation in
cherry juice. In the course of a short time there is nothing to be seen
but cells of comparatively small size, disjointed and round or oval, and
filaments comparatively short and slender. This appearance is indicated
in our drawing by the cells _a_, _a_, _a_. As these latter forms
multiply with great rapidity, we soon have to search widely over the
microscopic field before we find any of the long forms from which they
spring. Instead of the forms given in Plate X., we have only those
represented in Plate XI. In other words, the aspect of these ferments
changes daily, from the very commencement of fermentation. Thus the
yeast would appear to grow smaller, coincidently with the progress of
fermentation passing from a condition in which it consists of large
cells and long ramified filaments, to a condition in which the cells are
small and the filaments short. These changes are principally due to an
alteration in the method of budding and in the life-processes of the
yeast, which speedily exhibits itself when the air supply is reduced,
and not through any intermixture of foreign ferments. So, at least, all
our observations up to the present time lead us to believe. As soon as
the oxygen has been absorbed the cells which form are oval or globular,
and the filaments do not lengthen or become so plump.

[Illustration: Plate 11. accharomyces Pastorianus, in course of Regular
Growth.]

This is, however, not the only cause of these changes in form and
aspect, although the presence of air, in greater or less quantity, has a
marked influence on the earlier developments of yeast; there is another
circumstance to be taken into account, difficult indeed to state
shortly, but which is demonstrated clearly by the microscope, and is
connected with the actual state of the germ cells. As a general rule the
budding of a cell is not an identical process when the cell is quite
young, and when it has become exhausted from want of nourishment.
Between these two conditions there is a difference which may be compared
with that which exists, for example, between a newly-formed grain which
would not germinate, and the same grain matured by rest, if we may use
the expression, that is, which has been kept long enough for its
germination to be possible. In other words, and as far as our subject is
concerned, we are not to expect that, by reviving our old yeast cells
and putting them to grow with abundance of air, in a saccharine,
nutritive medium, we shall obtain the appearance of the earlier
developments of the germ-cells on the surface of sweet and acid fruits.
We see this clearly in Plate VII., the right-hand half of which
represents the recruited budding of cells, such as those represented in
the left-hand half, in a medium peculiarly adapted to their vitality,
_and in the presence of much air_. As regards the length and size of
filaments and cells, there is little appreciable difference between the
two sides. The principal difference consists in the relative freshness
and the budding going on in individuals in the right-hand half.

There is a simple means of transforming the small, disjointed forms of
the yeast as it occurs in a deposit, at the end of a fermentation, back
into the long, tubular, pear-shaped forms peculiar to the germination of
the germ-cells, which exist amongst the particles of dust spread over
the surface of fruits. Plate X. illustrates the result of the process.
For this purpose we must effect as complete an exhaustion as possible of
the ferment _saccharomyces pastorianus_, by leaving it to itself for a
very long time, without aliment, in contact with pure air, in a damp
state; or, better still, in presence of sweetened water. We cultivate
some yeast in wort, in one of our two-necked flasks, and then carefully
decanting the fermented liquid through the right-hand neck, leave the
deposit of yeast on the sides of the flask. The glass stopper which
closes the india-rubber tube must be replaced, and the moist yeast be
left thus, in contact with pure air. The cells will steadily continue
their activity, and so gradually age, without meanwhile losing their
vitality. We use the word _age_, as we have already observed, because
the period of rejuvenescence in the case of such a yeast is so much the
slower the longer the plant has remained in that state.

Under these conditions the yeast rarely dies. It becomes attenuated and
shrivelled but still preserves its vitality, that is, the power of
reproducing itself after a lapse of several months or even several
years. In the end, however, it dies, a fact which is proved by the
cells, when sown in a nutritive medium, remaining inert.

To exhaust yeast, without destroying it, sweetened water is preferable.
Having decanted the beer, we substitute in its place water sweetened
with 10 per cent. of pure sugar. By effecting the substitution in the
following manner, we escape the risk of introducing germs from floating
particles of dust, which would nullify all experiments of this kind. We
prepare, then, a flask containing sweetened water, free from all foreign
germs, which we attach to the other flask [_i.e._, two of M. Pasteur’s
flasks with two necks, one straight and wide, the other bent and narrow
(Fig. 8)]. This is done by taking the india-rubber tube off the flask
containing yeast, and removing the glass stopper from the other
india-rubber tube attached to the flask containing the sweetened water;
then, introducing the right-hand neck of the yeast flask into the
india-rubber tube connected with the other, we raise the latter flask so
as to pour the sweetened water on to the yeast. At the same time an
assistant passes the flame of a spirit-lamp over the bent part of the
curved tube attached to the water flask, with the object of destroying
the vitality of the germs in the floating particles of dust, which enter
the flask in proportion as it is emptied into the other.

The sweetened water, which is thus brought into contact with yeast of
greater or less freshness, soon begins to ferment. Fermentation
accomplished, the vinous liquid is decanted and replaced by fresh
sweetened water, which ferments in its turn, although even at this stage
with greater difficulty than the first; this second dose is again
decanted, and again replaced by fresh sweetened water, and this process
is repeated three or four times. The yeast becomes weaker and weaker,
and eventually is unable to cause any fermentation in sweetened water
poured on it.

This exhaustion of yeast in sweetened water may be produced more quickly
by the following means:—It is sufficient to sow a mere trace of pure
yeast in a large quantity of sweetened water, say 100 c.c. (nearly four
fluid ounces), that is, instead of pouring the contents of a bottle of
sweetened water upon the whole deposit of yeast in the flask which
contained the fermented wort, we simply take a little yeast, by means of
a fine tube, from the deposit at the bottom of the flask, and introduce
it into the flask of sweetened water. This large proportion of liquid is
itself sufficient to exhaust the small quantity of yeast, quickly
checking the feeble fermentation which it had induced, so feeble indeed
as frequently not to be detected by the eye, from the fact that the
amount of liquid present is more than sufficient to dissolve any bubbles
of carbonic acid gas that might otherwise have been liberated.

It is a remarkable fact that the yeast, which during its protracted stay
in the sweetened water becomes enfeebled to such a degree that it can no
longer excite the least fermentation in that water, but will remain in
its presence for an indefinite time in a state of inert dust, does not
die. In some of my experiments the yeast has remained alive in the
sweetened water for more than two years.[94] It is almost unnecessary to
point out that these results are altogether out of keeping with the
various properties that are usually attributed to yeast.[95]

In these experiments we may use yeast-water[96] instead of water
sweetened with sugar. Into some flasks of pure yeast-water we put a
little yeast, taking all precautions to prevent the introduction of
foreign germs. No fermentation results, there being no sugar present;
the yeast, however, begins to bud, and this budding is more or less
marked according to the quantity of carbohydrate food which we introduce
along with the specimen. An interior chemical action also goes on,
causing a gradual change in the aspect of the yeast. The plasma of the
cells collects about the centres, assuming a yellowish-brown colour,
becoming granular, and forming within the cells masses more or less
irregular in shape, very rarely spherical.

We may observe here that these conditions seem to be peculiarly adapted
to show the character of the interior sporulation of the cells
discovered by Dr. Rees. Notwithstanding this we have never succeeded in
finding it distinctly, under these circumstances.

The fact which should claim all our attention, we repeat, is, that this
exhausted, shrivelled-up, aged-looking yeast preserves its faculty of
germination for several years; that, moreover, this faculty may be
aroused by placing it in aerated nutritive media, in which case it will
exhibit all the peculiarities which, under similar conditions,
characterize some of the germ-cells found on the surface of our sweet
domestic fruits. In other words, this yeast, instead of multiplying, as
it always does in the course of several growths in saccharine musts, in
the form of cells which detach themselves readily as soon as they have
nearly attained the form and size of the mother-cells, begins to shoot
out into such beautiful forms as those of _dematium pullulans_,
producing like that ferment long, well-grown, branching filaments, as
well as plump and frequently pyriform cells, as represented in Plate X.

The following figures (33 to 37) and descriptions of the observations to
which they relate will furnish fresh proofs of our assertions. In these
figures we see _saccharomyces pastorianus_, which has been exhausted in
sweetened water or in yeast-water, undergo revival in saccharine musts,
give rise to elongated, branching, pear-shaped forms, such as belong to
the original ferments of fruits, and afterwards assume the most minute
forms that we find in fermentations progressing or completed.

Let us examine Fig. 33. The history of this growth is as follows:—

Some spontaneous yeast which, after repeated cultivation, had acquired
the aspect represented in Plate XI.—which aspect the _saccharomyces
pastorianus_ generally assumes under these circumstances—was exhausted
in sweetened water, and subsequently revived in must at 10° C. to 11° C.
(51° F.). At this temperature germination was not very marked before the
end of eight days; at a temperature of 20° C. (68° F.), it only took
three days, under similar conditions. The sketch includes but one of the
long branches from which the ferment cells and the budding joints took
rise, but there were a great number more. Some of the forms represented
in the figure bear a striking resemblance, it appears to us, to some of
those of _dematium_, in Plate IX.; and even we may trace out the several
peculiarities of form which distinguish the figures in the latter plate.

[Illustration: Fig. 33.]

The next figure (34) represents the earliest forms of germination of
another specimen of _saccharomyces pastorianus_ in wort, after it had
been exhausted by four successive growths in sweetened water. We here
see the large ferment-form which appears at the commencement of
fermentation, in acid fruits, such as cherries and gooseberries (Plate
IX.), associated with smaller forms, which follow it and emanate from
it, in proportion as the process of budding is repeated. The field was
covered with this minute form, and we had to search about considerably
before we could find any of the large cells and the long, branching,
jointed filaments which we have sketched. The reason of this was, that
these large, extended filaments only appear at the beginning, when there
is still an abundance of air, giving place, after repeated budding, to
minute cells or short filaments, the ever-increasing number of which
soon hides the others from sight.

[Illustration: Fig. 34.]

[Illustration: Fig. 35.]

Fig. 35 represents _saccharomyces pastorianus_ again, as it appears
after having been exhausted by two years preservation in yeast-water, in
contact with pure air. Strange to say, it has lost its elongated
appearance, and would appear to have originated from a round ferment.
The cells are much exhausted, and most of them seem to have a double
border; their interior is very granular and of a yellowish colour. One
might readily take the specimen to be a dead old ferment, which,
however, it by no means is.

Fig. 36 represents the germination of this ferment, which had previously
been revived in a flask of wort, at the temperature of the air, in May,
1875. The following are the details of our observation:—

We sowed a trace of the exhausted yeast (Fig. 35) in a flask of wort on
May 16th. The sketch (Fig. 36) was made on May 19th, but on the 18th
there was a sensible revival. It will be seen how much the little
ferment had developed in the course of three days from the time when the
process commenced. If we had waited a few days longer before taking our
sample, we should probably have had difficulty in finding any cells or
filaments of the large ferment form, as there would have been so few of
them in comparison with the others.

[Illustration: Fig. 36.]

In the above figure we should remark the chain of large cells and
long-jointed processes, _a_, _b_, _c_, _d_ : _d_ is one of the cells
that we sowed; it has become transparent, and its contents, which are
slightly granular, have lost their brownish tint; _c_ is a large cell
which sprang from the preceding one; its outline is clear, and it is
full of fine, yellowish granules, which present a perfect resemblance to
the large ferment-cells of fruits, proceeding from the germ-cells on the
surface of those fruits, when it begins to appear in sweet juices; _b_
is a long filament, sprung from the preceding cell; and, last of all,
_a_ is a joint and its bud, in which the border is not yet very clearly
defined; it has scarcely any granules, and is finer than the others,
belonging, in short, to the small ferment form represented in Plate XI.
Here, then, we see the transition of the large ferment to the small, on
the same branch, after two generations from the germination of the
germ-cell _d_. This observation corroborates the opinion maintained by
us, that in Figs. 33, 34, 36, as in Plate X., we have not a mixture of
two ferments, the one consisting of large, elongated filaments, the
other of small cells, but one and the same ferment, the differences in
the form and size of which depend on particular conditions. The smallest
ferment-form very soon becomes the only one visible, and it preserves
its peculiar appearance in successive growths from inability to return
to the full, elongated, filamentous forms before undergoing a prolonged
exhaustion. The ferment of _mucor_ would probably afford similar
indications: it would be very interesting to find out.

[Illustration: Fig. 37. 1 Div. = 1/450th of millimetre (1/11250th of
in.).]

The following is one of the most curious of the forms presented by
_saccharomyces pastorianus_, occurring after exhaustion in a sweet
mineral liquid. The ferment, taken from a closed vat, in which it had
been used for beer, was sown in the mineral liquid on July 4th, 1873.
The following days the ferment developed feebly, but perceptibly, and
gradually increased in bulk. The flask was left to itself in an oven at
25° C. (77° F.) until December 3rd, when we ascertained that all the
sugar had fermented. We then sowed a trace of the deposit, which had
become abundant, in a flask of pure wort. On December 4th there was no
perceptible change. On December 5th, however, fermentation was in active
progress; a large quantity of froth covered the surface of the liquid,
and a considerable deposit of ferment had already taken place at the
bottom. We made a microscopical examination of this deposit, a sketch of
which we append (Fig. 37). The dark, double-bordered cells are those
which were sown but did not rejuvenesce. We may notice in different
places several of these same cells, recognizable by their granular
contents, which they are beginning to lose, to make room for germinating
cells and joints, often numerous. For instance, in the group at the
bottom of our figure one of the cells is in course of rejuvenescence and
germination, and has given rise to no fewer than six cells, filaments,
or groups of filaments. In different fields of our microscope we met
with a crowd of branches, more or less ramified, and chains of cells, of
greater or less length, of which we have sketched a few. In proportion
as the budding of these branches is repeated, the cells and joints
become more readily disunited, grow small, and assume the appearance of
_saccharomyces pastorianus_ in ordinary growths, almost as represented
in Plate XI. At first, when the old, exhausted cells begin to germinate,
their appearance rather resembles that of _dematium pullulans_, as seen
in the germination of many of the corpuscles on the surface of clusters
of grapes or fruits, or their woody parts, some specimens of which are
to be found in Plate IX.

We may briefly summarize the leading facts demonstrated in the above
paragraph. We have seen that there are different alcoholic ferments. In
the fermentation of natural saccharine juices, which, especially when
acid, so readily undergo a decided alcoholic fermentation, the ferments
originate in certain germ-cells, which are spread in the form of minute
spherical bodies of a yellow or brown colour, isolated or in groups,
over the exterior surface of the epidermis of the plant, and which are
gifted with an extraordinary power of budding with ease and rapidity in
fermentable liquids. The presence of atmospheric oxygen is indispensable
to the germination of these germ-cells, a fact which explains
Gay-Lussac’s observation that atmospheric oxygen is necessary for the
commencement of spontaneous fermentation in must.[97] Of these various
ferments one deserves special mention—namely, the variety termed
_saccharomyces pastorianus_. As is the case with all ferments, when we
gather it from the deposits produced in must that has been fermented by
its action, it is composed entirely of oval or spherical cells or of
short joints. When again placed in a similar must it buds, like all the
ordinary ferments, and the buds detach themselves from the joints or
mother-cells as soon as they have attained the size of these latter,
from which time in the new deposit is reproduced the original
ferment-form from which it sprung, and so on. Under certain conditions
of exhaustion, however, which may be easily obtained, and which we have
already accurately described, the cells undergo an absolute change as
regards their capabilities of budding and germinating. Each cell,
modified in its structure by the conditions we have mentioned, shows a
tendency to shoot out all around its surface, with astonishing rapidity,
into a multitude of buds, from many of which spring branching chains,
covered in parts, and more especially at the internodes, with cells and
jointed filaments, which fall off and bud in their turn, soon to present
the forms of the yeast deposit. In this way _saccharomyces pastorianus_
seems to afford a kind of bond of union between the race of ferments on
the one hand, and certain kinds of ordinary fungoid growths on the
other. Of these latter the plant which De Bary has named _dematium_, and
which is generally found on the surface of leaves or dead wood, more
especially, however, on the wood of the vine at the end of autumn, the
time of the vintage, presents a striking example.

There seems every reason to believe that at this period of the year one
or more of the varieties of _dematium_ furnish cells of yeast, or even
that the ordinary aërobian varieties of _dematium_ produce at a certain
stage of their vegetation, in addition to aërobian cells and torulæ,
other cells and torulæ which are anaërobian, that is, alcoholic
ferments.

In this manner we arrive at the confirmation of an idea entertained by
most authors who have studied yeast closely—namely, that it must be an
organ detached from some more complex vegetable form. We may also add
that in the case of _saccharomyces_ the chains of filaments, both
tubular and fusiform, and septate cells more or less pyriform
originating in them, when attentively observed, remind us forcibly of
the filamentous chains and spore-balls, or conidia of _mucor racemosus_
when submerged, so that one might suppose that the spore-ferment of our
_dematium_ is itself an organ detached from some still more complex
vegetable form, in the same way that conidia-ferment of _mucor
racemosus_ belongs to that more complex fungoid growth.

In the following passage De Bary uses, for the first time, the words
_dematium pullulans_ (Hofmeister, vol. ii. p. 182, 1866). The German
naturalist begins by citing the opinions of Bail, Berkeley, and H.
Hoffmann, the first of whom maintains that _mucor mucedo_ becomes
transformed into the yeast of beer, the second that yeast is a peculiar
state of _penicillium_, and the third that it may be generated by fungi
of very different nature, and especially by _penicillium glaucum_ and
_mucor mucedo_. He goes on to say: “I have taken great pains to repeat
the experiments of Bail, Berkeley, and H. Hoffmann, but I have never
been able to confirm the results which they have stated, either in the
case of growths in microscopic cells or in experiments performed in
test-tubes with the purest possible substances—specially prepared
solutions or must of wine and spores of penicillium, _mucor mucedo_,
_botrytis cinerea_, &c.” On this point M. De Bary arrives at exactly the
same results which we communicated to the _Société Philomathique_ and
the _Société Chimique_ of Paris, as already given in Chap. IV. § 4, p.
128, note.

M. De Bary goes on to say: “In researches of this kind it is difficult
to eliminate two sources of error. On the one hand, it is beyond doubt
that cells of ferment are actually scattered over everything, and that,
consequently, they may easily get into the experimental liquid along
with the spores that we sow, and so occasion mistakes. On the other
hand, there are a great many fungi which develop budding processes
similar to yeast, but incapable of producing fermentation, which yet in
some cases spring directly from spores as well as from mycelium,
especially we may instance _exoascus_. This last observation is
especially applicable to the extraordinarily numerous variety of fungi
which rank under the Dematiei and Sphaeriacei, and which I shall term,
for convenience of naming, _dematium pullulans_.”[98]

We shall conclude this paragraph with a remark that has doubtless
presented itself to the minds of our readers, which is, that it would be
impossible to carry out the experiments we have described if we could
not make sure of dealing with pure ferments, or, at least, with mixtures
the components of which are sufficiently well known for us to assign to
each the effect produced by it in the total phenomena observed. It would
be extremely difficult to continue growths of yeast-deposit in sweetened
water or in a moist atmosphere if the little plant were mixed with
spores of other fungoid growths, a variety of ferment-forms, and germs
of bacteria, vibrios, or infusoria in general. All these foreign
organisms would tend to develop just in proportion as the conditions of
the media were more or less favourable to their growth, and, in a very
few days, our flasks would be filled with swarms of beings which, in
most cases, would entirely conceal the facts relating to those forms,
the separate study of which it was our object to follow out. We shall
have occasion, therefore, to examine, in a subsequent paragraph, the
preparation of ferments in a state of purity. At present we may state
that yeast, which in its ordinary condition is a mass of cells so liable
to change that its preservation in a moist state is impossible,
manifesting in the course of a few days during the winter, and in
twenty-four hours during the heat of summer, all the signs of incipient
putrefaction, thereby losing its distinctive characteristics, is
nevertheless capable, when pure, of enduring the highest atmospheric
temperatures for whole years without showing the least signs of putrid
change or contamination with any other microscopic organisms, and
without the cells losing their power of reproduction. In the presence of
facts like these, the theory of spontaneous generation must seem
chimerical. The hypothesis of the possible transformation of yeast into
_penicillium glaucum_, bacteria, and vibrios, or conversely, which the
theories of Turpin, H. Hoffmann, Berkeley, Trécul, Hallier, and Béchamp
involve, is equally refuted by these facts.


                    § II.—On “Spontaneous” Ferment.


The expression _spontaneous ferment_ may be applied to any ferment that
appears in a fermentable liquid without having been purposely sown in
it. In this respect the ferments mentioned in the preceding paragraph,
those of all saccharine juices of fruit which ferment when left to
themselves—the ferments of wine, for example—are spontaneous. The term,
however, is not altogether appropriate, because, after all, the process
is the same as if an actual sowing had been made, since, as we have
shown, it is absolutely necessary for the juice to come into contact
with the surfaces of the fruit, so that the ferment may be mixed with
it, and so produce subsequent fermentation. Therefore, although we may
apply the term _spontaneous ferment_ to the ferments of fruits, we
intend that expression to apply in this paragraph solely to those
ferments that are generated in a saccharine liquid, in which, by
previous boiling, we have destroyed all ferment germs, and which,
nevertheless, enters into fermentation after being exposed in free
contact with air. In such a case it is entirely from the particles of
dust floating in the air that the ferment germs that appear in the
liquid are derived. Such are typical _spontaneous_ fermentations, and it
is of the ferment so obtained that we are about to speak.

In the course of the researches which we undertook in order to ascertain
whether _mycoderma vini_, or vinous efflorescence, became transformed,
in the case of beer, into actual alcoholic ferment—researches which were
the more protracted and varied in consequence of their leading to the
condemnation as erroneous, on the faith of new and more precise
experiments, such as those given in Chap. IV. § 2, of that
transformation, in which we had for long believed—we had occasion to
observe several spontaneous fermentations of this kind in various
saccharine liquids. We then proceeded to describe our method of
conducting the experiments. Having brought about the development of a
film of _mycoderma vini_ or _cerevisiæ_ on the surface of a liquid,
fermented or not, we submerged that film in wort, which we afterwards
put into long-necked flasks, in which alcoholic fermentation generally
took place in the course of a few days. This fermentation in no way
resulted from the transformation of the cells constituting the
efflorescence into ferment. The mycodermic film merely acted as a
receptacle of true ferment germs, wafted thither with the particles of
dust floating in the air of the laboratory, which germs developed in the
liquid into actual alcoholic ferments amongst the cells of the submerged
mycoderma. By conducting experiments in this manner we brought about
several spontaneous fermentations, the germs of which could have been
introduced by nothing but the particles of dust in the air. These
fermentations, which we were obliged to follow very carefully with the
microscope from the time when they first manifested themselves, on
account of the transformation that we were seeking, which transformation
we thought might possibly be that of the cells of _mycoderma vini_ into
cells of ferment, generally gave us during the first days of
fermentation the large, elongated, branchy ferment represented in Plate
X., which was succeeded by the small ferment represented in Plate
XI.[99]

[Illustration: Plate 12. Ferment-cells from a Spontaneous Fermentation
just starting.]

Here let me describe one of these experiments. In the beginning of
March, 1872, we grew some _mycoderma vini_, obtained from wine, on some
wort contained in a shallow basin. On March 6th we submerged the
efflorescence and put it all together, liquid and film, into a
long-necked flask. On March 9th we detected incipient fermentation, and
on March 12th we took a sketch of the yeast of the deposit, as given in
Plate XII. This is the large and long branching, more or less
pear-shaped form, which occurs at the beginning of fermentation in the
sweet and acid musts of our domestic fruits. On March 16th we made
another sketch of the deposit, in which the proportion of cells, in the
form of elongated segments and filaments, reminded us, in some measure,
of the filamentous mycelium of typical fungoid growths much diminished.
In this case, however, the majority of cells were oval, round, and in
short segments. On this day, March 16th, we added some fresh wort to
that which had fermented, with the object of prolonging the duration of
fermentation and increasing the proportion of yeast. On March 19th we
made a fresh sketch, which it is not necessary either to reproduce;
suffice it to say, that the yeast was now considerably more regular and
uniform in appearance.

[Illustration: Fig. 38.]

Spontaneous ferment, therefore, very often occurs in this large
ferment-form, which, by repeated developments in the act of
fermentation, becomes reduced by degrees after successive generations to
the ferment which, following Dr. Rees, we have named _saccharomyces
pastorianus_, a polymorphous ferment which must be studied closely that
it may not be confounded with others, inasmuch as it is so universally
diffused that we very seldom fail to find it in any ferment which has
been exposed in contact with ordinary air, at least, we may repeat, in a
laboratory devoted to researches on fermentation. We have found the same
thing occur in a brewery, being there mixed with the ferments used in
brewing.

There are, no doubt, several varieties of this _saccharomyces_. We
sometimes find amongst the spontaneous ferments which repeated growths
have brought to a more or less uniform state, the forms represented in
Plate XI., but the cells and segments much smaller. Amongst others, Dr.
Rees has distinguished a _saccharomyces exiguus_.

Fig. 38 represents another spontaneous ferment, which appeared in a
boiled saccharine wort, which entered into fermentation after being
exposed to the air of the laboratory.

The sketch was made directly after the fermentation had commenced.
Probably this is simply one of the earlier forms assumed by the
_saccharomyces_, or by one of its varieties. It will be seen that the
alcoholic ferment is associated with another little filiform ferment,
probably the lactic. The spontaneous ferments are almost always impure,
a circumstance that may be readily understood if we bear in mind the
results described in Chapters III. and IV.


                  § III.—On “High” and “Low” Ferments.


The ferments mentioned in the preceding paragraphs do not belong,
properly speaking, to industrial products; that is to say, in actual
practice there are no operations in which the ferments of fruits and
spontaneous ferments are employed for the purpose. It is quite true that
these ferments are the cause of the fermentations from which wine,
cider, gin, rum, gentiana, mead, &c., are derived, but these
fermentations are spontaneous, they take place without the intervention
of man, and without man’s directing their production, or taking any
notice of the agent which starts them.

In the manufacture of beer, on the other hand, the practice is quite
different. We may say that the wort is never left to ferment
spontaneously, the fermentation being invariably produced by the
addition of yeast formed on the spot in a preceding operation, or
procured from some other working brewery, which, again, had at some time
been supplied from a third brewery, which itself had derived it from
another, and so on, as far back as the oldest brewery that can be
imagined. A brewer never prepares his own yeast. We have already had
occasion to remark that the interchange of yeasts amongst breweries is a
time-honoured custom, which has been observed in all countries at all
periods, as far back as we can trace the history of brewing. The yeasts
which in the present day produce beer in the brewery of Tourtel, near
Nancy, in that of Grüber, at Strasburg, that of Dreher, at Vienna, and
others, came originally from breweries, where and when it would be hard
to say. In the case of the first working brewery, the yeast was, no
doubt, derived from some spontaneous fermentation, which took place in
an infusion of barley that had been left to itself, or, from some
natural spontaneous ferment, and nothing could be easier than to realize
this fact again. In the brewing industry there are two distinct modes of
fermentation:—“high” fermentation and “low” fermentation, some of the
distinctive characteristics of which we have pointed out in Chapter I.
It may be questioned whether the spontaneous yeast employed in the first
brewery, or that which a wort left to itself in the present day would
yield, would be of the “high” or “low” type. It may be concluded from
what we have said on the subject of spontaneous fermentations in wort,
that wort, left to itself, would furnish ferments more or less
resembling those of wine. We have never obtained in spontaneous
fermentations of wort either a distinctly “high” ferment, or a
distinctly “low” one, properly so called; nor, further, have we ever
obtained either one of these distinct kinds, with its industrial
characteristics, in experiments on the ferments of fruits. What, then,
was the origin of the “high” and “low” ferments now used by brewers?
What was the nature of their original germs? These are questions which
we are unable to answer, but we are very much inclined to think that we
have here another example of the modifications which plants as well as
races of animals undergo, and which become hereditary in the course of
prolonged domestication. We know nothing of corn in its wild state, we
cannot tell what its first grain was like. We know nothing of the
silk-worm in its original state, and we are ignorant of the characters
of the race that furnished the first egg.

These reflections may seem to favour the supposition that there is a
real difference between “high” yeast and “low” yeast, and that both of
these differ from spontaneous ferments and the ferments of domestic
fruits. These are propositions demanding most careful consideration, for
it is generally admitted that these ferments become intermixed, that
their morphological differences are merely a question of medium, and
that the transition of one to the other is a simple matter. The
following facts seem to contradict such statements.

[Illustration: Fig. 39.]

“High” Ferment.—Fig. 39 represents some “high” yeast taken from a
deposit after fermentation, and Fig. 40 the same yeast in course of
propagation in some aerated wort. In comparing “high” yeast with other
alcoholic ferments at the same stage of development, there are three
points which are especially striking: the diameter of its cells is
relatively large, their general aspect is rounder, and when they are
undergoing propagation their mode of budding produces a markedly
ramified appearance, so that the cells always occur in clusters and
branches. Fig. 40 gives a very exact idea of these characters. To
investigate satisfactorily the branching habit of growth peculiar to
this ferment we should examine it during the first few hours of its
propagation, when, under the influence of the oxygen dissolved in the
fermentable liquid, its vital activity is greatest. Later on, often on
the day following the sowing, the groups become disconnected, and at the
end of the fermentation the cells have quite separated from each other,
not more than 2 or 3 per cent. remaining united, and even these in
groups of not more than two cells together. This is represented in Fig.
39.

To give an idea of the rapidity with which this ferment multiplies, we
may state that our sketch (Fig. 40) was made under the following
conditions:—On April 28th, 1874, we caused a flask of wort to ferment by
means of a trace of “high” yeast. On the morning of the next day, that
is fourteen hours afterwards, an appreciable deposit of yeast had
formed, and some frothy patches appeared on the surface of the liquid,
showing that fermentation had set in. On May 1st we decanted the beer,
substituting for it water sweetened with 10 per cent. of sugar. On May
2nd we decanted the sweetened water, and substituted a fresh quantity
containing the same percentage of sugar. On May 3rd, at mid-day, we took
some of the fermenting liquid from this flask and put it into a flask of
wort; five hours after the introduction of the ferment we made the
sketch in question. The field is covered with branching clusters, the
groups being sketched exactly as they occurred in the field. Their
activity was due to the condition of the ferment, and to the perfect
fitness of the nutritive medium for its vegetation. In sweetened water
the budding of the cells was considerably less active; no branching
groups of cells are to be found. Budding, nevertheless, occurs to a
considerable extent, but it is limited to one bud, or two at the most,
to each cell. Fermentation in pure sweetened water is mostly correlative
with the duration of vital activity in the globules already formed.

[Illustration: Fig. 40.]

Let us next suffer our yeast to exhaust itself by keeping it in a great
excess of sweetened water for a very long time; we shall then be able to
observe its process of revival, and see if we can find any facts
analogous to those presented by _saccharomyces pastorianus_ (Chapter V.
§ 1).

With this object in view, on May 6th, 1874, we impregnated two fresh
flasks of sweetened water with some of the contents of the
before-mentioned flask, which we had refilled with sweetened water on
May 2nd. On May 13th we decanted the liquid, which was still very sweet,
from one of these two fresh flasks, which could hardly be said to have
fermented at all—the quantity of yeast in them being so small—and
replaced it with some wort. Strange to say, on the morning of the 14th
we found an appreciable growth of yeast, and a froth of carbonic acid
gas on the surface of the liquid. The yeast therefore was not dead,
although its fermentative powers had been exhausted. There was, however,
no remarkable feature in connection with its revival, nor did we find
the slightest trace of any of the elongated ferment-form. What we got
was simply the ramified groups of “high” yeast again, in round cells,
but nothing more.

Fearing that our yeast might not have remained for a sufficient time in
the sweetened water for exhaustion, we set aside, for a whole year, the
other flask which we had prepared on May 6th. On May 16th, 1875, we
decanted the sweetened liquid and replaced it with wort. This time,
however, there was no revival of the yeast; it had perished.
Fortunately, we had also saved the flask of yeast and sweetened water
which was prepared on May 2nd, 1874, as already mentioned, and in this
case, as will be seen, the vitality of the yeast had not been
extinguished, doubtless, in consequence of the formation of what we
shall presently designate by the name of _aërobian ferment_. On May
16th, 1875, we decanted the liquid from this last flask, and replaced it
with wort. On the next day the surface of the wort was covered with a
thin froth, indicating the commencement of fermentation. The microscope
revealed nothing extraordinary, or indicative of the fermentation of any
special ferment. To assure ourselves that our ferment had remained
“high,” we sowed some of it in a fresh flask of wort on May 19th, and
then, seven hours after impregnation, submitting it to examination, we
could find nothing but ramified groups in fine condition, without a
single elongated cell, indeed, it would have been impossible to find a
more beautiful specimen of “high” yeast, or one of a more decided
character.

It would seem, therefore, that “high” yeast cannot, under any
circumstances, assume the form and character of the ferment
_saccharomyces pastorianus_, or of other known ferments. We are
justified, therefore, in regarding it as a distinct species of ferment,
an opinion which is supported by other circumstances.

1. In equal quantities of saccharine wort a considerably greater growth
of “high” yeast is obtained than of other yeasts. We need no very
rigorous proofs to convince ourselves of this fact: for by simply
causing equal volumes of the same wort to ferment, the one being pitched
with _saccharomyces pastorianus_, for example, the other with “high”
yeast, we shall obtain a perceptibly greater volume of “high” yeast than
of the other, in certain cases even five or six times as much.

2. “High” yeast is of a tougher texture than the others, separating,
when the fermented liquor and its deposit is shaken up, into lumps which
refuse to disappear; whereas _saccharomyces pastorianus_ diffuses
through the whole liquid with the greatest ease.

3. “High” yeast produces a special beer, with a peculiar flavour, well
known to consumers, but little esteemed at the present day. Hence the
gradual displacement of breweries worked on the old “high” fermentation
system by others in which “low” yeast (of which more anon) alone is
employed.

4. Lastly, one characteristic of “high” yeast, which it shares in common
with some other ferments, although not with all, and which, from a
practical point of view, deserves special mention, is that as
fermentation proceeds the yeast rises to the surface of the liquid.
Whilst the process of the manufacture of beer by this ferment is going
on, the yeast is seen to work out of the bung-holes, flowing over in
considerable quantity. The ferment named after the author, as well as
“low” yeast, does not possess this property: it remains at the bottom of
the vessels. When “high” fermentation takes place in vessels that are
not filled, the ferment forms a thick layer, a kind of cap on the
surface of the beer. This characteristic may be witnessed even in the
fermentation of very small quantities of liquid. In our flasks, in which
the volume of fermenting wort does not exceed 100 c.c. or 150 c.c.
(about 4 or 5 fluid ounces), we may perceive, as the violence of
fermentation subsides, and the head falls, the sides of the vessel
covered to a height of from 1 cm. to 2 cm. (about 3/4-in.) above the
surface of the liquid, with particles of yeasty matter, in little
masses, or in a thin film, raised to that height by the head, and left
behind when that fell.

“Low” Ferment.—Whilst high yeast performs its functions in the breweries
in which it is used at somewhat high temperatures—namely, between 16° C.
and 20° C. (60° F. to 68° F.)—“low” yeast is never employed at a higher
temperature than 10° C. (50° F.), and it is even thought preferable that
it should not be subjected to more than 6° C., 7° C., or 8° C. (43° F.
to 46° F.). At these comparatively low temperatures “high” yeast would
have no perceptible action, whereas it is at such temperatures that
“low” yeast best performs its functions.

In our Memoir on alcoholic fermentation, published in 1860, in the
_Annales de Chimie et de Physique_, the idea of the identity of the two
yeasts was accepted; but we had at that time made no special
observations of our own on the subject.

Upon closer investigation we are inclined to believe that the two yeasts
are quite distinct. We might keep our “high” yeast at the lowest
temperatures that it can bear, and repeat our growths under these
conditions; or, on the other hand, we might subject the “low” yeast to
temperatures higher than those at which it ordinarily grows, without
ever succeeding in changing the first into the second or the second into
the first, supposing, of course, that each of our yeasts was pure to
begin with. If they were intermixed the change in the conditions of
development would cause one or the other to preponderate, and incline us
to believe that a transformation had really occurred.

[Illustration: Fig. 41.]

It is true that brewers generally are of a different opinion. Most of
them assert that “low” yeast cultivated at a high temperature becomes
“high” yeast; and conversely, that “high” yeast becomes “low” by
repeated growths at a low temperature. Many have told us that they have
proved this. Nevertheless it is our belief that the success of such
transformation has been but apparent, attributable in each case to the
fact, as we have just stated, of their having operated on a mixture of
the two yeasts.

Mitscherlich, and various authors after him, have asserted that “high”
yeast propagates by budding, and “low” yeast, on the contrary, by
spores, formed by the endogenous division of the protoplasm of the
cells, and set free by the rupture of the cell-wall, which then,
increasing in size, assume the character of ordinary cells. But we have
never been able to confirm this.

Fig. 41 represents a field of low yeast, taken from the deposit in a vat
after the fermentation of the beer was finished. The granular matter
mixed with the cells is altogether amorphous although in many cases
perfectly spherical. It is a product in no way related to this yeast
(see Plate I., No. 7).[100] “High” yeast and all the ferments of beer
have this kind of deposit associated with them. There is no doubt that
confused observations as regards these minute bodies have been the cause
of the error which we had to deal with in connection with a particular
mode of reproduction of low yeast, as to which we have already fully
expressed our views (Chap. V. § 1, p. 146).

Comparing Fig. 41 with Fig. 40 (p. 189), it may be seen that the general
aspect of low yeast is distinguished, in its early stages, although in
no very decided manner, from that of “high” yeast, by being slightly
smaller and less round or spherical in its cells than the latter.[101]
These differences, however, would escape an unpractised eye.

As to the case of “high” yeast, the deposits of “low” yeast after
fermentation appear as scattered, isolated cells; we do not find more
than two or three per cent. of united cells. Nevertheless the two yeasts
present, as we shall see, quite marked differences in the character of
their budding and multiplication.

[Illustration: Fig. 42.]

On May 28th, 1875, we put a trace of pure, unicellular, “low” yeast,
taken at the end of a fermentation, into a flask of wort. On May 29th,
sixteen hours after impregnation, the temperature during the night
having been 15° C. (59° F.), we made a sketch of the yeast before its
development had become apparent to the naked eye. No perceptible
development, that is to say, no visible deposit at the bottom of the
liquid and formation of patches of froth on the surface, took place
before May 30th. A mere glance at Fig. 42 will be sufficient to enable
us to detect a considerable difference between it and Fig. 40, which
represents the multiplication of the cells of “high” yeast. The cells of
the “low” yeast are slightly smaller and rather more oval, as we have
already had occasion to notice, and the budding processes are
considerably less ramified, in consequence of which there is a
comparative absence of globular clusters which are so striking a feature
in the development of “high” yeast, when examined early enough.
Moreover, if we cause our “low” yeast to age, by leaving it for a longer
or shorter time in the beer which it has formed, or if we exhaust it in
sweetened water by leaving it for whole months in a volume of sweetened
water considerably larger than what it is capable of fermenting, and
then proceed to revive it and cause it to propagate in an aerated
saccharine wort capable of nourishing it, this yeast will resume its
original aspect, as sketched and described. At most we shall observe
certain minute differences in the size of the cells in successive
growths. A very remarkable industrial characteristic of this yeast is
the fact that it never rises to the surface, no matter at what
temperature it may be working, whether between 6° C. and 8° C. or 15° C.
and 20° C.;[102] in other words, it is not buoyed up by the carbonic
acid gas when the fermentation is at its height. At the end of the
fermentation, the surface of the liquid and the sides of the vessel
above the level of the liquid are clean and not covered with the yeast,
which remains altogether at the bottom of the fermented liquid.
Moreover, the weight of new yeast which it yields is always less than
that yielded by “high” yeast, for the same quantity of fermentable
liquid, although greater than that which _saccharomyces pastorianus_
would give. Lastly, the beer possesses a flavour and delicacy which
cause it to be held in higher esteem by consumers than beers produced by
means of other ferments.[103]


   § IV.—On the Existence and Production of Other Species of Ferment.


Our present knowledge of the alcoholic ferments embraces the following,
without taking into account the ferment-form of _mucor_:—

The ferment named after the author, which is found associated with the
ferments of the grape and other domestic fruits, and with spontaneous
ferments in general.

The ferment of “high” beer.

The ferment of “low” beer.

To these must be added the ordinary ferment of wine, and that called
_apiculatus_, although, indeed, these last are of little practical
importance, since, in general, they soon become lost amongst others of
greater vitality, in the spontaneous fermentation of fruits. These are
not the only alcoholic ferments; a study of the germ-cells diffused over
the surface of fruits, grains, and stalks of all vegetables in different
countries, would doubtless lead to the discovery of many new ones. We
are even inclined to believe that one ferment might give rise to a
multitude of others. The investigations which we have undertaken in this
direction are as yet not far advanced; we may, however, be allowed
merely to state the principle which governs them. A ferment is a
combination of cells, the individuals of which must differ more or less
from each other. Each of these cells has certain generic and specific
peculiarities which it shares with the neighbouring cells; but over and
above this, certain peculiar characteristics which distinguish it, and
which it is capable of transmitting to succeeding generations. If,
therefore, we could manage with some species of ferment to isolate the
different cells that compose it, and could cultivate each of these
separately, we should obtain as many specimens of ferments, which would,
probably, be distinct from one another, inasmuch as each of them would
inherit the individual peculiarities of the cell from which it
originated. Our endeavours are directed to realizing this result
practically, by first thoroughly drying a ferment and reducing it to
fine powder. We have seen (Chap. III. § 6) that this mode of experiment
is practicable, that in a powder composed of yeast and plaster the
ferment preserves its faculty of reproduction for a very long time. If
we now drop a small quantity of this powder from a sufficient height,
and then, at a certain distance below the cloud of dust so formed, open
several flasks previously deprived of air and containing a fermentable
liquid that has been boiled, immediately closing them all up again,
under such circumstances it is conceivable that some of the cells of
yeast diffused in the cloud of dust, and separated widely in the act of
falling, will enter some of our flasks singly, and there develop an
appreciable weight of ferment, all the cells of which will have sprung
from the same mother-cell. We have proved that flasks may be easily
impregnated under these conditions, and our preliminary observations,
although incomplete, seem to favour the idea that numerous varieties of
ferment are to be obtained by these means.

Spontaneous ferments, properly so called, of which we have already
spoken, are, after all, the result of sowings of this kind. Originating
in liquids which have been boiled, and then left to themselves in
contact with the air in a place where cells or germs must have existed,
these ferments must necessarily often spring from single germs or from a
limited few, and this also would probably be a means of developing
distinct varieties of ferments.

Without dwelling longer on the practical consequences likely to result
from the ideas which we have just expressed, we shall proceed to
describe two new alcoholic ferments, which differ widely from those
already mentioned.

_New “High” Ferment._—We met with this ferment accidentally, under the
following circumstances:—On February 12th, 1873, we had brewed in the
laboratory about 2-½ hectolitres (rather over 50 gallons) of wort, 10
litres (about two gallons) of which were set aside to cool in a
white-iron trough, and left during the night exposed to free contact
with air in the underground part of the laboratory, where we have a
small experimental brewery. Next day we put some of this latter into a
bottle; the wort soon began to show evidence of change, various
productions made their appearance on the surface of the liquid, and a
deposit of yeast settled at the bottom. On May 23rd, perceiving bubbles
of gas and a steady fermentation set up in the wort, which remained all
the time corked up, and fearing that the bottle might burst by the
increasing internal pressure, we drew the cork. A considerable
liberation of gas at once took place, accompanied by a voluminous foam
which half emptied the bottle. A microscopical examination of the
deposit from the disturbed liquid led to the discovery of a very
homogeneous yeast, associated with various other organisms; it was
clearly a yeast which we had not hitherto met with amongst the
spontaneous ferments which we had had occasion to study. Thinking that
this might be a new species of ferment which would probably produce a
beer that was also unknown, we set to work to purify it by cultivation
in flasks of pure wort, during the months of May, June, and July. Our
last growths, of August 4th, 1873, were preserved, in order that we
might assure ourselves of the purity of the beer, and, consequently, of
the ferment. On November 15th its purity was established. On that date
we made some beer with this ferment, which had now been left to itself
for several months in contact with pure air. The beer which we obtained
resembled no known variety; consequently the ferment must itself have
been a distinct one, differing from others, especially those which we
have been considering in this chapter.

[Illustration: Fig. 43.]

Fig. 43 represents the rejuvenescence of this ferment. Comparing this
figure with Fig. 42, we see that this ferment presents a considerable
resemblance to “low” yeast in dimensions, method of budding, and oval
shape; but the feature which distinguishes it essentially from “low”
yeast is that it rises to the surface, like “high” yeast. Buoyed up by
the gas during fermentation, it forms a layer of yeast on the surface of
the fermenting liquid, where it remains after the head has fallen. Some
of this head of yeast likewise adheres to the sides of the vessel above
the level of the liquid.

In short, by the greater regularity of its forms and the uniform
dimensions of its cells, this ferment is to be easily distinguished from
_saccharomyces pastorianus_; its aspect, which is oval instead of
spherical, and the ramified form of its chains of cells, which is less
marked than in the case of “high” yeast, also prevent our confounding it
with the latter ferment; in its rising character it differs absolutely
from “low” yeast; lastly, it may be distinguished from all other
ferments by the flavour of the beer that it produces.

The ferment which we discovered in this accidental way may be utilized.
Indeed, we may ask, is it not to be found already in our beer? We are
inclined to believe that it is. After the war of 1870, some Viennese
traders established at Maisons-Alfort, near Paris, a manufactory of
yeast for bakers. They saccharified by means of malt a mixture of the
meals of rye, maize, and barley, which they then caused to ferment. One
day we had occasion to study the yeast produced in this establishment,
and although we did not submit it to a sufficient number of consecutive
experiments to enable us to speak positively, we are under the
impression that the yeast produced at Maisons-Alfort is a “high” one,
differing from what may be properly termed the “high” yeast of breweries
in which “high” fermentation is practised, but presenting a great
resemblance to the “high” yeast of which we have been speaking. It would
be interesting to confirm the opinion of their possible identity by
fresh studies, and the best way of doing this would be to compare the
qualities of beer which the two yeasts could produce.

_Caseous Ferment._—We give the title caseous for a reason that will
presently appear, to a ferment which we came across also accidentally.
We were trying different methods of purifying yeasts, and for this
purpose had composed a liquid formed of:

      Ordinary wort                                 150 c.c.[104]
      Water saturated with bi-tartrate of potash    50 c.c.
      Alcohol of 90°                                25 c.c.

Quantities of this liquid were placed in several of our double-necked
flasks, submitted to boiling, then, after cooling, impregnated with
different ferments, and kept in a water-bath at 50° C. (122° F.) for one
hour.

In operating under these conditions with brewers’ “high” yeast, say, for
instance, with what is called Dutch yeast, a kind well known in
distilleries, fermentation shows itself in the course of a few days, in
spite of the increased temperature to which our liquid, which is hopped
and slightly acid and alcoholic, has been subjected. The time required
for the resumption of fermentation depends both upon the degree of
temperature to which the yeast has been exposed and upon the duration of
its exposure. These, however, are not the points upon which we now wish
to dwell. It is of greater importance to notice that the new yeast has
none of the characteristics of “high” ferment, of which Dutch yeast
seems to be exclusively composed, if we do not take into account
impurities which cannot be avoided in a commercial product of this
nature. Other specimens of Dutch yeasts would give the same results.

[Illustration: Fig. 44.]

[Illustration: Fig. 45.]

Figs. 44 and 45 represent this new ferment magnified to the same extent
as the other ferments have generally been, that is 400/1; it will
readily be seen how different its form is from that of “high” yeast, how
far it is from having the spherical aspect and mode of budding
characteristic of that ferment. In Fig. 45 the ferment is represented in
a mass; in Fig. 44 we see the ramified groups, the cells and segments of
which form, after separation, the yeast of the deposit. It thus appears
to be composed of jointed branches of greater or less length, which, at
the junctions of the segments, put forth similar cells or segments of a
round, oval, pyriform, cylindrical, or other shape; in all its
characters recalling the description of _dematium_. Moreover, the cells
and segments exhibit a greater sharpness of outline, as well as a more
marked transparency and refractive power than are found in the majority
of ferments; but the most curious physical characteristic of this
ferment is its plasticity and elasticity, if we may use those terms. It
can only be made to diffuse through water with great difficulty; when
shaken up in it, it sinks to the bottom quickly as a clotted sediment,
and the supernatant liquid appears scarcely at all charged with globules
in suspension. Again, when placed on a microscope slide and compressed
by the cover-glass, it returns to its original form on removal of the
pressure. It is from these considerations that we have given to it the
name of _caseous ferment_.[105] Lastly, this ferment produces a beer of
a peculiar kind, which cannot be confounded with other kinds of beer
known in the present day. We should add that it preserves its
characteristics in repeated growths, and that we have never found it
reproduce ordinary “high” yeast.

When caseous ferment is sown in a saccharine medium charged with mineral
salts, its aspect, form, and mode of budding differ completely from what
they are when the ferment exists in a natural medium, such as wort or
other liquid adapted to the nutrition and life of ferments.

[Illustration: Fig. 46.]

Fig. 46 represents this ferment in course of development, forty-eight
hours after it had been sown in a saline medium (we employed Raulin’s
fluid, substituting bi-tartrate for the nitrate of ammonia). It will be
seen how different its aspect is from that of the preceding figures; it
is still capable, however, of resuming the forms of the latter if
cultivated afresh in natural saccharine worts.

“High” yeast from a “high” fermentation brewery in the Ardennes, after
having been exposed to heat under the conditions given above, likewise
produced caseous ferment, without a trace of “high” ferment, just as
happened in the case of the Dutch yeast. All the “high” yeasts used in
brewing seem to behave in the same manner.

What conclusion are we to draw from these facts? Apparently that “high”
yeast is modified by heat in an acid and alcoholic medium, giving rise
to caseous ferment. On the other hand, it might be conceived that the
“high” yeasts on which we experimented were not pure, but contained, in
a state of intermixture, some caseous ferment, and that by the
application of a temperature of 50° C. (122° F.) to our alcoholic
medium, the high ferment was all killed and the caseous ferment alone
survived. It is a remarkable fact that this latter hypothesis,
improbable as it seems, inasmuch as the microscope revealed no
intermixture of ferments, seems, nevertheless, to be a true one. As a
matter of fact, if we subject to a temperature of 50° C. for one hour in
the medium in which it acts, not the “high” yeast of commerce but “high”
yeast that is _absolutely pure_, this will perish utterly, and the wort
after cooling may remain for years in an oven without either undergoing
fermentation or developing any growth whatever of “high” ferment or
“caseous” ferment.

On the other hand, if we impregnate this same alcoholic liquid with some
of the caseous ferment and then heat the vessel to 50° C. for one hour,
the caseous ferment will go on reproducing itself after the liquid has
cooled down.[106]

It seems, therefore, impossible to admit that caseous ferment results
from a modification of “high” ferment, and we are led to believe that in
the preceding experiments it must have been the progeny of cells of
caseous ferment present in the “high” yeasts of commerce, which cells,
probably in consequence of their scarcity, the microscope was unable to
reveal, but which, nevertheless, did exist, and went on reproducing
themselves alone after the heating.

[Illustration: Fig. 47.]

This conclusion is supported by the following fact, which also tends to
prove that in the case of the “high” English _pale ales_, caseous
ferment plays a most important part. In the medium already described, we
sowed the deposit from a bottle of good English pale ale. After having
been heated the yeast went on growing, and we obtained the very
beautiful specimen of caseous ferment represented in Fig. 47. The two
dark globules are dead cells which had been killed. Two minute segments
of lactic ferment are also visible in the sketch—the yeast which we
sowed was, of course, impure—and their presence proves, we may observe,
by the way, that lactic ferment also can withstand a temperature of 50°
C. (122° F.) in the medium which we here employed. The yeast as sowed is
represented in Fig. 48; it reminds us forcibly of certain forms of the
caseous ferment. Amongst the globules, which for the most part were
transparent and very young, there were some which appeared aged and of a
yellowish colour and granular. These latter probably belonged to the
yeast of manufacture. Their shape distinguishes them from “high” yeast,
properly so called, as on the other hand it causes them to appear more
like cells of a recent growth to which, there is no doubt, beer, after
it is put in bottle, owes its effervescence and head. These various
circumstances incline us to believe that the caseous ferment forms part
of certain commercial yeasts, especially those used in the celebrated
breweries of Bass and Allsopp, at Burton-on-Trent, in the manufacture of
pale ale. Caseous yeast is, moreover, a “high” ferment, that is to say,
it rises to the surface.

[Illustration: Fig. 48.]


      § V.—On a New Race of Alcoholic Ferments: Aërobian Ferments.


Mention has already been made of certain researches which we undertook
with the object of ascertaining whether _mycoderma vini_, or
efflorescence of wine, and _mycoderma cerevisiæ_, or efflorescence of
beer, which grow equally well in all fermented liquids, have the power
of becoming transformed into actual alcoholic ferment. The result of
those researches was stated to be that these mycodermata do not become
transformed into ferment, properly so called, and that whenever any such
transformation has been supposed to have taken place, the ferment
produced was derived from germs introduced by the air or by the utensils
employed. What we did ascertain of the ferment-producing power of
_mycoderma vini_, was merely that this plant, when submerged, is capable
of causing sugar to ferment, in consequence of a certain continuous life
possible to its cells, apart from the oxidations resulting from the
presence of free oxygen, but without any generation of new cells taking
place.

Whilst engaged in these researches, we were pursuing others in relation
to the converse of the proposition just discussed, that is to say,
respecting the possibility of ferment becoming transformed into
_mycoderma vini_ or _mycoderma cerevisiae_. Our experiments in
connection with this subject chiefly consisted in various endeavours by
way of exhausting the yeast and subsequent revival of its growth. This
exhaustion was effected by growing the yeast in excess of sweetened
water, and at other times in unsweetened yeast-water, our efforts being
directed to deprive it of all power of fermenting. We afterwards caused
it to develop afresh in highly aerated, nutritious liquids, in order
that we might see how it reproduced itself, and if its new form were
that of a mycoderma. The yeast after having lost its power as a ferment,
and being no longer able to act in pure sweetened water, nevertheless
reproduced itself when placed in fermentable media, holding in solution
materials adapted to its nutrition; yet we never succeeded in obtaining
any organism besides the ferment, and, indeed, the identical variety of
ferment on which we had operated. In no case was _mycoderma vini_ or
_cerevisiæ_ produced, and we concluded that we were justified in stating
that whenever the _mycoderma vini_ appeared on the surface of a
fermented or fermentable liquid, its germ must have been introduced by
the surrounding air, or have previously existed in the liquid, and that
the reason why this germ multiplied so abundantly was because the liquid
in question had been peculiarly adapted to the vitality of the plant.

In a laboratory where alcoholic fermentations are studied, these germs
of _mycoderma vini_ exist in great abundance on the surfaces of
different objects. This fact admits of easy proof; we have merely to
open in such a laboratory some flasks containing yeast-water deprived of
air, or yeast-water sweetened, or any natural saccharine medium, or any
fermented liquid, which till the moment when our flasks were closed had
been kept boiling (Chap. IV.); it would be a very rare thing, indeed, if
_mycoderma vini_ did not develop in most of these flasks after the air
was readmitted, especially if, shortly before this operation, the dust
lying on the surface of the tables or floor of the laboratory had been
stirred up by dusting or sweeping.

This series of experiments, the salient points of which we have just
given, conducted with a view to ascertain whether yeast could be
transformed into mycoderma, has led the way to certain results of
special interest, results which concern all alcoholic ferments, and
which in all probability will be found in the long run to apply to all
aërobian ferments.

It being necessary for the conduct of our experiments to preserve our
yeast in a state of purity for an indefinite period, often for a great
length of time, in contact with pure air, we discovered that yeast was
possessed of extraordinary vitality, and that it rarely perished
completely throughout, inasmuch as we could almost invariably cause it
to revive by bringing it into contact with fresh, fermentable liquid.
This revival of the yeast—and it is to this point that we are most
anxious to direct the attention of our readers—is effected from two
distinct sources:—

1. By those cells of yeast which have not perished.

2. By cells of new formation.

We may give an example to explain this more clearly. In one of our
two-necked flasks we cause some pure wort to ferment by employing yeast
also in a state of purity. Fermentation completed, we leave the liquid
to itself, not touching the flask again. The fermented liquor covers a
deposit of yeast, apparently inert, and no trace of _mycoderma vini_
makes its appearance on the surface of the liquid. Let us suppose that
we go on daily for a considerable time introducing a little of the yeast
from this flask to a different flask of wort: the fresh flasks will
begin to ferment. The only appreciable difference which these successive
flasks will present, their impregnation having been effected at
intervals of twenty-four hours, will be that, _ceteris paribus_,
fermentation in them will be more and more slow in making its
appearance. This difference, as we have already explained, will be due
to the fact that the yeast in the first flask will, in the course of
time, undergo, in each of its cells, a process which we cannot better
describe than as a progressive _senescence_. The cells gradually become
filled with amorphous granulations, their interior becomes yellow, and
the protoplasm collects, either at the centre or near the borders; in
short, the vitality of the yeast becomes feeble. When, however, it is
taken out of the liquid in which it has fermented and introduced into a
fresh saccharine wort, it gradually resumes its transparency, and then
begins to germinate. These effects are the less rapidly brought about
the longer the cells remain exhausting themselves in the first fermented
liquid. They might be left in that liquid for such a length of time that
they would eventually perish, a fact which would manifest itself in
their absolute sterility and quiescence when sown in a fresh medium. In
general, however, matters are not carried far enough for this to take
place, and the yeast, preserved in a state of purity in its fermented
liquid, retains the capacity of revival, which may then go on
indefinitely. As a matter of observation, the cells of yeast, after
causing the liquid to ferment, instead of remaining inactive, and so by
living at their own expense gradually passing into a state of
exhaustion, begin to bud again; at least this is true of many of them.
Multiplying afresh in the fermented medium, under the influence of the
air, they form a kind of mycodermic film on the liquid surface, or a
ring round the sides of the flask, on a level with the liquid. This
development might often be mistaken for _mycoderma vini_ or _cerevisiæ_;
in reality there is not a single cell of _mycoderma_ formed. If we sow a
trace of the new growth in a saccharine medium it will behave exactly as
yeast would, budding and multiplying, and setting up fermentation in the
liquid. And thus, in spite of its mycodermic aspect, this growth is
nothing but yeast, since it gives rise to true alcoholic fermentation;
but it is a kind of yeast which, under the foregoing conditions, lives
after the manner of fungoid growths, absorbing the oxygen of the air and
emitting carbonic acid gas. It appears on the surface of all fermented
liquids, especially those which, like beer, contain carbohydrates, and
its quantity is the greater, and its action the more rapid, in
proportion as it has more perfect access to the air. We have termed this
yeast _aërobian ferment_ or _fungoid ferment_.

It may easily be understood how this kind of production has escaped
notice up to the present time. The conditions of our experiment were, in
many respects, novel; a saccharine liquid had never before been caused
to ferment by means of pure yeast, absolutely free from foreign germs; a
fermented liquid had not previously been exposed to contact with pure
air for an indefinite time. On the other hand, all ordinary fermented
liquids, when left to themselves in contact with air, are a ready prey
to _mycoderma vini_ or _aceti_ at their surface, and then give rise to
true fungoid growths. The appearance of these organisms, which always
takes place soon, has thus constantly concealed or prevented the
development of the true aërobian ferments. In repeating the experiment
described any alcoholic ferment may be used, and each one will be found
to produce its own peculiar fungoid form of ferment. Another point
worthy of notice is that these aërobian ferments, when they put forth
buds in the act of fermentation, reproduce the forms of the original
ferment, at least apparently so. In this respect they cannot be
distinguished, notwithstanding the fact, surprising as it seems, that
the two kinds of ferments are not identical. If we operate on a “low”
yeast its aërobian ferment will differ physiologically from the ferment
from which it sprung, presenting various special peculiarities which are
not to be found in the original “low” yeast. In most of our experiments
we have found the new aërobian ferment to be similar in its action to
“high” yeast, rising to the surface, and producing a beer which
possesses a greater fragrance than beer brewed with the identical “low”
yeast from which it was derived. Lastly, the properties of an aërobious
ferment are not peculiar to first growth, but are hereditary; by
repeating the growth of the first aërobian ferment we do not cause them
to disappear, we find them again in succeeding generations.

Notwithstanding these facts, it would be difficult to discover any very
appreciable differences between the forms of the cells of any particular
yeast and those of its aërobian ferment in course of development. So
true is this, that the aërobian ferment of _saccharomyces pastorianus_
might even be caused to put on the forms of _dematium pullulans_, which
we have had occasion to observe specially characterize this ferment
after the cells have been subjected to a prolonged process of
senescence.[107] This is evident from the following example, which will
once again show the remarkable extent to which the forms of a particular
organism may be varied by changes in composition of the nutritive
medium:—

On August 6th, 1873, we took some of the ferment _saccharomyces
pastorianus_ from a flask of wort that had undergone fermentation, and
sowed a scarcely perceptible quantity of it in another flask containing
a saline medium, composed as follows:—

     Water containing about 10 per cent. of    150 c.c. (5-1/4 fl.
     sugar-candy                                              oz.)

     Ash of yeast                              0·5 gramme (8 grs.)

     Ammonic bitartrate                        0·2   “    (3 grs.)

     Ammonic sulphate                          0·2   ”    (3 grs.)

In the course of the following days the ferment began to develop,
although with difficulty, the fermentation revealing itself by
collections of bubbles appearing here and there on the surface of the
liquid. We left the flask undisturbed till the 25th of November
following. On that day we found a very white deposit of ferment covering
the yeast-ash that had not been taken into solution, and a ring of
aërobian ferment on a level with the surface of liquid; all the sugar
had disappeared; the liquid contained 5·2 per cent. of alcohol, by
volume, at a temperature of 15° C. (59° F.); and, lastly, in consequence
of the purity of the materials employed, there was no trace of the
formation of fungoid growths, whether of _mycoderma vini_ or of
_mycoderma cerevisiæ_, on the surface of the liquid, or of vibrios or
lactic-ferment below the surface.

Thus then we see—and several other examples throughout this work confirm
the fact—that saccharine liquids holding mineral salts in solution are
as capable of complete fermentation as any media of natural composition.
It is true that ferment develops slowly and with difficulty in them, and
at times takes on rather curious forms, but, nevertheless, it does
develop in the media and carry on a fermentation in which not the
minutest particle of sugar is left undecomposed. This is true, at least,
in the case of _saccharomyces pastorianus_, but there are other ferments
which in such media are checked in their multiplication and in their
continued action on sugar. One condition indispensable to the
accomplishment of fermentation in such a sweetened mineral medium, by
means of _saccharomyces pastorianus_, is the absolute purity of the
materials and of the ferment. It is necessary that the life and
physiological action of the latter should be in no way interfered with
by the presence of other microscopic organisms. We shall have occasion
to revert to this important detail in connection with our growths.

[Illustration: Fig. 49.]

Fig. 49 represents the ferment as it appeared when examined on August
11th, 1873. We can no longer recognize in it any _saccharomyces
pastorianus_. The general appearance is spherical, and there are a
number of clusters of budding cells which remind one at first sight of
the mode of germination of brewers’ “high” yeast. At _a_, _a_, _a_, we
see globules from which irregular abortive filaments have sprung, a
proof of difficult germination. No such monstrosities could ever have
occurred if we had used beer-wort or must as our nutritive medium.

On November 25th we made another examination and sketch of the ferment,
the appearance of which did not differ materially from that given above.
The general appearance was the same, consisting mostly of globules
joined together in clusters of two or three or more. No separation, such
as occurs in the case of ferment formed in natural worts, had taken
place. The ferment, moreover, was very irregular, and comprised cells of
all sizes. We sowed some of it in a flask of pure wort. On November 26th
there was no apparent development: on November 27th, however, not more
than forty-eight hours after impregnation, there was a considerable
deposit of white ferment at the bottom of the liquid, and fermentation
was so active that the surface of the liquid was covered with an
abundant froth. This shows us the wonderful vitality and recuperative
power possessed by germs which, left to themselves for about four
months, revived so readily. It proves too that the reviving influences
took effect on some aërobian ferment. From the mode of life of this
latter being similar to that of a surface fungoid growth, it does not
become exhausted as the cells of ordinary ferment do. Now the cells
which, sown on August 6th, had become exhausted by prolonged stay in the
mineral liquid, and were almost inert, would have required several days
for their revival; but in the experiment described the revival was
rapid, and this rapidity proves, as we have said, that the revival must
have taken place in cells of aërobian ferment.

[Illustration: Fig. 50.]

Taking some fresh yeast from the bottom of the liquid we examined and
made a sketch of it (Fig. 50). The field was filled with round and oval
cells, jointed and ramified filaments, budding and multiplying in the
most remarkable manner, reminding us of the germination of the cells of
yeast exhausted in sweetened water, and also of the germination in the
form of _dematium pullulans_ of certain germ-cells which are spread over
the surface of sweet, domestic fruits. We could never grow tired, as we
wrote it in our original notes, of sketching this beautiful plant, which
establishes very clearly a transition between one of the best defined
cellular ferments, viz., _saccharomyces pastorianus_, and certain forms
of very common fungoid growths, those of _dematium_, and even of the
most common mould, _mucor mucedo_ or _racemosus_, when it vegetates
beneath the surface of a liquid and acts as a ferment.[108] We have
here, as in these cases, filamentous chains branching into other similar
chains, composed of more or less elongated cells, which at length fall
off and germinate exactly as the conidia-bearing _hyphae_ of _mucor_ do.

The aërobian ferment of “high” yeast, in whatever medium we cultivated
it, presented no peculiarity, as far as its forms were concerned. It was
composed of cells of spherical shape, like ordinary “high” yeast, and
germinated in the same way as the latter.

[Illustration: Fig. 51.]

Fig. 51 represents the revival of this aërobian ferment. We recognize
here the branched mode of budding and spherical contour characteristic
of “high” yeast proper. Nor does the aërobian ferment of “low” yeast
present any special peculiarities, in forms, dimensions, and mode of
growth closely resembling the “low” yeast from which it is derived. At
the commencement of its restoration, however, if this is performed in
sweetened water, the cells in the groups are larger than those which are
subsequently developed.

[Illustration: Fig. 52.]

Fig. 52 represents the aërobian ferment of yeast used in
“low”-fermentation breweries, examined forty-eight hours after pitching.
We find that groups resembling that at a are of very rare occurrence.
They are to be seen only at the very beginning, generally only for the
first few hours of the renewed activity. Very soon, however, they
develop cells which are of the size of the oval cells budding at _b_.

[Illustration: Fig. 53.]

Fig. 53 represents the aërobian caseous yeast which forms rather
rapidly, in thick, greasy-looking pellicles, on the surface of liquids
which have been fermented by means of caseous ferment. The larger form
of cells, _a_ and _b_, is not often met with.

[Illustration: Fig. 54.]

On May 27th, 1875, we sowed, in a flask of wort, a trace of a pellicle
of this kind, which had formed on the surface of a flask in which
fermentation had been set up by means of caseous yeast in May of the
preceding year. On May 30th fermentation began to reveal its presence by
a voluminous froth, and the newly-formed yeast had reached the bottom of
the flask. A small quantity was taken out by a capillary glass tube, and
a sketch of the ferment made; this is given in Fig. 54. Amongst the
cells which occupy the field there are groups of some of larger size.
These are not distinct forms mixed with the others, but simply another
illustration of the fact that old cells in course of revival, especially
when they have been exhausted in sweetened water, as we have just
observed of the aërobian ferment of “low” yeast, commence with forms of
larger diameter or more elongated than the ordinary forms peculiar to
the ferment which at a later stage are developed from them. We have seen
how marked and exaggerated this feature was in the case of
_saccharomyces pastorianus_.

Let us again call attention to the forms of aërobian ferment furnished
by the yeast which we have already described under the name of _new
“high” yeast_. Fig. 55 represents this aërobian ferment, as taken on
November 27th, 1873, from a pellicle of rather greasy and moist
appearance, on the surface of a flask of fermented beer-wort which had
been impregnated on July 21st, 1872. It might readily be mistaken for
ordinary “high” yeast, yet no two ferments can be more distinct.

[Illustration: Fig. 55.]

[Illustration: Fig. 56.]

On November 27th, 1873, we sowed a trace of this ferment in a flask of
wort. From the 29th, with a continuous temperature of 25° C. (77° F.), a
considerable deposit of yeast began to form, and the froth of
fermentation covered the whole surface of the liquid. We took a little
of this deposit for examination; it is represented in Fig. 56. The field
is occupied with oval cells of great uniformity. We recognize the aspect
of the original yeast (Fig. 43). Here and there, indeed, we come across
some cells of larger size, such as those at _a_ and _b_, which is
another illustration of the remark that we have just made respecting the
forms which revived exhausted cells take on at the commencement of a new
germination.

The physical aspect of the several aërobian ferments is in general so
characteristic that we are often able by simple inspection to
distinguish between them as they occur on the surface of liquids.
_Saccharomyces pastorianus_ in its aërobian state forms a crown of cells
round the sides of the vessel at the surface of the liquid, which crown
is broken up by the least agitation of the liquid; its vitality
continues for years.

The aërobian ferment of “high” yeast appears in the form of small
isolated teats on the surface of the fermented liquid. It develops
rather sluggishly, and has no great vitality.

The aërobian form of “low” yeast develops as a somewhat fragile layer,
the least agitation precipitating it to the bottom of the vessel in a
cloud of very small irregular flocks, that do not diffuse through the
liquid as they fall. With free access to air it retains life for a long
time.

The aërobian ferment of caseous yeast forms a continuous greasy-looking
pellicle, gradually thickening, which breaks up into fragments when
shaken. With a supply of air it lives very long, and the pellicle
gradually increases in thickness.

In reviewing these ferments we may naturally ask ourselves the question
whether the “high” ferments of which we have spoken—the industrial one
concerned in the “high” fermentation of breweries, and the other which
we have termed _new “high” ferment_—are not aërobian ferments of “low”
yeasts. We are inclined to think that the ferment which in the preceding
paragraph we termed _new “high” ferment_, may, perhaps, be the aërobian
form of the “low” yeast employed by Alsatian and German brewers. We have
studied this new “high” ferment side by side with the aërobian ferment
of “low” yeast, and the result we have arrived at is, that in appearance
and mode of germination, as well as in the flavour and quality of the
beers which they produce, they greatly resemble one another. In the last
respect, however, we cannot say that the identity is quite absolute, and
hence it is with some doubt that we suggest the possible identity of the
two ferments. As regards the ordinary “high” yeast of breweries, it may
well be supposed, both from its power of rising to the surface during
fermentation and from the peculiar smell and flavour of its beer, that
we have in it the aërobian ferment of some “low” yeast, as to the
identity of which, however, we can say nothing, having no knowledge as
to where it is to be found; or, indeed, any certainty that such a yeast
actually exists.

In writing these lines an idea suggests itself which might be profitably
made the subject of serious experimental study. What would be the
peculiar properties of the aërobious ferment-form of an aërobian yeast?
Certain facts incline us to believe that these forms differ from each
other just as a “low” yeast differs from its aërobian ferment. If this
were actually the case it would be very interesting to compare the
peculiar properties of an indefinite series of aërobian ferments, all
derived from a common origin. We find recorded in our laboratory notes
that a certain aërobian ferment of the second generation produced a beer
different from that produced by the same ferment of the first
generation, being possessed of a fragrance so marked that, on entering
our laboratory, in which only a few litres of this beer were fermenting,
we were at once struck by the powerful odour which it emitted.


              § VI.—The Purification of Commercial Yeasts.


We have already stated that the researches detailed in the preceding
chapter require for their successful prosecution that the ferments on
which we experiment should be absolutely free from germs of other
organisms, and we have shown how impossible it would be, if this
condition were not complied with, to follow for weeks or months,
sometimes even years, the changes which occur in a yeast maintained in
contact with air, either in sweetened water or in a liquid which has
fermented under its influence. Equally necessary is it that the
saccharine worts employed should also be exempt from these impurities,
as well as the air, which is being constantly renewed at the surface of
the liquids. These last conditions may be realized by the adoption of
our double-necked flasks, with which a laboratory for research of this
kind should be furnished, always ready for use, filled with the
different kinds of liquids that may be required.

In general, the inconveniences resulting from the impurity of a yeast
employed do not immediately manifest themselves, in consequence of the
enormous preponderance of the true yeast, which, in comparison with the
foreign germs that contaminate it, may be so great that microscopical
examination fails to reveal even the presence of these latter. Again, it
is a well-known fact that the abundance of one growth in a limited
medium operates to the prejudice of a less abundant one, inasmuch as the
first consumes the nutritive materials at the expense of the second, and
more particularly the needful amount of oxygen. It follows, that when a
saccharine liquid is impregnated with commercial yeast, nothing but
yeast may be detected for a time, and one is led to believe in the
purity of the subsequent growth. This, however, supposes that the
external conditions, as well as those of the medium of growth, are
equally adapted to the life of the yeast and that of those organisms
present as impurities; for if these conditions rather favoured the
nutrition of the latter, we should be sure to find their proper
developments appearing at an early stage. For example, when the growth
of yeast becomes sluggish, we have invariably the development of such
after-growths. The principal germs, having exhausted the saccharine
liquid which has fermented under their influence and is no longer
adapted for their growth, cease to develop, and have their place taken
by ferments of disease, spores of moulds, mycodermata, &c., the growth
of which proceeds more or less rapidly, in proportion as the character
of the liquid and the surrounding temperature are more or less suited to
their growth.

Here, too, we have an explanation of the rapid change that occurs in
brewers’ yeast when left to itself after fermentation. In such a mass of
cells, kept apart from any food-supply, and only with difficulty able to
keep themselves in life by consuming their own soluble contents, we have
an excellent field for the development of foreign germs. In this way we
may have a rapid putrefaction in yeast, to which there will be a
correspondingly rapid growth of organisms in the liquid, where they
find, as well as in the yeast-cells, appropriate nourishment. Nothing
could better confirm this view of the matter than the array of facts, by
way of antithesis, already described, in which we have seen a pure yeast
remain for an indefinite time in contact with pure air, without
undergoing any putrefaction, or manifesting other changes than those
which result from the combustions peculiar to living cells when left to
support themselves, in a moist state, in contact with oxygen.

In the process of brewing, as soon as fermentation is finished, or
rather, as soon as certain physical effects are produced, for instance,
when the beer falls bright, or, as the French say technically, when the
yeast breaks up,[109] the beer is racked; subsequently the yeast, which
is left in a plastic layer at the bottom of the vessels, is collected,
washed, and kept under water in a cool place, to be used again in the
course of twenty-four or forty-eight hours. Brewers never care to keep
their yeast for a longer time before using it, especially in summer. We
can understand how this practice prevents the foreign germs which are
mixed with the yeast from living and reproducing; but although the
conditions of brewing, as far as the treatment of the yeast is
concerned, may, in a certain measure, prevent the development of these
germs of disease, nevertheless they are there, and from their extreme
minuteness, pass into the beer in greater or less number, however bright
it may have been rendered by racking. There they only await conditions
favourable to their existence to enable them to develop, and to affect
more or less injuriously the qualities of that delicate beverage.

On December 15th, 1872, we bought nine samples of beer in different
large cafés in Paris, which had all come from the best breweries of
Strasburg, Nancy, Vienna, and Burton. After leaving them for twenty-four
hours, we decanted all our samples, and then sowed a drop of the deposit
of each in flasks of pure wort. On January 2nd, 1873, we examined the
ferments formed in these worts, which had been kept in an oven at a
temperature of 20° C. (68° F.), and also tasted the beers produced; they
all had an abominable taste, and each contained diseased ferments.

At the same time, by way of comparison, we impregnated other flasks of
wort with pure ferments. None of the beers of this series acquired a bad
taste or produced foreign ferments; they only became flat.

When we review the operations of the brewer’s art, we are surprised by
the comparative perfection to which that art has been brought by the
laborious experience of years, and the more so when we consider that, as
regards the question of the diseases of beer, the brewer has never been
guided by any such rigorous principles as those which we have explained
in this work. We have already given proofs of this in our first chapter.

The beer is racked and separated from its yeast before fermentation has
entirely ceased. The principal reason for this is that it is necessary
that the beer, after being run into cask, should work again and undergo
a secondary fermentation, in order that it may not be invaded by the
parasites, of which we have already spoken, as would not fail to be the
case if the beer were suffered to remain in a state of perfect
quiescence. Not only is the beer racked before it has attained its limit
of attenuation, but in addition to this, and also with the view of
checking the development of parasites, it is placed in cellars sensibly
cooler than the temperature of fermentation, low as that is in the case
of “low” beers: the temperature of the cellars being not higher than 2°
or 3° C. (36° F.).

Unfortunately, the requirements of trade prevent our complying with
these exigencies to the end. When the beer is sold it is conveyed away,
no matter what the season may be, and deposited in the retailer’s
cellar, for a longer or shorter time, according to the variations of
consumption. On a warm day beer will be in great demand; the next day,
if rain or cold have come on, the demand will be very limited, since
beer is, in our climate at least, a drink for hot weather. From causes
of this nature, the beer may have to remain a long time in the cellars
of the retailers or consumers. By way of precaution, indeed, it is put
into very small casks, which permit of a frequent renewal of the supply,
and is conveyed to distances by express trains, and during the night; it
is even sent away in wagons provided with a kind of double case, the
outer jacket being filled with ice, which keeps the air surrounding the
casks constantly cold. Such are some of the troublesome measures taken
to obviate the danger that we have pointed out. They operate very
injuriously in restricting the trade and raising the price of beer. It
is a matter of extreme importance, then, that our produce should be
better removed from the action of those microscopic enemies which beer
contains; in other words, that this beverage should have less cause to
fear circumstances favourable to the development of the germs of
impurity with which it is always contaminated, as a natural consequence
of the methods of manufacture at present adopted. The question of
alteration in the flavour of beer should be regarded from another point
of view which merits equal attention. We have seen that there are
different kinds of beer, each of which corresponds to a special ferment
from which it derives its flavour and aroma, and, in a word, everything
which gives it a value in the eyes of the consumer. It very often
happens, especially in badly-managed breweries, and more particularly in
those in which several beers are manufactured, that the yeast is a
mixture of different ferments. The evil effects of such a mixture are
experienced in the course of manufacture, and still more so in the beer
after manufacture. Brewers in good “low” fermentation breweries, who
brew what is called _stock_ beer, during the winter months, for
consumption in summer, up to August and September, are very anxious to
prevent the development of a _vinous_ flavour in beers of this kind.
According to our observations, this vinous flavour seems to be
principally due to an intermixture with the pitching yeast of
_saccharomyces pastorianus_ or its varieties, one of the peculiarities
of which ferment is that in the course of time it imparts a decided
vinous flavour to beer. If this ferment were not present amongst the
yeast-cells—and here we are speaking of an absolute, so to say,
mathematically absolute absence—the beer produced would gradually grow
old in the store cellars, without ever acquiring any vinous flavour,
properly so called.

This vinous flavour develops more especially in English beers when these
are kept. It is an easy matter to show that in English beers, after
their manufacture, _saccharomyces pastorianus_ and the ferment which we
have termed _caseous_, which also imparts a peculiar flavour, form
almost exclusively, notwithstanding the fact that the yeast used in the
manufacture of English beer is a ferment essentially distinct from
_saccharomyces pastorianus_.

The secondary fermentation which takes place in “high” and “low” beers
stored in cask after manufacture, is very often due to this same
ferment, which may be recognized by elongated jointed cells, at times
more or less ramified, as well as by the influence which it exercises
upon the flavour of the beer.

We may add that the general result of our researches has convinced us
that “high” yeast cannot transform itself, any more than “low” yeast
can, into the ferment of which we are speaking, and that whenever a beer
produced by means of “high” or “low” yeast develops a foreign ferment,
this ferment must have existed in the original yeast in the form of
germs, which, from their extreme scarcity, often fail to be detected by
means of the microscope. The best proof that we can give of this is the
fact that a beer produced by means of “high” or “low” yeast, if left to
itself for months or years, will never contain in its deposit anything
besides the yeast that was used in its manufacture, provided that that
was pure to begin with. Now this can never be the case in dealing with
actual commercial beers, no matter what they may be or in what brewery
they may be produced. In all beers, in the course of time, in addition
to diseased ferments, ferments essentially different from those used in
their manufacture will appear, and notably _saccharomyces pastorianus_;
this result must be attributed to the general impurity of commercial
yeasts.

In certain cases the intermixture of ferments is to be feared almost as
much as the presence of diseased ferments, when these latter have not
developed to any great extent. We have often seen our fermentations
invaded by ferments differing absolutely from those which we originally
employed. The repetition of growths, and more particularly changes in
the composition of our fermentable media, purposely made with the view
of attaining certain results, often produce complications of this kind.
For a long time we were unable to realize the true significance of the
results of some of our experiments, in consequence of the facts which we
have just explained, as well as those detailed in the preceding
paragraph, having escaped our notice; indeed, our ignorance of those
facts added greatly to the difficulty and length of our researches. Our
labours from the commencement of this work to the date of its
publication have extended over not less than five years, and no one can
know better than ourselves with what advantage we might devote a still
longer time to it; but, as Lavoisier says, one would never give anything
to the world if he delayed doing so until he fully attained unto his
ideal aims, which always seem more distant the more one increases one’s
efforts in the attempt.

Our preceding observations show how extremely important it is to employ
pure yeasts to obtain, on the one hand, well flavoured beers, whilst
adhering to the processes at present existing in breweries, and on the
other, beers of good keeping qualities, less liable to injury, less
dependent on actual commercial requirements, capable, that is, of
withstanding conditions favourable to the development of ferments
prejudicial to the soundness of the produce, what we have named ferments
of disease.

In the case of intermixture of alcoholic ferments, we may sometimes
manage to effect their separation by taking advantage of their unequal
vitalities in different media of cultivation. On December 17th, 1872, we
made a powder of commercial Dutch yeast and plaster, as described in
Chapter III. § 6. The Dutch yeast was a “high” ferment.

On July 25th, 1873, we sowed a portion of this dried mixture in a flask
of pure wort. From July 27th patches of bubbles from fermentation were
visible on the surface.

On August 2nd the fermentation was completed. The yeast, examined under
the microscope, was _apparently_ pure, formed of spherical cells of a
fine “high” ferment. We poured away the fermented liquid, observing
every necessary precaution, and left in the flask almost all the deposit
of yeast, and not more than one or two cubic centimetres (about half a
tea-spoonful) of beer.

On November 15th following the yeast, examined afresh, still seemed pure
and still exhibited the form of round cells of “high” yeast, only that
they had taken on a very aged aspect, showing a double contour, and
filled with granulations collected irregularly about the centre. Such
are the precise characteristics of dead cells; nevertheless it was still
possible that some living cells yet remained. To assure ourselves of
this we took some of the yeast and placed it in a flask of pure wort. On
the 19th a little froth from fermentative action appeared on the
surface. We then examined the yeast and discovered that it was no longer
“high” yeast, but a small ferment of rather irregular appearance, in
which the jointed cells of _saccharomyces pastorianus_, as it usually
appears after a succession of growths, predominated. It must not be
imagined here that what we saw was a transformation of one yeast into
another. The phenomena are to be explained much more simply. The Dutch
yeast employed being very impure must have contained traces of foreign
ferments, especially of _saccharomyces pastorianus_. Reduced to a dry
powder on December 17th, 1872, the two or more varieties of cells
comprising it had preserved their vitality in consequence of the
plaster, and this vitality had continued at all events until July 25th,
1873. Subsequently, when cultivated in wort, they had multiplied in that
medium. The _saccharomyces_ had revived like the rest, but its quantity,
compared with the high Dutch yeast, was so small that the microscopical
observations made on August 2nd, when the flask was decanted, failed to
discover its presence. Between August 2nd and November 15th the high
yeast must have perished entirely: the cells of _saccharomyces_, on the
contrary, still maintained their vitality, and these alone multiplied in
the flask of wort impregnated on November 15th. Here we have an example
of the separation of alcoholic ferments, through the unequal resistance
they sometimes offer to adverse conditions to which they may be
subjected. We may also conclude that if we had prepared a quantity of
beer with the “high” yeast, which in our experiment of August 2nd, 1873,
seemed to have developed in a state of entire purity, this beer when
made and stored in cask or bottle could not have failed to undergo a
secondary fermentation, in consequence of a development of
_saccharomyces pastorianus_.

Let us take, as another example of purification of the same kind, the
case of the different ferments of the vintage. When must begins to
ferment the apiculated ferment invariably appears, and becomes
afterwards associated, more or less, with the _saccharomyces
pastorianus_, in the presence of which the multiplication of the
apiculated ferment soon ceases. _Saccharomyces pastorianus_, in its
turn, is gradually displaced by the ferment which we have termed the
ordinary ferment of wine, and which Dr. Rees has named _saccharomyces
ellipsoïdeus_. On the subject of these changes in the proportion of the
ferments of wine, the Note which we published in 1862 in the _Bulletin
de la Société chimique_ may be consulted. Now, these various ferments
mutually interfere with each other: whereas if _saccharomyces
apiculatus_ were there alone it would multiply to a greater extent, and
with greater advantage to the fermentation of the must. This result is
obtained by filtering the must, as we have already observed.

It is evident from what we have just said that the principal part of the
deposits of yeast in the sediment of fermented grapes, at the time when
the wine is first racked, which in the Jura, is called _l’entonnaison_,
is composed of the ordinary ferment of wine, the _saccharomyces
ellipsoïdeus_, and that the cells of apiculated ferment are scarcely
discoverable with the microscope, being scattered amongst an infinite
multitude of other ferments.[110]

[Illustration: Fig. 57.]

We procured from Arbois, on January 20th, 1875, some wine yeast taken
from a large barrel of the preceding vintage, racked on January 18th.
The ferment was very irregular. Some of its cells were very old, of a
yellowish colour, and full of granulations—amongst these a certain
number formed jointed segments, rather elongated, and probably belonging
to _saccharomyces pastorianus_. The other cells were transparent, and
apparently still young. This mixture of the two ferments is represented
in Fig. 57. No doubt if we had searched carefully we should also have
found some cells of _saccharomyces apiculatus_. On January 21st we sowed
a small quantity of this raw yeast in a flask of sweetened water. On the
24th we poured off the liquid, and supplied the deposit with fresh
sweetened water. The exterior temperature was 12° C. (54° F.). On the
27th we took some of the deposit and put it into a flask of wort. The
following days there was a development of yeast, accompanied by
fermentation. We obtained, however, neither the large forms of the
ferments of fruits, nor those of the more minute ferments represented in
Plate XI. The _saccharomyces pastorianus_, represented in the yeast
which we sowed by aged, granular, elongated cells, had, therefore, not
revived. Fearing that this result might have been attributable to
insufficiency of the exhaustion, which had only lasted for a few days,
we raised the temperature of the flask of sweetened water to 25° C. (77°
F.), at which we kept it until February 20th. On that day we sowed some
of this yeast in wort. There was a very perceptible revival the next
day, but it was still impossible to detect with the microscope the forms
we have just mentioned, nor did _saccharomyces pastorianus_ appear in
fresh, succeeding growths.

[Illustration: Fig. 58.]

Fig. 58 represents the yeast formed, which evidently had sprung from the
transparent cells seen in Fig. 57, and doubtless belonging to the
ordinary ferment of wine, _saccharomyces ellipsoïdeus_. Here we have
another example of the natural separation of ferments brought about by
the death of one or two of them, or by extreme differences in the time
of their revival.

We cultivated this yeast (Fig. 58), to some considerable extent, in
beer-wort. It produced a peculiar beer, of vinous character, in fact a
true _barley wine_. This proves, we may here remark, that ordinary wine,
in its flavour and quality, depends to a great extent on the specific
nature of the ferments which develop during the fermentation of the
vintage; and we may fairly assume that if we were to subject the same
must to the action of different ferments we should obtain wines of
different characters. With a view to the practical application of this
idea, it would be well to undertake new studies in this direction; and
the methods of cultivating and managing ferments, explained in this
work, would be of great value in such researches.

The purification of ferments may be accomplished by various methods,
according as we have to deal with an intermixture of ferments, or to
regard as our principal object the expulsion of ferments of disease,
such as vibrio germs, lactic ferment, the filamentous ferment of turned
beer, _mycoderma aceti_ or _mycoderma vini_.

One method of easy application consists in sowing the yeast in water
sweetened with 10 per cent. of sugar. This liquid should be first
boiled, and preserved in the two-necked flasks which we have so often
described. Sweetened water is a very exhaustive medium for ferments, and
the organisms mixed with them. A great many cells perish in it, and the
chances are that the foreign germs, which are always scarce in
comparison with the great number of cells of ferment, may be amongst
those which die, or those which become so exhausted that when the yeast,
after this treatment, is sown in wort, they disappear, and allow those
cells which have remained vigorous enough to develop alone. The addition
of a little tartaric acid to the saccharine solution—say, from 1/1000 to
2/1000 part by weight—often facilitates the destruction of certain germs
of impurity. _Mycoderma aceti_ and _mycoderma vini_ do not find suitable
life-conditions in the sweetened water; they soon disappear if
cultivated alternately in sweetened water and wort.

In the place of flasks we may make use simply of shallow basins, covered
with sheets of glass, such as we have already had occasion to describe,
for cultivating yeast in wort after it has been for a longer or shorter
time in the sweetened water. The success of these methods of
purification is mainly due to the fact that wort is highly aerated, and
experience shows that the principal disease-ferments of beer are as much
checked in their development by the presence of air as they are favoured
by its absence, the inverse of which holds good in the case of alcoholic
ferments. So true is this that, working with commercial yeast, which is
invariably impure, it would be impossible in our opinion to make beer in
closed vessels; and, indeed, as a matter of fact, one has never
succeeded in doing this, although the attempt has often been made. To do
so requires, much more than in methods actually in use, the employment
of pure yeast.

There is, therefore, this advantage in cultivating yeasts in shallow
basins, that the multiplication of the alcoholic ferments is promoted,
and that of most of the disease-ferments is checked. There is an
exception, indeed, in the case of mycodermata; but of all
disease-ferments these are the most easily got rid of, by repeating our
growths before they make their appearance. Notwithstanding this, our
two-necked flasks, which also contain much air at first, are to be
preferred to the shallow basins, inasmuch as they are a perfect
safeguard against the germs floating in the surrounding air, as well as
those of the ferment _saccharomyces pastorianus_.

Another method is suggested to us by the curious results of which we
have already spoken, obtained by sowing yeasts in a wort rendered acid
and alcoholic by the addition of bi-tartrate of potash and alcohol.
Experience proves that many disease-ferments find great difficulty in
withstanding a succession of growths in wort to which 1-½ per cent. of
tartaric acid and from 2 to 3 per cent. of alcohol have been added. Such
a mixture, however, is equally well adapted to the requirements of
_saccharomyces pastorianus_, and we must always assure ourselves that
this organism has not taken the place of the yeast we are endeavouring
to purify. Growths at a very low temperature are of great help in
enabling us to get rid of all ferments that are foreign to “low” yeast,
and should be resorted to in all cases where this yeast is to be
purified.

Another method of purification, which is perhaps quicker, although
inferior in other respects, consists in the employment of carbolic
acid—that is to say, in purifying our yeast by successive growths, we
may add to every 100 c.c. (3-½ fluid ounces) of wort that we employ from
ten to twelve drops of phenol water, containing 10 per cent. of the
acid. The action of the phenol, which at first is invariably combined
with that of the oxygen of the air, tends to destroy the vitality of
many of the cells sown, involving to some extent also the yeast which we
are interested in preserving. But amongst the number of cells that are
affected those which are less abundant, that is to say, those which are
present as impurities, are paralyzed relatively in much greater
proportion. If the acid does not destroy them it greatly checks their
development, and the cells of yeast, which multiply continuously in vast
numbers (for the fermentation goes on in spite of the phenol, if this is
added in small quantity), gradually choke the foreign germs in a
succession of growths.

By these different means, which are employed separately or combined with
one another, we generally manage to obtain the yeast which we wish to
purify in a very pure state. We need scarcely add that it is always
well, in the case of our purifications, to begin with specimens which
are already as pure as it is possible to obtain them. In making our
choice the microscope is our best guide, but it is not a sufficient one.
We should be strangely deceived if we believed in the purity of a yeast
for the sole reason that when examined under the microscope it appeared
to contain nothing of a foreign nature. The best means of assuring
ourselves of the purity of a yeast consists in making some beer in one
of our two-necked flasks, and leaving this flask, after fermentation, in
an oven at a temperature of 20° or 25° C. (68° to 77° F.). If the beer,
in the course of a few weeks, does not thicken, or become covered with
efflorescence, if its deposit is microscopically pure, if, in short, it
only tastes flat, we may have every confidence in the purity of the
yeast which produced it. After we have purified a yeast we are,
unfortunately, never sure that it has not undergone some change in the
course of the manipulations to which it has been subjected in
purification. It is indispensable, therefore, that we should test it,
and see if the flavour of the beer produced by it is really the one that
we want—viz., that of the beer from which we took the yeast that we
submitted to purification.

In the course of a series of practical experiments that we were carrying
out in the large brewery of Tourtel, at Tantonville, in 1875, in
connection with the new process of brewing, which will be explained in
Chap. VII., the following circumstance occurred. We had purified some of
the yeast of the brewery, by means of a succession of growths and adding
a few drops of phenol, and had obtained a yeast of irreproachable
purity. It happened that this yeast, which was repeatedly cultivated in
the brewery during the summer of 1875, from six to ten hectolitres (130
to 220 gallons) of wort being used on each occasion, always produced a
beer that had a yeast-bitten flavour and defective clarifying powers,
notwithstanding that it possessed remarkable keeping properties, which
it owed to the pureness of the ferment employed. As a matter of fact,
the beer suffered no injury from journeys of more than 300 miles, by
slow trains, in ordinary casks, containing from 50 to 100 litres (10 to
20 gallons), during the great heats of June and July, or from being
subsequently stored for two months in a cellar, the temperature of which
rose during that time from 12° to 18° C. (54° to 65° F.). The
temperature of fermentation had been 13° C. (55° F.). Beer from the same
brewery, made with the same wort by the ordinary process, did not remain
sound for three weeks in this same cellar.

To what may we attribute the peculiarity of the beer as just described?
It is probable that during our processes of purification some ferment
had taken the place of the principal yeast. Commercial yeasts, even
those with which the brewer is thoroughly satisfied, generally contain
various ferments, which are maintained in their relative proportions, or
very nearly so, by the uniform conditions under which work is carried on
in a brewery; but these proportions, it is obvious, might be very
seriously affected by any radical change in the conditions of growth.

Footnote 82:

  Extract from a Note which I inserted in 1862 in the _Bulletin de la
  Société chimique_ of Paris.

Footnote 83:

  Schützenberger, in his work on “Fermentation,” following Dr. de
  Vauréal. Paris, 1875, p. 278. [See pp. 61, 62 English version in
  International Scientific Series (H. S. King & Co., London, 1876). This
  appears to be the only reference to this subject in the English
  copy.—D. C. R.]

Footnote 84:

  The principal result of Dr. Rees’ labours consists in the discovery of
  a sporulation peculiar to yeast cells, that is to say, to a formation
  in the interior of these cells, and under particular conditions—such
  as when the growth occurs on slices of cooked potatoes, carrots,
  &c.—of two, three, or four smaller cells, which, when placed in
  fermentable liquids, act like the germinating spores of ferments. The
  mother-cell may be regarded as an _ascus_, and the daughter-cells as
  _ascospores_, and so the genus _saccharomyces_ may be classified among
  the group of fungi termed _ascomycetes_. These facts have been
  frequently confirmed, notably by Dr. Engel, professor of the Faculty
  of Medicine, at Nancy. Previously to Dr. Rees’ discovery, M. de Seynes
  (_Comptes rendus_, t. lxvii., 1868) had described an endogenous
  formation of spores in _mycoderma vini_, particularly in the elongated
  cells, followed by the rupture of the mother-cell, and subsequent
  absorption of cell-walls and other contents after the issue of the
  endospores, which we have just termed _ascospores_. We ourselves had
  also previously called attention to those refractive corpuscles which
  appear amongst vibrios as probably being reproductive corpuscles, and
  we had likewise witnessed the reabsorption of the parts surrounding
  them. The plate on page 228 of our “Studies on the Silkworm Disease”
  represents the phenomena in question.

Footnote 85:

  See _Comptes rendus de l’Académie des Sciences_, vol. lviii. p. 144.

Footnote 86:

  The plates referred to in this paragraph were exhibited at a meeting
  of the Academy of Sciences, November 18, 1872, and commented upon by
  the perpetual secretary, M. Dumas.

Footnote 87:

  For these observations, we employed small glass cells, which we made
  out of some St. Gobain glass by punching holes through it, and then
  cementing on one side one of the little glasses used for covering
  objects in microscopical examinations. In this manner we made small
  troughs, in which we placed some wort that had been boiled, and a drop
  of the water in which grapes had been washed. To prevent evaporation
  we covered the cells with a sheet of glass. We examined the liquid in
  these cells by inclining our microscope to the angle required.[88]

  [Illustration: Fig. 29.]

  We also made use of cells similar to those employed by MM. Van Tieghem
  and Lemonnier[89] in their researches on _mucorines_ (Fig. 30).

  [Illustration: Fig. 30.]

  An apparatus similar to that employed by M. Duclaux in 1853[90] would
  do equally well. We should be able to work with even greater facility
  if we employed bulbs like some which we ordered in Germany, some
  twelve years ago, of the well-known glass-blower, Geissler. We have
  heard that these bulbs now sold by that maker are much used by German
  microscopists. They consist of a tube blown out into a flat bulb, the
  sides of which, in the centre, come sufficiently close together to
  enclose but a very thin layer of liquid, and to admit of microscopical
  examination. We may fill these tube-bulbs completely with liquid, to
  the exclusion of air or we may surround the central drop with air.

  [Illustration: Fig. 31.]

Footnote 88:

  In our essay on acetic fermentation, published in 1864, we have
  already described this apparatus, which we employed to follow the
  multiplication of the jointed filaments of _mycoderma aceti_. See
  PASTEUR, _Etudes sur le vinaigre_, p. 64, Paris, 1868.

Footnote 89:

  VAN TIEGHEM and LEMONNIER, _Annales des Sciences naturelles_, 5th
  series, _Botanique_, t. xvii. 1873.

Footnote 90:

  DUCLAUX, _Comptes rendus des séances de l’Académie des Sciences_, t.
  lvi. p. 1225.

Footnote 91:

  In experiments of this kind there is always a slight increase in the
  volume of air in the jar. This increase may be very perceptible even
  when the experiment made with fresh grapes, in August, for instance,
  causes no fermentation due to the action of yeast. After the oxygen of
  the air has been absorbed and replaced by carbonic acid gas, either by
  direct oxidation or by the action of moulds, the grapes, although
  crushed, act like fruits plunged into carbonic acid gas[92], and this
  effect is even more marked in the case of imperfectly crushed grapes.
  The reason is, that the crushing is never so perfect as to injure all
  the cells of the parenchyma. We may easily convince ourselves that the
  experiment on the liberation of carbonic acid gas and the formation of
  alcohol by grapes and fruits in general when plunged into carbonic
  acid succeeds very well in the case of fragments of fruits or grapes,
  and succeeds better the less the parts are crushed.

Footnote 92:

  See paragraph: Fermentation in saccharine fruits immersed in carbonic
  acid gas, Chap. vi, § 2, p. 266.

Footnote 93:

  Dr. Rees has given the name _saccharomyces ellipsoideus_ to the
  ferment of wine represented in Plates VIII. to XI. of our “Studies on
  Wine,” which we have termed the ordinary ferment of wine, from its
  being the most abundant of the ferments found at the end of the
  fermentation that produces the wine.

Footnote 94:

  The alcoholic ferments in general, subjected to these weakening
  influences, have not all the same power of resistance. That one which
  seems to possess this power in the highest degree is the
  _saccharomyces pastorianus_, which ferment we had in view in writing
  the above.

Footnote 95:

  The term _exhaustion_ (_épuisement_), which we have just used, was,
  perhaps, not altogether felicitously chosen. No doubt we exhaust the
  cells of yeast when we sow an imponderable weight of them in a large
  quantity of sweetened water; it might, however, be better to say that
  in such a case we adopt a particular method of preserving the vitality
  of the cells, without suffering them to die of exhaustion, or to
  multiply by budding. We may remark that the yeast, in this case,
  exists in a state of latent life, which resembles that of cells on the
  surface of fruit. The cells on the surface of fruits, bunches, or
  barks, can no more find around them sufficient aliment for their
  propagation than can our yeast-cells in a great excess of sweetened
  water. We would not, however, say of the spores on the surface of
  fruits, or their woods, that they are in a state of exhaustion; the
  term would be misapplied.

Footnote 96:

  See foot note p. 79.—D. C. R.

Footnote 97:

  M. Béchamp (_Comptes rendus_, November 18th, 1872) asserts that _the
  air has no direct influence on the production of ferment or on the
  process of alcoholic fermentation_. That experienced chemist deduces
  this erroneous assertion from experiments on sweetened water, to which
  bunches of grapes, petals of corn-poppies and petals of _robinia
  pseudo-acacia_ had been added. As may be seen in our “Studies on Wine”
  (p. 7, 1st edition, 1866), these experiments conducted by M. Béchamp
  in 1872 were merely a reproduction of those made long before with vine
  leaves, petals of elder-flowers, leaves of sorrel, &c., by the Marquis
  de Bullion, Fabroni, and other experimentalists. M. Béchamp has
  modified his later experiments by not adding the bunches of grapes,
  leaves, &c., to the sweetened water before having introduced carbonic
  acid gas into the liquid. Fermentation having still taken place in
  spite of this change, M. Béchamp wrongly concluded that _air has no
  direct influence on the production of yeast on an alcoholic
  fermentation_. The introduction of the carbonic acid gas could not
  remove all the air imparted to the sweetened water by the objects
  placed in it, and it was this air which remained adhering to these
  objects that permitted the production of fermentation. We may avail
  ourselves of the opportunity here presented to add that, in this same
  Note of November, 1872, M. Béchamp commences by making various
  assertions concerning the forms assumed by cells of the alcoholic
  ferment of the grape when in process of fermentation. This question
  was discussed by us ten years before, and our conclusions supported by
  sketches, in a Note which appeared in the _Bulletin de la Société
  chimique de Paris_, for 1862.

Footnote 98:

  The germs of ferments are less widely diffused than M. de Bary
  supposes, as may be seen from our observations in Chap. III. See, too,
  our Memoir of 1862, _Sur les Générations dites Spontanées_, p. 49. It
  is only in a laboratory devoted to researches on fermentation, or
  places such as vaults, cellars, and breweries, that the air holds
  appreciably in suspension cells of ferments, ready to germinate in
  saccharine media. If we except these particular circumstances, ferment
  is not very largely diffused, save on the surface of fruits and the
  wood of the trees which bear them, and perhaps, also, on some other
  plants. The particles of dust held in suspension in any atmosphere
  whatever rarely produce fermentation in pure must even when we take
  all possible precautions, so that the action be not overlooked; for
  true fermentation may be hidden by fungoid growths, when there is much
  air and but a small quantity of saccharine liquid present.

Footnote 99:

  In these experiments the apiculated ferment appeared sometimes, but
  much less frequently than _saccharomyces pastorianus_. We also met
  with the ellipsoidal ferment. We should probably have a greater
  variety of ferments if our experiments could be conducted in the open
  air, but insects and particles of dust of all kinds brought by the
  wind render experiments under such conditions difficult and
  untrustworthy. In a laboratory we have not these difficulties to
  contend against, but, unfortunately, the operations ordinarily carried
  on there cause the results of our experiments to be of a less general
  character than they would be if obtained in free contact with country
  air.

Footnote 100:

  [A rather serious clerical error appears to have here crept into the
  original, for on referring to Plate I. and the letterpress descriptive
  of No. 7 (p. 5), we find it applies to a very formidable species of
  diseased ferment, whereas the author is here speaking of an amorphous
  deposit, harmless in character, and more or less associated with all
  yeasts. Doubtless No. 7 should stand No. 6, see p. 6.—D. C. R.]

Footnote 101:

  [We would here call the reader’s attention to the following extract
  from Dr. Graham’s appreciative review of this work in “Nature,”
  January 11th, 1877. He says: “M. Pasteur seems to be in error in
  stating (p. 190, Fr. ed.) that the bottom yeast may be distinguished
  by being less spherical than top yeast. It is true that in London and
  Edinburgh yeast, the cells will be found usually round; hard water,
  however, such as that at Burton, or artificially made so, yields yeast
  in which the cells are distinctly ovoid in appearance, resembling very
  closely Bavarian bottom yeast.”—D. C. R.]

Footnote 102:

  [43° F. to 46° F. or 59° F. to 68° F.]

Footnote 103:

  [On this point again Dr. Graham expresses some dissent (“Nature,” loc.
  cit.): “Here surely M. Pasteur must be thinking rather of the inferior
  products of the surface fermentation in France and Germany, than of
  those of England and Scotland.”—D. C. R.]

Footnote 104:

  [28·4 c.c. = 1 fl. oz. approximately.]

Footnote 105:

  [M. Pasteur has evidently employed the word “caseous” to express the
  curdy nature of the ferment he is describing, its plasticity and other
  peculiarities of physical character; but we are, nevertheless, tempted
  to suggest that he may have had in mind also the peculiar “cheesy”
  odour given off by these very yeasts, which he refers to in the text
  as containing a considerable intermixture of “caseous ferment.”—F. F.]

Footnote 106:

  The caseous ferment, however, must not be exposed to heat, under the
  afore-mentioned conditions, when it is too young. At the commencement
  of its development, for instance, within a few days of having been
  sown. In such case, it would be in danger of perishing, probably in
  consequence of the tenderness of its tissues. At the end of a
  fermentation, and even several months afterwards, it might be safely
  heated to 50° C. (122° Fahr.) without any harm to it. “Low” yeast also
  can withstand a temperature of 50° C. in the medium in question.

Footnote 107:

  Although we believe that the aërobian ferment of a particular yeast
  may be produced by a kind of transformation of the cells of the
  latter, yet we admit that this question is open to some doubt. The
  facts which we unexpectedly discovered in connection with the
  _caseous_ ferment should make us extremely careful, and disposed to
  inquire whether aërobian ferments do not originally, in a state of
  intermixture, form part of the ferments from which they spring. One
  reason which might incline us to believe this, is the fact that a
  ferment sometimes perishes without the appearance of aërobian ferment
  on the surface. There is nothing very natural indeed in the hypothesis
  that we advance, which sets aside the supposed intermixture; but, on
  the other hand, if the aërobian ferment is a particular ferment,
  simply intermixed with some other variety and developed by change of
  conditions, how are we to account for its great resemblance in
  appearance and mode of budding to the ferment on the surface of which
  it appears? This resemblance, however, might be accounted for very
  naturally if the two ferments were originally related.

Footnote 108:

  We insist on this fact, that Fig. 50 represents the forms on revival
  of the aërobian ferment of _saccharomyces pastorianus, when this has
  grown in a mineral medium_. When produced on the surface of fermented
  wort, the aërobian ferment of which we are speaking presents no
  peculiarity, nor is there any irregularity in its forms or in its
  development, and when we proceed to cultivate it in a natural
  saccharine medium, or in wort, it does not produce any forms of
  _dematium_, as in the preceding case; but the reason of this is that,
  in consequence of the nature of the first medium, which is better
  adapted to its nutrition, it assumes at once, in the second medium,
  the forms of deposit-yeast in the course of ordinary germination.

Footnote 109:

  “La cassure de la levûre.”

Footnote 110:

  We have reason to believe that the ratio of the proportions of these
  ferments depends greatly on the climatic conditions preceding the
  period of vintage, on the state of dryness or humidity, as well as the
  temperature at the time of gathering the grapes, and also on the
  nature of the vines.




                              CHAPTER VI.
               THE PHYSIOLOGICAL THEORY OF FERMENTATION.


        § I.—On the Relations existing between Oxygen and Yeast.


The object of all science is a continuous reduction of the number of
unexplained phenomena. It is observed, for instance, that fleshy fruits
are not liable to fermentation so long as their epidermis remains
uninjured. On the other hand, they ferment very readily when they are
piled up in heaps, more or less open, and immersed in their saccharine
juice. The mass becomes heated and swells; carbonic acid gas is
disengaged, and the sugar disappears and is replaced by alcohol. Now, as
to the question of the origin of these spontaneous phenomena, so
remarkable in character as well as usefulness for man’s service, modern
knowledge has taught us that fermentation is the consequence of a
development of vegetable cells, the germs of which do not exist in the
saccharine juices within fruits; that many varieties of these cellular
plants exist, each giving rise to its own particular fermentation. The
principal products of these various fermentations, although resembling
each other in their nature, differ in their relative proportions and in
the accessory substances that accompany them, a fact which alone is
sufficient to account for wide differences in the quality and commercial
value of alcoholic beverages.

Now that the discovery of ferments and their living nature, and our
knowledge of their origin, may have solved the mystery of the
spontaneous appearance of fermentations in natural saccharine juices, we
may ask whether we must still regard the reactions that occur in these
fermentations as phenomena inexplicable by the ordinary laws of
chemistry. We can readily see that fermentations occupy a special place
in the series of chemical and biological phenomena. What gives to
fermentations certain exceptional characters, of which we are only now
beginning to suspect the causes, is the mode of life in the minute
plants designated under the generic name of _ferments_, a mode of life
which is essentially different from that in other vegetables, and from
which result phenomena equally exceptional throughout the whole range of
the chemistry of living beings.

The least reflection will suffice to convince us that the alcoholic
ferments must possess the faculty of vegetating and performing their
functions out of contact with air. Let us consider, for instance, the
method of vintage practised in the Jura. The bunches are laid at the
foot of the vine in a large tub, and the grapes there stripped from
them. When the grapes, some of which are uninjured, others bruised, and
all moistened by the juice issuing from the latter, fill the tub—where
they form what is commonly called the _vintage_—they are conveyed in
barrels to large vessels fixed in cellars of a considerable depth. These
vessels are not filled to more than three-quarters of their capacity.
Fermentation soon takes place in them, and the carbonic acid gas finds
escape through the bunghole, the diameter of which, in the case of the
largest vessels, is not more than ten or twelve centimetres (about four
inches). The wine is not drawn off before the end of two or three
months. In this way it seems highly probable that the yeast which
produces the wine under such conditions must have developed, to a great
extent at least, out of contact with oxygen. No doubt oxygen is not
entirely absent from the first; nay, its limited presence is even a
necessity to the manifestation of the phenomena which follow. The grapes
are stripped from the bunch in contact with air, and the must which
drops from the wounded fruit takes a little of this gas into solution.
This small quantity of air so introduced into the must, at the
commencement of operations, plays a most indispensable part, it being
from the presence of this that the spores of ferments which are spread
over the surface of the grapes and the woody part of the bunches derive
the power of starting their vital phenomena.[111] This air, however,
especially when the grapes have been stripped from the bunches, is in
such small proportion, and that which is in contact with the liquid mass
is so promptly expelled by the carbonic acid gas, which is evolved as
soon as a little yeast has formed, that it will readily be admitted that
most of the yeast is produced apart from the influence of oxygen,
whether free or in solution. We shall revert to this fact, which is of
great importance. At present we are only concerned in pointing out that,
from the mere knowledge of the practices of certain localities, we are
induced to believe that the cells of yeast, after they have developed
from their spores, continue to live and multiply without the
intervention of oxygen, and that the alcoholic ferments have a mode of
life which is probably quite exceptional, since it is not generally met
with in other species, vegetable or animal.

Another equally exceptional characteristic of yeast and fermentation in
general consists in the small proportion which the yeast that forms
bears to the sugar that decomposes. In all other known beings the weight
of nutritive matter assimilated corresponds with the weight of food used
up, any difference that may exist being comparatively small. The life of
yeast is entirely different. For a certain weight of yeast formed, we
may have ten times, twenty times, a hundred times as much sugar, or even
more decomposed, as we shall experimentally prove by-and-bye; that is to
say, that whilst the proportion varies in a precise manner, according to
conditions which we shall have occasion to specify, it is also greatly
out of proportion to the weight of the yeast. We repeat, the life of no
other being, under its normal physiological conditions, can show
anything similar. The alcoholic ferments, therefore, present themselves
to us as plants which possess at least two singular properties: they can
live without air, that is, without oxygen, and they can cause
decomposition to an amount which, though variable, yet, as estimated by
weight of product formed, is out of all proportion to the weight of
their own substance. These are facts of so great importance, and so
intimately connected with the theory of fermentation, that it is
indispensable to endeavour to establish them experimentally, with all
the exactness of which they will admit.

The question before us is whether yeast is in reality an anaërobian
plant, and what quantities of sugar it may cause to ferment, under the
various conditions under which we cause it to act.

The following experiments were undertaken to solve this double
problem:—We took a double-necked flask, of three litres (five pints)
capacity, one of the tubes being curved and forming an escape for the
gas; the other one, on the right hand side (Fig. 59), being furnished
with a glass tap. We filled this flask with pure yeast-water, sweetened
with 5 per cent. of sugar candy, the flask being so full that there was
not the least trace of air remaining above the tap or in the escape
tube; this artificial wort had, however, been itself aerated. The curved
tube was plunged in a porcelain vessel full of mercury, resting on a
firm support. In the small cylindrical funnel above the tap, the
capacity of which was from 10 c.c. to 15 c.c. (about half a fluid ounce)
we caused to ferment, at a temperature of 20° or 25° C. (about 75° F.),
five or six cubic centimetres of the saccharine liquid, by means of a
trace of yeast, which multiplied rapidly, causing fermentation, and
forming a slight deposit of yeast at the bottom of the funnel above the
tap. We then opened the tap, and some of the liquid in the funnel
entered the flask, carrying with it the small deposit of yeast, which
was sufficient to impregnate the saccharine liquid contained in the
flask. In this manner it is possible to introduce as small a quantity of
yeast as we wish, a quantity the weight of which, we may say, is hardly
appreciable. The yeast sown multiplies rapidly and produces
fermentation, the carbonic acid gas from which is expelled into the
mercury. In less than twelve days all the sugar had disappeared, and the
fermentation had finished. There was a sensible deposit of yeast
adhering to the sides of the flask; collected and dried it weighed 2·25
grammes (34 grains). It is evident that in this experiment the total
amount of yeast formed, if it required oxygen to enable it to live,
could not have absorbed, at most, more than the volume which was
originally held in solution in the saccharine liquid, when that was
exposed to the air before being introduced into the flask.

[Illustration: Fig. 59.]

[Illustration: Fig. 60.]

Some exact experiments conducted by M. Raulin in our laboratory have
established the fact that saccharine worts, like water, soon become
saturated when shaken briskly with an excess of air, and also that they
always take into solution a little less air than saturated pure water
contains under the same conditions of temperature and pressure. At a
temperature of 25° C. (77° F.) therefore, if we adopt the coefficient of
the solubility of oxygen in water given in Bunsen’s tables, we find that
1 litre (1-3/4 pints) of water saturated with air contains 5·5 c.c. (0·3
cubic inch) of oxygen. The three litres of yeast-water in the flask,
supposing it to have been saturated, contained less than 16·5 c.c. (1
cubic inch) of oxygen, or, in weight, less than 23 milligrammes (0·35
grains). This was the maximum amount of oxygen, supposing the greatest
possible quantity to have been absorbed, that was required by the yeast
formed in the fermentation of 150 grammes (4·8 Troy ounces) of sugar. We
shall better understand the significance of this result later on. Let us
repeat the foregoing experiment, but under altered conditions. Let us
fill, as before, our flask with sweetened yeast-water, but let this be
first boiled, so as to expel all the air it contains. To effect this we
arrange our apparatus as represented in the accompanying sketch (Fig.
60). We place our flask, A, on a tripod above a gas flame, and in place
of the vessel of mercury substitute a porcelain dish, under which we can
put a gas flame, and which contains some fermentable, saccharine liquid,
similar to that with which the flask is filled. We boil the liquid in
the flask and that in the basin simultaneously, and then let them cool
down together, so that as the liquid in the flask cools some of the
liquid is sucked from the basin into the flask. From a trial experiment
which we conducted, determining the quantity of oxygen that remained in
solution in the liquid after cooling, according to M. Schützenberger’s
valuable method, by means of hydrosulphite of soda,[112] we found that
the three litres in the flask, treated as we have described, contained
less than one milligramme (0·015 grain) of oxygen. At the same time we
conducted another experiment, by way of comparison (Fig. 61). We took a
flask, B, of larger capacity than the former one, which we filled about
half with the same volume as before of a saccharine liquid of
identically the same composition. This liquid had been previously freed
from alterative germs by boiling. In the funnel surmounting A, we put a
few cubic centimetres of saccharine liquid in a state of fermentation,
and when this small quantity of liquid was in full fermentation, and the
yeast in it was young and vigorous, we opened the tap, closing it again
immediately, so that a little of the liquid and yeast still remained in
the funnel. By this means we caused the liquid in A to ferment. We also
impregnated the liquid in B with some yeast taken from the funnel of A.
We then replaced the porcelain dish in which the curved escape tube of A
had been plunged, by a vessel filled with mercury.

[Illustration: Fig. 61.]

The following is a description of two of these comparative fermentations
and the results they gave.

The fermentable liquid was composed of yeast-water sweetened with 5 per
cent. of sugar-candy; the ferment employed was _saccharomyces
pastorianus_.

The impregnation took place on January 20th. The flasks were placed in
an oven at 25° C. (77° F.).


_Flask A, without air._


    January 21st.—Fermentation commenced; a little frothy liquid issued
    from the escape-tube and covered the mercury.

    The following days, fermentation was active. Examining the yeast
    mixed with the froth that was expelled into the mercury by the
    evolution of carbonic acid gas, we found that it was very fine,
    young, and actively budding.

    February 3rd.—Fermentation still continued, showing itself by a
    number of little bubbles rising from the bottom of the liquid, which
    had settled bright. The yeast was at the bottom in the form of a
    deposit.

    February 7th.—Fermentation still continued, but very languidly.

    February 9th.—A very languid fermentation still went on, discernible
    in little bubbles rising from the bottom of the flask.


_Flask B, with air._


    January 21st.—A sensible development of yeast.

    The following days, fermentation was active, and there was an
    abundant froth on the surface of the liquid.

    February 1st.—All symptoms of fermentation had ceased.

As the fermentation in A would have continued for a long time, being so
very languid, and as that in B had been finished for several days, we
brought to a close our two experiments on February 9th. To do this we
poured off the liquids in A and B, collecting the yeasts on tared
filters. Filtration was an easy matter, more especially in the case of
A. Examining the yeasts under the microscope, immediately after
decantation, we found that both of them remained very pure. The yeast in
A was in little clusters, the globules of which were collected together,
and appeared by their well defined borders to be ready for an easy
revival in contact with air.

As might have been expected, the liquid in the flask B did not contain
the least trace of sugar; that in the flask A still contained some, as
was evident from the non-completion of fermentation, but not more than
4·6 grammes (71 grains). Now, as each flask originally contained 3
litres of liquid, holding in solution 5 per cent. of sugar, it follows
that 150 grammes (2,310 grains) of sugar had fermented in the flask B,
and 145·4 grammes (2,239·2 grains) in the flask A. The weights of yeast
after drying at 100° C. (212° F.) were—

     For the flask B, with air      1·970 grammes (30·4 grains).
     For the flask A, without air   1·368 grammes.[113]

The proportions were 1 of yeast to 76 of fermented sugar in the first
case, and 1 of yeast to 89 of fermented sugar in the second.

From these facts the following consequences may be deduced:

1. The fermentable liquid (flask B), which since it had been in contact
with air, necessarily held air in solution, although not to the point of
saturation, inasmuch as it had been once boiled to free it from all
foreign germs, furnished a weight of yeast sensibly greater than that
yielded by the liquid which contained no air at all (flask A), or, at
least, which could only have contained an exceedingly minute quantity.

2. This same slightly aerated fermentable liquid fermented much more
rapidly than the other. In eight or ten days it contained no more sugar;
while the other, after twenty days, still contained an appreciable
quantity.

Is this last fact to be explained by the greater quantity of yeast
formed in B? By no means. At first, when the air has access to the
liquid, much yeast is formed and little sugar disappears, as we shall
prove immediately; nevertheless the yeast formed in contact with the air
is more active than the other. Fermentation is correlative, first to the
development of the globules, and then to the continued life of those
globules once formed. The more oxygen these last globules have at their
disposal during their formation, the more vigorous, transparent, and
turgescent, and, as a consequence of this last quality, the more active
they are in decomposing sugar. We shall revert hereafter to these facts.

3. In the airless flask the proportion of yeast to sugar was 1/89; it
was only 1/76 in the flask which had air at first.

The proportion that the weight of yeast formed bears to the weight of
the sugar is, therefore, variable, and this variation depends, to a
certain extent, upon the presence of air and the possibility of oxygen
being absorbed by the yeast. We shall presently show that yeast
possesses the power of absorbing that gas and emitting carbonic acid,
like ordinary fungi, that even oxygen may be reckoned amongst the number
of food-stuffs that may be assimilated by this plant, and that this
fixation of oxygen in yeast, as well as the oxidations resulting from
it, have the most marked effect on the life of yeast, on the
multiplication of its cells, and on their activity as ferments acting
upon sugar, whether immediately or afterwards, apart from supplies of
oxygen or air.

In the preceding experiment, conducted without the presence of air,
there is one circumstance particularly worthy of notice. This experiment
succeeds, that is to say, the yeast sown in the medium deprived of
oxygen develops, only when this yeast is in a state of great vigour. We
have already explained the meaning of this last expression. But we wish
now to call attention to a very evident fact in connection with this
point. We impregnate a fermentable liquid; yeast develops and
fermentation appears. This lasts for several days and then ceases. Let
us suppose that, from the day when fermentation first appears in the
production of a minute froth, which gradually increases till it whitens
the surface of the liquid, we take, every twenty-four hours, or at
longer intervals, a trace of the yeast deposited on the bottom of the
vessel and use it for starting fresh fermentations. Conducting these
fermentations all under precisely the same conditions of temperature,
character, and volume of liquid, let us continue this for a prolonged
time, even after the original fermentation is finished. We shall have no
difficulty in seeing that the first signs of action in each of our
series of second fermentations appear always later and later in
proportion to the length of time that has elapsed from the commencement
of the original fermentation. In other words, the time necessary for the
development of the germs and the production of that amount of yeast
sufficient to cause the first appearance of fermentation varies with the
state of the impregnating cells, and is longer in proportion as the
cells are further removed from the period of their formation. It is
essential, in experiments of this kind, that the quantities of yeast
successively taken should be as nearly as possible equal in weight or
volume, since, _ceteris paribus_, fermentations manifest themselves more
quickly the larger the quantity of yeast employed in impregnation.

If we compare under the microscope the appearance and character of the
successive quantities of yeast taken, we shall see plainly that the
structure of the cells undergoes a progressive change. The first sample
which we take, quite at the beginning of the original fermentation,
generally gives us cells rather larger than those later on, and
possessing a remarkable tenderness. Their walls are extremely thin, the
consistency and softness of their protoplasm is akin to fluidity, and
their granular contents appear in the form of scarcely visible spots.
The borders of the cells soon become more marked, a proof that their
walls undergo a thickening; their protoplasm also becomes denser, and
the granulations more distinct. Cells of the same organ, in the states
of infancy and old age, should not differ more than the cells of which
we are speaking, taken in their extreme states. The progressive changes
in the cells, after they have acquired their normal form and volume,
clearly demonstrate the existence of a chemical work of a remarkable
intensity, during which their weight increases, although in volume they
undergo no sensible change, a fact that we have often characterized as
“the continued life of cells already formed.” We may call this work a
process of maturation on the part of the cells, almost the same that we
see going on in the case of adult beings in general, which continue to
live for a long time, even after they have become incapable of
reproduction, and long after their volume has become permanently fixed.

This being so it is evident, we repeat, that, to multiply in a
fermentable medium, quite out of contact with oxygen, the cells of yeast
must be extremely young, full of life and health, and still under the
influence of the vital activity which they owe to the free oxygen which
has served to form them, and which they have perhaps stored up for a
time. When older, they reproduce themselves with much difficulty when
deprived of air, and gradually become more languid; and if they do
multiply, it is in strange and monstrous forms. A little older still,
they remain absolutely inert in a medium deprived of free oxygen. This
is not because they are dead; for in general they may be revived in a
marvellous manner in the same liquid if it has been first aerated before
they are sown. It would not surprise us to learn that at this point
certain preconceived ideas suggest themselves to the mind of an
attentive reader on the subject of the causes that may serve to account
for such strange phenomena in the life of these beings which our
ignorance hides under the expressions of _youth_ and _age_; this,
however, is a subject that we cannot pause to consider here.

At this point we must observe—for it is a matter of great
importance—that, in the operations of the brewer there is always a time
when the yeasts are in this state of vigorous youth of which we have
been speaking, acquired under the influence of free oxygen, since all
the worts and all the yeasts of commerce are necessarily manipulated in
contact with air, and so impregnated more or less with oxygen. The yeast
immediately seizes upon this gas and acquires a state of freshness and
activity, which permits it to live afterwards out of contact with air,
and to act as a ferment. Thus, in ordinary brewery practice, we find the
yeast already formed in abundance even before the earliest external
signs of fermentation have made their appearance. In this first phase of
its existence, yeast lives chiefly like an ordinary fungus.

From the same circumstances it is clear that the brewer’s fermentations
may, speaking quite strictly, last for an indefinite time, in
consequence of the unceasing supply of fresh wort, and from the fact,
moreover, that the exterior air is constantly being introduced during
the work, and that the air contained in the fresh worts keeps up the
vital activity of the yeast, as the act of breathing keeps up the vigour
and life of cells in all living beings. If the air could not renew
itself in any way, the vital activity which the cells originally
received, under its influence, would become more and more exhausted, and
the fermentation eventually come to an end.

We may recount one of the results obtained in other experiments similar
to the last, in which, however, we employed yeast which was still older
than that used for our experiment with flask A (Fig. 60), and moreover
took still greater precautions to prevent the presence of air. Instead
of leaving the flask, as well as the dish, to cool slowly, after having
expelled all air by boiling, we permitted the liquid in the dish to
continue boiling whilst the flask was being cooled by artificial means;
the end of the escape tube was then taken out of the still boiling dish
and plunged into the mercury trough. In impregnating the liquid, instead
of employing the contents of the small cylindrical funnel whilst still
in a state of fermentation, we waited until this was finished. Under
these conditions, fermentation was still going on in our flask, after a
lapse of three months. We stopped it and found that 0·255 gramme (3·9
grains) of yeast had been formed, and that 45 grammes (693 grains) of
sugar had fermented, the ratio between the weights of yeast and sugar
being thus 0·255/45 = 1/176. In this experiment the yeast developed with
much difficulty, by reason of the conditions to which it had been
subjected. In appearance the cells varied much, some were to be found
large, elongated, and of tubular aspect, some seemed very old and were
extremely granular, whilst others were more transparent. All of them
might be considered abnormal cells.

In such experiments we encounter another difficulty. If the yeast sown
in the non-aerated fermentable liquid is in the least degree impure,
especially if we use sweetened yeast-water, we may be sure that
alcoholic fermentation will soon cease, if, indeed, it ever commences,
and that accessory fermentations will go on. The vibrios of butyric
fermentation, for instance, will propagate with remarkable facility
under these circumstances. Clearly then, the purity of the yeast at the
moment of impregnation, and the purity of the liquid in the funnel, are
conditions indispensable to success.

To secure the latter of these conditions, we close the funnel, as shown
in Fig. 60, by means of a cork pierced with two holes, through one of
which a short tube passes, to which a short length of india-rubber
tubing provided with a glass stopper is attached; through the other hole
a thin curved tube is passed. Thus fitted, the funnel can answer the
same purposes as our double-necked flasks. A few cubic centimetres of
sweetened yeast-water are then put in it and boiled, so that the steam
may destroy any germs adhering to the sides. When cold the liquid is
impregnated by means of a trace of pure yeast, introduced through the
glass-stoppered tube. If these precautions are neglected it is scarcely
possible to secure a successful fermentation in our flasks, because the
yeast sown is immediately held in check by a development of anaërobian
vibrios. For greater security, we may add to the fermentable liquid, at
the moment when it is prepared, a very small quantity of tartaric acid,
which will prevent the development of butyric vibrios.

[Illustration: Fig. 62.]

The variation of the ratio between the weight of the yeast and that of
the sugar decomposed by it now claims special attention. Side by side
with the experiments which we have just described, we conducted a third
lot by means of the flask C (Fig. 62), holding 4·7 litres (8-1/4 pints),
and fitted up like the usual two-necked flasks, with the object of
freeing the fermentable liquid from foreign germs, by boiling it to
begin with, so that we might carry on our work under conditions of
purity. The volume of yeast-water (containing 5 per cent. of sugar) was
only 200 c.c. (7 fl. oz.), and consequently, taking into account the
capacity of the flask, it formed but a very thin layer at the bottom. On
the day after impregnation the deposit of yeast was already
considerable, and forty-eight hours afterwards the fermentation was
completed. On the third day we collected the yeast, after having
analyzed the gas contained in the flask. This analysis was easily
accomplished by placing the flask in a hot-water bath, whilst the end of
the curved tube was plunged under a cylinder of mercury. The gas
contained 41·4 per cent. of carbonic acid, and, after the absorption,
the remaining air contained—

                         Oxygen           19·7
                         Nitrogen         80·3
                                           ——-
                                         100·0

Taking into consideration the volume of the flask, this shows a minimum
of 50 c.c. (3·05 cub. in.) of oxygen to have been absorbed by the yeast.
The liquid contained no more sugar, and the weight of the yeast, dried
at a temperature of 100° C. (212° F.), was 0·44 gramme (6·8 grains). The
ratio between the weight of the yeast and that of the sugar was,
therefore, 0·44/[114]10 = 1/22·7. On this occasion, where we had
increased the quantity of oxygen held in solution, so as to yield itself
for assimilation at the beginning and during the earlier developments of
the yeast, we found instead of the previous ratio of 1/76 that of 1/23.

[Illustration: Fig. 63.]

The next experiment was to increase the proportion of oxygen to a still
greater extent, by rendering the diffusion of gas a more easy matter
than it is in a flask, the air in which is in a state of perfect
quiescence. Such a state of matters hinders the supply of oxygen,
inasmuch as the carbonic acid, as soon as it is liberated, at once forms
an immovable layer on the surface of the liquid, and so separates off
the oxygen. To effect the purpose of our present experiment, we used
flat basins having glass bottoms and low sides, also of glass, in which
the depth of the liquid is not more than a few millimetres (less than
1/4-inch) (Fig. 63). The following is one of our experiments so
conducted:—On April 16th, 1860, we sowed a trace of beer yeast (“high”
yeast) in 200 c.c. (7 fl. oz.) of a saccharine liquid containing 1·720
grammes (26·2 grains) of sugar-candy. From April 18th our yeast was in
good condition and well developed. We collected it, after having added
to the liquid a few drops of concentrated sulphuric acid, with the
object of checking the fermentation to a great extent, and facilitating
filtration. The sugar remaining in the filtered liquid, determined by
Fehling’s solution, showed that 1·04 grammes (16 grains) of sugar had
disappeared. The weight of the yeast, dried at 100° C. (212° F.), was
0·127 gramme (2 grains), which gives us the ratio between the weight of
the yeast and that of the fermented sugar

[Illustration: Formula 1: 0·127/1·04 = 1/8·1.]

which is considerably higher than the preceding ones.

We may still further increase this ratio by making our estimation as
soon as possible after the impregnation, or the addition of the ferment.
It will be readily understood why yeast, which is composed of cells that
bud and subsequently detach themselves from one another, soon forms a
deposit at the bottom of the vessels.

In consequence of this habit of growth, the cells constantly covering
each other prevents the lower layers from having access to the oxygen
held in solution in the liquid, which is absorbed by the upper ones.
Hence, those which are covered and deprived of this gas act on the sugar
without deriving any vital benefit from the oxygen—a circumstance which
must tend to diminish the ratio of which we are speaking. Once more
repeating the preceding experiment, but stopping it as soon as we think
that the weight of yeast formed may be determined by the balance (we
find that this may be done twenty-four hours after impregnation with an
inappreciable quantity of yeast) in this case the ratio between the
weights of yeast and sugar is

[Illustration: Formula 2: (0^{gr}·024 yeast)/(0^{gr}·098 sugar) = 1/4.]

This is the highest ratio that we have been able to obtain.

Under these conditions the fermentation of sugar is extremely languid:
the ratio obtained is very nearly the same that ordinary fungoid growths
would give. The carbonic acid evolved is principally formed by the
decompositions which result from the assimilation of atmospheric oxygen.
The yeast, therefore, lives and performs its functions after the manner
of ordinary fungi: so far it is no longer a ferment, so to say;
moreover, we might expect to find it cease to be a ferment at all if we
could only surround each cell separately with all the air that it
required. This is what the preceding phenomena teach us; we shall have
occasion to compare them later on with others which relate to the vital
action exercised on yeast by the sugar of milk.

We may here be permitted to make a digression.

In his work on fermentations, which M. Schützenberger has recently
published, the author criticises the deductions that we have drawn from
the preceding experiments, and combats the explanation which we have
given of the phenomena of fermentation.[115] It is an easy matter to
show the weak point of M. Schützenberger’s reasoning. We determined the
power of the ferment by the relation of the weight of sugar decomposed
to the weight of yeast produced. M. Schützenberger asserts that in doing
this we lay down a doubtful hypothesis, and he thinks that this power,
which he terms _fermentative energy_, may be estimated more correctly by
the quantity of sugar decomposed by the unit-weight of yeast in
unit-time; moreover, since our experiments show that yeast is very
vigorous when it has a sufficient supply of oxygen, and that, in such a
case, it can decompose much sugar in a little time, M. Schützenberger
concludes that it must then have great power as a ferment, even greater
than it has when it performs its functions without the aid of air, since
under this condition it decomposes sugar very slowly. In short, he is
disposed to draw from our observations the very opposite conclusion to
that which we arrived at.

M. Schützenberger has failed to notice that the power of a ferment is
independent of the time during which it performs its functions. We
placed a trace of yeast in one litre of saccharine wort; it propagated,
and all the sugar was decomposed. Now, whether the chemical action
involved in this decomposition of sugar had required for its completion
one day, or one month, or one year, such a factor was of no more
importance in this matter than the mechanical labour required to raise a
ton of materials from the ground to the top of a house would be affected
by the fact that it had taken twelve hours instead of one. The notion of
time has nothing to do with the definition of work. M. Schützenberger
has not perceived that in introducing the consideration of time into the
definition of the power of a ferment, he must introduce, at the same
time, that of the vital activity of the cells, which is independent of
their character as a ferment. Apart from the consideration of the
relation existing between the weight of fermentable substance decomposed
and that of ferment produced, there is no occasion to speak of
fermentations or of ferments. The phenomena of fermentation and of
ferments have been placed apart from others, precisely because, in
certain chemical actions, that ratio has been out of proportion; but the
time that these phenomena require for their accomplishment has nothing
to do either with their existence proper, or with their power. The cells
of a ferment may, under some circumstances, require eight days for
revival and propagation, whilst, under other conditions, only a few
hours are necessary; so that, if we introduce the notion of time into
our estimate of their power of decomposition, we may be led to conclude
that in the first case that power was entirely wanting, and that in the
second case it was considerable, although all the time we are dealing
with the same organism—the identical ferment.

M. Schützenberger is astonished that fermentation can take place in the
presence of free oxygen, if, as we suppose, the decomposition of the
sugar is the consequence of the nutrition of the yeast, at the expense
of the combined oxygen, which yields itself to the ferment. At all
events, he argues, fermentation ought to be slower in the presence of
free oxygen. But why should it be slower? We have proved that in the
presence of oxygen the vital activity of the cells increases, so that,
as far as rapidity of action is concerned, its power cannot be
diminished. It might, nevertheless, be weakened as a ferment, and this
is precisely what happens. Free oxygen imparts to the yeast an increased
vital activity, but at the same time impairs rapidly its power as
yeast—_quâ_ yeast, inasmuch as under this condition it approaches the
state in which it can carry on its vital processes after the manner of
an ordinary fungus; the mode of life, that is, in which the ratio
between the weight of sugar decomposed and the weight of the new cells
produced will be the same as holds generally among organisms which are
not ferments. In short, varying our form of expression a little, we may
conclude with perfect truth, from the sum total of observed facts, that
the yeast which lives in the presence of oxygen and can assimilate as
much of that gas as is necessary to its perfect nutrition, ceases
absolutely to be a ferment at all. Nevertheless, yeast formed under
these conditions and subsequently brought into the presence of sugar,
_out of the influence of air_, would decompose more _in a given time_
than in any other of its states. The reason is that yeast which has
formed in contact with air, having the maximum of free oxygen that it
can assimilate, is fresher and possessed of greater vital activity than
that which has been formed without air or with an insufficiency of air.
M. Schützenberger would associate this activity with the notion of time
in estimating the power of the ferment; but he forgets to notice that
yeast can only manifest this maximum of energy under a radical change of
its life-conditions; by having no more air at its disposal and breathing
no more free oxygen. In other words, when its respiratory power becomes
null, its fermentative power is at its greatest. M. Schützenberger
asserts exactly the opposite (p. 151 of his work—Paris, 1875),[116] and
so gratuitously places himself in opposition to facts.

[Illustration: Fig. 64.]

In presence of abundant air-supply, yeast vegetates with extraordinary
activity. We see this in the weight of new yeast, comparatively large,
that may be formed in the course of a few hours. The microscope still
more clearly shows this activity in the rapidity of budding, and the
fresh and active appearance of all the cells. Fig. 64 represents the
yeast of our last experiment at the moment when we stopped the
fermentation. Nothing has been taken from imagination, all the groups
have been faithfully sketched as they were.[117]

In passing it is of interest to note how promptly the preceding results
were turned to good account practically. In well-managed distilleries,
the custom of aerating the wort and the juices, to render them more
adapted to fermentation, has been introduced. The molasses, mixed with
water, is permitted to run in thin threads through the air at the moment
when the yeast is added. Manufactories have been erected, in which the
manufacture of yeast is almost exclusively carried on. The saccharine
worts, after the addition of yeast, are left to themselves, in contact
with air, in shallow vats of large superficial area, realizing thus on
an immense scale the conditions of the experiments which we undertook in
1861, and which we have already described in determining the rapid and
easy multiplication of yeast in contact with air.

The next experiment attempted was to determine the volume of oxygen
absorbed by a known quantity of yeast, the yeast living in contact with
air, and under such conditions that the absorption of air was
comparatively easy and abundant.

[Illustration: Fig. 65.]

With this object we repeated the experiment that we performed with the
large-bottomed flask (Fig. 62), employing a vessel shaped like Fig. B.
(Fig. 65), which is, in point of fact, the flask A with its neck drawn
out and closed in a flame, after the introduction of a thin layer of
some saccharine juice impregnated with a trace of pure yeast. The
following are the data and results of an experiment of this kind.

We employed 60 c.c. (about 2 fluid ounces) of yeast-water, sweetened
with 2 per cent. of sugar and impregnated with a trace of yeast. After
having subjected our vessel to a temperature of 25° C. (77° F.) in an
oven for fifteen hours, the drawn-out point was brought under an
inverted jar filled with mercury and the point broken off. A portion of
the gas escaped and was collected in the jar.

For 25 c.c. of this gas we found, after absorption by potash, 20·6, and
after absorption by pyrogallic acid, 17·3. Taking into account the
volume which remained free in the flask, which held 315 c.c., there was
a total absorption of 14·5 c.c. (0·88 cub. in.) of oxygen.[118] The
weight of yeast, in a state of dryness, was 0·035 gramme.

It follows that in the production of 35 milligrammes (0·524 grain) of
yeast there was an absorption of 14 or 15 c.c. (about 7/8 cubic inch) of
oxygen, even supposing that the yeast was formed entirely under the
influence of that gas: this is equivalent to not less than 414 c.c. for
1 gramme of yeast (or about 33 cubic inches for every 20 grains).[119]

Such is the large volume of oxygen necessary for the development of one
gramme of yeast when the plant can assimilate this gas after the manner
of an ordinary fungus.

Let us now return to the first experiment described in this paragraph
(page 238), in which a flask of three litres capacity was filled with
fermentable liquid, which, when caused to ferment, yielded 2·25 grammes
of yeast, under circumstances where it could not obtain a greater supply
of free oxygen than 16·5 c.c. (about one cubic inch). According to what
we have just stated, if this 2·25 grammes (34 grains) of yeast had not
been able to live without oxygen, in other words, if the original cells
had been unable to multiply otherwise than by absorbing free oxygen, the
amount of that gas required could not have been less than 2·25 × 414
c.c., that is, 931·5 c.c. (56·85 cubic inches). The greater part of the
2·25 grammes, therefore, had evidently been produced as the growth of an
anaërobian plant.

Ordinary fungi likewise require large quantities of oxygen for their
development, as we may easily prove by cultivating any mould in a closed
vessel full of air, and then taking the weight of plant formed and
measuring the volume of oxygen absorbed. To do this, we take a flask of
the shape shown in Fig. 66, capable of holding about 300 c.c. (10-½
fluid ounces), and containing a liquid adapted to the life of moulds. We
boil this liquid and seal the drawn-out point, after the steam has
expelled the air wholly or in part; we then open the flask in a garden
or in a room. Should a fungus-spore enter the flask, as will invariably
be the case in a certain number of flasks out of several used in the
experiment, except under special circumstances, it will develop there
and gradually absorb all the oxygen contained in the air of the flask.
Measuring the volume of this air, and weighing, after drying, the amount
of plant formed, we find that for a certain quantity of oxygen absorbed
we have a certain weight of mycelium, or of mycelium together with its
organs of fructification. In an experiment of this kind, in which the
plant was weighed a year after its development, we found for 0·008
gramme (0·123 grain) of _mycelium_, dried at 100° C. (212° F.), an
absorption that amounted to not less than 43 c.c. (1·5 cubic inches) of
oxygen, at 25°. These numbers, however, must vary sensibly with the
nature of the mould employed, and also with the greater or less activity
of its development, because the phenomenon is complicated by the
presence of accessory oxidations, such as we find in the case of
_mycoderma vini_ and _aceti_, to which cause the large absorption of
oxygen in our last experiment may doubtless be attributed.[120]

[Illustration: Fig. 66.]

The conclusions to be drawn from the whole of the preceding facts can
scarcely admit of doubt. As for ourselves, we have no hesitation in
finding in them the foundation of the true theory of fermentation. In
the experiments which we have described, fermentation by yeast, that is
to say, by the type of ferments properly so called, is presented to us,
in a word, as the direct consequence of the processes of nutrition,
assimilation, and life, when these are carried on without the agency of
free oxygen. The heat required in the accomplishment of that work must
necessarily have been borrowed from the decomposition of the fermentable
matter, that is from the saccharine substance which, like other unstable
substances, liberates heat in undergoing decomposition. Fermentation by
means of yeast appears, therefore, to be essentially connected with the
property possessed by this minute cellular plant of performing its
respiratory functions, somehow or other, with oxygen existing combined
in sugar. Its fermentative power—which power must not be confounded with
the fermentative activity or the intensity of decomposition in a given
time—varies considerably between two limits, fixed by the greatest and
least possible access to free oxygen which the plant has in the process
of nutrition. If we supply it with a sufficient quantity of free oxygen
for the necessities of its life, nutrition, and respiratory combustions,
in other words, if we cause it to live after the manner of a mould,
properly so called, it ceases to be a ferment, that is, the ratio
between the weight of the plant developed and that of the sugar
decomposed, which forms its principal food, is similar in amount to that
in the case of fungi.[121] On the other hand, if we deprive the yeast of
air entirely, or cause it to develop in a saccharine medium deprived of
free oxygen, it will multiply just as if air were present, although with
less activity, and under these circumstances its fermentative character
will be most marked; under these circumstances, moreover, we shall find
the greatest disproportion, all other conditions being the same, between
the weight of yeast formed and the weight of sugar decomposed. Lastly,
if free oxygen occurs in varying quantities, the ferment-power of the
yeast may pass through all the degrees comprehended between the two
extreme limits of which we have just spoken. It seems to us that we
could not have a better proof of the direct relation that fermentation
bears to life, carried on in the absence of free oxygen, or with a
quantity of that gas insufficient for all the acts of nutrition and
assimilation.

Another equally striking proof of the truth of this theory is the fact,
demonstrated in Chapter IV., that the ordinary moulds assume the
character of a ferment when compelled to live without air, or with
quantities of air too scant to permit of their organs having around them
as much of that element as is necessary for their life as aërobian
plants. Ferments, therefore, only possess in a higher degree a character
which belongs to many common moulds, if not to all, and which they
share, probably, more or less, with all living cells, namely the power
of living either an aërobian or anaërobian life, according to the
conditions under which they are placed.

It may be readily understood how, in their state of aërobian life, the
alcoholic ferments have failed to attract attention. Those ferments are
only cultivated out of contact with air, at the bottom of liquids which
soon become saturated with carbonic acid gas. Air is only present in the
earlier developments of their germs, and without attracting the
attention of the operator, whilst in their state of anaërobian growth
their life and action are of prolonged duration. We must have recourse
to special experimental apparatus to enable us to demonstrate the mode
of life of alcoholic ferments under the influence of free oxygen; it is
their state of existence apart from air, in the depths of liquids that
attracts all our attention. The results of their action are, however,
marvellous, if we regard the products resulting from them, in the
important industries of which they are the life and soul. In the case of
ordinary moulds, the opposite holds good. What we want to use special
experimental apparatus for with them is to enable us to demonstrate the
possibility of their continuing to live for a time out of contact with
air, and all our attention, in their case, is attracted by the facility
with which they develop under the influence of oxygen. Thus the
decomposition of saccharine liquids, which is the consequence of the
life of fungi without air, is scarcely perceptible, and so is of no
practical importance. Their aerial life, on the other hand, in which
they respire and accomplish their process of oxidation under the
influence of free oxygen, is a normal phenomenon, and one of prolonged
duration which cannot fail to strike the least thoughtful of observers.
We are convinced that a day will come when moulds will be utilized in
certain industrial operations, on account of their power of destroying
organic matter. The conversion of alcohol into vinegar in the process of
acetification, and the production of gallic acid by the action of fungi
on wet gall-nuts, are already connected with this kind of
phenomena.[122] On this last subject, the important work of M. Van
Tieghem (_Annales Scientifiques de l’École Normale_, vol. vi.) may be
consulted.

The possibility of living without oxygen, in the case of ordinary
moulds, is connected with certain morphological modifications which are
more marked in proportion as this faculty is itself more developed.
These changes in the vegetative forms are scarcely perceptible in the
case of _penicillium_ and _mycoderma vini_, but they are very evident in
the case of _aspergillus_, consisting of a marked tendency on the part
of the submerged mycelial filaments to increase in diameter, and to
develop cross partitions at short intervals, so that they sometimes bear
a resemblance to chains of conidia. In _mucor_, again, they are very
marked, the inflated filaments which, closely interwoven, present chains
of cells which fall off and bud, gradually producing a mass of cells. If
we consider the matter carefully, we shall see that yeast presents the
same characteristics. For instance, what can more closely resemble the
mucor of Plates V. and VI. than the _saccharomyces_ of Figs. 33 and 37?
Have we not in each case ramified chains of elongated cells or joints,
more or less narrowed in the middle, and shorter segments or cells
dropping off at the constrictions, and proceeding to bud in the liquid
on their own account? Moreover, the less oxygen there is present, the
more marked is the tendency to the formation of these budding cells,
which isolate themselves and soon drop off. Who could ever imagine, in
examining the ferment of _mucor_ represented in Plate VI., that its
first germ was the ordinary _mucor_ that is found everywhere, with fine
filaments, straight or ramified according to the variety, which send up
aerial _hyphae_, terminating in little round heads bearing spores. So
was it that in the ferment of Plate XI. we could scarcely recognize the
ramified filaments of Figs. 33 and 37.

It is a great presumption in favour of the truth of theoretical ideas
when the results of experiments undertaken on the strength of those
ideas are confirmed by various facts more recently added to science, and
when those ideas force themselves more and more on our minds, in spite
of a _primâ facie_ improbability. This is exactly the character of those
ideas which we have just expounded. We propounded them in 1861, and not
only have they remained unshaken since, but they have served to
foreshadow new facts, so that it is much easier to defend them in the
present day than it was to do so fifteen years ago. We first called
attention to them in various notes, which we read before the Chemical
Society of Paris, notably at its meetings of April 12th and June 28th,
1861, and in papers in the _Comptes rendus de l’Académie des Sciences_.
It may be of some interest to quote here, in its entirety, our
communication of June 28th, 1861, entitled, “Influences of Oxygen on the
Development of Yeast and on Alcoholic Fermentation,” which we extract
from the _Bulletin de la Société Chimique de Paris_:—

“M. Pasteur gives the results of his researches on the fermentation of
sugar and the development of yeast-cells, according as that fermentation
takes place apart from the influence of free oxygen or in contact with
that gas. His experiments, however, have nothing in common with those of
Gay-Lussac, which were performed with the juice of grapes, crushed under
conditions where they would not be affected by air, and then brought in
contact with oxygen.

“Yeast, when perfectly developed, is able to bud and grow in a
saccharine and albuminous liquid, in the complete absence of oxygen or
air. In this case but little yeast is formed, and a comparatively large
quantity of sugar disappears—sixty or eighty parts for one of yeast
formed. Under these conditions fermentation is very sluggish.

“If the experiment is made in contact with the air, and with a great
surface of liquid, fermentation is rapid. For the same quantity of sugar
decomposed much more yeast is formed. The air with which the liquid is
in contact is absorbed by the yeast. The yeast develops very actively,
but its fermentative character tends to disappear under these
conditions; we find, in fact, that for one part of yeast formed, not
more than from four to ten parts of sugar are transformed. The
fermentative character of this yeast, nevertheless, continues, and
produces even increased effects, if it is made to act on sugar apart
from the influence of free oxygen.

“It seems, therefore, natural to admit that when yeast functions as a
ferment by living apart from the influence of air, it derives oxygen
from the sugar, and that this is the origin of its fermentative
character.

“M. Pasteur explains the fact of the immense activity at the
commencement of fermentations by the influence of the oxygen of the air
held in solution in the liquids, at the time when the action commences.
The author has found, moreover, that the yeast of beer sown in an
albuminous liquid, such as yeast-water, still multiplies, even when
there is not a trace of sugar in the liquid, provided always that
atmospheric oxygen is present in large quantities. When deprived of air,
under these conditions, yeast does not germinate at all. The same
experiments may be repeated with albuminous liquid, mixed with a
solution of non-fermentable sugar, such as ordinary crystallized
milk-sugar. The results are precisely the same.

“Yeast formed thus in the absence of sugar does not change its nature;
it is still capable of causing sugar to ferment, if brought to bear upon
that substance apart from air. It must be remarked, however, that the
development of yeast is effected with great difficulty when it has not a
fermentable substance for its food. In short, the yeast of beer acts in
exactly the same manner as an ordinary plant, and the analogy would be
complete if ordinary plants had such an affinity for oxygen as permitted
them to breathe by appropriating this element from unstable compounds,
in which case, according to M. Pasteur, they would appear as ferments
for those substances.

“M. Pasteur declares that he hopes to be able to realize this result,
that is to say, to discover the conditions under which certain inferior
plants may live apart from air in the presence of sugar, causing that
substance to ferment as the yeast of beer would do.”

This summary and the preconceived views that it set forth have lost
nothing of their exactness; on the contrary, time has strengthened them.
The surmises of the last two paragraphs have received a valuable
confirmation from recent observations made by Messrs. Lechartier and
Bellamy, as well as by ourselves, an account of which we must put before
our readers. It is necessary, however, before touching upon this curious
feature in connection with fermentations to insist on the accuracy of a
passage in the preceding summary, the statement, namely, that yeast
could multiply in an albuminous liquid, in which it found a
non-fermentable sugar, milk-sugar for example. The following is an
experiment on this point:—On August 15th, 1875, we sowed a trace of
yeast in 150 c.c. (rather more than 5 fluid ounces) of yeast-water,
containing 2-½ per cent. of milk-sugar. The solution was prepared in one
of our double-necked flasks, with the necessary precautions to secure
absence of germs, and the yeast sown was itself perfectly pure. Three
months afterwards, November 15th, 1875, we examined the liquid for
alcohol; it contained only the smallest trace; as for the yeast, which
had sensibly developed, collected and dried on a filter paper, it
weighed 0·050 gramme (0·76 grain). In this case we have the yeast
multiplying without giving rise to the least fermentation, like a
fungoid growth, absorbing oxygen, and evolving carbonic acid, and there
is no doubt that the cessation of its development in this experiment was
due to the progressive deprivation of oxygen that occurred. As soon as
the gaseous mixture in the flask consisted entirely of carbonic acid and
nitrogen, the vitality of the yeast was dependent on, and in proportion
to, the quantity of air which entered the flask in consequence of
variations of temperature. The question now arose, was this yeast, which
had developed wholly as an ordinary fungus, still capable of manifesting
the character of a ferment? To settle this point we had taken the
precaution, on August 15th, 1875, of preparing another flask, exactly
similar to the preceding one in every respect, and which gave results
identical with those described. We decanted this on November 15th,
pouring some wort on the deposit of the plant, which remained in the
flask. In less than five hours from the time when we placed it in the
oven, the plant had started fermentation in the wort, as we could see by
the bubbles of gas rising to form patches on the surface of the liquid.
We may add that yeast in the medium which we have been discussing will
not develop at all without air.

The importance of these results can escape no one; they prove clearly
that the fermentative character is not an invariable phenomenon of
yeast-life, they show that yeast is a plant which does not differ from
ordinary plants, and which manifests its fermentative power solely in
consequence of particular conditions under which it is compelled to
live. It may carry on its life as a ferment or not, and after having
lived without manifesting the slightest symptom of fermentative
character, it is quite ready to manifest that character when brought
under suitable conditions. The fermentative property, therefore, is not
a power peculiar to cells of a special nature. It is not a permanent
character of a particular structure, like, for instance, the property of
acidity or alkalinity. It is a peculiarity dependent on external
circumstances and on the nutritive conditions of the organism.


 § II.—Fermentation in Saccharine Fruits Immersed in Carbonic Acid Gas.


The theory which we have, step by step, evolved, on the subject of the
causes of the chemical phenomena of fermentation, may claim a character
of simplicity and generality that is well worthy of attention.
Fermentation is no longer one of those isolated and mysterious phenomena
which do not admit of explanation. It is the consequence of a peculiar
vital process of nutrition which occurs under certain conditions,
differing from those which characterize the life of all ordinary beings,
animal or vegetable, but by which the latter may be affected, more or
less, in a way which brings them, to some extent. within the class of
ferments, properly so called. We can even conceive that the fermentative
character may belong to every organized form, to every animal or
vegetable cell, on the sole condition that the chemico-vital acts of
assimilation and excretion must be capable of taking place in that cell
for a brief period, longer or shorter it may be, without the necessity
for recourse to supplies of atmospheric oxygen; in other words, the cell
must be able to derive its needful heat from the decomposition of some
body which yields a surplus of heat in the process.

As a consequence of these conclusions it should be an easy matter to
show, in the majority of living beings, the manifestation of the
phenomena of fermentation; for there are, probably, none in which all
chemical action entirely disappears, upon the sudden cessation of life.
One day, when we were expressing these views in our laboratory, in the
presence of M. Dumas, who seemed inclined to admit their truth, we
added: “We would make a wager that if we were to plunge a bunch of
grapes into carbonic acid gas, there would be immediately produced
alcohol and carbonic acid, in consequence of a renewed action starting
in the interior cells of the grapes, in such a way that these cells
would assume the function of yeast-cells. We will make the experiment,
and when you come to-morrow—it was our good fortune to have M. Dumas
working in our laboratory at that time—we will give you an account of
the result.” Our predictions were realized. We then endeavoured to find,
in the presence of M. Dumas, who assisted us in our endeavour, cells of
yeast in the grapes; but it was quite impossible to discover any.[123]

Encouraged by this result, we undertook fresh experiments on grapes, on
a melon, on oranges, on plums, and on rhubarb leaves, gathered in the
garden of the _École Normale_, and, in every case, our substance when
immersed in carbonic acid gas, gave rise to the production of alcohol
and carbonic acid. We obtained the following surprising results from
some _prunes de Monsieur_[124]:—On July 31st, 1872, we placed
twenty-four of these plums under a glass cylinder, which we immediately
filled with carbonic acid gas. The plums had been gathered on the
previous day. By the side of the cylinder we placed other twenty-four
plums, which were left there uncovered. Eight days afterwards, in the
course of which time there had been a considerable evolution of carbonic
acid from the cylinder, we withdrew the plums and compared them with
those which had been left exposed to the air. The difference was
striking, almost incredible. Whilst the plums which had been surrounded
with air (the experiments of Bérard have long since taught us that,
under this latter condition, fruits absorb oxygen from the air and emit
carbonic acid gas in almost equal volume) had become very soft and
watery and sweet, the plums taken from under the jar had remained very
firm and hard, the flesh was by no means watery, but they had lost much
sugar. Lastly, when submitted to distillation, after crushing, they
yielded 6·5 grammes (99·7 grains) of alcohol, more than 1 per cent. of
the total weight of the plums. What better proof could we have than
these facts of the existence of a considerable chemical action in the
interior of fruit, an action which derives the heat necessary for its
manifestation from the decomposition of the sugar present in the cells?
Moreover, and this circumstance is especially worthy of our attention,
in all these experiments we found that there was a liberation of heat,
of which the fruits and other organs were the seat, as soon as they were
plunged in the carbonic acid gas. This heat is so considerable that it
may at times be detected by the hand, if the two sides of the cylinder,
one of which is in contact with the objects, are touched alternately. It
also makes itself evident in the formation of vapour, which condenses in
little drops on those parts of the bell which are less directly exposed
to the influence of the heat resulting from the decomposition of the
sugar of the cells.[125]

In short, fermentation is a very general phenomenon. It is life without
air, or life without free oxygen, or, more generally still, it is the
result of a chemical process accomplished on a fermentable substance,
_i.e._ a substance capable of producing heat by its decomposition, in
which process the entire heat used up is derived from a part of the heat
that the decomposition of the fermentable substance sets free. The class
of fermentations, properly so called, is, however, restricted by the
small number of substances capable of decomposing with the production of
heat, and at the same time of serving for the nourishment of lower forms
of life, when deprived of the presence and action of air. This, again,
is a consequence of our theory, which is well worthy of notice.

The facts that we have just mentioned in reference to the formation of
alcohol and carbonic acid in the substance of ripe fruits, under certain
special conditions, and apart from the action of ferment, are already
known to science. They were discovered in 1869 by M. Lechartier,
formerly a pupil in the _École Normale Supérieure_, and his coadjutor,
M. Bellamy.[126] In 1821, in a very remarkable work, especially when we
consider the period when it appeared, Bérard demonstrated several
important propositions in connection with the maturation of fruits:—

I. All fruits, even those that are still green, and likewise even those
that are exposed to the sun, absorb oxygen and set free an almost equal
volume of carbonic acid gas. This is a condition of their proper
ripening.

II. Ripe fruits placed in a limited atmosphere, after having absorbed
all the oxygen and set free an almost equal volume of carbonic acid,
continue to emit that gas in notable quantity, even when no bruise is to
be seen—“as though by a kind of fermentation,” as Bérard actually
observes—and lose their saccharine particles, a circumstance which
causes the fruits to appear more acid, although the actual weight of
their acid may undergo no augmentation whatever.

In this beautiful work, and in all subsequent ones of which the ripening
of fruits has been the subject, two facts of great theoretical value
have escaped the notice of the authors; these are the two facts which
Messrs. Lechartier and Bellamy pointed out, for the first time, namely,
the production of alcohol and the absence of cells of ferments. It is
worthy of remark that these two facts, as we have shown above, were
actually foreshadowed in the theory of fermentation that we advocated as
far back as 1861, and we are happy to add that Messrs. Lechartier and
Bellamy, who, at first, had prudently drawn no theoretical conclusions
from their work, now entirely agree with the theory we have
advanced.[127] Their mode of reasoning is very different from that of
the savants with whom we discussed the subject before the Academy, on
the occasion when the communication which we addressed to the Academy,
in October, 1872, attracted attention once more to the remarkable
observations of Messrs. Lechartier and Bellamy.[128] M. Fremy, in
particular, was desirous of finding in those observations a confirmation
of his views on the subject of _hemi-organism_, and a condemnation of
ours, notwithstanding the fact that the preceding explanations and, more
particularly our Note of 1861, which we have quoted word for word in the
last paragraph, furnish the most conclusive evidence in favour of those
ideas which we advocate. Indeed, as far back as 1861 we pointed out very
clearly that if we could find plants able to live when deprived of air,
in the presence of sugar, they would bring about a fermentation of that
substance, in the same manner as yeast does. Such is the case with the
fungi already studied in Chapter IV.; such, too, is the case with the
fruits employed in the experiments of Messrs. Lechartier and Bellamy,
and in our own experiments, the results of which not only confirm those
obtained by these gentlemen, but even extend them, in so far as we have
shown that fruits, when surrounded with carbonic acid gas, immediately
produce alcohol. When surrounded with air, they live in their aërobian
state, and we have no ferment-action; immersed immediately afterwards in
carbonic acid gas, they now assume their anaërobian state, and at once
begin to act upon the sugar in the manner of ferments, and emit heat. As
for seeing in these facts anything like a confirmation of the theory of
hemi-organism, imagined by M. Fremy, the idea of such a thing is absurd.
The following, for instance, is the theory of the fermentation of the
vintage, according to M. Fremy.[129]

“To speak here of alcoholic fermentation alone,”[130] our author says,
“I hold that in the production of wine it is the juice of the fruit
itself that, in contact with air, produces grains of ferment, by the
transformation of the albuminous matter; M. Pasteur, on the other hand,
maintains that the fermentation is produced by germs existing outside
the skin of the grapes.”

Now what bearing on this purely imaginary theory can the fact have, that
a whole fruit, immersed in carbonic acid gas, immediately produces
alcohol and carbonic acid? In the preceding passage, which we have
borrowed from M. Fremy, an indispensable condition of the transformation
of the albuminous matter is the contact with air and the crushing of the
grapes. Here, however, we are dealing with _uninjured fruits in contact
with carbonic acid gas_. Our theory, on the other hand, which, we may
repeat, we have advocated since 1861, maintains that all cells become
fermentative when their vital action is protracted in the absence of
air, which are precisely the conditions that hold in the experiment on
fruits immersed in carbonic acid gas. The vital energy is not
immediately suspended in their cells, and the latter are deprived of
air. Consequently, fermentation must result. Moreover, we may add, if we
destroy the fruit, or crush it before immersing it in the gas, it no
longer produces alcohol or fermentation of any kind, a circumstance that
may be attributed to the fact of the destruction of vital action in the
crushed fruit. On the other hand, in what way ought this crushing to
affect the hypothesis of hemi-organism? The crushed fruit ought to act
quite as well, or even better than that which is uncrushed. In short,
nothing can be more directly opposed to the theory of the mode of
manifestation of that hidden force to which the name of hemi-organism
has been given, than the discovery of the production of these phenomena
of fermentation in fruits surrounded with carbonic acid gas; whilst the
theory, which sees in fermentation a consequence of vital energy in
absence of air, finds in these facts the strictest confirmation of an
express prediction, which from the first formed an integral part of its
statement.

We should not be justified in devoting further time to opinions which
are not supported by any serious experiment. Abroad, as well as in
France, the theory of the transformation of albuminous substances into
organized ferments had been advocated long before it was taken up by M.
Fremy. It no longer commands the slightest credit, nor do any observers
of note any longer give it the least attention; it might even be said
that it has become a subject of ridicule.

An attempt has also been made to prove that we have contradicted
ourselves, inasmuch as in 1860 we published our opinion that alcoholic
fermentation can never occur without a simultaneous occurrence of
organization, development, and multiplication of globules; or continued
life, carried on from globules already formed.[131] Nothing, however,
can be truer than that opinion, and at the present moment, after fifteen
years of study devoted to the subject, since the publication to which we
have referred, we need no longer say “we think,” but instead, “we
affirm” that it is correct. It is, as a matter of fact, to alcoholic
fermentation, properly so called, that the charge to which we have
referred relates—to that fermentation which yields, besides alcohol,
carbonic acid, succinic acid, glycerine, volatile acids, and other
products. This fermentation undoubtedly requires the presence of
yeast-cells, under the conditions that we have named. Those who have
contradicted us have fallen into the error of supposing that the
fermentation of fruits is an ordinary alcoholic fermentation, identical
with that produced by beer-yeast, and that, consequently, the cells of
that yeast must, according to our own theory, be always present. There
is not the least authority for such a supposition. When we come to exact
quantitative estimations—and these are to be found in the figures
supplied by Messrs. Lechartier and Bellamy—it will be seen that the
proportions of alcohol and carbonic acid gas produced in the
fermentation of fruits differ widely from those that we find in
alcoholic fermentations, properly so called, as must necessarily be the
case, since, in the former, the ferment-action is effected by the cells
of a fruit, but in the latter by cells of ordinary alcoholic ferment.
Indeed we have a strong conviction that each fruit would be found to
give rise to a special action, the chemical equation of which would be
different from that in the case of other fruits. As for the circumstance
that the cells of these fruits cause fermentation, without multiplying,
this comes under the kind of activity, which we have already
distinguished by the expression _continuous life in cells already
formed_.

We will conclude this paragraph with a few remarks on the subject of the
equations of fermentations, which have been suggested to us principally
in attempts to explain the results derived from the fermentation of
fruits immersed in carbonic acid gas.

Originally, when fermentations were put amongst the class of
decompositions by contact-action, it seemed probable, and, in fact, was
believed, that every fermentation had its own well-defined equation,
which never varied. In the present day, on the contrary, it must be
borne in mind that the equation of a fermentation varies essentially
with the conditions under which that fermentation is accomplished, and
that a statement of this equation is a problem no less complicated than
that in the case of the nutrition of a living being. To every
fermentation may be assigned an equation in a general sort of way, an
equation, however, which, in numerous points of detail, is liable to the
thousand variations connected with the phenomena of life. Moreover,
there will be as many distinct fermentations brought about by one
ferment as there are fermentable substances capable of supplying the
carbon element of the food of that same ferment, in the same way that
the equation of the nutrition of an animal will vary with the nature of
the food which it consumes. As regards fermentation producing alcohol,
which may be effected by several different ferments, there will be, in
the case of a given sugar, as many general equations as there are
ferments, whether they be ferment-cells, properly so called, or cells of
the organs of living beings functioning as ferments. In the same way the
equation of nutrition varies in the case of different animals nourished
on the same food. And it is from the same reason that ordinary wort
produces such a variety of beers when treated with the numerous
alcoholic ferments which we have described. These remarks are applicable
to all ferments alike; for instance, butyric ferment is capable of
producing a host of distinct fermentations, in consequence of its
ability to derive the carbonaceous part of its food from very different
substances, from sugar, or lactic acid, or glycerine, or mannite, and
many others.

When we say that every fermentation has its own peculiar ferment, it
must be understood that we are speaking of the fermentation considered
as a whole, including all the accessory products. We do not mean to
imply that the ferment in question is not capable of acting on some
other fermentable substance and giving rise to fermentation of a very
different kind. Moreover, it is quite erroneous to suppose that the
presence of a single one of the products of a fermentation implies the
co-existence of a particular ferment. If, for example, we find alcohol
among the products of a fermentation, or even alcohol and carbonic acid
gas together, this does not prove that the ferment must be an alcoholic
ferment, belonging to alcoholic fermentations, in the strict sense of
the term. Nor, again, does the mere presence of lactic acid necessarily
imply the presence of lactic ferment. As a matter of fact, different
fermentations may give rise to one or even several identical products.
We could not say with certainty, from a purely chemical point of view,
that we were dealing, for example, with an alcoholic fermentation,
properly so called, and that the yeast of beer must be present in it, if
we had not first determined the presence of all the numerous products of
that particular fermentation, and that they were present in those
proportions, characteristic of that fermentation under conditions
similar to those under which the fermentation in question had occurred.
In works on fermentation, the reader will often find those confusions
against which we are now attempting to guard him. It is precisely in
consequence of not having had their attention drawn to such observations
that some have imagined that the fermentation in fruits, immersed in
carbonic acid gas, is in contradiction to the assertion which we
originally made in our Memoir on alcoholic fermentation, published in
1860, the exact words of which we may here repeat:—“The chemical
phenomena of fermentation are related essentially to a vital activity,
beginning and ending with the latter; we believe that alcoholic
fermentation never occurs”—we were discussing the question of ordinary
alcoholic fermentation produced by the yeast of beer—“without the
simultaneous occurrence of organization, development, and multiplication
of globules, or continued life, carried on by means of globules already
formed. The general results of the present Memoir seem to us to be in
direct opposition to the opinions of MM. Liebig and Berzelius.” These
conclusions, we repeat, are as true now as they ever were, and are as
applicable to the fermentation of fruits, of which nothing was known in
1860, as they are to the fermentation produced by means of yeast. Only,
in the case of fruits, it is the cells of the parenchyma that function
as ferment, _by a continuation of their vital activity in carbonic acid
gas_, whilst in the other case the ferment consists of the cells of
yeast.

There should be nothing very surprising in the fact that fermentation
can originate in fruits and form alcohol, without the presence of yeast,
if the fermentation of fruits were not confounded completely with
ordinary alcoholic fermentation, yielding the same products and in the
same proportions. It is through the misuse of words that the
fermentation of fruits has been termed _alcoholic_, in a way which has
misled many persons.[132] In this fermentation, neither alcohol nor
carbonic acid gas exists in those proportions in which they are found in
fermentations produced by yeast; and although we may determine in it the
presence of succinic acid, glycerine, and a small quantity of volatile
acids,[133] the relative proportions of these substances will be
different from what they are in the case of alcoholic fermentation.


§ III.—Reply to certain Critical Observations of the German Naturalists,
                    Oscar Brefeld and Moritz Traube.


The essential point of the theory of fermentation, which we have been
concerned in proving in preceding paragraphs, may be briefly put in the
statement that ferments, properly so called, constitute a class of
beings possessing the faculty of living out of contact with free oxygen;
or, more concisely still, we may say, fermentation is a result of life
without air.

If our affirmation were inexact, if ferment-cells did require for their
growth or for their increase in number or weight, as all other vegetable
cells do, the presence of oxygen, whether gaseous or held in solution in
liquids, this new theory would lose all value, its very _raison d’être_
would be gone, at least as far as the most important part of
fermentations is concerned. This is precisely what M. Oscar Brefeld has
endeavoured to prove, in a Memoir read to the Physico-Medical Society of
Wurzburg, on July 26th, 1873, in which, although we have ample evidence
of the great experimental skill of its author, he has, nevertheless, in
our opinion, arrived at conclusions entirely opposed to fact.

“From the experiments which I have just described,” he says, “it
follows, in the most indisputable manner, that _a ferment cannot
increase without free oxygen_. Pasteur’s supposition that a ferment,
unlike all other living organisms, can live and increase at the expense
of oxygen held in combination, is, consequently, altogether wanting in
any solid basis of experimental proof. Moreover, since, according to the
theory of Pasteur, it is precisely this faculty of living and increasing
at the expense of the oxygen held in combination that constitutes the
phenomenon of fermentation, it follows that the whole theory, commanding
though it does such general assent, is shown to be untenable; it is
simply inaccurate.”

The experiments to which Dr. Brefeld alludes, consisted in keeping under
continued study with the microscope, in a room specially prepared for
the purpose, one or more cells of ferment in wort, in an atmosphere of
carbonic acid gas, free from the least traces of free oxygen. We have,
however, recognized the fact that the increase of a ferment out of
contact with air is only possible in the case of a very young specimen;
but our author employed brewer’s yeast taken after fermentation, and to
this fact we may attribute the non-success of his growths. Dr. Brefeld,
without knowing it, operated on yeast in one of the states in which it
requires gaseous oxygen to enable it to germinate again. A perusal of
what we have previously written on the subject of the revival of yeast,
according to its age, will show how widely the time required for such
revival may vary in different cases. What may be perfectly true of the
state of a yeast to-day may not be so to-morrow, since yeast is
continually undergoing modifications. We have already shown the energy
and activity with which a ferment can vegetate in the presence of free
oxygen, and we have pointed out the great extent to which a very small
quantity of oxygen held in solution in fermenting liquids can operate at
the beginning of fermentation. It is this oxygen that produces revival
in the cells of the ferment and enables them to resume the faculty of
germinating and continuing their life, and of multiplying when deprived
of air.

In our opinion, a simple reflection should have guarded Dr. Brefeld
against the interpretation which he has attached to his observations. If
a cell of ferment cannot bud or increase without absorbing oxygen,
either free or held in solution in the liquid, the ratio between the
weight of ferment formed during fermentation and that of oxygen used up
must be constant. We had, however, clearly established, as far back as
1861, the fact that this ratio is extremely variable, a fact, moreover,
which is placed beyond doubt by the experiments described in the
preceding paragraph. Though but small quantities of oxygen are absorbed,
a considerable weight of ferment may be generated; whilst if the ferment
has abundance of oxygen at its disposal, it will absorb much, and the
weight of yeast formed will be still greater. The ratio between the
weight of ferment formed and that of sugar decomposed may pass through
all stages between certain very wide limits, the variations depending on
the greater or less absorption of free oxygen. And in this fact, we
believe, lies one of the most essential supports of the theory which we
advocate. In denouncing the impossibility, as he considered it, of a
ferment living without air or oxygen, and so acting in defiance of that
law which governs all living beings, animal or vegetable, Dr. Brefeld
ought also to have borne in mind the fact which we have pointed out,
that alcoholic yeast is not the only organized ferment which lives in an
anaërobian state. It is really a small matter that one more ferment
should be placed in a list of exceptions to the generality of living
beings, for whom there is a rigid law in their vital economy which
requires for continued life a continuous respiration, a continuous
supply of free oxygen. Why, for instance, has Dr. Brefeld omitted the
facts bearing on the life of the vibrios of butyric fermentation?
Doubtless he thought we were equally mistaken in these: a few actual
experiments would have put him right.

These remarks on the criticisms of Dr. Brefeld are also applicable to
certain observations of M. Moritz Traube’s, although, as regards the
principal object of Dr. Brefeld’s attack, we are indebted to M. Traube
for our defence. This gentleman maintained the exactness of our results
before the Chemical Society of Berlin, proving by fresh experiments that
yeast is able to live and multiply without the intervention of oxygen.
“My researches,” he said, “confirm in an indisputable manner M.
Pasteur’s assertion that the multiplication of yeast can take place in
media which contain no trace of free oxygen.... M. Brefeld’s assertion
to the contrary is erroneous.” But, immediately afterwards, M. Traube
adds: “Have we here a confirmation of Pasteur’s theory? By no means. The
results of my experiments demonstrate, on the contrary, that this theory
has no sure foundation.” What were these results? Whilst proving that
yeast could live without air, M. Traube, as we ourselves did, found that
it had great difficulty in living under these conditions; indeed he
never succeeded in obtaining more than the first stages of true
fermentation. This was doubtless for the two following reasons—first, in
consequence of the accidental production of secondary and diseased
fermentations, which frequently prevent the propagation of alcoholic
ferment; and, secondly, in consequence of the original exhausted
condition of the yeast employed. As long ago as 1861 we pointed out the
slowness and difficulty of the vital action of yeast when deprived of
air, and a little way back, in the preceding paragraph, we have called
attention to certain fermentations that cannot be completed under such
conditions without going into the causes of these peculiarities. M.
Traube expresses himself thus: “Pasteur’s conclusion, that yeast in the
absence of air is able to derive the oxygen necessary for its
development from sugar, is erroneous; its increase is arrested, even
when the greater part of the sugar still remains undecomposed. _It is in
a mixture of albuminous substances that yeast, when deprived of air,
finds the materials for its development._” This last assertion of M.
Traube’s is entirely disproved by those fermentation experiments in
which, after suppressing the presence of albuminous substances, the
action, nevertheless, went on in a purely inorganic medium, out of
contact with air, a fact of which we shall give irrefutable proofs.[134]


          § IV.—Fermentation of Dextro-Tartrate of Lime.[135]


Tartrate of lime, in spite of its insolubility in water, is capable of
complete fermentation in a mineral medium.

If we put some pure tartrate of lime, in the form of a granulated,
crystalline powder, into pure water, together with some sulphate of
ammonia and phosphates of potassium and magnesium, in very small
proportions, a spontaneous fermentation will take place in the deposit
in the course of a few days, although no germs of ferment have been
added. A living, organized ferment, of the vibrionic type, filiform,
with tortuous motions, and often of immense length, forms spontaneously
by the development of some germs derived in some way from the inevitable
particles of dust floating in the air or resting on the surface of the
vessels or materials which we employ. The germs of the vibrios concerned
in putrefaction are diffused around us on every side, and, in all
probability, it is one or more of these germs that develop in the medium
in question. In this way they effect the decomposition of the tartrate,
from which they must necessarily obtain the carbon of their food,
without which they cannot exist, while the nitrogen is furnished by the
ammonia of the ammoniacal salt, the mineral principles by the phosphate
of potassium and magnesium, and the sulphur by the sulphate of ammonia.
How strange to see organization, life, and motion originating under such
conditions! Stranger still to think that this organization, life, and
motion are effected without the participation of free oxygen. Once the
germ gets a primary impulse on its living career by access of oxygen, it
goes on reproducing indefinitely, absolutely without atmospheric air.
Here then we have a fact which it is important to establish beyond the
possibility of doubt, that we may prove that yeast is not the only
organized ferment able to live and multiply when out of the influence of
free oxygen.

Into a flask, like that represented in Fig. 67, of 2·5 litres (about
four pints) in capacity, we put:—

         Pure, crystallized, neutral tartrate of  100 grammes.
         lime

         Phosphate of ammonia                        1 gramme.

         Phosphate of magnesium                      1 gramme.

         Phosphate of potassium                    0·5 gramme.

         Sulphate of ammonia                       0·5 gramme.

         (1 gramme=15·43 grains.)

To this we added pure distilled water, so as to entirely fill the flask.

In order to expel all the air dissolved in the water and adhering to the
solid substances, we first placed our flask in a bath of chloride of
calcium, in a large cylindrical white iron pot, set over a flame. The
exit-tube of the flask was plunged in a test-tube of Bohemian glass
three-quarters full of distilled water, and also heated by a flame. We
boiled the liquids in the flask and test-tube for a sufficient time to
expel all the air contained in them. We then withdrew the heat from
under the test-tube, and immediately afterwards covered the water which
it contained with a layer of oil, and then permitted the whole apparatus
to cool down.

[Illustration: Fig. 67.]

Next day we applied a finger to the open extremity of the exit-tube,
which we then plunged in a vessel of mercury. In this particular
experiment which we are describing, we permitted the flask to remain in
this state for a fortnight. It might have remained for a century without
ever manifesting the least sign of fermentation, the fermentation of the
tartrate being a consequence of life, and life after the boiling no
longer existed in the flask. When it was evident that the contents of
the flask were perfectly inert, we impregnated them rapidly, as
follows:—All the liquid contained in the exit-tube was removed by means
of a fine caoutchouc tube, and replaced by about 1 c.c. (about 17
minims) of liquid and deposit from another flask, similar to the one we
have described, but which had been fermenting spontaneously for twelve
days; we lost no time in refilling completely the exit-tube with water
which had been first boiled and then cooled down in carbonic acid gas.
This operation lasted only a few minutes. The exit-tube was again
plunged under mercury. Subsequently the tube was not moved from under
the mercury, and as it formed part of the flask, and there was neither
cork nor india-rubber, any introduction of air was consequently
impossible. The small quantity of air introduced during the impregnation
was insignificant, and it might even be shown that it injured rather
than assisted the growth of the organisms, inasmuch as these consisted
of adult individuals which had lived without air and might be liable to
be damaged or even destroyed by it. Be this as it may, in a subsequent
experiment we shall find the possibility removed of any aeration taking
place in this way, however infinitesimal, so that no doubt may linger on
this subject.

The following days the organisms multiplied, the deposit of tartrate
gradually disappeared, and a sensible ferment action was manifest on the
surface, and throughout the bulk of the liquid. The deposit seemed
lifted up in places, and was covered with a layer of a dark-grey colour,
puffed up, and having an organic and gelatinous appearance. For several
days, in spite of this action in the deposit, we detected no
disengagement of gas, except when the flask was slightly shaken, in
which case rather large bubbles adhering to the deposit rose, carrying
with them some solid particles, which quickly fell back again, whilst
the bubbles diminished in size as they rose, from being partially taken
into solution, in consequence of the liquid not being saturated. The
smallest bubbles had even time to dissolve completely before they could
reach the surface of the liquid. In course of time the liquid was
saturated, and the tartrate was gradually displaced by mammillated
crusts, or clear, transparent crystals of carbonate of lime at the
bottom and on the sides of the vessel.

The impregnation took place on February 10th, and on March 15th the
liquid was nearly saturated. The bubbles then began to lodge in the bent
part of the exit-tube, at the top of the flask. A glass measuring-tube
containing mercury was now placed with its open end over the point of
the exit-tube under the mercury in the trough, so that no bubble might
escape. A steady evolution of gas went on from the 17th to the 18th,
17·4 c.c. (1·06 cubic inches) having been collected. This was proved to
be nearly absolutely pure carbonic acid, as indeed might have been
suspected from the fact that the evolution did not begin before a
distinct saturation of the liquid was observed.[136]

The liquid, which was turbid on the day after its impregnation, had, in
spite of the liberation of gas, again become so transparent that we
could read our handwriting through the body of the flask.
Notwithstanding this, there was still a very active operation going on
in the deposit, but it was confined to that spot. Indeed, the swarming
vibrios were bound to remain there, the tartrate of lime being still
more insoluble in water saturated with carbonate of lime than it is in
pure water. A supply of carbonaceous food, at all events, was absolutely
wanting in the bulk of the liquid. Every day we continued to collect and
analyze the total amount of gas disengaged. To the very last, it was
composed of pure carbonic acid gas. Only during the first few days did
the absorption by the concentrated potash leave a very minute residue.
By April 26th all liberation of gas had ceased, the last bubbles having
risen in the course of April 23rd. The flask had been all the time in
the oven, at a temperature between 25° C. and 28° C. (77° F. and 83°
F.). The total volume of gas collected was 2·135 litres (130·2 cubic
inches). To obtain the whole volume of gas formed we had to add to this
what was held in the liquid in the state of acid carbonate of lime. To
determine this we poured a portion of the liquid from the flask into
another flask of similar shape, but smaller, up to a gauge-mark on the
neck.[137] This smaller flask had been previously filled with carbonic
acid. The carbonic acid of the fermented liquid was then expelled by
means of heat, and collected over mercury. In this way we found a volume
of 8·322 litres (508 cubic inches) of gas in solution, which, added to
2·135 litres, gave a total of 10·457 litres (638·2 cubic inches) at 20°
and 760, which calculated to 0° C. and 760 mm. atmospheric pressure (32°
F. and 30 inches) gave a weight of 19·70 grammes (302·2 grains) of
carbonic acid.

Exactly half of the lime of the tartrate employed got used up in the
soluble salts formed during fermentation; the other half was partly
precipitated in the form of carbonate of lime, partly dissolved in the
liquid by the carbonic acid. The soluble salts seemed to us to be a
mixture or combination of 1 equivalent of metacetate of lime, with 2
equivalents of the acetate, for every 10 equivalents of carbonic acid
produced, the whole corresponding to the fermentation of 3 equivalents
of neutral tartrate of lime.[138] This point, however, is worthy of
being studied with greater care: the present statement of the nature of
the products formed is given with all reserve. For our point, indeed,
the matter is of little importance, since the equation of the
fermentation does not concern us.

[Illustration: Fig. 68.]

After the completion of fermentation there was not a trace of tartrate
of lime remaining at the bottom of the vessel: it had disappeared
gradually as it got broken up into the different products of
fermentation, and its place was taken by some crystallized carbonate of
lime—the excess, namely, which had been unable to dissolve by the action
of the carbonic acid. Associated, moreover, with this carbonate of lime
there was a quantity of some kind of animal matter, which, under the
microscope, appeared to be composed of masses of granules mixed with
very fine filaments of varying lengths, studded with minute dots, and
presenting all the characteristics of a nitrogenous organic
substance.[139] That this was really the ferment is evident enough from
all that we have already said. To convince ourselves more thoroughly of
the fact, and at the same time to enable us to observe the mode of
activity of the organism, we instituted the following supplementary
observation. Side by side with the experiment just described, we
conducted a similar one, which we intermitted after the fermentation was
somewhat advanced, and about half of the tartrate dissolved. Breaking
off, with a file, the exit tube at the point where the neck began to
narrow off, we took some of the deposit from the bottom by means of a
long, straight piece of tubing, in order to bring it under microscopical
examination. We found it to consist of a host of long filaments of
extreme tenuity, their diameter being about 1/1000th of a millimetre
(0·000039 in.); their length varied, in some cases being as much as
1/20th of a millimetre (0·0019 in.). A crowd of these long vibrios were
to be seen creeping slowly along, with a sinuous movement, showing
three, four, or even five flexures. The filaments that were at rest had
the same aspect as these last, with the exception that they appeared
punctate, as though composed of a series of granules arranged in
irregular order. No doubt these were vibrios in which vital action had
ceased, exhausted specimens which we may compare with the old granular
ferment of beer, whilst those in motion may be compared with young and
vigorous yeast. The absence of movement in the former seems to prove
that this view is correct. Both kinds showed a tendency to form
clusters, the compactness of which impeded the movements of those which
were in motion. Moreover, it was noticeable that the masses of these
latter rested on tartrate not yet dissolved, whilst the granular
clusters of the others rested directly on the glass, at the bottom of
the flask, as if, having decomposed the tartrate, the only carbonaceous
food at their disposal, they had then died at the spot where we captured
them from inability to escape, precisely in consequence of that state of
entanglement which they combined to form, during the period of their
active development. Besides these we observed vibrios of the same
diameter, but of much smaller length, whirling round with great
rapidity, and darting backwards and forwards; these were probably
identical with the longer ones, and possessed greater freedom of
movement, no doubt in consequence of their greater shortness. Not one of
these vibrios could be found throughout the mass of the liquid.

We may remark that as there was a somewhat putrid odour from the deposit
in which the vibrios swarmed, the action must have been one of
reduction, and no doubt to this fact was due the greyish coloration of
the deposit. We suppose that the substances employed, however pure,
always contain some trace of iron, which becomes converted into the
sulphide, the black colour of which would modify the originally white
deposit of insoluble tartrate and phosphate.

But what is the nature of these vibrios? We have already said that we
believe that they are nothing but the ordinary vibrios of putrefaction,
reduced to a state of extreme tenuity by the special conditions of
nutrition involved in the fermentable medium used; in a word, we think
that the fermentation in question might be called putrefaction of
tartrate of lime. It would be easy enough to determine this point by
growing the vibrios of such a fermentation in media adapted to the
production of the ordinary forms of vibrio; but this is an experiment
which we have not ourselves tried.

One word more on the subject of these curious beings. In a great many of
them there appears to be something like a clear spot, a kind of bead, at
one of their extremities. This is an illusion arising from the fact that
the extremity of these vibrios is curved, hanging downwards, thus
causing a greater refraction at that particular point, and leading us to
think that the diameter is greater at that extremity. We may easily
undeceive ourselves if we watch the movements of the vibrio, when we
will readily recognize the bend, especially as it is brought into the
vertical plane passing over the rest of the filament. In this way we
will see the bright spot, the head disappear, and then reappear.

The chief inference that it concerns us to draw from the preceding facts
is one which cannot admit of doubt, and which we need not insist on any
further—namely, that vibrios, as met with in the fermentation of neutral
tartrate of lime, are able to live and multiply when entirely deprived
of air.


  § V.—Another Example of Life Without Air—Fermentation of Lactate of
                                 Lime.


As another example of life without air, accompanied by fermentation
properly so called, we may lastly cite the fermentation of lactate of
lime in a mineral medium.

In the experiment described in the last paragraph, it will be remembered
that the ferment-liquid and the germs employed in its impregnation came
in contact with air, although only for a very brief time. Now,
notwithstanding that we possess exact observations which prove that the
diffusion of oxygen and nitrogen in a liquid absolutely deprived of air,
so far from taking place rapidly, is, on the contrary, a very slow
process indeed; yet we were anxious to guard the experiment that we are
about to describe from the slightest possible trace of oxygen at the
moment of impregnation.

We employed a liquid prepared as follows: Into from 9 to 10 litres
(somewhat over 2 gallons) of pure water the following salts[140] were
introduced successively, viz:—

              Pure lactate of lime           225 grammes
              Phosphate of ammonia           0·75 grammes
              Phosphate of potassium         0·4 grammes
              Sulphate of magnesium          0·4 grammes
              Sulphate of ammonia            0·2 grammes
              [1 gramme=15·43 grains.]

[Illustration: Fig. 69.]

On March 23rd, 1875, we filled a 6 litre (about 11 pints) flask, of the
shape represented in Fig. 69, and placed it over a heater. Another flame
was placed below a vessel containing the same liquid, into which the
curved tube of the flask was plunged. The liquids in the flask and in
the basin were raised to boiling together, and kept in this condition
for more than half-an-hour, so as to expel all the air held in solution.
The liquid was several times forced out of the flask by the steam, and
sucked back again; but the portion which re-entered the flask was always
boiling. On the following day, when the flask had cooled, we transferred
the end of the delivery tube to a vessel full of mercury and placed the
whole apparatus in an oven at a temperature varying between 25° C. and
30° C. (77° F. and 86° F.); then, after having refilled the small
cylindrical tap-funnel with carbonic acid, we passed into it with all
necessary precautions 10 c.c. (0·35 fl. oz.) of a liquid similar to that
described, which had been already in active fermentation for several
days out of contact with air and now swarmed with vibrios. We then
turned the tap of the funnel, until only a small quantity of liquid was
left, just enough to prevent the access of air. In this way the
impregnation was accomplished without either the ferment-liquid or the
ferment-germs having been brought in contact, even for the shortest
space, with the external air. The fermentation, the occurrence of which
at an earlier or later period depends for the most part on the condition
of the impregnating germs, and the number introduced in the act, in this
case began to manifest itself by the appearance of minute bubbles from
March 29th. But not till April 9th did we observe bubbles of larger size
rise to the surface. From that date onward they continued to come in
increasing number, from certain points at the bottom of the flask, where
a deposit of earthy phosphates existed; and at the same time the liquid,
which for the first few days remained perfectly clear, began to grow
turbid in consequence of the development of vibrios. It was on the same
day that we first observed a deposit on the sides of carbonate of lime
in crystals.

It is a matter of some interest to notice here that, in the mode of
procedure adopted, everything combined to prevent the interference of
air. A portion of the liquid expelled at the beginning of the
experiment, partly because of the increased temperature in the oven and
partly also by the force of the gas, as it began to be evolved from the
fermentative action, reached the surface of the mercury, where, being
the most suitable medium we know for the growth of bacteria, it speedily
swarmed with these organisms.[141] In this way any passage of air, if
such a thing were possible, between the mercury and the sides of the
delivery-tube was altogether prevented, since the bacteria would consume
every trace of oxygen which might be dissolved in the liquid lying on
the surface of the mercury. Hence it is impossible to imagine that the
slightest trace of oxygen could have got into the liquid in the flask.

Before passing on we may remark that in this ready absorption of oxygen
by bacteria we have a means of depriving fermentable liquids of every
trace of that gas with a facility and success equal or even greater than
by the method of preliminary boiling. Such a solution as we have
described, if kept at summer heat, without any previous boiling, becomes
turbid in the course of twenty-four hours from a _spontaneous_
development of bacteria; and it is easy to prove that they absorb all
the oxygen held in solution.[142] If we completely fill a flask of a few
litres capacity (about a gallon) (Fig. 67) with the liquid described,
taking care to have the delivery-tube also filled, and its opening
plunged under mercury, and, forty-eight hours afterwards, by means of a
chloride of calcium bath, expel from the liquid on the surface of the
mercury all the gas which it holds in solution, this gas, when analyzed,
will be found to be composed of a mixture of nitrogen and carbonic acid
gas, _without the least trace of oxygen_. Here, then, we have an
excellent means of depriving the fermentable liquid of air; we have
simply to completely fill a flask with the liquid, and place it in the
oven, merely avoiding any addition of butyric vibrios before the lapse
of two or three days. We may wait even longer; and then, if the liquid
does not become impregnated spontaneously with vibrio germs, the liquid,
which at first was turbid from the presence of bacteria, will become
bright again, since the bacteria when deprived of life, or, at least, of
the power of moving, after they have exhausted all the oxygen in
solution, will fall inert to the bottom of the vessel. On several
occasions, we have determined this interesting fact, which tends to
prove that the butyric vibrios cannot be regarded as another form of
bacteria, inasmuch as, on the hypothesis of an original relation between
the two productions, butyric fermentation ought in every case to follow
the growth of bacteria.

We may also call attention to another striking experiment, well suited
to show the effect of differences in the composition of the medium upon
the propagation of microscopic beings. The fermentation which we last
described commenced on March 27th and continued until May 10th; that to
which we are now to refer, however, was completed in four days, the
liquid employed being similar in composition and quantity to that
employed in the former experiment. On April 23rd, 1875, we filled a
flask of the same shape as that represented in Fig. 69, and of similar
capacity, viz., 6 litres, with a liquid composed as described at page
293. This liquid had been previously left to itself for five days in
large open flasks, in consequence of which it had developed an abundant
growth of bacteria. On the fifth day a few bubbles, rising from the
bottom of the vessels, at long intervals, betokened the commencement of
butyric fermentation, a fact, moreover, confirmed by the microscope, in
the appearance of the vibrios of this fermentation in specimens of the
liquid taken from the bottom of the vessels, the middle of its mass, and
even in the layer on the surface that was swarming with bacteria. We
transferred the liquid so prepared to the 6-litre flask arranged over
the mercury. By evening a tolerably active fermentation had begun to
manifest itself. On the 24th this fermentation was proceeding with
astonishing rapidity, which continued during the 25th and 26th. During
the evening of the 26th it slackened, and on the 27th all signs of
fermentation had ceased. This was not, as might be supposed, a sudden
stoppage, due to some unknown cause; the fermentation was actually
completed, for when we examined the fermented liquid on the 28th we
could not find the smallest quantity of lactate of lime. If the needs of
industry should ever require the production of large quantities of
butyric acid, there would, beyond doubt, be found in the preceding fact
valuable information in devising an easy method of preparing that
product in abundance.[143]

Before we go any further, let us devote some attention to the vibrios of
the preceding fermentations.

On May 27th, 1862, we completely filled a flask, capable of holding
2·780 litres (about five pints), with the solution of lactate and
phosphates.[144] We refrained from impregnating it with any germs. The
liquid became turbid from a development of bacteria, and then underwent
butyric fermentation. By June 9th the fermentation had become
sufficiently active to enable us to collect in the course of twenty-four
hours, over mercury, as in all our experiments, about 100 cc. (about 6
cubic inches) of gas. By June 11th, judging from the volume of gas
liberated in the course of twenty-four hours, the activity of the
fermentation had doubled. We examined a drop of the turbid liquid. Here
are the notes accompanying the sketch (Fig. 70) as they stand in our
note-book:—“A swarm of vibrios, so active in their movements that the
eye has great difficulty in following them. They may be seen in pairs
throughout the field, apparently making efforts to separate from each
other. The connection would seem to be by some invisible, gelatinous
thread, which yields so far to their efforts that they succeed in
breaking away from actual contact, but yet are, for a while, so far
restrained that the movements of one have a visible effect on those of
the other. By and by, however, we see a complete separation effected,
and each moves on its separate way with an activity still greater than
it had before.”

[Illustration: Fig. 70.]

One of the best methods that can be employed for the microscopical
examination of these vibrios, quite out of contact with air, is the
following:—After butyric fermentation has been going on for several days
in a flask, A (Fig. 71), we connect this flask by an india-rubber tube
with one of the flattened bulbs previously described, page 156 (Fig.
31), which we then place on the stage of the microscope (Fig. 71). When
we wish to make an observation we close, under the mercury, at the point
_b_, the end of the drawn-out and bent delivery-tube. The continued
evolution of gas soon exerts such a pressure within the flask, that when
we open the tap _r_, the liquid is driven into the bulb _l l_, until it
becomes quite full and the liquid flows over into the glass V. In this
manner we may bring the vibrios under observation without their coming
into contact with the least trace of air, and with as much success as if
the bulb, which takes the place of an object glass, had been plunged
into the very centre of the flask. The movements and fissiparous
multiplication of the vibrios may thus be seen in all their beauty, and
it is indeed a most interesting sight. The movements do not immediately
cease when the temperature is suddenly lowered, even to a considerable
extent, 15° C. (59° F.) for example; they are only slackened.
Nevertheless, it is better to observe them at the temperatures most
favourable to fermentation, even in the oven where the vessels employed
in the experiment are kept at a temperature between 25° C. and 30° C.
(77° F. and 86° F.).

[Illustration: Fig. 71.]

We may now continue our account of the fermentation which we were
studying when we made this last digression. On June 17th that
fermentation produced three times as much gas as it did on June 11th,
when the residue of hydrogen, after absorption by potash, was 72·6 per
cent.; whilst on the 17th it was only 49·2 per cent. Let us again
discuss the microscopic aspect of the turbid liquid at this stage.
Appended is the sketch we made (Fig. 72) and our notes on it:—“A most
beautiful object: vibrios all in motion, advancing or undulating. They
have grown considerably in bulk and length since the 11th; many of them
are joined together into long sinuous chains, very mobile at the
articulations, visibly less active and more wavering in proportion to
the number that go to form the chain, or the length of the individuals.”
This description is applicable to the majority of the vibrios which
occur in cylindrical rods and are homogeneous in aspect. There are
others, of rare occurrence in chains, which have a clear corpuscle, that
is to say, a portion more refractive than the other parts of the
segments, at one of their extremities. Sometimes the foremost segment
has the corpuscle at one end, sometimes at the other. The long segments
of the commoner kind attain a length of from 10 to 30 and even 45
thousandths of a millimetre. Their diameter is from 1-½ to 2, very
rarely 3, thousandths of a millimetre.[145]

[Illustration: Fig. 72.]

[Illustration: Fig. 73.]

On June 28th, fermentation was quite finished; there was no longer any
trace of gas, nor any lactate in solution. All the infusoria were lying
motionless at the bottom of the flask. The liquid clarified by degrees,
and in the course of a few days became quite bright. Here we may
inquire, were these motionless infusoria, which from complete exhaustion
of the lactate, the source of the carbonaceous part of their food, were
now lying inert at the bottom of the fermenting vessel—were they dead
beyond power of revival?[146] The following experiment leads us to
believe that they were not perfectly lifeless, and that they behave in
the same manner as the yeast of beer, which, after it has decomposed all
the sugar in a fermentable liquid, is ready to revive and multiply in a
fresh saccharine medium. On April 22nd, 1875, we left in the oven, at a
temperature of 25° C. (77° F.), a fermentation of lactate of lime that
had been completed. The delivery tube of the flask, A, (Fig. 73) in
which it had taken place had never been withdrawn from under the
mercury. We kept the liquid under observation daily, and saw it
gradually become brighter; this went on for fifteen days. We then filled
a similar flask, B, with the solution of lactate, which we boiled, not
only to kill the germs of vibrios which the liquid might contain, but
also to expel the air that it held in solution. When the flask, B, had
cooled, we connected the two flasks, avoiding the introduction of
air[147], after having slightly shaken the flask, A, to stir up the
deposit at the bottom. There was then a pressure, due to carbonic acid
at the end of the delivery tube of this latter flask, at the point _a_,
so that on opening the taps _r_ and _s_, the deposit at the bottom of
flask A was driven over into flask B, which in consequence was
impregnated with the deposit of a fermentation that had been completed
fifteen days before. Two days after impregnation, the flask B began to
show signs of fermentation. It follows, that the deposit of vibrios of a
completed butyric fermentation may be kept, at least for a certain time,
without losing the power of causing fermentation. It furnishes a butyric
ferment, capable of revival and action in a suitable, fresh, fermentable
medium.

The reader who has attentively studied the facts which we have placed
before him cannot, in our opinion, entertain the least doubt on the
subject of the possible multiplication of the vibrios of a fermentation
of lactate of lime out of contact with atmospheric oxygen. If fresh
proofs of this important proposition were necessary, they might be found
in the following observations, from which it may be inferred that
atmospheric oxygen is capable of suddenly checking a fermentation
produced by butyric vibrios, and rendering them absolutely motionless,
so that it cannot be necessary to enable them to live. On May 7th, 1862,
we placed in the oven a flask holding 2·580 litres (4-½ pints), and
filled with the solution of lactate of lime and phosphates, which we had
impregnated on the 9th with two drops of a liquid in butyric
fermentation. In the course of a few days fermentation declared itself:
on the 16th it was in progress, but feebly; on the 18th it was active;
on the 30th it was very active. On June 1st it yielded hourly 35 c.c.
(2·3 cubic inches) of gas, containing ten per cent. of hydrogen. On the
2nd we began the study of the action of air on the vibrios of this
fermentation. To do this we cut off the delivery-tube on a level with
its point of junction to the flask, then with a 50 c.c. pipette we took
out that quantity (1-3/4 fl. oz.) of liquid which was, of course,
replaced at once by air. We then reversed the flask with the opening
under the mercury, and shook it every ten minutes for more than an hour.
Wishing to make sure, to begin with, that the oxygen had been absorbed,
we connected under the mercury the beak of the flask by means of a thin
india-rubber tube filled with water, with a small flask, the neck of
which had been drawn out, and was filled with water; we then raised the
large flask with the smaller kept above it. A Mohr’s clip, which closed
the india-rubber tube, and which we then opened, permitted the water
contained in the small flask to pass into the large one, whilst the gas,
on the contrary, passed upwards from the large flask into the small one.
We analyzed the gas immediately, and found that, allowing for carbonic
acid and hydrogen, it did not contain more than 14·2 per cent. of
oxygen, which corresponds to an absorption of 6·6 c.c., or of 3·3 c.c.
(0·2 cubic inch) of oxygen for the 50 c.c. (3·05 cubic inches) of air
employed. Lastly, we again established connection by an india-rubber
tube between the flasks, after having seen by microscopical examination
that the movements of the vibrios were very languid. Fermentation had
become less vigorous without having actually ceased, no doubt because
some portions of the liquid had not been brought into contact with the
atmospheric oxygen, in spite of the prolonged shaking that the flask had
undergone after the introduction of the air. Whatever the cause might
have been, the significance of the phenomenon is not doubtful. To assure
ourselves further of the effect of air on the vibrios, we half filled
two test tubes with the fermenting liquid taken from another
fermentation which had also attained its maximum of intensity, into one
of which we passed a current of air, into the other carbonic acid gas.
In the course of half an hour, all the vibrios in the aerated tube were
dead, or at least motionless, and fermentation had ceased. In the other
tube, after three hours’ exposure to the effects of the carbonic acid
gas, the vibrios were still very active, and fermentation was going on.

There is a most simple method of observing the deadly effect of
atmospheric air upon vibrios. We have seen in the microscopical
examination made by means of the apparatus represented in Fig. 71, how
remarkable were the movements of the vibrios when absolutely deprived of
air, and how easy it was to discern them. We will repeat this
observation, and at the same time make a comparative study of the same
liquid, under the microscope, in the ordinary way, that is to say, by
placing a drop of the liquid on an object-glass, and covering it with a
thin glass slip, a method which must necessarily bring the drop into
contact with air, if only for a moment. It is surprising what a
remarkable difference is observed immediately between the movements of
the vibrios in the bulb and of those under the glass. In the case of the
latter we generally see all movement at once cease near the edges of the
glass, where the drop of liquid is in direct contact with the air; the
movements continue for a longer or shorter time about the centre, in
proportion as the air is more or less intercepted by the vibrios at the
circumference of the liquid. It does not require much skill in
experiments of this kind to enable one to see plainly that immediately
after the glass has been placed on the drop, which has been affected all
over by atmospheric air, the whole of the vibrios seem to languish and
to manifest symptoms of illness—we can think of no better expression to
explain what we see taking place—and that they gradually recover their
activity about the centre, in proportion as they find themselves in a
part of the medium that is less affected by the presence of oxygen.

Some most curious facts are to be found in connection with an
observation, the correlative and inverse of the foregoing, on the
ordinary aërobian bacteria. If we examine below the microscope a drop of
liquid full of these organisms under a coverslip, we very soon observe a
cessation of motion in all the bacteria which lie in the central portion
of the liquid, where the oxygen rapidly disappears to supply the
necessities of the bacteria existing there; whilst, on the other hand,
near the edges of the cover-glass the movements are very active, in
consequence of the constant supply of air. In spite of the speedy death
of the bacteria beneath the centre of the glass, we see life prolonged
there if by chance a bubble of air has been enclosed. All round this
bubble a vast number of bacteria collect in a thick, moving circle, but
as soon as all the oxygen of the bubble has been absorbed they fall
apparently lifeless, and are scattered by the movement of the
liquid.[148]

We may here be permitted to add, as a purely historical matter, that it
was these two observations just described, made successively one day in
1861, on vibrios and bacteria, that first suggested to us the idea of
the possibility of life without air, and caused us to think that the
vibrios which we met so frequently in our lactic fermentations must be
the true butyric ferment.

We may pause a moment to consider an interesting question in reference
to the two characters under which vibrios appear in butyric
fermentations. What is the reason that some vibrios exhibit refractive
corpuscles, generally of a lenticular form, such as we see in Fig. 72?
We are strongly inclined to believe that these corpuscles have to do
with a special mode of reproduction in the vibrios, common alike to the
anaërobian forms which we are studying, and the ordinary aërobian forms
in which also the corpuscles of which we are speaking may occur. The
explanation of the phenomenon, from our point of view, would be that,
after a certain number of fissiparous generations, and under the
influence of variations in the composition of the medium, which is
constantly changing through fermentation as well as through the active
life of the vibrios themselves, cysts, which are simply the refractive
corpuscles, form along them at different points. From these gemmules we
have ultimately produced vibrios, ready to reproduce others by the
process of transverse division for a certain time, to be themselves
encysted later on. Various observations incline us to believe that, in
their ordinary form of minute, soft, exuberant rods, the vibrios perish
when submitted to desiccation, but when they occur in the corpuscular or
encysted form they possess unusual powers of resistance, and may be
brought to the state of dry dust and be wafted about by winds. None of
the matter which surrounds the corpuscle or cyst seems to take part in
the preservation of the germ, when the cyst is formed, for it is all
re-absorbed, gradually leaving the cyst bare. The cysts appear as masses
of corpuscles, in which the most practised eye cannot detect anything of
an organic nature, or anything to remind one of the vibrios which
produced them; nevertheless, these minute bodies are endowed with a
latent vital action, and only await favourable conditions to develop
long rods of vibrios. We are not, it is true, in a position to adduce
any very forcible proofs in support of these opinions. They have been
suggested to us by experiments, none of which, however, have been
absolutely decisive in their favour. We may cite one of our observations
on this subject.

In a fermentation of glycerine in a mineral medium—the glycerine was
fermenting under the influence of butyric vibrios—after we had
determined the, we may say, exclusive presence of lenticular vibrios,
with refractive corpuscles, we observed the fermentation, which, for
some unknown reason, had been very languid, suddenly become extremely
active, but now through the influence of ordinary vibrios. The gemmules
with brilliant corpuscles had almost disappeared; we could see but very
few, and those now consisted of the refractive bodies alone, the bulk of
the vibrios accompanying them having undergone some process of
re-absorption.

Another observation which still more closely accords with this
hypothesis is given in our work on the silkworm disease (vol. i., page
256). We there demonstrate that, when we place in water some of the dust
formed of desiccated vibrios, containing a host of these refractive
corpuscles, in the course of a very few hours large vibrios appear,
well-developed rods fully grown, in which the brilliant points are
absent; whilst in the water no process of development from smaller
vibrios is to be discerned, a fact which seems to show that the former
had issued fully grown from the refractive corpuscles, just as we see
_colpoda_ issue with their adult aspect from the dust of their cysts.
This observation, we may remark, furnishes one of the best proofs that
can be adduced against the spontaneous generation of vibrios or
bacteria, since it is probable that the same observation applies to
bacteria. It is true that we cannot say of mere points of dust, examined
under the microscope, that one particular germ belongs to vibrio,
another to bacterium; but how is it possible to doubt that the vibrios
issue, as we see them, from an ovum of some kind, a cyst, or germ, of
determinate character, when, after having placed some of these
indeterminate motes of dust into clean water, we suddenly see, after an
interval of not more than one or two hours, an adult vibrio crossing the
field of the microscope, without our having been able to detect any
intermediate state between its birth and adolescence?

It is a question whether differences in the aspect and nature of
vibrios, which depend upon their more or less advanced age, or are
occasioned by the influence of certain conditions of the medium in which
they propagate, do not bring about corresponding changes in the course
of the fermentation and the nature of its products. Judging at least
from the variations in the proportions of hydrogen and carbonic acid gas
produced in butyric fermentations, we are inclined to think that this
must be the case; nay, more, we find that hydrogen is not even a
constant product in these fermentations. We have met with butyric
fermentations of lactate of lime which did not yield the minutest trace
of hydrogen, or anything besides carbonic acid. Fig. 74 represents the
vibrios which we observed in a fermentation of this kind. They present
no special features. Butyl alcohol is, according to our observations, an
ordinary product, although it varies and is by no means a necessary
concomitant of these fermentations. It might be supposed, since butylic
alcohol may be produced, and hydrogen be in deficit, that the proportion
of the former of these products would attain its maximum when the latter
assumed a minimum. This, however, is by no means the case; even in those
few fermentations that we have met with in which hydrogen was absent,
there was no formation of butylic alcohol.

[Illustration: Fig. 74.]

From a consideration of all the facts detailed in this paragraph we can
have no hesitation in concluding that, on the one hand, in cases of
butyric fermentation, the vibrios which abound in them and constitute
their ferment, live without air or free oxygen; and that, on the other
hand, the presence of gaseous oxygen operates prejudicially against the
movements and activity of those vibrios. But now does it follow that the
presence of minute quantities of air brought into contact with a liquid
undergoing butyric fermentation would prevent the continuance of that
fermentation, or even exercise any check upon it? We have not made any
direct experiments upon this subject; but we should not be surprised to
find that, so far from hindering, air may, under such circumstances,
facilitate the propagation of the vibrios and accelerate fermentation.
This is exactly what happens in the case of yeast. But how could we
reconcile this, supposing it were proved to be the case, with the fact
just insisted on as to the danger of bringing the butyric vibrios into
contact with air? It may be possible that _life without air_ results
from habit, whilst _death through air_ may be brought about by a sudden
change in the conditions of the existence of the vibrios. The following
remarkable experiment is well known: A bird is placed in a glass jar of
one or two litres (60 to 120 cubic inches) in capacity, which is then
closed. After a time the creature exhibits every sign of intense
uneasiness and asphyxia long before it dies; a similar bird of the same
size is introduced into the jar; the death of the latter takes place
instantaneously, whilst the life of the former may still be prolonged
under these conditions for a considerable time, and there is no
difficulty even in restoring the bird to perfect health by taking it out
of the jar. It seems impossible to deny that we have here a case of the
adaptation of an organism to the gradual contamination of the medium;
and so it may likewise happen that the anaërobian vibrios of a butyric
fermentation, which develop and multiply absolutely without free oxygen,
perish immediately when suddenly taken out of their airless medium, and
that the result might be different if they had been gradually brought
under the action of air in small quantities at a time.

We are compelled here to admit that vibrios frequently abound in liquids
exposed to the air, and that they appropriate the atmospheric oxygen,
and could not withstand a sudden removal from its influence. Must we,
then, believe that such vibrios are absolutely different from those of
butyric fermentations? It would, perhaps, be more natural to admit that
in the one case there is an adaptation to life with air, and in the
other case an adaptation to life without air; each of these varieties
perishing when suddenly transferred from its habitual condition to that
of the other, whilst by a series of progressive changes one might be
modified into the other.[149] We know that in the case of alcoholic
ferments, although these can actually live without air, propagation is
wonderfully assisted by the presence of minute quantities of air; and
certain experiments, which we have not yet published, lead us to believe
that, after having lived without air, they cannot be suddenly exposed
with impunity to the influence of large quantities of oxygen.

We must not forget, however, that aërobian torulæ and anaërobian
ferments present an example of organisms apparently identical, in which,
however, we have not yet been able to discover any ties of a common
origin. Hence we were forced to regard them as distinct species; and so
it is possible that there may likewise be aërobian and anaërobian
vibrios without any transformation of the one into the other.

The question has been raised whether vibrios, especially those which we
have shown to be the ferment of butyric and many other fermentations,
are, in their nature, animal or vegetable. M. Ch. Robin attaches great
importance to the solution of this question, of which he speaks as
follows[150]:—“The determination of the nature, whether animal or
vegetable, of organisms, either as a whole or in respect to their
anatomical parts, assimilative or reproductive, is a problem which has
been capable of solution for a quarter of a century. The method has been
brought to a state of remarkable precision, experimentally, as well as
in its theoretical aspects, since those who devote their attention to
the organic sciences consider it indispensable in every observation and
experiment to determine accurately, before anything else, whether the
object of their study is animal or vegetable in its nature, whether
adult or otherwise. To neglect this is as serious an omission for such
students, as for chemists would be the neglecting to determine whether
it is nitrogen or hydrogen, urea or stearine that has been extracted
from a tissue, or which it is whose combinations they are studying in
this or that chemical operation. Now, scarcely any one of those who
study fermentations, properly so called, and putrefactions, ever pay
attention to the preceding data.... Among the observers to whom I allude
even M. Pasteur is to be found, who, even in his most recent
communications, omits to state definitely what is the nature of many of
the ferments which he has studied, with the exception, however, of those
which belong to the cryptogamic group called _torulaceæ_. Various
passages in his works seem to show that he considers the cryptogamic
organisms called _bacteria_, as well as those known as _vibrios_, as
belonging to the animal kingdom (see _Bulletin de l’Académie de
Médecine_, Paris, 1875, pp. 249, 251, especially 256, 266, 267, 289, and
290). These would be very different, at least physiologically, the
former being _aërobian_, whilst the vibrios are _anaërobian_, that is to
say, requiring no air to enable them to live, and being killed by
oxygen, should it be dissolved in the liquid to any considerable
extent.”

We are unable to see the matter in the same light as our learned
colleague does; to our thinking, we should be labouring under a great
delusion were we to suppose “that it is quite as serious an omission not
to determine the animal or vegetable nature of a ferment as it would be
to confound nitrogen with hydrogen, or urea with stearine.” The
importance of the solutions of disputed questions often depends upon the
point of view from which these are regarded. As far as the result of our
labours is concerned, we devoted our attention to these two questions
exclusively:—1. Is the ferment, in every fermentation properly so
called, an organized being? 2. Can this organized being live without
air? Now, what bearing can the question of the animal or vegetable
nature of the ferment, of the organized being, have upon the
investigation of these two problems? In studying butyric fermentation,
for example, we endeavoured to establish these two fundamental
points:—1. _The butyric ferment is a vibrio._ 2. _This vibrio may
dispense with air in its life, and, as a matter of fact, does dispense
with it in the act of producing butyric fermentation._ We did not
consider it at all necessary to pronounce any opinion as to the animal
or vegetable nature of this organism, and, even up to the present
moment, the idea that vibrio is an animal and not a plant is, in our
minds, a matter of sentiment rather than of conviction.

M. Robin, however, would have no difficulty in determining the limits of
the two kingdoms. According to him, “every variety of cellulose is, we
may say, insoluble in ammonia, as also are the reproductive elements of
plants, whether male or female. Whatever phase of evolution the elements
which reproduce a new individual may have reached, treatment with this
reagent, either cold or raised to boiling, leaves them absolutely intact
under the eyes of the observer, except that their contents, from being
partially dissolved, become more transparent. Every vegetable, whether
microscopic or not, every mycelium, and every spore thus preserves in
its entirety its special characteristics of form, volume, and structural
arrangements; whilst in the case of microscopic animals, or the ova and
microscopic embryos of different members of the animal kingdom, the very
opposite is the case.”

We should be glad to learn that the employment of a drop of ammonia
would enable us to pronounce an opinion, with this degree of confidence,
on the nature of the lowest microscopic beings; but is M. Robin
absolutely correct in his assumptions? That gentleman himself remarks
that spermatozoa, which belong to animal organisms, are insoluble in
ammonia, the effect of which is merely to make them paler. If a
difference of action in certain reagents, in ammonia, for example, were
sufficient to determine the limits of the animal and vegetable kingdoms,
might we not argue that there must be a very great and natural
difference between moulds and bacteria, inasmuch as the presence of a
small quantity of acid in the nutritive medium facilitates the growth
and propagation of the former, whilst it is able to prevent the life of
bacteria and vibrios? Although, as is well known, movement is not an
exclusive characteristic of animals, yet we have always been inclined to
regard vibrios as animals, on account of the peculiar character of their
movements. How greatly they differ in this respect from the diatomacæ,
for example! When the vibrio encounters an obstacle it turns, or after
having assured itself by some visual effort or other that it cannot
overcome it, it retraces its steps. The colpoda—undoubted
infusoria—behave in an exactly similar manner. It is true one may argue
that the zoospores of certain cryptogamia exhibit similar movements; but
do not these zoospores possess as much of an animal nature as do the
spermatozoa? As far as bacteria are concerned, when, as already
remarked, we see them crowd round a bubble of air in a liquid to prolong
their life, oxygen having failed them everywhere else, how can we avoid
believing that they are animated by an instinct for life, of the same
kind as that which we find in animals. M. Robin seems to us to be wrong
in supposing that it is possible to draw any absolute line of separation
between the animal and vegetable kingdoms. The settlement of this line,
however, we repeat again, no matter what it may be, has no serious
bearing upon the questions that have been the subject of our researches.

In like manner the difficulty which M. Robin has raised in objecting to
the employment of the word _germ_, when we cannot specify whether the
nature of that germ is animal or vegetable, is in many respects an
unnecessary one. In all the questions which we have discussed, whether
we were speaking of fermentation or spontaneous generation, the word
_germ_ has been used in the sense of _origin of living organism_. If
Liebig, for example, said of an albuminous substance that it gave birth
to ferment, could we contradict him more plainly than by replying: “No;
ferment is an organized being, the germ of which is always present, and
the albuminous substance merely serves by its occurrence to nourish the
germ and its successive generations.”

In our Memoir of 1862, on so-called _spontaneous_ generations, would it
not have been an entire mistake to have attempted to assign specific
names to the microscopic organisms which we met with in the course of
our observations? Not only would we have met with extreme difficulty in
the attempt, arising from the state of extreme confusion which even in
the present day exists in the classification and nomenclature of these
microscopic organisms, but we should have been forced to sacrifice
clearness in our work besides; at all events, we should have wandered
from our principal object, which was the determination of the presence
or absence of life in general, and had nothing to do with the
manifestation of a particular kind of life in this or that species,
animal or vegetable. Thus we have systematically employed the vaguest
nomenclature, such as _mucors_, _torulæ_, _bacteria_, and _vibrios_.
There was nothing arbitrary in our doing this, whereas there is much
that is arbitrary in adopting a definite system of nomenclature, and
applying it to organisms but imperfectly known, the differences or
resemblances between which are only recognizable through certain
characteristics, the true signification of which is obscure. Take, for
example, the extensive array of widely different systems that have been
invented during the last few years for the species of the genera
bacterium and vibrio in the works of Cohn, H. Hoffmann, Hallier, and
Billroth. The confusion which prevails here is very great, although we
do not of course by any means place these different works on the same
footing as regards their respective merits.

M. Robin is, however, right in recognizing the impossibility of
maintaining in the present day, as he formerly did, “that fermentation
is an exterior phenomenon, going on outside cryptogamic cells, a
phenomenon of contact. It is probably,” he adds, “an interior and
molecular action at work in the inmost recesses of the substance of each
cell.” From the day when we first proved that it is possible for all
organized ferments, properly so called, to spring up and multiply from
their respective germs, sown, whether consciously or by accident, in a
mineral medium free from organic and nitrogenous matters other than
ammonia, in which medium the fermentable matter alone is adapted to
provide the ferment with whatever carbon enters into its composition,
from that time forward the theories of Liebig, as well as that of
Berzelius, which M. Robin formerly defended, have had to give place to
others more in harmony with facts. We trust that the day will come when
M. Robin will likewise acknowledge that he has been in error on the
subject of the doctrine of spontaneous generation, which he continues to
affirm, without adducing any direct proofs in support of it, at the end
of the article to which we have been here replying.

We have devoted the greater part of this chapter to the establishing
with all possible exactness the extremely important physiological fact
of life without air, and its correlation to the phenomena of
fermentations properly so called—that is to say, of those which are due
to the presence of microscopic cellular organisms. This is the chief
basis of the new theory that we propose for the explanation of these
phenomena. The details into which we have entered were indispensable on
account of the novelty of the subject no less than on account of the
necessity we were under of combating the criticisms of the two German
naturalists, Drs. Oscar Brefeld and Traube, whose works had cast some
doubts on the correctness of the facts upon which we had based the
preceding propositions. We have much pleasure in adding that at the very
moment when we were revising the proofs of this chapter, we received
from M. Brefeld an essay, dated from Berlin, January, 1876, in which,
after describing his later experimental researches, he owns with
praiseworthy frankness that Dr. Traube and he were both of them
mistaken. Life without air is now a proposition which he accepts as
perfectly demonstrated. He has witnessed it in the case of _mucor
racemosus_, and has also verified it in the case of yeast. “If,” he
says, “after the results of my previous researches, which I conducted
with all possible exactness, I was inclined to consider Pasteur’s
assertions as inaccurate, and to attack them, I have no hesitation now
in recognizing them as true, and in proclaiming the service which
Pasteur has rendered to science in being the first to indicate the exact
relation of things in the phenomenon of fermentation.” In his later
researches. Dr. Brefeld has adopted the method which we have long
employed for demonstrating the life and multiplication of butyric
vibrios in the entire absence of air, as well as the method of
conducting growths in mineral media associated with the fermentable
substance. We need not pause to consider certain other secondary
criticisms of Dr. Brefeld. A perusal of the present work will, we trust,
convince him that they are based on no surer foundation than were his
former criticisms.

To bring one’s self to believe in a truth that has just dawned upon one
is the first step towards progress; to persuade others is the second.
There is a third step, less useful perhaps, but highly gratifying
nevertheless, which is, to convince one’s opponents.

We, therefore, have experienced great satisfaction in learning that we
have won over to our ideas an observer of singular ability, on a subject
which is of the utmost importance to the physiology of cells.


    § VI.—Reply to the Critical Observations of Liebig, Published in
                               1870.[151]


In the Memoir which we published, in 1860, on alcoholic fermentation,
and in several subsequent works, we were led to a different conclusion
on the causes of this very remarkable phenomenon from that which Liebig
had adopted. The opinions of Mitscherlich and Berzelius had ceased to be
tenable in the presence of the new facts which we had brought to light.
From that time we felt sure that the celebrated chemist of Munich had
adopted our conclusions, from the fact that he remained silent on this
question for a long time, although it had been until then the constant
subject of his study, as is shown by all his works. Suddenly there
appeared in the _Annales de Chimie et de Physique_ a long essay,
reproduced from a lecture delivered by him before the Academy of Bavaria
in 1868 and 1869. In this Liebig again maintained, not, however, without
certain modifications, the views which he had expressed in his former
publications, and disputed the correctness of the principal facts
enunciated in our Memoir of 1860, on which were based the arguments
against his theory.

“I had admitted,” he says, “that the resolution of fermentable matter
into compounds of a simpler kind must be traced to some process of
decomposition taking place in the ferment, and that the action of this
same ferment on the fermentable matter must continue or cease according
to the prolongation or cessation of the alteration produced in the
ferment. The molecular change in the sugar would, consequently, be
brought about by the destruction or modification of one or more of the
component parts of the ferment, and could only take place through the
contact of the two substances. M. Pasteur regards fermentation in the
following light:—The chemical action of fermentation is essentially a
phenomenon correlative with a vital action, beginning and ending with
it. He believes that alcoholic fermentation can never occur without the
simultaneous occurrence of organization, development, and multiplication
of globules, or continuous life, carried on from globules already
formed. But the idea that the decomposition of sugar during fermentation
is due to the development of the cellules of the ferment, is in
contradiction with the fact that the ferment is able to bring about the
fermentation of a pure solution of sugar. The greater part of the
ferment is composed of a substance that is rich in nitrogen and contains
sulphur. It contains, moreover, an appreciable quantity of phosphates,
hence it is difficult to conceive how, in the absence of these elements
in a pure solution of sugar undergoing fermentation, the number of cells
is capable of any increase.”

Notwithstanding Liebig’s belief to the contrary, the idea that the
decomposition of sugar during fermentation is intimately connected with
a development of the cellules of the ferment, or a prolongation of the
life of cellules already formed, is in no way opposed to the fact that
the ferment is capable of bringing about the fermentation of a pure
solution of sugar. It is manifest to any one who has studied such
fermentation with the microscope, even in those cases where the
sweetened water has been absolutely pure, that ferment-cells do
multiply, the reason being that the cells carry with them all the
food-supplies necessary for the life of the ferment. They may be
observed budding, at least many of them, and there can be no doubt that
those which do not bud still continue to live; life has other ways of
manifesting itself besides development and cell-proliferation.

If we refer to the figures on page 81 of our Memoir of 1860, Experiments
D, E, F, G, H, I, we shall see that the weight of yeast, in the case of
the fermentation of a pure solution of sugar, undergoes a considerable
increase, even without taking into account the fact that the sugared
water gains from the yeast certain soluble parts, since, in the
experiments just mentioned, the weights of solid yeast, washed and dried
at 100° C. (212° F.), are much greater than those of the raw yeast
employed, dried at the same temperature.

In these experiments we employed the following weights of yeast,
expressed in grammes (1 gramme = 15·43 grains)—

                                 2·313
                                 2·626
                                 1·198
                                 0·699
                                 0·326
                                 0·476

which became after fermentation, we repeat, without taking into account
the matters which the sugared water gained from the yeast—

                                     grammes. grains.

                   2·486   [Increase 0·173 =  2·65
                   2·963   “         0·337 =  5·16
                   1·700   ”         0·502 =  7·7
                   0·712   “         0·013 =  0·2
                   0·335   ”         0·009 =  0·14
                   0·590   “         0·114 =  1·75

Have we not in this marked increase in weight a proof of life, or, to
adopt an expression which may be preferred, a proof of a profound
chemical work of nutrition and assimilation?

We may cite on this subject one of our earlier experiments, which is to
be found in the _Comptes rendus de l’Académie_ for the year 1857, and
which clearly shows the great influence exerted on fermentation by the
soluble portion that the sugared water takes up from the globules of
ferment:—

“We take two equal quantities of fresh yeast that have been washed very
freely. One of these we cause to ferment in water containing nothing but
sugar, and, after removing from the other all its soluble particles—by
boiling it in an excess of water and then filtering it to separate the
globules—we add to the filtered liquid as much sugar as was used in the
first case along with a mere trace of fresh yeast, insufficient, as far
as its weight is concerned, to affect the results of our experiment. The
globules which we have sown bud, the liquid becomes turbid, a deposit of
yeast gradually forms, and, side by side with these appearances, the
decomposition of the sugar is effected, and in the course of a few hours
manifests itself clearly. These results are such as we might have
anticipated. The following fact, however, is of importance. In effecting
by these means the organization into globules of the soluble part of the
yeast that we used in the second case, we find that a considerable
quantity of sugar is decomposed. The following are the results of our
experiment: 5 grammes of yeast caused the fermentation of 12·9 grammes
of sugar in six days, at the end of which time it was exhausted. The
soluble portion of a like quantity of 5 grammes of the same yeast caused
the fermentation of 10 grammes of sugar in nine days, after which the
yeast developed by the sowing was likewise exhausted.”

How is it possible to maintain that, in the fermentation of water
containing nothing but sugar, the soluble portion of the yeast does not
act, either in the production of new globules or the perfection of old
ones, when we see, in the preceding experiment, that after this
nitrogenous and mineral portion has been removed by boiling, it
immediately serves for the production of new globules, which, under the
influence of the sowing of a mere trace of globules, causes the
fermentation of much sugar?[152]

In short, Liebig is not justified in saying that the solution of pure
sugar, caused to ferment by means of yeast, contains none of the
elements needed for the growth of yeast, neither nitrogen, sulphur, nor
phosphorus, and that, consequently, it should not be possible, by our
theory, for the sugar to ferment. On the contrary, the solution does
contain all these elements, as a consequence of the introduction and
presence of the yeast.

Let us proceed with our examination of Liebig’s criticisms:—

“To this,” he goes on to say, “must be added the decomposing action
which yeast exercises on a great number of substances, and which
resembles that which sugar undergoes. I have shown that malate of lime
ferments readily enough through the action of yeast, and that it splits
up into three other calcareous salts, namely the acetate, the carbonate,
and the succinate. If the action of yeast consists in its increase and
multiplication, it is difficult to conceive this action in the case of
malate of lime and other calcareous salts of vegetable acids.”

This statement, with all due deference to the opinion of our illustrious
critic, is by no means correct. Yeast has no action on malate of lime,
or on other calcareous salts formed by vegetable acids. Liebig had
previously, much to his own satisfaction, brought forward urea as being
capable of transformation into carbonate of ammonia during alcoholic
fermentation in contact with yeast. This has been proved by us to be
erroneous. It is an error of the same kind that Liebig again brings
forward here. In the fermentation of which he speaks (that of malate of
lime), certain spontaneous ferments are produced, the germs of which are
associated with the yeast, and develop in the mixture of yeast and
malate. The yeast merely serves as a source of food for these new
ferments without taking any direct part in the fermentations of which we
are speaking. Our researches leave no doubt on this point, as is evident
from the observations on the fermentations of tartrate of lime
previously given.

It is true that there are circumstances under which yeast brings about
modifications in different substances. Doebereiner and Mitscherlich,
more especially, have shown that yeast imparts to water a soluble
material, which liquefies cane-sugar and produces inversion in it by
causing it to take up the elements of water, just as diastase behaves to
starch or emulsin to amygdalin.

M. Berthelot also has shown that this substance may be isolated by
precipitating it with alcohol, in the same way as diastase is
precipitated from its solutions.[153] These are remarkable facts, which
are, however, at present but vaguely connected with the alcoholic
fermentation of sugar by means of yeast. The researches in which we have
proved the existence of special forms of living ferments in many
fermentations, which one might have supposed to have been produced by
simple contact action, had established beyond doubt the existence of
profound differences between those fermentations, which we have
distinguished as fermentations proper, and the phenomena connected with
soluble substances. The more we advance, the more clearly we are able to
detect these differences. M. Dumas has insisted on the fact that the
ferments of fermentation proper multiply and reproduce themselves in the
process, whilst the others are destroyed.[154] Still more recently M.
Müntz has shown that chloroform prevents fermentations proper, but does
not interfere with the action of diastase (_Comptes rendus_, 1875.) M.
Bouchardat had already established the fact that “hydrocyanic acid,
salts of mercury, ether, alcohol, creosote, and the oils of turpentine,
lemon, cloves, and mustard destroy or check alcoholic fermentation,
whilst in no way interfering with the glucoside fermentations” (_Annales
de Chimie et de Physique_, 3rd series, t. xiv., 1845.) We may add, in
praise of M. Bouchardat’s sagacity, that that skilful observer has
always considered these results as a proof that alcoholic fermentation
is dependent on the life of the yeast-cell, and that a distinction
should be made between the two orders of fermentation.

M. Paul Bert, in his remarkable studies on the influence of barometric
pressure on the phenomena of life, has recognized the fact that
compressed oxygen is fatal to certain ferments, whilst under similar
conditions it does not interfere with the action of those substances
classed under the name of _soluble ferments_, such as diastase (the
ferment which inverts cane sugar), emulsin, and others. During their
stay in compressed air, ferments proper ceased their activity, nor did
they resume it, even after exposure to air at ordinary pressures,
provided the access of germs was prevented.

We now come to Liebig’s principal objection, with which he concludes his
ingenious argument, and to which no less than eight or nine pages of the
_Annales_ are devoted.

Our author takes up the question of the possibility of causing yeast to
grow in sweetened water, to which a salt of ammonia and some yeast-ash
have been added—a fact which is evidently incompatible with his theory
that a ferment is always an albuminous substance on its way to
decomposition. In this case the albuminous substance does not exist; we
have only the mineral substances which will serve to produce it. We know
that Liebig regarded yeast, and, generally speaking, any ferment
whatever, as being a nitrogenous, albuminous substance which, in the
same way as emulsin, for example, possesses the power of bringing about
certain chemical decompositions. He connected fermentation with the easy
decomposition of that albuminous substance, and imagined that the
phenomenon occurred in the following manner:—“The albuminous substance
on its way to decomposition possesses the power of communicating to
certain other bodies that same state of mobility by which its own atoms
are already affected; and through its contact with other bodies it
imparts to them the power of decomposing or of entering into other
combinations.” Here Liebig failed to perceive that the ferment, in its
capacity of a living organism, had anything to do with the fermentation.

This theory dates back as far as 1843. In 1846 Messrs. Boutron and
Fremy, in a Memoir on lactic fermentation, published in the _Annales de
Chimie et de Physique_, strained the conclusions deducible from it to a
most unjustifiable extent. They asserted that one and the same
nitrogenous substance might undergo various modifications in contact
with air, so as to become successively alcoholic, lactic, butyric, and
other ferments. There is nothing more convenient than purely
hypothetical theories, theories which are not the necessary consequences
of facts; when fresh facts which cannot be reconciled with the original
hypothesis are discovered, new hypotheses can be tacked on to the old
ones. This is exactly what Liebig and Fremy have done, each in his turn,
under the pressure of our studies, commenced in 1857. In 1864 Fremy
devised the theory of _hemi-organism_, which meant nothing more than
that he gave up Liebig’s theory of 1843, together with the additions
which Boutron and he had made to it in 1846; in other words, he
abandoned the idea of albuminous substances being ferments, to take up
another idea, that albuminous substances, in contact with air, are
peculiarly adapted to undergo organization into new beings—that is, the
living ferments which we had discovered—and that the ferments of beer
and of the grape have a common origin.

This theory of hemi-organism was word for word the antiquated opinion of
Turpin, as may be readily seen by referring to Chapter IV., section III.
of the present work. The public, especially a certain section of the
public, did not go very deeply into an examination of the subject. It
was the period when the doctrine of spontaneous generation was being
discussed with much warmth. The new word hemi-organism, which was the
only novelty in M. Fremy’s theory, deceived people. It was thought that
M. Fremy had really discovered the solution of the question of the day.
It is true that it was rather difficult to understand the process by
which an albuminous substance could become all at once a living and
budding cell. This difficulty was readily solved by M. Fremy, who
declared that it was the result of some power that was not yet
understood, the power of “organic impulse.”[155]

Liebig, who, as well as M. Fremy, was compelled to renounce his original
opinions concerning the nature of ferments, devised the following
obscure theory (Memoir by Liebig, 1870, already cited):—

“There seems to be no doubt as to the part which the vegetable organism
plays in the phenomenon of fermentation. It is through it alone that an
albuminous substance and sugar are enabled to unite and form this
particular combination, this unstable form under which alone, as a
component part of the mycoderm, they manifest an action on sugar. Should
the mycoderm cease to grow, the bond which unites the constituent parts
of the cellular contents is loosened, and it is through the motion
produced therein that the cells of yeast bring about a disarrangement or
separation of the elements of the sugar into other organic molecules.”

One might easily believe that the translator for the _Annales_ has made
some mistake, so great is the obscurity of this passage.

Whether we take this new form of the theory or the old one, neither can
be reconciled at all with the development of yeast and fermentation in a
saccharine mineral medium, for in the latter experiment fermentation is
correlative to the life of the ferment and to its nutrition, a constant
change going on between the ferment and its food-matters, since all the
carbon assimilated by the ferment is derived from sugar, its nitrogen
from ammonia, and phosphorus from the phosphates in solution. And even
all said, what purpose can be served by the gratuitous hypothesis of
contact-action or communicated motion? The experiment of which we are
speaking is thus a fundamental one; indeed, it is its possibility that
constitutes the most effective point in the controversy. No doubt Liebig
might say, “but it is the motion of life and of nutrition which
constitutes your experiment, and this is the communicated motion that my
theory requires.” Curiously enough, Liebig does endeavour, as a matter
of fact, to say this, but he does so timidly and incidentally: “From a
chemical point of view, which point of view I would not willingly
abandon, a _vital action_ is a phenomenon of motion, and, in this double
sense of _life_ M. Pasteur’s theory agrees with my own, and is not in
contradiction with it (page 6).” This is true. Elsewhere Liebig says:—

“It is possible that the only correlation between the physiological act
and the phenomenon of fermentation is the production, in the living
cell, of the substance which, by some special property analogous to that
by which emulsin exerts a decomposing action on salicin and amygdalin,
may bring about the decomposition of sugar into other organic molecules;
the physiological act, in this view, would be necessary for the
production of this substance, but would have nothing else to do with the
fermentation (page 10).” To this, again, we have no objection to raise.

Liebig, however, does not dwell upon these considerations, which he
merely notices in passing, because he is well aware that, as far as the
defence of his theory is concerned, they would be mere evasions. If he
had insisted on them, or based his opposition solely upon them, our
answer would have been simply this: “If you admit with us that
fermentation is correlated with the life and nutrition of the ferment,
we agree upon the principal point. So agreeing, let us examine, if you
will, the actual cause of fermentation;—this is a second question, quite
distinct from the first. Science is built up of successive solutions
given to questions of ever-increasing subtlety, approaching nearer and
nearer towards the very essence of phenomena. If we proceed to discuss
together the question of how living, organized beings act in decomposing
fermentable substances, we will be found to fall out once more on your
hypothesis of communicated motion, since, according to our ideas, the
actual cause of fermentation is to be sought, in most cases, in the fact
of life without air, which is the characteristic of many ferments.”

Let us briefly see what Liebig thinks of the experiment in which
fermentation is produced by the impregnation of a saccharine mineral
medium, a result so greatly at variance with his mode of viewing the
question.[156] After deep consideration he pronounces this experiment to
be inexact, and the result ill-founded. Liebig, however, was not one to
reject a fact without grave reasons for his doing so, or with the sole
object of evading a troublesome discussion. “I have repeated this
experiment,” he says, “a great number of times, with the greatest
possible care, and have obtained the same results as M. Pasteur,
excepting as regards the formation and increase of the ferment.” It was,
however, the formation and increase of the ferment that constituted the
point of the experiment. Our discussion was, therefore, distinctly
limited to this: Liebig denied that the ferment was capable of
development in a saccharine mineral medium, whilst we asserted that this
development did actually take place, and was comparatively easy to
prove. In 1871 we replied to M. Liebig before the Paris Academy of
Sciences in a Note, in which we offered to prepare in a mineral medium,
in the presence of a commission to be chosen for the purpose, as great a
weight of ferment as Liebig could reasonably demand.[157] We were bolder
than we should, perhaps, have been in 1860; the reason was that our
knowledge of the subject had been strengthened by ten years of renewed
research. Liebig did not accept our proposal, nor did he even reply to
our Note. Up to the time of his death, which took place on April 18th,
1873, he wrote nothing more on the subject.[158]

When we published, in 1860, the details of the experiment in question,
we pointed out at some length the difficulties of conducting it
successfully, and the possible causes of failure. We called attention
particularly to the fact that saccharine mineral media are much more
suited for the nutrition of bacteria, lactic ferment, and other lowly
forms, than they are to that of yeast, and in consequence readily become
filled with various organisms from the spontaneous growth of germs
derived from the particles of dust floating in the atmosphere. The
reason why we do not observe the growth of alcoholic ferments,
especially at the commencement of the experiments, is because of the
unsuitableness of those media for the life of yeast. The latter may,
nevertheless, form in them subsequent to this development of other
organized forms, by reason of the modification produced in the original
mineral medium by the albuminous matters that they introduce into it. It
is interesting to peruse, in our Memoir of 1860, certain facts of the
same kind relating to fermentation by means of albumens—that of the
blood, for example, from which, we may mention incidentally, we were led
to infer the existence of several distinct albumens in the serum, a
conclusion which, since then, has been confirmed by various observers,
notably by M. Béchamp. Now, in his experiments on fermentation in
sweetened water, with yeast-ash and a salt of ammonia, there is no doubt
that Liebig had failed to avoid those difficulties which are entailed by
the spontaneous growth of other organisms than yeast. Moreover, it is
possible that, to have established the certainty of this result, Liebig
should have had recourse to a closer microscopical observation than from
certain passages in his Memoir he seems to have adopted. We have little
doubt that his pupils could tell us that Liebig did not even employ that
instrument without which any exact study of fermentation is not merely
difficult but well-nigh impossible. We ourselves, for the reasons
mentioned, did not obtain a simple alcoholic fermentation any more than
Liebig did. In that particular experiment, the details of which we gave
in our Memoir of 1860, we obtained lactic and alcoholic fermentation
together; an appreciable quantity of lactic acid formed and arrested the
propagation of the lactic and alcoholic ferments, so that more than half
of the sugar remained in the liquid without fermenting. This, however,
in no way detracted from the correctness of the conclusion which we
deduced from the experiment, and from other similar ones; it might even
be said that, from a general and philosophical point of view—which is
the only one of interest here—the result was doubly satisfactory,
inasmuch as we demonstrated that mineral media were adapted to the
simultaneous development of several organized ferments, instead of only
one. The fortuitous association of different ferments could not
invalidate the conclusion that all the nitrogen of the cells of the
alcoholic and lactic ferments was derived from the nitrogen in the
ammoniacal salts, and that all the carbon of those ferments was taken
from the sugar, since, in the medium employed in our experiment, the
sugar was the only substance that contained carbon. Liebig carefully
abstained from noticing this fact, which would have been fatal to the
very groundwork of his criticisms, and thought that he was keeping up
the appearance of a grave contradiction by arguing that we had never
obtained a simple alcoholic fermentation. It would be unprofitable to
dwell longer upon the subject of the difficulties which the propagation
of yeast in a saccharine mineral medium formerly presented. As a matter
of fact, the progress of our studies has imparted to the question an
aspect very different from that which it formerly wore; it was this
circumstance which emboldened us to offer, in our reply to Liebig before
the Academy of Sciences in 1871, to prepare, in a saccharine mineral
medium, in the presence of a commission to be appointed by our opponent,
any quantity of ferment that he might require, and to effect the
fermentation of any weight of sugar whatsoever.

Our knowledge of the facts detailed in the preceding chapters concerning
pure ferments and their manipulation in the presence of pure air,
enables us to completely disregard those causes of embarrassment that
result from the fortuitous occurrence of the germs of organisms,
different in character from the ferments, introduced by the air or from
the sides of vessels, or even by the ferment itself.

Let us once more take one of our double-necked flasks (Fig. 22, p. 110),
which we will suppose is capable of containing three or four litres (six
to eight pints).

Let us put into it the following:—

                   Pure distilled
                   water.

                   Sugar candy          200 grammes.

                   Bitartrate of        1·0  “
                   potassium

                   ”      “  ammonia    0·5  ”

                   Sulphate of ammonia  1·5  “

                   Ash of yeast         1·5  ”

                   [1 gramme = 15·43
                   grains.]

Let us boil the mixture, to destroy all germs of organisms that may
exist in the air or liquid or on the sides of the flask, and then permit
it to cool, after having placed, by way of extra precaution, a small
quantity of asbestos in the end of the fine, curved tube. Let us next
introduce a trace of ferment into the liquid, through the other neck,
which, as we described, is terminated by a small piece of india-rubber
tube closed with a glass stopper.

Here are the details of such an experiment:—

On December 9, 1873, we sowed some pure ferment—_saccharomyces
pastorianus_. From December 11, that is, within so short a time as
forty-eight hours after impregnation, we saw a multitude of extremely
minute bubbles rising almost continuously from the bottom, indicating
that at this point the fermentation had commenced. On the following
days, several patches of froth appeared on the surface of the liquid. We
left the flask undisturbed in the oven, at a temperature of 25° C. (77°
F.). On April 24, 1874, we tested some of the liquid, obtained by means
of the straight tube, to see if it still contained any sugar. We found
that it contained less than two grammes, so that 198 grammes (4·2 oz.
Troy) had already disappeared. Some time afterwards the fermentation
came to an end; we carried on the experiment, nevertheless, until April
18, 1875.

There was no development of any organism absolutely foreign to the
ferment, which was itself abundant, a circumstance that, added to the
persistent vitality of the ferment, in spite of the unsuitableness of
the medium for its nutrition, permitted the perfect completion of
fermentation. There was not the minutest quantity of sugar remaining.
The total weight of ferment, after washing and drying at 100° C. (212°
F.), was 2·563 grammes (39·5 grains).

In experiments of this kind, in which the ferment has to be weighed, it
is better not to use any yeast-ash that cannot be dissolved completely,
so as to be capable of easy separation from the ferment formed. Raulin’s
liquid, the composition of which we have already given (p. 89,
footnote), may be used in such cases with success.

All the alcoholic ferments are not capable to the same extent of
development by means of phosphates, ammoniacal salts, and sugar. There
are some whose development is arrested a longer or shorter time before
the transformation of all the sugar. In a series of comparative
experiments, 200 grammes of sugar-candy being used in each case, we
found that whilst _saccharomyces pastorianus_ effected a complete
fermentation of the sugar, the caseous ferment did not decompose more
than two-thirds, and the ferment which we have designated _new “high”
ferment_ not more than one-fifth: and keeping the flasks for a longer
time in the oven had no effect in increasing the proportions of sugar
fermented in these two last cases.

We conducted a great number of fermentations in mineral media, in
consequence of a circumstance which it may be interesting to mention
here. A person who was working in our laboratory asserted that the
success of our experiments depended upon the impurity of the sugar-candy
which we employed, and that if this sugar had been pure—much purer than
was the ordinary, white, commercial sugar-candy, which up to that time
we had always used—the ferment could not have multiplied. The persistent
objections of our friend, and our desire to convince him, caused us to
repeat all our previous experiments on the subject, using sugar of great
purity, which had been specially prepared for us, with the utmost care,
by a skilful confectioner, Seugnot. The result only confirmed our former
conclusions. Even this did not satisfy our obstinate friend, who went to
the trouble of preparing some pure sugar for himself, in little
crystals, by repeated crystallizations of carefully-selected commercial
sugar-candy; he then repeated our experiments himself. This time all his
doubts were overcome. It even happened that the fermentations with the
perfectly pure sugar instead of being slow were very active, when
compared with those which we had conducted with the commercial
sugar-candy.

We may here add a few words on the non-transformation of yeast into
_penicillium glaucum_.

If at any time during fermentation we pour off the fermenting liquid,
the deposit of yeast remaining in the vessel may continue there, in
contact with air, without our ever being able to discover the least
formation of _penicillium glaucum_ in it. We may keep a current of pure
air constantly passing through the flask; the experiment will give the
same result. Nevertheless, this is a medium peculiarly adapted to the
development of this mould, inasmuch as if we introduce merely a few
spores of _penicillium_, an abundant vegetation of that growth will
afterwards appear on the deposit. The descriptions of Messrs. Turpin,
Hoffmann, and Trécul have, therefore, been based on one of these
illusions which we meet with so frequently in microscopical
observations. When we laid these facts before the Academy,[159] M.
Trécul professed his inability to comprehend them:[160]

“According to M. Pasteur,” he said, “the yeast of beer is _anaërobian_,
that is to say, it lives in a liquid deprived of free oxygen; and to
become _mycoderma_ or _penicillium_ it is above all things necessary
that it should be placed in air, since, without this, as the name
signifies, an aërobian being cannot exist. To bring about the
transformation of the yeast of beer into _mycoderma cerevisiæ_ or into
_penicillium glaucum_, we must accept the conditions under which these
two forms are obtained. If M. Pasteur will persist in keeping his yeast
in media which are incompatible with the desired modification, it is
clear that the results which he obtains must be always negative.”

Contrary to this perfectly gratuitous assertion of M. Trécul’s, we do
not keep our yeast in media which are calculated to prevent its
transformation into _penicillium_. As we have just seen, the principal
aim and object of our experiment was to bring this minute plant into
contact with air, and under conditions that would allow the
_penicillium_ to develop with perfect freedom. We conducted our
experiments exactly as Turpin and Hoffmann conducted theirs, and exactly
as they stipulate that such experiments should be conducted—with the one
sole difference, indispensable to the correctness of our observations,
that we carefully guarded ourselves against those causes of error which
they did not take the least trouble to avoid. It is possible to produce
a ready entrance and escape of pure air in the case of the double-necked
flasks which we have so often employed in the course of this work,
without having recourse to the continuous passage of a current of air.
Having made a file-mark on the thin curved neck at a distance of two or
three centimetres (an inch) from the flask, we must cut round the neck
at this point with a glazier’s diamond, and then remove it, taking care
to cover the opening immediately with a sheet of paper which has been
passed through the flame, and which we must fasten with a thread round
the part of the neck still left. In this manner we may increase or
prolong the fructification of fungoid growths, or the life of aërobian
ferments in our flasks.

What we have said of _penicillium glaucum_ will apply equally to
_mycoderma cerevisiæ_. Notwithstanding what Turpin and Trécul may assert
to the contrary, yeast, in contact with air as it was under the
conditions of the experiment just described, will not yield _mycoderma
vini_ or _mycoderma cerevisiæ_ any more than it will _penicillium_.

The experiments described in the preceding paragraphs on the increase of
organized ferments in mineral media of the composition described, are of
great physiological interest. Amongst other results, they show that all
the proteic matter of ferments may be produced by the vital activity of
the cells, which, apart altogether from the influence of light or free
oxygen (unless, indeed, we are dealing with aërobian moulds which
require free oxygen), have the power of developing a chemical activity
between carbo-hydrates, ammoniacal salts, phosphates and sulphates of
potassium and magnesium. It may be admitted with truth that a similar
effect obtains in the case of the higher plants, so that in the existing
state of science we fail to conceive what serious reason can be urged
against our considering this effect as general. It would be perfectly
logical to extend the results of which we are speaking to all plants,
and to believe that the proteic matter of vegetables, and perhaps of
animals also, is formed exclusively by the activity of the cells
operating upon the ammoniacal and other mineral salts of the sap or
plasma of the blood, and the carbo-hydrates, the formation of which, in
the case of the higher plants, requires only the concurrence of the
chemical impulse of green light.

Viewed in this manner, the formation of the proteic substances would be
independent of the great act of reduction of carbonic acid gas under the
influence of light. These substances would not be built up from the
elements of water, ammonia, and carbonic acid gas, after the
decomposition of this last; they would be formed where they are found in
the cells themselves, by some process of union between the
carbo-hydrates imported by the sap, and the phosphates of potassium and
magnesium and salts of ammonia. Lastly, in vegetable growth, by means of
a carbo-hydrate and a mineral medium, since the carbo-hydrate is capable
of many variations, and it would be difficult to understand how it could
be split up into its elements before serving to constitute the proteic
substances, we may hope to obtain as many distinct proteic substances,
and even cellulose substances, as there are carbo-hydrates. We have
commenced certain studies in this direction.

If solar radiation is indispensable to the decomposition of carbonic
acid and the building up of the primary substances in the case of
higher vegetable life, it is still possible that certain inferior
organisms may do without it and nevertheless yield the most complex
substances, fatty or carbo-hydrate, such as cellulose, various organic
acids, and proteic matter; not, however, by borrowing their carbon
from the carbonic acid which is saturated with oxygen, but from other
matters still capable of acquiring oxygen, and so of yielding heat in
the process, such as alcohol and acetic acid, for example, to cite
merely carbon compounds most removed from organization. As these last
compounds, and a host of others equally adapted to serve as the
carbonaceous food of _mycoderms_ and the mucedines, may be produced
synthetically by means of carbon and the vapour of water, after the
methods that science owes to Berthelot, it follows that, in the case
of certain inferior beings, life would be possible even if it should
be that the solar light was extinguished.[161]

Footnote 111:

  It has been remarked in practice that fermentation is facilitated by
  leaving the grapes on the bunches. The reason of this has not yet been
  discovered. Still we have no doubt that it may be attributed,
  principally, to the fact that the interstices between the grapes, and
  the spaces which the bunch leaves throughout, considerably increase
  the volume of air placed at the service of the germs of ferment.

Footnote 112:

  [NaHSO_{2}, now called _Sodium hyposulphite_. See p. 355,
  footnote.—D.C.R.]

Footnote 113:

  [This appears to be a misprint for 1·638 grammes = 25·3
  grains.—D.C.R.]

Footnote 114:

  [200 c.c. of liquid were used, which, as containing 5 per cent., had
  in solution 10 grammes of sugar.—D. C. R.]

Footnote 115:

  [International Science Series, vol. xx., pp. 179-182. London, 1876.—D.
  C. R.]

Footnote 116:

  Page 182, English edition.

Footnote 117:

  This figure is on a scale of 300 diameters, most of the figures in
  this work being of 400 diameters.

Footnote 118:

  [It may be useful for the non-scientific reader to put it thus:—that
  the 25 c.c. which escaped, being a fair sample of the whole gas in the
  flask, and containing (1) 25 - 20·6 = 4·4 c.c., absorbed by potash and
  therefore due to carbonic acid, and (2) 20·6 - 17·3 = 3·3 c.c.,
  absorbed by pyrogallate, and therefore due to oxygen, and the
  remaining 17·3 c.c. being nitrogen, the whole gas in the flask, which
  has a capacity of 315 c.c., will contain oxygen in the above
  proportion, and therefore its amount may be determined, provided we
  know the total gas in the flask before opening. On the other hand, we
  know that air normally contains, approximately, 1/5th its volume of
  oxygen, the rest being nitrogen, so that, by ascertaining the
  diminution of the proportion in the flask, we can find how many cubic
  centimetres have been absorbed by the yeast. The author, however, has
  not given all the _data_ necessary for accurate calculation.—D.C.R.]

Footnote 119:

  This number is probably too small; it is scarcely possible that the
  increase of weight in the yeast, even under the exceptional conditions
  of the experiment described, was not to some extent at least due to
  oxidation apart from free oxygen, inasmuch as some of the cells were
  covered by others. The increased weight of the yeast is always due to
  the action of two distinct modes of vital energy—activity, namely, in
  presence and activity in absence of air. We might endeavour to shorten
  the duration of the experiment still further, in which case we would
  still more assimilate the life of the yeast to that of ordinary
  moulds.

Footnote 120:

  In these experiments, in which the moulds remain for a long time in
  contact with a saccharine wort out of contact with oxygen—the oxygen
  being promptly absorbed by the vital action of the plant (see our
  _Mémoire sur les Générations dites Spontanées_, p. 54, note)—there is
  no doubt that an appreciable quantity of alcohol is formed because the
  plant does not immediately lose its vital activity, after the
  absorption of oxygen.

  A 300-c.c. (10-oz.) flask, containing 100 c.c. of must, after the air
  in it had been expelled by boiling, was opened and immediately
  re-closed, on August 15th, 1873. A fungoid growth—a unique one, of
  greenish-grey colour—developed from spontaneous impregnation, and
  decolorized the liquid, which originally was of a yellowish-brown.
  Some large crystals, sparkling like diamonds, of neutral tartrate of
  lime, were precipitated. About a year afterwards, long after the death
  of the plant, we examined this liquid. It contained 0·3 gramme (4·6
  grains) of alcohol, and 0·053 gramme (0·8 grain) of vegetable matter,
  dried at 100° C. (212° F.). We ascertained that the spores of the
  fungus were dead at the moment when the flask was opened. When sown,
  they did not develop in the least degree.

Footnote 121:

  We find in M. Raulin’s Note, already quoted, that “the minimum ratio
  between the weight of sugar and the weight of organized matter, that
  is, the weight of fungoid growth which it helps to form, may be
  expressed 10/3·2 = 3·1.” JULES RAULIN, _Études chimiques sur la
  végétation. Recherches sur le développement d’une mucédinée dans un
  milieu artificiel_, p. 192, Paris, 1870. We have seen, in the case of
  yeast, that this ratio may be as low as 4/1.

Footnote 122:

  We shall show, some day, that the processes of oxidation due to growth
  of fungi cause, in certain decompositions, liberation of ammonia to a
  considerable extent, and that by regulating their action we might
  cause them to extract the nitrogen from a host of organic _débris_, as
  also, by checking the production of such organisms, we might
  considerably increase the proportion of nitrates in the artificial
  nitrogenous substances. By cultivating various moulds on the surface
  of damp bread in a current of air, we have obtained an abundance of
  ammonia, derived from the decomposition of the albuminoids effected by
  the fungoid life. The decomposition of asparagus, and several other
  animal or vegetable substances, has given similar results.

Footnote 123:

  To determine the absence of cells of ferment in fruits that have been
  immersed in carbonic acid gas, we must first of all carefully raise
  the pellicle of the fruit, taking care that the subjacent parenchyma
  does not touch the surface of the pellicle, since the organized
  corpuscles existing on the exterior of the fruit might introduce an
  error into our microscopical observations. Experiments on grapes have
  given us an explanation of a fact generally known, the cause of which,
  however, had hitherto escaped our knowledge. We all know that the
  taste and aroma of the vintage, that is, of the grapes stripped from
  the bunches and thrown into tubs, where they get soaked in the juice
  that issues from wounded specimens, are very different from the taste
  and aroma of an uninjured bunch. Now grapes that have been immersed in
  an atmosphere of carbonic acid gas have exactly the flavour and smell
  of the vintage; the reason is that, in the vintage tub, the grapes are
  immediately surrounded by an atmosphere of carbonic acid gas, and
  undergo, in consequence, the fermentation peculiar to grapes that have
  been plunged in this gas. These facts deserve to be studied from a
  practical point of view. It would be interesting, for example, to
  learn what difference there would be in the quality of two wines, the
  grapes of which, in the one case, had been perfectly crushed, so as to
  cause as great a separation of the cells of the parenchyma as
  possible; in the other case, left, for the most part, whole, as in the
  case in the ordinary vintage. The first wine would be deprived of
  those fixed and fragrant principles produced by the fermentation of
  which we have just spoken, when the grapes are immersed in carbonic
  acid gas. By such a comparison as that which we suggest, we should be
  able to form an _à priori_ judgment on the merits of the new system,
  which has not been carefully studied, although already widely adopted,
  of milled, cylindrical crushers, for pressing the vintage.

Footnote 124:

  We have sometimes found small quantities of alcohol in fruits and
  other vegetable organs, surrounded with ordinary air, but always in
  small proportion, and in a manner which suggested its accidental
  character. It is easy to understand how, in the thickness of certain
  fruits, certain parts of those fruits might be deprived of air, under
  which circumstance they would have been acting under conditions
  similar to those under which fruits act when wholly immersed in
  carbonic acid gas. Moreover it would be useful to determine whether
  alcohol is not a normal product of vegetation.

Footnote 125:

  In these studies on plants living immersed in carbonic acid gas, we
  have come across a fact which corroborates those which we have already
  given in reference to the facility with which lactic and viscous
  ferments, and, generally speaking, those which we have termed the
  disease-ferments of beer, develop when deprived of air, and which
  shows, consequently, how very marked their aërobian character is. If
  we immerse beetroots or turnips in carbonic acid gas, we produce
  well-defined fermentations in those roots. Their whole surface readily
  permits the escape of the highly acid liquids, and they become filled
  with lactic, viscous, and other ferments. This shows us the great
  danger which may result from the use of pits, in which the beetroots
  are preserved, when the air is not renewed, and that the original
  oxygen is expelled by the vital processes of fungi, or other
  deoxidizing chemical actions. We have directed the attention of the
  manufacturers of beetroot sugar to this point.

Footnote 126:

  LECHARTIER and BELLAMY, _Comptes rendus de l’Académie des Sciences_,
  vol. lxix., pp. 366 and 466, 1869.

Footnote 127:

  Those gentlemen express themselves thus: “In a note presented to the
  Academy in November, 1872, we published certain experiments which
  showed that carbonic acid and alcohol may be produced in fruits kept
  in a closed vessel, out of contact with atmospheric oxygen, without
  our being able to discover alcoholic ferment in the interior of those
  fruits.

  “M. Pasteur, as a logical deduction from the principles which he has
  established in connection with the theory of fermentation, considers
  that _the formation of alcohol may be attributed to the fact that the
  physical and chemical processes of life in the cells of fruit continue
  under new conditions, in a manner similar to those of the cells of
  ferment_. Experiments, continued during 1872, 1873, and 1874, on
  different fruits, have furnished results all of which seem to us to
  harmonize with this proposition, and to establish it on a firm basis
  of proof.” _Comptes rendus_, t. lxxix., p. 949, 1874.

Footnote 128:

  PASTEUR, _Faites nouveaux pour servir à la connaissance de la théorie
  des fermentations proprement dites_. (_Comptes rendus de l’Académie
  des Sciences_, t. lxxv., p. 784). See, in the same volume, the
  discussion that followed; also, PASTEUR, _Note sur la production de
  l’alcool par les fruits_, same volume, p. 1054, in which we recount
  the observations anterior to our own, made by Messrs. Lechartier and
  Bellamy in 1869.

Footnote 129:

  _Comptes rendus_, meeting of January 15th, 1872.

Footnote 130:

  As a matter of fact, M. Fremy applies his theory of hemi-organism, not
  only to the alcoholic fermentation of grape juice, but to all other
  fermentations. The following passage occurs in one of his Notes
  (_Comptes rendus de l’Académie_, t. lxxv., p. 979, October 28th,
  1872):

  “_Experiments on Germinated Barley._—The object of these was to show
  that, when barley, left to itself in sweetened water, produces in
  succession alcoholic, lactic, butyric, and acetic fermentations, these
  modifications are brought about by ferments which are produced inside
  the grains themselves, and not by atmospheric germs. More than forty
  different experiments were devoted to this part of my work.” Need we
  add that this assertion is based on no substantial foundation? The
  cells belonging to the grains of barley, or their albuminous contents,
  never do produce cells of alcoholic ferment, or of lactic ferment, or
  butyric vibrios. Whenever those ferments appear they may be traced to
  germs of those organisms, diffused throughout the interior of the
  grains, or adhering to their exterior surface, or existing in the
  water employed, or on the sides of the vessels used. There are many
  ways of demonstrating this, of which the following is one: since the
  results of our experiments have shown that sweetened water,
  phosphates, and chalk very readily give rise to lactic and butyric
  fermentations, what reason is there for supposing that if we
  substitute grains of barley for chalk, the lactic and butyric ferments
  will spring from those grains, in consequence of a transformation of
  their cells or albuminous substances? Surely, there is no ground for
  maintaining that they are produced by hemi-organism, since a medium
  composed of sugar, or chalk, or phosphates of ammonia, potash, or
  magnesia contains no albuminous substances. This is an indirect but
  irresistible argument against the hemi-organism theory.

Footnote 131:

  PASTEUR, _Mémoire sur la fermentation alcoolique_, 1860; _Annales de
  Chimie et de Physique_. The word _globules_ is here used for _cells_.
  In our researches we have always endeavoured to prevent any confusion
  of ideas. We stated at the beginning of our Memoir of 1860, that: “We
  apply the term _alcoholic_ to that fermentation which sugar undergoes
  under the influence of the ferment known as _beer yeast_.” This is the
  fermentation which produces wine and all alcoholic beverages. This,
  too, is regarded as the type for a host of similar phenomena,
  designated, by general usage, under the generic name of
  _fermentation_, and qualified by the name of one of the essential
  products of the special phenomenon under observation. Bearing in mind
  this fact in reference to the nomenclature that we have adopted, it
  will be seen that the expression _alcoholic fermentation_ cannot be
  applied to every phenomenon of fermentation in which alcohol is
  produced, inasmuch as there may be a number of phenomena having this
  character in common. If we had not at starting defined that particular
  one amongst the number of very distinct phenomena, which, to the
  exclusion of the others, should bear the name _alcoholic
  fermentation_, we should inevitably have given rise to a confusion of
  language that would soon pass from words to ideas, and tend to
  introduce unnecessary complexity into researches which are already, in
  themselves, sufficiently complex to necessitate the adoption of
  scrupulous care to prevent their becoming still more involved. It
  seems to us that any further doubt as to the meaning of the words
  _alcoholic fermentation_, and the sense in which they are employed, is
  impossible, inasmuch as Lavoisier, Gay-Lussac, and Thénard have
  applied this term to the fermentation of sugar by means of beer yeast.
  It would be both dangerous and unprofitable to discard the example set
  by those illustrious masters, to whom we are indebted for our earliest
  knowledge of this subject.

Footnote 132:

  See, for example, the communications of MM. Colin and Poggiale, and
  the discussion on them, in the _Bulletin de l’Académie de Médicine_,
  March 2nd, 9th, and 30th, and February 16th and 23rd, 1875.

Footnote 133:

  We have elsewhere determined the formation of minute quantities of
  volatile acids in alcoholic fermentation. M. Béchamp, who studied
  these, recognized several belonging to the series of fatty acids,
  acetic acid, butyric acid, &c. “The presence of succinic acid is not
  accidental, but constant; if we put aside volatile acids that form in
  quantities which we may call infinitely small, we may say that
  succinic acid is the only normal acid of alcoholic fermentation.”
  PASTEUR, _Comptes rendus de l’Académie_, t. xlvii. p. 224, 1858.

Footnote 134:

  Traube’s conceptions were governed by a theory of fermentation
  entirely his own, a hypothetical one, as he admits, of which the
  following is a brief summary: “We have no reason to doubt,” Traube
  says, “that the protoplasm of vegetable cells is itself, or contains
  within it, a chemical ferment which causes the alcoholic fermentation
  of sugar; its efficacy seems closely connected with the presence of
  the cell, inasmuch as, up to the present time, we have discovered no
  means of isolating it from the cells with success. In the presence of
  air, this ferment oxidizes sugar, by bringing oxygen to bear upon it;
  in the absence of air it decomposes the sugar by taking away oxygen
  from one group of atoms of the molecule of sugar and bringing it to
  act upon other atoms; on the one hand yielding a product of alcohol by
  reduction, on the other hand a product of carbonic acid by oxidation.”

  Traube supposes that this chemical ferment exists in yeast and in all
  sweet fruits, but only when the cells are intact, for he has proved
  for himself that thoroughly crushed fruits give rise to no
  fermentation whatever in carbonic acid gas. In this respect this
  imaginary chemical ferment would differ entirely from those which we
  call _soluble ferments_, since diastase, emulsine, &c., may be easily
  isolated.

  For a full account of the views of Brefeld and Traube, and the
  discussion which they carried on on the subject of the results of our
  experiments, our readers may consult the _Journal of the Chemical
  Society of Berlin_, vii. p. 872. The numbers for September and
  December, 1874, in the same volume, contain the replies of the two
  authors.

Footnote 135:

  See PASTEUR, _Comptes rendus de l’Académie des Sciences_, t. lvi. p.
  416.

Footnote 136:

  [Carbonic acid being considerably more soluble than other gases
  possible under the circumstances.—ED.]

Footnote 137:

  We had to avoid filling the small flask completely, for fear of
  causing some of the liquid to pass on to the surface of the mercury in
  the measuring tube. The liquid condensed by boiling forms pure water,
  the solvent affinity of which for carbonic acid, at the temperature we
  employ, is well known.

Footnote 138:

  The following is a curious consequence of these numbers and of the
  nature of the products of this fermentation. The carbonic acid
  liberated being quite pure, especially when the liquid has been boiled
  to expel all air from the flask, and capable of perfect solution, it
  follows that, the volume of liquid being sufficient and the weight of
  tartrate suitably chosen—we may set aside tartrate of lime in an
  insoluble, crystalline powder, along with phosphates at the bottom of
  a closed vessel full of water, and find soon afterwards in their place
  carbonate of lime, and, in the liquid, soluble salts of lime, with a
  mass of organic matter at the bottom, without any liberation of gas or
  appearance of fermentation ever taking place, except as far as the
  vital action and transformation in the tartrate are concerned. It is
  easy to calculate that a vessel or flask of five litres (rather more
  than a gallon) would be large enough for the accomplishment of this
  remarkable and singularly quiet transformation, in the case of fifty
  grammes (767 grains) of tartrate of lime.

Footnote 139:

  We treated the whole deposit with dilute hydrochloric acid, which
  dissolved the carbonate of lime, and the insoluble phosphates of
  calcium and magnesium; afterwards filtering the liquid through a
  weighed filter paper. Dried at 100° C. (212° F.), the weight of
  organic matter thus obtained was 0·54 gramme (8·3 grains), which was
  rather more than 1/200th of the weight of fermentable matter.

Footnote 140:

  Should the solution of lactate of lime be turbid, it may be clarified
  by filtration, after previously adding a small quantity of phosphate
  of ammonia, which throws down phosphate of lime. It is only after this
  process of clarification and filtration that the phosphates of the
  formula are added. The solution soon becomes turbid, if left in
  contact with air, in consequence of the spontaneous formation of
  bacteria.

Footnote 141:

  The naturalist Cohn, of Breslau, who published an excellent work on
  bacteria in 1872, described, after Mayer, the composition of a liquid
  peculiarly adapted to the propagation of these organisms, which it
  would be well to compare for its utility in studies of this kind with
  our solution of lactate and phosphates. The following is Cohn’s
  formula:—

             Distilled water          20 c.c. (0·7 fl. oz.)

             Phosphate of          0·1 gramme (1·5 grains).
             potassium

             Sulphate of           0·1 gramme (1·5 grains).
             magnesium

             Tribasic phosphate   0·01 gramme (0·15 grain).
             of lime

             Tartrate of ammonia     0·2 gramme (3 grains).

  This liquid, the author says, has a feeble acid reaction and forms a
  perfectly clear solution.

Footnote 142:

  On the rapid absorption of oxygen by bacteria, see also our _Mémoire_
  of 1872, _sur les Générations dites Spontanées_, especially the note
  on page 78.

Footnote 143:

  In what way are we to account for so great a difference between the
  two fermentations that we have just described? Probably, it was owing
  to some modification effected in the medium by the previous life of
  the bacteria, or to the special character of the vibrios used in
  impregnation. Or, again, it might have been due to the action of the
  air, which, under the conditions of our second experiment, was not
  absolutely eliminated, since we took no precaution against its
  introduction at the moment of filling our flask, and this would tend
  to facilitate the multiplication of anaërobian vibrios, just as, under
  similar conditions, would have been the case if we had been dealing
  with a fermentation by ordinary yeast.

Footnote 144:

  In this case the liquid was composed as follows:—a saturated solution
  of lactate of lime, at a temperature of 25° C. (77° F.) was prepared,
  containing for every 100 c.c. (3-½ fl. oz.) 25·65 grammes (394 grains)
  of the lactate, C_{6}H_{5}O_{5}_Ca_O [_new notation_,
  C_{6}H_{10}_Ca_O_{6}]. This solution was rendered very clear by the
  addition of one gramme of phosphate of ammonia and subsequent
  filtration. For a volume of 8 litres (14 pints) of this clear,
  saturated solution, we used [1 gramme = 15·43 grains]:—

                   Phosphate of ammonia 2 grammes.

                   Phosphate of         1 grammes.
                   potassium

                   Phosphate of         1 grammes.
                   magnesium

                   Sulphate of ammonia  0·5 gramme.

Footnote 145:

  [1 millimetre = 0·039 inch: hence the dimensions indicated will
  be—length, from 0·00039 to 0·00117, or even 0·00176 in.; diameter,
  from 0·000058 to 0·000078, rarely 0.000117 in.]

Footnote 146:

  The carbonaceous supply, as we remarked, had failed them, and to this
  failure the absence of vital action, nutrition, and multiplication was
  attributable. The liquid, however, contained butyrate of lime, a salt
  possessing properties similar to those of the lactate. Why could not
  this salt equally well support the life of the vibrios? The
  explanation of the difficulty seems to us to lie simply in the fact
  that lactic acid produces heat by its decomposition, whilst butyric
  acid does not, and the vibrios seem to require heat daring the
  chemical process of their nutrition.

Footnote 147:

  To do this, it is sufficient first to fill the curved ends of the
  stop-cocked tubes of the flasks, as well as the india-rubber tube _c
  c_, which connects them, with boiling water that contains no air.

Footnote 148:

  We find this fact, which we published as long ago as 1863, confirmed
  in a work of H. Hoffmann’s published in 1869, under the title _Mémoire
  sur les bactéries_, which has appeared in French (_Annales des
  Sciences naturelles_, 5th series, vol. xi.). On this subject we may
  cite an observation that has not yet been published. Aërobian bacteria
  lose all power of movement when suddenly plunged into carbonic acid
  gas; they recover it, however, as if they had only been suffering from
  anæsthesia, as soon as they are brought into the air again.

Footnote 149:

  These doubts might easily be removed by putting the matter to the test
  of direct experiment.

Footnote 150:

  ROBIN, _Sur la nature des fermentations_, &c. (_Journal de l’Anatomie
  et de la Physiologie_, July and August, 1875, p. 386).

Footnote 151:

  LIEBIG, _Sur la fermentation et la source de la force musculaire_
  (_Annales de Chimie et de Physique_, 4th series, t. xxiii. p. 5,
  1870.)

Footnote 152:

  It is important that we should here remark that, in the fermentation
  of pure solution of sugar by means of yeast, the oxygen originally
  dissolved in the water, as well as that appropriated by the globules
  of yeast in their contact with air, has a considerable effect on the
  activity of fermentation. As a matter of fact, if we pass a strong
  current of carbonic acid through the sugared water and the water in
  which the yeast has been treated, the fermentation will be rendered
  extremely sluggish, and the few new cells of yeast which form will
  assume strange and abnormal aspects. Indeed this might have been
  expected, for we have seen that yeast, when somewhat old, is incapable
  of development or of causing fermentation, even in a fermentable
  medium containing all the nutritive principles of yeast, if the liquid
  has been deprived of air; much more should we expect this to be the
  case in pure sugared water, likewise deprived of air.

Footnote 153:

  DOEBEREINER, _Journal de Chimie de Schweigger_, vol. xii. p. 129, and
  _Journal de Pharmacie_, vol. i. p. 342.

  MITSCHERLICH, _Monatsberichte d. Kön. Preuss. Akad. d. Wissen. zu
  Berlin_, and _Rapports annuels de Berzelius_, Paris, 1843, 3rd year.
  On the occasion of a communication on the inversion of cane sugar, by
  H. Rose, published in 1840, M. Mitscherlich observed: “The inversion
  of cane sugar in alcoholic fermentation is not due to the globules of
  yeast, but to a soluble matter in the water with which they mix. The
  liquid obtained by straining off the ferment on a filter paper,
  possesses the property of converting cane sugar into uncrystallizable
  sugar.”

  BERTHELOT, _Comptes rendus de l’Académie_. Meeting of May 28th, 1860.
  M. Berthelot confirms the preceding experiment of Mitscherlich, and
  proves, moreover, that the soluble matter of which that author speaks
  may be precipitated with alcohol without losing its invertive power.

  M. Béchamp has applied Mitscherlich’s observation, concerning the
  soluble fermentative part of yeast, to fungoid growths, and has made
  the interesting discovery that fungoid growths, like yeast, yield to
  water a substance that inverts sugar. When the production of fungoid
  growths is prevented by means of an antiseptic the inversion of sugar
  does not take place.

  We may here say a few words respecting M. Béchamp’s claim to priority
  of discovery. It is a well-known fact that we were the first to
  demonstrate that living ferments might be completely developed, if
  their germs were placed in pure water, together with sugar, ammonia,
  and phosphates. Relying on this established fact, that moulds are
  capable of development in sweetened water, in which, according to M.
  Béchamp, they invert the sugar, our author asserts that he has proved
  that, “living organized ferments may originate in media which contain
  no albuminous substances.” (See _Comptes rendus_, vol. lxxv. p. 1519.)
  To be logical, M. Béchamp might say that he has proved that certain
  moulds originate in pure sweetened water, without nitrogen or
  phosphates or other mineral elements, for such a deduction might very
  well be drawn from his work, in which we do not find the least
  expression of astonishment at the possibility of moulds developing in
  pure water, containing nothing but sugar without other mineral or
  organic principles.

  M. Béchamp’s first Note on the inversion of sugar was published in
  1855. In it we find nothing relating to the influence of moulds. His
  second, in which that influence is noticed, was published in January,
  1858, that is, subsequently to our work on lactic fermentation, which
  appeared in November, 1857. In that work we established, for the first
  time, that the lactic ferment is a living organized being, that
  albuminous substances have no share in the production of fermentation,
  and that they only serve as the food of the ferment. M. Béchamp’s Note
  was even subsequent to our first work on alcoholic fermentation, which
  appeared on December 21st, 1857. It is since the appearance of these
  two works of ours that the preponderating influence of the life of
  microscopic organisms, in the phenomena of fermentation, has been
  better understood. Immediately after their appearance M. Béchamp, who,
  from 1855, had made no observation on the action of fungoid growths on
  sugar, although he had remarked their presence, modified his former
  conclusions. (_Comptes rendus_, January 4th, 1858.)

Footnote 154:

  “There are two classes of ferments; the first, of which the yeast of
  beer may be taken as the type, perpetuate and renew themselves if they
  can find in the liquid in which they produce fermentation food enough
  for their wants; the second, of which diastase is the type, always
  sacrifice themselves in the exercise of their activity.” (DUMAS,
  _Comptes rendus de l’Académie_, t. lxxv. p. 277, 1872.)

Footnote 155:

  FREMY, _Comptes rendus de l’Académie_, vol. lviii. p. 1065, 1864.

Footnote 156:

  See our Memoir of 1860 (_Annales de Chimie et de Physique_, vol.
  lviii.) p. 61, and following, and especially pp. 69 and 70, where the
  details of the experiment will be found.

Footnote 157:

  PASTEUR, _Comptes rendus de l’Académie des Sciences_, vol. lxxiii. p.
  1419, 1871.

Footnote 158:

  In his Memoir of 1870, Liebig has made a remarkable admission: “My
  late friend Pelouze,” he says, “had communicated to me, nine years
  ago, certain results of M. Pasteur’s researches on fermentation. I
  told him that, just then, I was not disposed to alter my opinion on
  the cause of fermentation, and that if it were possible by means of
  ammonia to produce or multiply the yeast in fermenting liquors,
  industry would soon avail itself of the fact, and that I would wait to
  see if it did so; up to the present time, however, there has not been
  the least change in the manufacture of yeast.” We do not know what M.
  Pelouze’s reply was; but it is not difficult to conceive so sagacious
  an observer remarking to his illustrious friend, that the possibility
  of deriving pecuniary advantage from the wide application of a new
  scientific fact had never been regarded as the criterion of the
  exactness of that fact. We could prove, moreover, by the undoubted
  testimony of very distinguished practical men, notably by that of M.
  Pezeyre, director of distilleries, that upon this point also Liebig
  was mistaken.

Footnote 159:

  PASTEUR, _Comptes rendus de l’Académie_, vol. lxxviii. pp. 213-216.

Footnote 160:

  TRÉCUL, _Comptes rendus de l’Académie_, vol. lxxviii. pp. 217, 218.

Footnote 161:

  See on this subject the verbal observations which we addressed to the
  Academy of Sciences, at its meetings of April 10th and 24th, 1876.




                              CHAPTER VII.
                New Process for the Manufacture of Beer.


The principles established in the course of this work implicitly involve
the conditions of a new process of manufacture, the essential feature of
which would consist in the production of a beer of excellent keeping
qualities, we might even say a beer that could not undergo alteration.
It will not be difficult now to make ourselves clear on the point.

We have shown in the first place, that the changes which take place in
the ferment, the wort, and the beer itself, are due to the presence of
microscopic organisms of an entirely different character to that of the
ferment-cells properly so called, which organisms, by simultaneously
giving rise, in the course of their multiplication in the wort, ferment,
or beer, to other products, make the materials difficult to keep or
effect their deterioration. Again, we have seen that these
change-producing organisms, the ferments of disease, never arise
spontaneously in the wort or beer, but, whenever they make their
appearance in these fluids, have been imported from without, either in
company with the yeast, or from accession of atmospheric dust, or from
contact with the vessels, or from the materials themselves which the
brewer uses in his manufacture. Moreover, we know that these
disease-ferments, or their germs, are destroyed when the wort has its
temperature raised to the boiling-point. And, following up the
inferences from such facts, we have seen that wort exposed to pure air,
after having been heated to boiling, remains absolutely free of any sort
of fermentation.

Inasmuch then as the disease-germs of wort and beer are destroyed in the
copper in which the wort is boiled, and as, by employing a perfectly
pure ferment, we guard against the admission of any foreign ferment of
an evil character, we have it in our power to prepare a beer which shall
be incapable of undergoing any pernicious fermentation whatsoever. This
we shall have effected provided we can take the wort as run off from the
coppers, cool and manipulate it out of contact with ordinary air or in
contact with pure air, charge it with a pure yeast, and, lastly, store
the beer when the fermentation is complete in vessels thoroughly
purified from disease-ferments.[162]


                     § I.—Preliminary Experiments.


We may readily satisfy ourselves as to the truth of these inferences.
The following is one of the earliest experiments which I devised with a
view to establish their certainty. Into a flask with a straight neck of
about a litre (1-3/4 pints) capacity, a quantity of wort from a brewery
was introduced and there raised to boiling, and whilst the vapour still
issued from the neck of the flask, connection was made with a two-necked
flask in which the cultivation of pure yeast had been carried on. The
cork and glass tube used for this purpose had previously been treated
with boiling water.

[Illustration: Fig. 75.]

When the wort had cooled down in the flask and matters were arranged as
represented in Fig. 75, I raised the two-necked flask so as to cause a
little of the liquid and yeast to flow into the wort. Thereupon
fermentation was set up, and the resulting carbonic acid gas made its
escape by the drawn-out end of the doubled-necked flask. The entire
arrangement with its supporting stand remained in this connection for
eighteen months, sometimes on a stove, sometimes in the laboratory,
exposed to all the variations of external temperature. At the end of
that time I tasted the beer in the flask; it was perfectly sound, and
the ferment, submitted to the microscope, showed not the slightest trace
of any foreign ferments: and, doubtless, the experiment might have been
protracted over any number of years with the same result.

The only change that occurs in course of time is the appearance in the
neck of the flask at the surface of the beer of a deposit of small
prominences resembling a crystallization, but which really consists of
those forms of ferment to which in Chapter V. I attached the name of
_aërobian ferment_. The beer, after being transferred to a bottle that
had been washed with hot water, was kept for several months in the heat
of summer, without exhibiting the slightest trace of deterioration.

[Illustration: Fig. 76.]

[Illustration: Fig. 77.]

The essential conditions of the preceding experiment can readily be
realized on the large scale. For this purpose we may employ the
apparatus in the above sketch (Figs. 76 and 77) constructed of tin or
tinned copper. As appears from the sketch, this consists of a
cylindrical tub resting on a support, and closed at the top by a cover,
whose lower edge fits into a gutter containing water. The wort prepared
in the copper is led into the cylinder, a process which does not
materially lower its temperature. Now we know that wort in breweries
which has been cooled in contact with the air, and so got charged with
disease-germs, will, nevertheless, recover its faculty of keeping for
any length of time in pure air, if we again raise its temperature to 80°
C. (176° F.) or even 70° or 75° C. (158°, 167° F.) Having filled the tub
with the hot wort and put on the lid, we then connect, by means of a
caoutchouc tube _c d_, the metal tube _a c_ (which opens into one of the
tubulures projecting above the lid) with the system of tubes _d e_, _f
g_, of which _d e_ is fixed to the cylinder; _e f_ is a caoutchouc
junction connecting _e_ with the bent glass tube _g_. We then dash over
the apparatus, lid, tubulures, and their corks a quantity of boiling
water. This collects in the gutter in which the lid rests, and any
excess overflows into a second gutter outside the first, where, however,
it cannot remain, but passes away by means of a ring of small holes
between the base of the outer trough _i i_ and the cylinder. The
overflow is collected in a third trough at the bottom, whence it can be
removed by a pipe M. T is a bent thermometer to indicate the temperature
of the wort; its bulb is protected by an inlet socket _d d_, pierced
with holes; _r_ is a stopcock for discharging the water in the gutter,
which serves as a hydraulic junction between the cylinder and its lid;
R, V, are stopcocks, or openings for the discharge of the liquid in the
cylinder and its deposit. The next process is to cool the vessel, which
may be done either by leaving it to itself, or by introducing a current
of cold water through the tubulure E, soldered on to the lid. This
tubulure is of the form of an inverted funnel, and is pierced at the
bottom with a close row of holes, through which the cold water issues in
a sheet over the surface of the cover. In whichever way the cooling is
effected, the external air continues all the time to enter the vessel
beneath the lid by way of the long, narrow passage _g f e d c a_, and
must necessarily get purified by depositing in its course all
fungoid-germs, just as happened in the case of the two-necked flask of
air experiments. This, however, may be still further secured by
introducing a small plug of cotton wool, or asbestos, into the end of
the tube _g_.

The experiments which we have carried out with this apparatus have
proved that, by adopting such an arrangement, beer, a liquid peculiarly
liable to change, may be kept as long as we wish, for weeks or months,
in contact with air, since the tube _g_ is open, without evincing the
least symptom of disease. It matters little whether the leaves and
strobiles of the hops are introduced with the hot wort or strained off;
the result is the same. On the other hand, a leak in the apparatus from
which the wort gets mixed with ordinary water from outside during
cooling, will speedily effect a change in the wort and cause it to swarm
with vibrios, or butyric ferment, lactic ferment, and other germs of
disease, whilst its taste will be rendered extremely nauseous. It can
only be through one’s own fault, that is, from want of skill in carrying
out the operation, that any change can be brought about by the water in
the gutter not being kept out of the fermenting vessel. That water may
even become putrid without the organisms contained in it being able to
reach the wort in the fermenting vessel. The apparatus may be of any
size whatever; we have worked with vessels containing 12 hectolitres
with as much ease and certainty as when we used an apparatus of 1
hectolitre (22 gallons).

It is easy to carry out the process of cooling in the presence of
carbonic acid gas if we fit a bent tube, similar to _a c d e f g_, to
the second tubulure D. Through this tube, or its companion, the gas can
be passed as it issues from an apparatus in which it is generated, or
from a gasometer filled with it, or from a vessel of beer undergoing
fermentation.

However, there is no necessity that the cooling should take place in the
fermenting vessel. It may be effected separately, in vessels of greater
or less depth, in spiral coils surrounded with cold water, or in any
kind of refrigerator, provided always that the conditions of purity are
satisfied, and that the flow of the cooled wort takes place under the
same conditions. Jets of steam, which are already extensively used for
the cleansing of pipes in breweries, may be employed here with great
advantage.

The pitching may be effected in various ways. A two-necked flask of a
capacity of from 200 to 300 cc. (about 7 to 10 fl. ozs.), in which not
more than 100 cc. (3-½ fl. ozs.) of wort has been fermented, will be
sufficient for an apparatus of 1 hectolitre (22 gallons), although the
flask may not contain more than 1 or 2 decigrammes (1-½ to 3 grains) of
yeast. In the manufacture of beer, as at present conducted, the
employment of so minute a quantity of yeast would lead to most
disastrous results. The fermentation would unfailingly become lactic and
butyric, since the foreign germs with which commercial worts and yeasts
are always contaminated would have ample time to develop during the
first twenty-four or forty-eight hours, whilst the small quantity of
yeast used in the pitching could scarcely do more than begin to develop
during that time. It is simply with the object of avoiding these
secondary fermentations that the brewer uses large quantities of yeast
for pitching.

After the wort and yeast have been _pulled up_,[163] a process which
every practical brewer adopts after pitching, every part of the liquid
is occupied by a multitude of yeast-cells, which seize upon the oxygen
in solution, germinate with activity, turn to their own account the
food-supplies most easily assimilated, and prevent the growth of the
germs of disease-ferments. In the new process which we are now
explaining, things happen quite differently. Our wort is pure, and our
yeast is pure, and if only a single cell of yeast were introduced into
the wort, the vital activity of this would be sufficient to bring about
alcoholic fermentation, and to transform the wort into beer, without our
having the least reason to apprehend the simultaneous development of any
other organisms whatsoever. In short, the new process enables us to
pitch with as small a quantity of yeast as we like. It is, nevertheless,
inexpedient to employ too minute a quantity, since by doing so we should
retard the commencement of fermentation.

[Illustration: Fig. 78.]

[Illustration: Fig. 79.]

In the case of an apparatus of 5 hectolitres (110 gallons) or double
that capacity, the pitching may be accomplished by means of flasks
holding from 4 to 9 litres (from 7 to 10 or 11 pints), (Fig. 79), or
copper cans, tinned inside, holding from 10 to 15 litres (2-1/4 to 3-1/4
gallons), and provided at the upper conical end with glass tubes (Fig.
78). The vessel must be half or two-thirds filled with wort. For this
purpose it will be well always to employ wort that has been preserved in
bottles by Appert’s process. We must use a stopper provided with tubes,
as represented in Fig. 79: _a b_ is a glass stopper which closes the
india-rubber tube _b c_; _m n p_ is a fine glass tube, or, better still,
made of copper.

The tap R being closed, a long india-rubber tube is attached to the
extremity of the curved tube, and the flask is completely immersed in a
hot-water bath; the india-rubber tube projects from the bath and keeps
the interior of the vessel in communication with the external
atmosphere. If the tube _m n p_ is of copper, we may avail ourselves of
its flexibility and bend it upwards, so as to place its open extremity
outside the bath. The water in the bath is then gradually raised to a
temperature of 100° C. (212° F.), at which it is kept for a quarter or
half an hour. In the case of copper cans, it is more convenient to place
them over a gas-heater. They may be treated in the same manner as the
flasks with curved necks. Vessels prepared in this manner may remain in
a laboratory, or in any part of a brewery, for an indefinite time,
without the wort in them undergoing the least change. It gradually
darkens in colour through a direct oxidation of a purely chemical
nature, but no tendency to disease will manifest itself.

Some days before we require to pitch an apparatus of several
hectolitres, we impregnate one of these flasks or cans.[164] For this
purpose we pass the flame of a spirit lamp over the tubes _c b a_ and _m
n p_, to destroy the particles of dust that might pass inside at the
moment when the stopper _a b_ is taken out, and then by means of a long,
straight glass tube we take some of the liquid from a flask or vessel
containing pure beer in a state of fermentation, and let a few drops of
this, with the yeast that it holds in suspension, fall into the flask or
can; the stopper _a b_ is once more passed through the flame and then
replaced; generally in the course of one or two days the yeast develops
in the flask sufficiently for the fermentation to show itself. We may
shorten the operation still further by emptying into the can the
contents of one of those double-necked flasks. To do this, we have
simply to attach the straight tube of the flask to the india-rubber _b
c_, and pour the liquid in. In a similar manner we introduce, through
one of the tubulures surmounting the lid of the fermenting apparatus,
the contents of the flasks or cans, either whilst they are still in
active fermentation, or after fermentation is over. For this purpose,
the tap R is connected by means of an india-rubber tube (Fig. 79), with
a tube passing through a cork fixed in one of the tubulures of the large
apparatus. All this may be done in considerably less time than we have
taken to describe it; and the operation may be performed accurately and
safely by any one who has witnessed it a few times, even though he may
not be skilled in chemical manipulations, especially if he takes care to
bear in mind the very simple principles which we have explained.

Since certain parts of the apparatus—the outer opening of the tap, or
the india-rubber tubing, for example—may contract particles of dust from
the air, those parts, before being used, must be boiled in water, or
washed with boiling water, or passed through the flame of a spirit lamp,
to destroy the germs mixed with the particles of dust that settle upon
them.

The method of cooling the wort in contact with carbonic acid prevents
access of oxygen to the latter up to the time of pitching, so that the
development of the yeast takes place apart from the influence of oxygen.
Now, we know that these conditions necessitate the employment of a very
young yeast—a yeast that is in course of active germination, such as may
be taken from an incipient preparatory fermentation. Nevertheless, even
with this, the development of the yeast under such conditions is
extremely slow, and the fermentation takes from fifteen to twenty-five
days; whilst, under the same circumstances, but with an aerated wort, it
would be finished in from eight to twelve days. This is a considerable
drawback, but, perhaps, a still more serious inconvenience is that the
beer takes much longer to clarify, and does so with greater difficulty
than those beers which are made with aerated worts. At the same time,
this is largely compensated by the superior quality of the beer, which
is stronger and has greater fulness on the palate, whilst the aroma of
the hops is preserved to an extent never found in beers brewed by the
ordinary process. Besides this, the yeast deposited at the bottom of the
fermenting vessel is much less active, and, being of an older type, is
revived with greater difficulty than that which forms in aerated worts.
This, which might be considered a disadvantage, if we had to employ the
yeast afterwards for pitching, has the great advantage of giving a beer
which, when racked, undergoes its secondary fermentation only slowly,
and with difficulty.[165] A beer of this kind is better adapted than
ordinary beer to stand a long journey without developing great pressure
inside the casks, and, if bottled, it will contain very little deposit,
and will not froth violently when uncorked. The reason is, that a yeast
is the more active, the more ready to multiply rapidly, and to work
vigorously the more highly aerated the wort was in which it was grown.
On the other hand, a yeast formed apart from air readily gets exhausted,
and may even perish in the liquid in which it ferments, when that is
kept out of contact with air; in other words, the vital action of yeast
is more restricted when it has not been subjected to the action of
oxygen during its formation.

If a great depth of wort, the surface of which alone is in contact with
atmospheric air, is left to cool down, it will act in almost exactly the
same manner as that which is cooled under an atmosphere of carbonic acid
gas, because the oxygen of the air is very slow in pervading wort that
is undisturbed. The gas will be taken into solution by the upper layer
only, whilst the bulk of the liquid will remain unaffected by it. In
some experiments which we conducted in a vessel which contained wort to
a depth of 70 centimetres (27·5 inches), and which was provided with a
tap that enabled us to draw off some of the liquid every day, until we
had reduced the depth to 35 centimetres, we found, at the end of eight
days, that there was not a trace of oxygen in solution at the latter
depth. It is even probable that, considering the slow diffusion of the
oxygen, on the one hand, and the combination that may take place between
it and certain components of the malt, on the other hand, it would take
a long time for all the wort, if undisturbed and of a certain depth, to
become saturated with oxygen. In the vessel represented in Figs. 76 and
77 there is a considerable depth of wort to cool down. Nevertheless, the
mere fact of the possibility of an aeration from the surface, whilst the
wort is cooling down in contact with pure air, is enough to account for
a certain effect that is produced on the yeast, later on, for the more
youthful appearance of the yeast of the deposit, compared with that
which we find in the case of wort cooled in the presence of carbonic
acid gas. The difference between the results is particularly striking
if, in both cases, we follow up microscopically the development of the
yeast during the first few days succeeding the pitching.

The influence of the air on fermentation is considerable. In the
ordinary process of brewing, fermentations would be almost impossible,
and in every case most defective, if the wort, before being run into the
fermenting vessels, were not aerated by its passage over the “coolers,”
where the aeration is more or less effective, according as the liquid is
more or less shallow. Worts and yeasts being impure, that is containing
the germs of foreign ferments, those germs would have time to germinate
in the fermenting vessels during the delay that the want of aeration in
the wort would cause in the development of the yeast. We are aware that
several inventions have been proposed to do away with the coolers, and
we feel convinced that the object has been to remedy irregularities in
fermentation. Considering the facts which we have published[166] on the
development of yeast in the presence of air, and its inactivity in
non-aerated media, such inventions ought to be supplemented by some
means of further aeration for the prevention of the mischief that they
must otherwise cause. In the existing process of brewing, the employment
of coolers is a necessity.

The influence of the air on the vital action of the yeast may be proved
in ways innumerable. The following is an experiment which we have often
carried out with surprising results. A fermentation is going on; we draw
off the liquid as rapidly as we please, and pour it back again into the
vessel immediately. Within an hour we find a marked increase in the
fermentation, evidenced by the liberation of a greater quantity of
carbonic acid gas. This experiment may be performed with especial ease
if we use the fermenting apparatus that we have described, for, by
fitting a gas measurer to the escape tube _a b c d e f g_, the number of
litres produced before the drawing off of the liquid may be compared
with those obtained after. The least physical change in the running of
the fermenting liquid whilst it is being drawn off, modifies the effect
in question; such as change in the diameter of the stream, the height
from which it falls, its greater or less scattering in falling, all
influence it. Again, as might be expected from such results,
corresponding modifications take place in the cells of yeast which come
under the influence of the air. They become firmer in aspect and
outline, their plasma becomes fuller, assumes a younger and more
transparent aspect, and the vacuoles disappear. The molecular
granulations, too, are less apparent. At a certain focus they disappear;
at another they reappear, not as black spots, however, but as brilliant
points so small as to be scarcely perceptible. If germination has been
suspended it is resumed; in short, everything tends to prove—and having
the yeast actually under our eyes we cannot doubt the fact—that the life
of the cells is more decided, and the work of nutrition more active
after they have been brought into contact with the oxygen of the air,
and have absorbed a greater or less quantity of that gas.

Under the ordinary conditions of brewing, the atmospheric air is present
in very varying quantities, whether introduced by the wort which holds
more or less in solution, or by diffusion over the surface of the
vessels, so that the same cells of yeast live by turns without air and
with air. At first they absorb all the oxygen held in solution, and
multiply under the influence of this absorption. Afterwards, when the
supply has been exhausted, and various assimilations have resulted from
it, they are deprived of it. Their life continues apart from oxygen, and
if the vessel were closed, fermentation would be accomplished under
these conditions, although more slowly. The vessel being open, a small
quantity of air diffuses continuously through the layer of carbonic acid
gas on the surface, and supports the vitality of the cells.

It is interesting to observe that, in the working of breweries, there
are several empirical practices the explanation of which is to be found
wholly in the fact that the aeration of wort or beer exercises a great
influence on fermentation. In many breweries we have seen the pitching
performed in the following manner: the brewer, having mixed his yeast in
many times its volume of wort, pours all the thick liquid from a height
from one bucket into another, and from that back again into the first,
and so on a great many times, until the two buckets are filled with the
froth enclosing air. In certain London breweries we have seen a bucket
suspended by a pulley over the fermenting tun, which is 3 or 4 metres
(10 or 12 feet) in depth; this the brewer, by means of a cord, can lower
into the tun and pull up again at will, giving it a kind of see-saw
movement which agitates the surface of the liquid and aerates it. The
use of the fermenting tun itself and the racking of the wort from that
tun into casks have the effect of aerating the beer and the yeast, and
imparting to the latter a greater vigour and activity.

The resumption of fermentation in cask, after the beer has been run out
of the tuns in “low” fermentation breweries is, in our opinion,
principally due to the aeration of the beer at the moment when it is
racked. The brewer ought to bear in mind that, during racking, every
detail is of importance; it makes a great difference whether when the
beer is run into the casks it falls from a height or is conducted by a
tube to the bottom of the casks, whether it passes directly into the
casks, or is poured into them from buckets, and whether it runs in a
stream of small or large diameter, since these different methods cause
the introduction of corresponding different quantities of air into the
beer.

We have devised a simple arrangement for bringing the fermenting liquid
into contact with various proportions of atmospheric air. Appended is a
sketch of this apparatus (Fig. 80). Instead of one tube serving alike
for the entrance and escape of gas, there are two similar ones, each of
which opens into one of the tubulures on the cover. Round the other end
of one of the tubes is fitted a kind of muff or bag, composed of a
cylindrical cage of metallic gauze, over which a layer of well-combed
cotton wool is placed, the whole being covered with a muslin bag. The
object of this arrangement is to act as an air-filter for retaining the
particles of dust. When fermentation has commenced in the apparatus, we
have simply to press momentarily the india-rubber connection between the
tube from the lid and the tube with the bag. This will at once cause a
regular stream of carbonic acid to issue from the end of the uncovered
tube, whilst the air will enter by the filtering tube to take its place;
and this arrangement will be maintained throughout the whole course of
the fermentation, even if we omit the precaution of increasing the power
of the syphon by making the tube for the escape of this gas longer than
the other one.[167]

[Illustration: Fig. 80.]

It will be readily understood how, whether by this last method, or by
the diameter of the tubes, we may vary the conditions of this
circulation of air in the apparatus, on the surface of the beer.


    § II.—Method of Estimating the Oxygen held in Solution in Wort.


The use of carbonic acid gas and the cooling of the wort, in contact
with that gas or in contact with very limited quantities of pure air,
are by no means necessary to the application of the new process. There
is only one thing that is absolutely essential—which is, the _purity_ of
the gases in the presence of which the wort is cooled and treated. If,
therefore, it is well to aerate our wort, either before or during
fermentation, this may be done, on the sole condition that the air
employed does not introduce any germs of disease that are likely to
develop in the beer during fermentation or afterwards. The question of
aerating the wort is not, however, so simple a matter as it seems at
first sight. A very simple observation will show that wort cannot be
safely oxygenated by exposure, without precaution, to the air, even
leaving out of account the germs of disease which that air may contain.
It is easy to show that finished wort has a decided flavour and aroma of
hops, as well as a sweet taste, and that it leaves a certain pleasant,
bitter after-taste on the palate. When we taste it in this condition we
cannot help thinking that a liquor of the kind, after fermentation,
ought to constitute a very valuable beverage, as wholesome as it is
pleasant. Now all this pleasant and refreshing sensation that the wort
leaves on the palate, which is due as much to the aroma as to the
bitterness of the hop, disappears absolutely, we may say, if the wort is
left exposed to contact with air for a sufficient time, and that whether
the air be warm or cold. We may easily perform the experiment in one of
our two-necked flasks, in which we can preserve the wort, in contact
with pure air, without any fear of change. The oxygen of the air enters
into combination with the substances that the hop introduces into the
wort, and the wort, in consequence of this oxidation, gradually becomes
transformed into a saccharine decoction, without odour, in which even
the bitter flavour is destroyed or hidden. In other words, the wort
grows weak and flat, in just the same way that beer and wine do, as well
as all the various natural or artificial worts which serve to produce
them. Thus it is evident that considerable care is necessary in
subjecting wort and beer, whether in course of manufacture or finished,
to the action of atmospheric air. If, therefore, it is a good thing to
supply wort with oxygen, as we have already pointed out, in order to
facilitate the fermentation and nourish the yeast, it is, on the other
hand, important that the quantity supplied to it should not be too
great, otherwise we may injure the quality of the beer, and particularly
its fulness on the palate, that is its apparent strength, which has very
little to do with the proportion of alcohol in it. The strength of a
beer is intimately connected with those substances introduced by the
hops into the wort and thus into the beer, to which we previously
alluded, and of which too little is known; their properties and the
palatableness resulting from them are very readily affected by the
oxygen of the air.[168]

We have, therefore, to ascertain the measure in which air occurs during
the process of brewing, and whether, in the actual process, there may
not be too great a proportion of active oxygen present. The study of
this subject requires that we should know what quantities of oxygen may
be held in solution in the wort or absorbed by direct combination.
Fortunately this has been rendered a comparatively easy matter by a
rapid method of estimating the oxygen held in solution in liquids of
various kinds, devised by M. Schützenberger in 1872. As soon as this
method was made known, we requested M. Raulin, who was attached to our
laboratory as assistant-director, to apply it to the determination of
oxygen in wort. This he did with his accustomed skill, devising certain
alterations of details which rendered the method at the same time surer
and more expeditious.

The principal feature in M. Schützenberger’s process consists in the
employment of a salt, the properties of which that chemist was the first
to recognize; he has named it _hydrosulphite of soda_, and it is
obtained by the action of zinc filings on a solution of bisulphite of
soda, out of contact with air.

Hydrosulphite of soda S^2O^2,NaO,HO, which is isomeric with hyposulphite
of soda, only differs from the bisulphite by two equivalents of
oxygen.[169] When brought into contact with free oxygen, it absorbs that
gas instantaneously and becomes converted into bisulphite; similarly
when mixed with water, it immediately absorbs the oxygen held in
solution. Again there are colouring matters, such as M. Coupier’s
soluble aniline blue, that are instantaneously decolourized by
hydrosulphite of soda, whilst they resist the action of the bisulphite.
If, taking care to avoid the access of air, we add hydrosulphite of soda
to a certain volume of water—a litre, for example—that has been deprived
of air and faintly coloured with Coupier’s blue, we shall see that a few
drops will be sufficient to effect the decoloration. If, on the
contrary, the water is aerated, the decoloration will not be effected
before a sufficient quantity of the hydrosulphite has been added to
absorb the oxygen in solution, and the volume of the reagent required is
in proportion to the quantity of oxygen in solution in the water. To
render the process sensitive, we must dilute the hydrosulphite to such
an extent that 10 c.c., for example, may correspond very nearly with 1
c.c. of oxygen. If the reagent would keep we should only have to
determine directly, once for all, the volume of oxygen that a known
volume of the liquid could absorb; but, in consequence of its extreme
liability to change through contact with air, it is necessary to titrate
the liquid every time before using it. This is easily done in the
following manner:—

According to the observations of Messrs. Schützenberger and Lalande, the
hydrosulphite decolourizes an ammoniacal solution of sulphate of copper,
reducing the copper to a lower state of oxidation; the sulphite and
bisulphite having no action as long as there is an excess of ammonia. We
prepare a strongly ammoniacal solution of sulphate of copper, containing
such a quantity of copper that 10 c.c. of the liquid will correspond, as
far as action on the hydrosulphite is concerned, with 1 c.c. of oxygen.
Calculation by equivalents gives us the correct value verified by direct
experiment.[170]

The object of the modification which M. Raulin has introduced, is to
avoid the loss of time thus occasioned by the changes which take place
in the titrated liquids by long keeping, as well as certain errors which
may arise from the acidity of the wort. On this latter point M.
Schützenberger has remarked that the quantities of hydrosulphite of soda
corresponding with one and the same volume of oxygen vary with the
acidity of the liquid operated upon, a phenomenon which that skilful
chemist explains by the formation of oxygenated water, of varying
stability in media of different acidity.

Instead of determining the strength of the titrated solution of
hydrosulphite before each operation, we take the solution as it happens
to be, and determine its strength by causing it to act on a known volume
of pure water saturated with oxygen at a certain temperature. The tables
of solubility of oxygen in water give the exact volume of oxygen on
which the measured volume of hydrosulphite used has acted. According to
Bunsen, about one minute’s brisk shaking in a closed bottle, with excess
of air, will be sufficient to effect the maximum saturation of the water
at the temperature at which we operate.

For experiments on wort we require:—

1. A 2-litre (3-½ pints) flask, A, containing _saturated_ hydrosulphite
of soda,[171] of such strength that 2·5 c.c. will be sufficient to
absorb almost all the oxygen in 50 c.c. of water saturated with air at
the ordinary temperature (that is, 1 volume of hyposulphite must equal
20 volumes of water).

2. A 2-litre flask, B, containing a solution of indigo-carmine, 50 c.c.
of which will be decolourized by about 20 c.c. of the hydrosulphite.
This solution contains about 20 grammes (30·7 grains) of commercial
indigo-carmine per litre (1·76 pints).

3. An apparatus, C, for the production of hydrogen.

4. An experimental apparatus composed of a burette, D, graduated in
tenths of a cubic centimetre, and a three-necked Wollf’s bottle, E.

5. A flask, F, holding about 100 c.c. provided with a straight tube
divided into tenths of a cubic centimetre, and containing a solution of
ammonia of such strength that about ten drops of it will neutralize the
acidity in 50 c.c. (1·76 fl. oz.) of wort.

To perform the operation we shake about 150 c.c. (5·3 fl. oz.) of
distilled water, at the existing temperature, in a 1-litre flask for a
minute or so; this saturates it with air, and we must at the same time
note the temperature. To be extremely precise, we should note also the
barometrical pressure.

[Illustration: Fig. 81.]

Into the bottle E we introduce about 50 c.c. of the indigo solution, and
200 c.c. of water at about 60° C. (140° F.), and fill the tube _e_ to
the point _b_ with water saturated with air; we then expel the air from
the bottle E by a current of hydrogen. The blue colour of the liquid in
the bottle is then very carefully brought to a yellow tint, by running
in, drop by drop, the hydrosulphite with which the burette D is filled.

We next pour 50 c.c. of distilled water saturated with air into the
funnel _a_, and pass it into the flask; the blue colour reappears. We
must then bring back the colour to exactly the same tint of yellow. Let
_n_ represent the number of divisions on the burette denoting the volume
of hydrosulphite employed for this purpose.

We repeat this last operation immediately, taking 50 c.c. of the wort,
the oxygen of which we wish to determine, having first introduced into
the funnel _a_ a sufficient number of drops of the ammoniacal solution
to neutralize the acidity of the wort. Let _n´_ represent the number of
divisions of hydrosulphite employed to restore the yellow tint in the
case of the wort.

We once more perform the experiment with 50 c.c. of saturated water; let
_n´´_ be the number found.[172]

The ratio which the quantity of oxygen held in solution in the wort
bears to the quantity of oxygen contained in the same volume of water
saturated with air, at the temperature _t_, and under the pressure H,
will be

[Illustration: Formula 3: _n´_/((_n_ + _n´´_)/2).]

it will be sufficient in most cases to bear in mind this ratio.

When we want to deduce the absolute quantity of oxygen held in solution
in a volume V of the wort, we have merely to multiply this ratio by the
quantity of oxygen contained in the same volume of water saturated with
air, at the temperature _t_ and under the pressure H, a very simple
problem if we know the coefficients of the solubility of oxygen in water
at different temperatures. These coefficients are given for ordinary
temperatures in the following table, which was compiled by Bunsen. We
have restricted the numbers to three places of decimals:—

                  Temperatures.        Coefficients.
                  0° C. (32° F.)       0·040
                  1° C. (33·8° F.)     0·040
                  2° C. (35·6° F.)     0·039
                  3° C. (37·4° F.)     0·038
                  4° C. (39·2° F.)     0·037
                  5° C. (41·0° F.)     0 036
                  6° C. (42·8° F.)     0·035
                  7° C. (44·6° F.)     0·035
                  8° C. (46·4° F.)     0·034
                  9° C. (48·2° F.)     0·033
                  10° C. (50·0° F.)    0·033
                  11° C. (51·8° F.)    0·032
                  12° C. (53·6° F.)    0 031
                  13° C. (55 4° F.)    0·031
                  14° C. (57·2° F.)    0·030
                  15° C. (59·0° F.)    0·030
                  16° C. (60·8° F.)    0·029
                  17° C. (62·4° F.)    0·029
                  18° C. (64·4° F.)    0·029
                  19° C. (66·2° F.)    0·028
                  20° C. (68·0° F.)    0·028

The primary condition which enables us to rely on the exactness of this
method is the fact which we have mentioned above, that a liquid if
shaken up with air for one minute will become perfectly saturated with
oxygen. Substantially this is the case. In estimating the oxygen in
different parts of a liquid treated thus, we have invariably obtained
the same figures to within about 1/50th.

It is true that the variable quantity of the oxygen held in solution in
the liquid contained in the part of the tube _eb_, as well as the oxygen
absorbed during the treatment of the liquid in contact with air,
constitute causes of error. Experience, however, proves that these
causes of error are insignificant, as long as we have to deal with a
liquid the aeration of which is not very far removed from the point of
saturation, and whose solubility-coefficient for oxygen is not widely
different from that of water for the same gas. Under such conditions we
have always found a constant ratio, to within about 1/40th between the
same liquid and air-saturated distilled water, placed under the same
circumstances.

If, on the other hand, we have to deal with a liquid which holds but a
minute quantity of oxygen in solution, the causes of error mentioned may
very seriously affect our results, and it will be absolutely necessary
to avoid them. The liquid experimented on must be treated out of contact
with air, by aspirating it directly from the vessel that contains it
into the pipette H, which is graduated for 50 c.c., and causing it to
pass thence into the flask E, by substituting the pipette for the funnel
_a_. Finally, before arranging the pipette, we cause a small quantity of
the liquid in the flask, which has been previously brought to the exact
yellow tint, to pass, by pressure, through the tube _eb_, so as to avoid
the cause of error that is likely to result from the air held in
solution in the liquid of that tube.

The liquid, the oxygen of which has to be determined, may also be passed
directly from the vessel containing it into the flask E; the rest of the
operation being performed as already described.

It was by this method that the oxygen held in saturate solution in wort
was determined. The following are the principal results obtained by M.
Raulin:—

1. At different pressures the ratio between the quantities of oxygen
held in solution in water and in wort is, all other conditions being
similar, constant. This ratio has been found equal to 1·20 in the case
of wort and water saturated with air at the ordinary pressure, and 1·24
in the case of wort and water saturated with pure oxygen.

2. The ratio between the coefficient of the solubility of oxygen in
water and that of its solubility in wort is very nearly constant at
different temperatures, increasing, however, slightly as the temperature
diminishes.

This ratio has been found to be—

                   Temperatures.
                   26° C.           (78·8° F.)   1·20
                   19·5° C.         (67·1° F.)   1·25
                   4° C.            (39·2° F.)   1·37

Another wort gave the following results:—

                   Temperatures.
                   9° C.            (48·2° F.)   1·15
                   21° C.           (69·8° F.)   1·10
                   25° C.           (77·0° F.)   1·07

3. The ratio between the quantities of oxygen held in solution in water
and those held in solution in wort increases with the concentration of
the wort. By evaporating the same wort to different degrees of
concentration, and afterwards saturating it with air, at the same
temperature, we obtained the following figures for the ratio in
question:—

                 Weak wort                      1·06
                 The same evaporated to half    1·15
                 “        ”      “  2/5         1·27
                 ”        “      ”  3/10        1·45
                 “        ”      “  1/6         1·96

4. Worts of different origin, but of the same density and temperature,
when saturated with oxygen, always contain very nearly the same quantity
of that gas.

Two portions of the same wort, shaken up with air, one being hot the
other cold, then left to themselves for some time, and afterwards
saturated with air, at the same temperature, gave the figures 1·22 for
the ratio between the oxygen in the water and that in the wort.

Different worts of the same density, saturated at a temperature of 15°
C. (59° F.), gave the following ratios:—

             Wort kept in a bottle with air for 19    1·140
             months

             Wort recently prepared                   1·142

             Wort kept in a bottle without air for 20 1·142
             months, aerated for 18 days

             Wort evaporated to dryness and made up   1·126
             with water

5. The solubility of oxygen in wort differs very little from the
solubility of oxygen in sweetened water of the same density.

An experiment was made with a solution of sugar on the one hand, and
with wort more or less diluted with water on the other hand, at the same
temperature of 11° C. (51·8° F.). The following figures were obtained
for the ratios of solubility:—

                                         Solution   Wort.
                                         of Sugar.

               Marking 17·9°             1·278      1·27
               Balling[173]

               “    14·0°    ”           1·190      1·15

               “     7·0°    ”           1·092      1·06

6. From the preceding results it is easy to deduce a general formula
which shall give the coefficient of solubility of oxygen in any wort,
marking B° by _Balling_, and at temperature _t_°.

From the figures of (2) it follows that above and below the temperature
of 15° C. (59° F.), the ratio which the coefficient of solubility of
oxygen in water bears to that of the solubility of the same gas in wort
varies about 0·006 for each degree of the thermometer. From the figures
of (3) it follows that the same ratio varies about 0·002 for each degree
of _Balling_ above and below the 15th degree on the instrument.

By taking _c_ for the coefficient of solubility of oxygen in water at
_t_° C., and _c´_ for that of oxygen in wort also at _t_° C., and having
a density B, by _Balling_ at 15° C.; and taking X for the ratio
_c_/_c´_, at 15° C. and 15° _Balling_, we shall have

    _c_/_c´_ = X + (B - 15) 0·022 - (_t_ - 15) 0·006.

By carefully ascertaining the ratio _c_/_c´_ for different worts, and
adopting the preceding formula, we have found for X a mean value of
1·16.

The definitive formula, therefore, is:

    (1) _c_/_c´_ = 1·16 + (B-15) 0·022 - (_t_ - 15) 0·006,

or again,

    (2) _c_/_c´_ = 0·86 - (B - 15) 0·016 + (_t_ - 15) 0·004.

The coefficient _c_ of the solubility of oxygen in water will be found
in the table given a few pages back.


  § III.—On the Quantity of Oxygen existing in a state of Solution in
                          Brewers’ Worts.[174]


The wort, when it comes from the copper in which it is boiled with the
hops, remains exposed upon the coolers for a time, the length of which
varies according to circumstances, the most important of which is the
exterior temperature. The average time is from seven to eight hours,
during which the volume of the wort diminishes, whilst its density
increases; at the same time, it deposits its proteinaceous matters and
absorbs oxygen from the air, either by way of solution or of
combination.

In the present paragraph we shall confine ourselves to the uncombined
oxygen held in a state of solution in wort, recognizable by the change
of colour produced by its action on white indigo.

The use of the coolers enables the brewer to obtain his wort in two
distinct states of limpidity—filtered wort and unfiltered wort. At the
same time there is a further difference between these worts, namely, in
the quantity of oxygen held in solution. The unfiltered wort comes
direct from the coolers; the wort to be filtered, mixed with a part of
the deposit, is run into a special vessel, from which it is distributed
over the filtering surfaces, which are generally of felt; filtered
bright, it is then received in a reservoir, from which it is distributed
amongst special fermenting vessels. Falling through the air in a thin
stream of drops, it must necessarily have become charged with a greater
quantity of oxygen than ordinary wort. In good breweries it is put apart
by itself to ferment, and the yeast which it yields is firmer and
deposits more easily than that of unfiltered wort. As for the
fermentation, it is, under similar conditions, quicker by a day or a day
and a half than in the case of ordinary wort. The difference in the
quantity of oxygen held in solution in the two kinds of wort is greater
in proportion as the external temperature is lower; in winter it may be
twice as great as in summer. The reason is that in summer a boiling wort
does not obtain a minimum temperature of 20° C. (68° F.), on the best
coolers, in less than six or seven hours. After leaving the coolers it
is passed over a refrigerator. In winter it attains that temperature in
about three hours, or less, which then goes on sinking on the coolers.
During the last two or three hours which are employed in bringing the
temperature still lower, as also during the running off, the wort
absorbs an appreciable quantity of oxygen. In other words, wort in
winter remains for a longer time at low temperatures, in free contact
with air.

Another circumstance unites with this exposure upon the coolers to
increase the aeration of the wort; the wort is run into the fermenting
tuns through pipes of large sectional area, more or less bent, and
carries with it by suction considerable quantities of air, which, from
the continual agitation, gets well mixed with it. The effect of this
mixing in the pipes is to considerably increase the proportion of air in
solution in the wort, especially in winter, when the temperature of the
wort is lower; and from the figures given below we may, although it is
very variable, put the average increase at a quarter of the whole
amount. The calculation has been made by comparing the quantities of air
held in solution in two samples of the same wort, one of which was taken
from the coolers at the moment of “turning out,”[175] and the other from
the fermenting vessel after it was filled.

Let us call the ratio between the quantity of oxygen held in solution by
a wort, and that which the same wort would hold in solution if saturated
at the same temperature, the _degree of saturation_ of that wort at the
temperature _t_.

The determination of degrees of saturation is reduced to a comparison of
the number of divisions of hydrosulphite _n_ which satisfies the wort in
the first case, with the number _n´_ corresponding with the same wort
saturated at the same temperature. The ratio _n_/_n´_ gives the degree
of saturation at the temperature _t_.

In experiments made with a wort at 14·5° _Balling_ as mean density, we
found the following results:—

In summer, in the case of worts reduced to the temperature of 5° C. (41°
F.) by a refrigerator, the degrees of saturation may be set down as—

                       For unfiltered worts 0·500
                       For filtered worts   0·800

In winter, in the case of some worts which were racked at a temperature
of from 3° to 4° C. (37·4° to 39·2° F.), without the use of a
refrigerator, we found the saturation complete in both worts. In the
case of a very low external temperature, however (-10° C., 14° F.), we
have failed to determine the saturation in an unfiltered wort. As
regards the mean winter figures, in the case of worts racked at a
temperature of 5° C. (41° F.), they may be fixed at these:—

                       For unfiltered wort  0·850
                       For filtered wort    0·950

In autumn and spring we find the mean figures to be intermediate between
those given above:—

                  For unfiltered wort  0·500 to 0·850
                  For filtered wort    0·800 to 0·950

From these ratios it is easy to find the quantity of oxygen contained in
brewers’ worts, if we also refer to Bunsen’s Tables and the formula (2)
given in the preceding section. At the temperature of 5° C. (41° F.), at
which the above worts were “gathered,”[176] and not taking into account
the very small correction that should be made for the difference of half
a degree on Balling, we find, by this formula, as the ratio of the
coefficients of the solubility of oxygen in saturated wort and in water—

    _c´_/_c_ = 0·82

Now, at the temperature of 5° C., the quantity of oxygen held in
solution in 1 litre of water is, according to Bunsen, 0·036 litre, at
the atmospheric pressure, and therefore at the pressure of 1/5th
atmosphere, which is that of the oxygen in atmospheric air, it will be—

    0·036/5 litre = 7·2 c.c.—[that is, 2 cubic inches per gallon.]

And, consequently, in the case of saturated wort, it will be—

    7·2 c.c. × 0·82 = 5·904 c.c.—[that is, 1·62 cub. inches per gall.]

Multiplying this last number of c.c. by the different _degrees of
saturation_ found, we shall obtain the volumes of oxygen held in
solution in 1 litre of different worts:—

          Summer worts {Unfiltered 0·500 × 5·904 c.c. = 2·952 c.c.
                       {Filtered   0·800 × 5·904  “   = 4·723  ”

          Winter worts {Unfiltered 0·850 × 5·904  “   = 5·018  ”
                       {Filtered   0·950 × 5·904  “   = 5·609  ”

It is important to notice that we are here dealing with wort taken from
the fermenting vessel just before it was pitched; that is to say, when
the quantity of oxygen held in solution was as large as the treatment to
which it had been subjected allowed of its being. The mode of taking it
for examination is as follows:—A burette, H (Fig. 81), is plunged into
the fermenting vessel, the temperature of which at the time is
ascertained very exactly, the upper part of the burette being fitted
with an india-rubber tube, _a b_, longer than itself. The liquid is then
sucked up the tube, and soon completely fills the apparatus and runs out
at _b_ (Fig. 82). By lowering the tube the whole arrangement thus forms
a syphon, and enables us to let the wort that we are experimenting on
flow for some minutes; when every trace of air has been thus expelled,
the lower tap is closed and the liquid is introduced into
Schützenberger’s apparatus.

[Illustration: Fig. 82.]

As for the saturated wort, the value of which in oxygen serves to
determine one of the elements of the degree of saturation, it is readily
obtained by introducing a volume of from 100 c.c. to 150 c.c. of wort
into a 2-litre or 3-litre flask, and shaking it briskly so as to
saturate it with air; it is then poured into a settling-glass, to
separate it from the great quantity of froth formed in the shaking, and
then, by means of a graduated pipette, 50 c.c. is taken for examination.

We have spoken of the influence that oxygen has on the activity of
yeast, on its development and, consequently, on the progress of
fermentation. Moreover, we know, from experiments already mentioned,
which we communicated to the Academy and the Chemical Society in 1861,
that the rapid development of yeast in contact with air is in reciprocal
relation to the disappearance of the oxygen from the air. Knowing the
conditions of the aeration of wort from the moment when it arrives on
the coolers until the moment when, in the fermenting tun, it is about to
be pitched, it would be interesting to ascertain what happens to the
oxygen dissolved in the wort at the moment of pitching, how yeast is
affected when suddenly brought into contact with that oxygen; what part,
in short, that gas plays in fermentation.

Let us therefore follow up, hour by hour, the degree of saturation after
pitching, in Tourtel’s brewery. On November 4th, 1875, some wort at 14°
Balling was pumped on to the coolers at 7 p.m., and at 4 a.m. went down
to a 32-hectolitre (700 gallons) tun, its temperature then being 6° C.
(42·8° F.) The pitching, in which about 100 grammes (3·2 oz. troy) of
pressed yeast was used per hectolitre (22 gallons), took place at 5 a.m.
The following is the curve of the degrees of saturation of the oxygen,
as drawn by Messrs. Calmettes and Grenet.

[Illustration: Fig. 83.]

The abscissæ represent the time expressed in hours, and the ordinates
give the degrees of saturation of the wort with oxygen. It will be seen
that about twelve hours after the pitching, and at a temperature of 6°
C., all the oxygen had disappeared, absorbed by the yeast. We shall find
that wort by itself, unassociated with yeast, would also have combined
with oxygen; but in the course of twelve hours, at 6° C., this
combination would have been scarcely appreciable in absence of yeast. It
follows, therefore, that the oxygen in solution is taken up by the
yeast, under the conditions of which we are speaking. This has been
proved directly by an experiment. A double quantity of yeast was
employed for a tun similar to the preceding one, and it was found that
the oxygen in solution disappeared completely in less than half the time
that it took to disappear in the first case.[177] It is very important
to notice that in our 32-hectolitre tun, at the moment when we
determined the complete disappearance of the oxygen in solution, the
cells of yeast had assumed a younger and fuller appearance than they had
at first; but they had not multiplied at all up to that time, nor were
there even any buds then visible on them. The oxygen, therefore, must be
stored up somehow in the cells, taken up by their oxidizable matters to
be brought into work subsequently, or to act as a _primum movens_ of
life and nutrition, spreading its influence over several successive
generations of cells.


             § IV.—On the Combination of Oxygen with Wort.


The atmospheric oxygen is not merely taken into solution by wort; it
also combines with it, as a very simple experiment will suffice to show.
If we place in a tinned iron vessel some boiling wort, separated from
the hops in the copper, and cool it suddenly by plunging it into iced
water, and after having cooled it down in this manner to 15° or 20° C.
(59° or 68° F.), saturate it with oxygen, by shaking it briskly in a
large flask, and then completely fill a vessel with it and close it up
for twelve hours, we shall find at the end of that time, if we test it
with the hydrosulphite of soda, as we have described in § II., that it
does not contain a trace of free oxygen. The whole of the gas which was
originally held in solution will have entered into combination, that is
to say, the liquid, first coloured blue with the indigo-carmine, and
then brought to a yellow tint by means of the hydrosulphite of soda,
will not regain its original blue colour through the action of this
wort. The following experiments were undertaken with the object of
studying this property of wort, and in order that we might form some
idea of its importance, and of the total quantity of oxygen that wort
can absorb under certain special circumstances. The experiments were
performed in our own laboratory on wort from Tourtel’s brewery, which M.
Calmettes had forwarded to us in bottles prepared in the brewery at
Tantonville, in the following manner: Each bottle was filled with
boiling wort taken from the copper and closed with a bored cork, through
which the neck of a funnel passed; the funnel also was filled with the
wort, and the whole preserved from contact with air by a layer of oil.
The next day the bottles were corked full by the help of a bottling
needle,[178] previously heated, with perfect corks that had been passed
through the flame. The bottles arrived in Paris in very good condition,
quite full of the liquid up to the corks. They were left undisturbed for
one or two days at the same temperature as that to which they had been
exposed during the corking and the journey. The object of this was to
afford time for a deposit of the wort to form at the bottom of each
bottle. As a matter of fact, we know that wort boiling in the copper is
charged with proteinaceous matters and other floating and insoluble
substances. The wort above the deposit was turbid and opaline; it was in
this state when we used it for our experiments. It may be taken for
granted, without risk of appreciable error, that the wort had been
absolutely deprived of oxygen in solution, inasmuch as it had been
bottled when boiling, and had cooled down out of contact with air. As
for the quantity of oxygen that it might have held in combination, this
must have been insignificant, although there must have been some, since
the wort had been exposed to the air in the copper; the oxygen in
combination, however, could have had no appreciable influence on the
results which we obtained. Let us call this wort _boiled wort_.

_First Experiment._—Into a straight-necked flask we introduced a certain
measured quantity of this wort by means of a syphon, taking care that
the syphon should only act on the opaque wort, and should not reach the
deposit at the bottom of the bottle. We then drew out the neck to a fine
tube in the flame and boiled the wort; and during ebullition we sealed
the end of the fine tube. After it had cooled, we arranged that pure air
should enter the flask. To do this we made a file mark near the fine
closed point of the flask, and connected the point by a piece of
india-rubber tubing with a glass tube containing a column of asbestos,
which we heated. We then broke off the point of the flask inside the
india-rubber tube, so that the air entered the flask after being
filtered through the asbestos. We removed the india-rubber tube and
sealed up once more the fine end of the neck at the point where we had
broken it off. Finally, to aerate the wort to saturation, we shook the
flask briskly for some minutes, and then placed it in a hot-water bath,
where we left it for about a quarter of an hour. We afterwards removed
it to an oven at 25° (77° F.). We repeated the same operation next day
and the four succeeding days.

The wort, which at first was scarcely coloured, gradually assumed a
reddish-brown tint, and deposited an amorphous matter, but without
brightening. It became clear, however, when filtered, which was not the
case with the turbid, opaline wort in the bottles when they arrived.

The following is an analysis of the air in the flask, made immediately
after a renewed and vigorous shaking, the object of which was to
saturate the wort with air before analyzing the supernatant air:—

    November 29th.

          Temperature at which the flask was refilled with air 4° C.
        (29·2° F.)

          Atmospheric pressure 751 mm. (29·6 ins.)

                  Total volume of flask 333 c.c. (20·32 cub. in.)

                  Volume occupied by the wort 120 “ ( 7·32 ” )

    December 8th.

          Volume of gas analyzed 27·6 c.c. (1·68 cub. ins.)

                  After treatment with potash 27·4 c.c. (1·67 “ )

                    ” “ pyrogallol 22·4 ” (1·36 “ )

                     Oxygen 5·0 c.c. (0·305 cub. in.)

    Composition of the gas:— Per cent.

        Oxygen 18·25

        Nitrogen 81·57

The formula which we deduced above (§ II.) allows us to conclude that at
the temperature of 8° C. (46·4° F.), which was the temperature at which
the wort was saturated before the analysis given above, the quantity of
oxygen in solution in the 120 c.c. (4·2 fl. oz.) of wort was 0·84 c.c.
(0·051 cub. in.).

At the moment when the flask was closed, the total volume of oxygen,
calculated to zero and 760 mm. (30 in.) pressure, was 44·73 c.c. (2·729
cub. in.).

At the moment when the analysis was finished, the volume of oxygen was
calculated to the same conditions of temperature and pressure, 38·86
c.c. (2·355 cub. in.); 5·87 c.c. (0·374 cub. in.) has, therefore,
disappeared. Now, as there is 0·84 c.c. (0·051 cub. in.) in solution,
there has, consequently, been an absorption, by combination with 120
c.c. of wort, of 5·03 c.c. (0·32 cub. in.) of oxygen, or 41·7 c.c. per
litre (11·6 cub. ins. per gallon).

_Second Experiment._—In a similar experiment, in which, however, the
flask was kept for five days at a rigorously constant temperature of 55°
C. (131° F.), day and night, and in which the supernatant air was not
shaken up with the wort, we found—

    Volume of gas analyzed 28·5

        After treatment with potash 28·3

          “ ” “ pyrogallol 23·0

           Oxygen 5·3

    Composition of the gas:— Per cent.

        Oxygen 18·6

        Nitrogen 81·4

    Total oxygen at first 29·40

      “ ” remaining 26·04

      “ ” that has disappeared 3·36

      “ ” in solution 0·54

      “ ” in combination 2·82

      Or per litre, 35·2 c.c. (9·8 cub. ins. per gallon).

The colour of the wort in this experiment had become sensibly similar to
that of the wort in the preceding experiment.

_Third Experiment._—In another experiment we left the flask, for the
same length of time again, after it had been refilled with air and
reclosed, at a temperature which varied between 2° and 4°C. (35·6° and
39·2° F.). In this case we found—

    Volume of air analyzed 27·8

    After the action of potash 27·8

    After pyrogallic acid 22·3

         Oxygen 5·5


    Composition of the gas:— Per cent.

          Oxygen 19·7

          Nitrogen 80·3

          Total oxygen at first 29·40 c.c.

            “ ” remaining 27·58

            “ ” that has disappeared 1·82

            “ ” in solution 0·44

            “ ” in combination 1·38

            Or per litre, 17·20 c.c. (4·8 cub. ins. per gallon).

In this last experiment the wort was scarcely darker in colour. Its
colour, compared with that of wort cooled on the coolers in the brewery,
was slightly darker; but the difference, although it existed, was
scarcely appreciable. We shall revert to this fact, which is of
importance, presently.

_Fourth Experiment._—The following series of experiments were undertaken
to enable us to form some idea of the rapidity with which oxygen is
absorbed by wort.

We employed three flasks. A, B, C, of the following capacities:—

    A = 234

    B = 214

    C = 203

into which we introduced the following quantities of wort (boiled wort,
without air):—

    Into A 96 c.c.

     “ B 84 ”

     “ C 84 ”

The necks of the flasks were then drawn out and sealed in a flame, the
liquid being at a temperature of 5° C. (41° F.). The flasks were then
placed in a hot-water bath and kept at 100° C. (212° F.) for a quarter
of an hour. The flask A was repeatedly shaken during cooling, as also
was the flask B, this being omitted in the case of the flask C.

The contents of flask A were submitted to analysis as soon as it was
quite cooled—that is to say, in about three hours. The analysis of
contents of B and C was delayed for about twenty-four hours. We took the
precaution of not commencing the analysis before we had shaken the
flasks for a few minutes, so that the wort in all of them might be
saturated at a fixed temperature, and thus enable us to ascertain the
exact quantity of oxygen in solution.

The analyses showed that the worts in the three flasks contained:—

    Flask A, oxygen in combination, per litre 20 c.c.

      “ B, ” “ ” 21·4 c.c.

      “ C, ” “ ” 16·8 c.c.

Several facts may be deduced from these experiments: the shaking up of
the wort with air has a marked effect on the absorption; a very
appreciable absorption immediately follows the shaking up of the wort
when warm; whereas, in the case of cold wort that has remained
undisturbed, the absorption takes place slowly.

The results of the preceding experiments plainly show that the wort,
which is very hot when it comes on to the coolers, where it remains for
several hours, must absorb an appreciable quantity of oxygen by
combination; but these same experiments teach us nothing definite
concerning the volume of oxygen that is actually absorbed. We can only
gather from the remark which concludes the third experiment given above,
that the total quantity of oxygen absorbed by the wort in Tourtel’s
brewery, during the time that it remains on the coolers, must be less
than 17 c.c. per litre (4·7 cubic inches per gallon), inasmuch as the
coloration effected by combined oxygen in the proportion of 17 c.c. per
litre was considerably greater than that of the wort taken from the
backs in the brewery.

If we knew the curve of cooling on the Tourtonville coolers we might
easily, in experiments conducted in our laboratory, assimilate the
conditions of our experiments to those of the oxidation of the wort in
the brewery, by exposing wort in contact with air in closed flasks to
temperatures varying according to the indications of the curve in
question. For this purpose, we induced M. Calmettes to study the process
of cooling upon the coolers at Tantonville. In Fig. 84 the figures found
in one of that gentleman’s experiments are given.

The abscissæ represent the time expressed in hours; the ordinates, the
degrees of temperature. The exterior temperature was 0° C. (32° F.); the
atmosphere was calm. The wort was pumped on to the coolers at 5.20 p.m.,
its temperature then being 85° C. (185° F.), and the operation of
pumping lasted from 5.20 to 5.30 p.m. The first determination was made
at 5.30 p.m., and was repeated every ten minutes until 7.30 p.m. Between
7.30 and 8.30 p.m. it was repeated every twenty minutes; after that, it
was repeated every half-hour until 2 a.m., when the wort went down to
the fermenting vessels. The mean depth of the wort was 8·5 centimetres
(3·1 inches).

[Illustration: Fig. 84. Curve of cooling of the wort on the coolers
(December 18th, 1875).]

Having determined the rate of cooling in the brewery, we made the
following experiment: a known quantity of wort from the copper—deprived,
consequently, of oxygen—in the same condition as when it comes on the
coolers, was put into a graduated, cylindrical vessel, which was then
closed with an india-rubber cork, and placed immediately, without being
shaken, in a hot water bath at 85° C. (185° F.). Another vessel similar
to the preceding one, and having a thermometer passed through the cork,
and immersed in the wort, enabled us to observe the temperature. The
temperature was gradually reduced, in exact accordance with the data of
the preceding curve, until the water, in the course of eight hours and a
half, was brought down to 10° C. (50° F.). It is true, that we cannot
pretend to have realized all the conditions of the coolers, in this
manner, but we approached them very nearly; moreover, it was an
approximation rather than a rigorous determination that we desired to
obtain. We then collected over mercury the air which remained in the
flask, and analyzed it very carefully; at the same time, with
Schützenberger’s apparatus, we determined the oxygen held in solution in
the wort so treated. From the results thus obtained we easily found the
quantity of oxygen that had disappeared—that is, the oxygen which the
wort had acquired from the atmosphere of the flask, and which had
combined with the oxidizable matters of the wort.

The volume of the flask being 815 c.c., that of the wort 391 c.c., and
the depth of the liquid 8 cm., we found an absorption by combination of
9·49 c.c. of oxygen per litre of wort (2·63 cub. ins. per gallon).
Another flask treated in the same manner gave us similar results.

As the oxygen in solution has so great an influence on fermentation, it
is important that we should, likewise, know the effect produced by the
oxygen in combination. The following considerations and experiments may
throw some light on this subject:—

We have already remarked that natural saccharine worts oxidize, and
acquire colour in contact with air, and that this coloration disappears
when these worts are caused to ferment. This furnishes one presumption,
that the oxygen in combination disappears then, from, being abstracted
by the ferment. A similar phenomenon is observable in the case of wort.
After having acquired a marked dark shade by remaining in contact with
pure air, it loses this colour very appreciably during fermentation; and
if the wort does not quite regain the colour which it originally had
when it came from the copper, this circumstance is probably owing to the
fact that the quantity of oxygen in combination with the wort is larger
than that which is abstracted by the yeast. We have seen that yeast
absorbs oxygen, since, in the case of a saccharine wort, more or less
saturated with oxygen in solution, when fermentation commences, the
first effect of the ferment is to cause that oxygen to combine with its
own substance. We should, therefore, expect to find the oxygen in
combination, as well as that held in solution, in wort, abstracted by
the yeast and contributing to the activity of fermentation. As a matter
of fact, this is proved by direct experiments, for the fermentation of a
wort that has oxidized in contact with air, or of one from which all the
oxygen that was held in solution in it has disappeared by direct
combination, is much more easy, rapid, and complete than the
fermentation of the same wort when it contains no oxygen, whether free
or combined. These experiments were as follows: we boiled some _copper
wort_ in a large double-necked flask, like those shown in Fig. 73; all
the air being expelled, pure air was allowed to enter the flask; and
when the wort was cool it was saturated with this air, by being shaken
briskly for a quarter of an hour. The wort was then forced by a pressure
of air, applied to the extremity of the S-shaped tube, into smaller
flasks, similar to the preceding ones; these we filled completely, and
then plunged the end of their sinuous tubes under mercury. After waiting
for two or three days, a longer time than was required for the oxygen in
solution to enter into combination—a fact which we confirmed by means of
a similar flask, which served as a standard—we caused the wort, so
prepared, to ferment in the flasks, and side by side, for the sake of
comparison, some _copper wort_ that contained no air in solution or
combination.

In other experiments we operated on pure wort, saturated with oxygen in
combination, by being allowed to remain for one year in an open flask in
contact with pure air. This wort was deprived of air in solution by a
protracted boiling over mercury. It was then pitched, out of contact of
air, with an old yeast. The yeast underwent no development at all, a
proof that oxygen in combination cannot act like oxygen that is free, or
simply in solution, in effecting the revival of the yeast; nevertheless,
after the revival has been once started by means of a small quantity of
air, fermentation declares itself with much greater facility than in the
case of copper wort, placed under the same conditions, but deprived of
oxygen in combination.


 § V. On the Influence of Oxygen in Combination on the Clarification of
                                 Wort.


Oxygen in combination has another effect which it is essentially
important to point out, for it concerns the clarification of beer. One
of the most valued properties of this beverage is its limpidity and
brilliancy. We know from the results of the fourth experiment in the
preceding paragraph that in the case of a wort shaken up when hot with
air, and examined as soon as cold, that is, after an interval of only
three hours, we find a notable volume of oxygen to have been absorbed by
combination; in the experiment to which we allude, this volume was not
less than 20 c.c. of oxygen per litre of wort. The shaking up of the
wort when cold with air saturated it with oxygen in solution, but the
quantity of oxygen which under these conditions entered into
combination, in the course of three hours, is insignificant, although
saturation by solution may be attained in the course of one minute’s
shaking. If two samples of the same wort are shaken up with air, one of
them being hot and the other cold, and both filtered after having been
left undisturbed for twenty-four hours, or even immediately after the
agitation, we cannot fail to be struck with the great difference that
they will present in point of brightness. The wort that was shaken up
hot will have more colour, and will be brilliant; the other will be
turbid, and will not become clear for five or six days, when left to
itself in contact with air and filtered again. This explains a fact that
may be easily verified in practice: Boiled wort, if cooled down
suddenly, or slowly but out of contact with air, or shaken up cold in
contact with air, is opaque when filtered; whilst the same wort, cooled
down on the coolers where it has taken a certain quantity of oxygen into
combination, generally passes through the filter very bright. The
intelligent brewer is uneasy when this is not the case, for it cannot be
denied that the easy clarification of wort has a favourable influence on
the easy clarification of beer.

It would, nevertheless, be a grave error to suppose that the
clarification of beer must necessarily follow that of wort, and we may
be permitted to make a digression here on the subject, to prove this
statement.

On February 3rd, 1874, we brewed 2 hectolitres (44 gallons) of beer. The
boiling wort, hops and all, was run into a vessel like that represented
in Fig. 80, but provided in addition with a false bottom, pierced with
holes and fixed at 1 centimetre (0·39 inch) above the true bottom of the
vessel; this was meant to retain the spent hops. The temperature of the
wort in the vessel after it was filled, February 3rd, 4 p.m., was 90° C.
(194° F.), that of the room was 10° C. (50° F.). We permitted the wort
to cool down gently, without running cold water over the vessel. The
wort indicated a density of 14° Balling.

The following temperatures were taken:—

                       Temp. of               Temp. of
                       Wort.                  Room.

    Feb. 4,    11 a.m. 38°   C.   (100·4° F.) 9° C.      (48·2° F.)

                7 p.m. 30°   C.   ( 86°   F.) 9° C.      (48·2° F.)

            11.30 p.m. 26·3° C.   ( 79·3° F.) 9° C.      (48·2° F.)

    Feb. 5,     9 a.m. 21°    C.  ( 69·8° F.) 8°   C.    (46·4° F.)

               12 a.m. 19·75° C.  ( 66·6° F.) 8°   C.    (46·4° F.)

                4 p.m. 18°    C.  ( 64·4° F.) 8·5° C.    (47·3° F.)

    Feb. 6,    11 a.m. 14°    C.  ( 57·2° F.) 8°   C.    (46·4° F.)

    Feb. 7,     2 p.m. 11°    C.  ( 51·8° F.) 7°   C.    (44·6° F.)

At the end of this time the wort drawn from the smaller tap half-way up
the vessel had already become very bright, although it was taken from
the bulk of the liquid above the deposit of hops.

On February 8th the temperature of the wort was 9·5° C. (49·1° F.), and
that of the room 5° C. (41° F.); the wort was again very bright. Taken
from the small tap and tested by Schützenberger’s process it gave no
evidence of free oxygen in solution, although its surface was in contact
with air. It continued absolutely pure, the arrangements of our vessel,
as we have already explained, allowing only such air to enter as was
first deprived of its disturbing germs.

Not till February 12th, after we had again determined the purity and
brilliant clearness of the wort, a brilliancy which we can compare with
nothing so well as Cognac, without the faintest trace of cloudiness, did
we set it to ferment in a vessel similar to that in which it had cooled,
but without the false bottom. In the process of transfer we effected its
aeration by causing it to fall on a small inverted tinned iron capsule
some 4 or 5 centimetres (1-½ to 2 inches) in diameter. By this
arrangement the wort took up air to the extent of rather more than a
third of its saturate capacity, that is to say, by spreading over the
capsule, and falling from it in a kind of sheet, it absorbed a volume of
oxygen more than a third of the total amount of oxygen which it was
capable of absorbing at the existing temperature; this was 12° C. (53·6°
F.) at the moment when the wort was drawn off. The pitching was
accomplished with a 6-litre flask containing about 4 litres (7·04 pints)
of beer that had been in “low” fermentation from February 3rd. The beer
was cleansed on February 24th, and had a density of 5-1/4° Balling. We
collected 2·345 kilos (75·39 oz. troy) of yeast, containing 56 per
cent., that is, 1·313 kilos (42·21 oz. troy) of pressed yeast,
containing 36·7 per cent. of yeast dried at 100° C. (212° F.), that is
482 grammes (15·49 oz. troy) for the brew, which would give 241 grammes
(7·748 oz. troy) of yeast formed per hectolitre (22 gallons).

The beer was turbid when drawn off, and the small glassful that we
removed did not brighten in twenty-four or even forty-eight hours. The
samples for some days previously had been in the same condition. The
yeast existed as a fine deposit without any straggling yeast about the
sides. The want of brightness was dependent rather on spurious colour
than on any actual turbidity. We may here remark that if in the
preceding experiment the wort had taken up oxygen into combination as
well as into solution at the time that it was aerated, the other
conditions being the same, the beer would have been bright and better.

It follows from this experiment that a wort may be _perfectly bright_ at
the moment when it is pitched, yet fail to produce a beer which shall be
bright when racked, or one that will brighten subsequently otherwise
than with great difficulty. We may add that when we repeated this same
experiment, cooling the wort, however, as rapidly as the conditions of
our apparatus permitted, and employing iced water, the beer appeared
very nearly bright when it was racked, and brightened pretty quickly in
cask and in bottle. The total duration of cooling was not longer than
two hours.

The question here arises what part does the oxygen combined with wort
play in the clarification of the latter, or in the clarification of
beer? Although it may be difficult to give a definite answer to this
question, we must bear in mind that in cases where the beer brightens
best, if we examine it under the microscope during fermentation, we see,
besides the clusters of yeast-cells, floating amorphous particles, which
are larger and more compact than those to which the turbidity of worts
and muddy beers is due, a circumstance which should lead us to suppose
that the oxygen in combination with the wort has the effect of modifying
the nature of the amorphous deposit which is produced during the
fermentation of the wort. During boiling, the hop yields to the wort a
variety of resinous, odorous, and astringent substances, which, for the
most part, are held in solution by the presence of sugar and dextrin. At
the moment when, under the influence of the yeast, which is itself more
or less oxidized, the sugar becomes transformed into alcohol and
carbonic acid, a portion of the bitter and resinous matters of the hop
becomes insoluble and remains in a state of suspension in the liquid. It
is very probable that at this point it is when the combined oxygen
assumes its function of modifying the physical structure of these
insoluble particles, agglomerating them, so that they become more easily
deposited.[179]

Moreover, oxidation tends to form a special precipitate in the wort,
which precipitate contributes towards the collection and deposition of
the very fine particles suspended in the wort, by a mechanical action,
similar to that which we notice in fining operations. On the coolers an
effect of this kind is produced. The wort in the copper contains
insoluble matters which pass on to the coolers. Very bright when
boiling, it grows turbid as it cools, and then contains two kinds of
insoluble substances: 1. Substances insoluble alike in the hot and cold
liquid, some of which even, as we have just seen, are formed under the
influence of heat and air: all these substances precipitating rapidly to
the bottom of the vessels. 2. Very fine particles insoluble in the cold,
but soluble in the hot liquid, appearing as the wort cools down, and
giving it a milky appearance. If the air does not come into play they
remain in suspension for an indefinite time, so to say. Wort taken
boiling from the copper and cooled down, therefore, forms a considerable
deposit at the bottom of the bottles. Now, if we put this wort into
bottles without filling them, putting into some only the milky wort from
above the deposit, and into others the same wort along with some of the
deposit, then raise it to 100° C. (212° F.), and before it has time to
cool down shake it up with air a good many times, it will be readily
seen that the wort in the bottles containing the deposit will brighten
more rapidly and satisfactorily than those in the bottles without the
deposit. The deposits which are insoluble in the copper have, therefore,
an influence on the clarification. We must add, however, that this
influence cannot be compared with that of direct oxidation.

The “turning out” of the wort and its stay upon the coolers to a certain
extent exhibit the different conditions which take part in its
clarification, inasmuch as the wort charged with its insoluble matters
is run off very hot, and with more or less violence against the external
air.


 § VI.—Application of the Principles of the New Process of Brewing with
                 the Use of Limited Quantities of Air.


We have now an idea of the quantities of oxygen which occur, free or
combined, in the actual processes of manufacture. We know, moreover,
that an excess of air may be injurious, especially to the aroma of the
beer, and to that quality which consumers prize so highly, which goes by
the name of _bouche_. It must, therefore, be important to ascertain
whether in existing processes the proportion of active oxygen may not be
excessive.

The best practical means of determining this would consist in comparing
the products of different processes with progressively increasing access
of air, starting from none at all, as in the case of cooling in the
presence of an atmosphere of carbonic acid gas. The following
arrangement (Fig. 85) permits us to realize these conditions:—

[Illustration: Fig. 85.]

The wort brought to a temperature between 75° and 80° C. (167° and 176°
F.) in the double-bottomed vessel C, passes by the tube _a b_ into a
refrigerator, such as Baudelot’s, for example, but acting in an inverse
manner to the ordinary mode of using Baudelot’s; that is to say, the
wort is made to circulate inside the tubes, whilst the cold water plays
on the outside.[180] The wort when cooled, its temperature being
indicated by a thermometer _c_, passes down by the tube _c_DD to fill
the fermenting vessel A. This vessel is made of tinned iron, or, better
still, tinned copper, and has a cover provided with a man-hole and
eye-hole; _m n_ one of the tubes for the circulation of air during
fermentation; its connecting-tube is not represented, it would be behind
the vessel.

At the point _d_ there is a pipe for admission of pure air; this is
represented on a larger scale at T. The wort, as it runs through the
large tube, carries with it air from outside, and this air is calcined
on its way in by means of a flame which plays on the copper tube through
which it passes. This arrangement supplies a third or more of the total
quantity of oxygen that the wort is capable of acquiring by solution at
the temperature at which we work.

F represents the arrangement of the reversed funnel in which the tube _m
n_ terminates. Its mouth is closed with cotton-wool held in place
between two pieces of wire gauze, for the purpose of purifying the air
that enters by it into the fermenting vessel during fermentation.

_v_ is an entrance tap for steam, by means of which the vessel and
refrigerator are cleansed from all extraneous germs before each
fermentation, and before the wort passes into the refrigerator.

When the fermenting vessel A is at work, we may start a fermentation in
a second vessel in the following manner: opening a small tap situated at
about a third of the height of the vessel, we pass a few litres of the
fermenting beer into a can of tinned copper, previously purified by a
current of steam, and filled with pure air. This can is then emptied
into the fresh vessel, an operation of no difficulty, since we have
merely to connect the tap of the can with the small tap of the vessel,
and lastly, the vessel is filled with wort, which then mixes with the
fermenting liquid. These various manipulations, it is evident, are
performed under conditions of complete purity, without the slightest
contact of the liquids either with the exterior air or with utensils
contaminated by disturbing germs.[181]

It is seldom that an industry adopts at once in their entirety new
practices which would necessitate a re-arrangement of plant, and the
process of which we are speaking would require such re-arrangement, as
far as the fermenting vessels and the method of cooling the wort are
concerned. The new process would, however, be of great value if once
introduced, simply for the manufacture of pure ferment and pure wort, or
even for that of pure ferment alone. In other words, we might retain the
ordinary methods employed in low fermentation, use the same method of
cooling or the new one, the same fermenting vessels, and the process of
fermentation at low temperatures; the yeast, however, would be prepared
in a state of purity in the closed vessel which we have described,
collected in those vessels, aerated, and then employed after the
old-established custom; better still, the pitching might be performed
with beer in the act of undergoing pure fermentation.

Above the fermenting-stage there might be arranged a room for the
vessels used in the new process, from which the pure beer could be run
for pitching purposes into the large tuns in the brewery below. It is
true that beer prepared in this manner would not be perfectly pure, but
from the results which have been obtained by working on this system,
there is no doubt that it would possess keeping qualities far superior
to those of beer made with ordinary yeast, even supposing that beer to
have been treated with every possible precaution, and to be as pure as
any produced in the best regulated breweries.

In the month of September, 1874, we conducted an experiment at
Tantonville, in a closed vessel capable of holding 6 hectolitres (132
gallons). The deposit of yeast served to pitch an open vessel, the wort
of which had, moreover, been cooled under conditions of purity. The
cooling had been effected by means of the Baudelot refrigerator,
represented in Fig. 85, the wort in the closed vessel having been
similarly treated. For shortness sake, we may designate the closed
vessel and its beer by the letter K, and use the letter M for the open
vessel and its beer, and T for the corresponding beer of the brewery.
The vessel K was pitched on September 4th, and racked on September 17th,
the beer then showing a density of 5·5° Balling.

The beers K and M were sent to Paris at the same time as some barrels of
the beer T, brewed by the ordinary process; and samples of these
different beers, which arrived on October 22nd, were procured from five
different cafés for purposes of examination.

The beer M did not suffer by comparison with the beer T. The similarity
between the flavours of these two was so close as to puzzle even
experienced judges. In both cases the beer was brilliantly clear. In two
cafés the beer M was even preferred to T, being considered softer on the
palate (_moelleuse_) and of more decided character (_corsée_) than T, a
circumstance which may be explained by the fact that its wort had been
less aerated.

The beer K, although very clear and bright, was considered inferior to
M, but the sole reason of this was that at the date when it was
tasted—November 3rd—it did not froth. As we have already remarked, a
peculiarity of the beers made in closed vessels is that their secondary
fermentation takes a longer time to develop. The yeast held in
suspension in the beer, at the moment when it is drawn off, is, in the
case of all beers, the yeast of a supplementary fermentation, if we may
use that expression. In the ordinary process of brewing, this yeast, in
consequence of the greater aeration of the wort at the commencement of
fermentation, is more active, or, rather, more ready to revive and
multiply than is that which develops in closed vessels. If the barrels
of the K beer had been tapped on the 12th or 15th of November, instead
of on the 3rd, it is probable that they would have contained as much
carbonic acid gas as the beer M contained at the earlier date. This
delay in the resumption of fermentation, which characterizes beer made
in closed vessels, is an advantage, inasmuch as it facilitates the
transmission of the beer to long distances, besides giving us the
smallest deposits of yeast in cask or bottle, as we have already pointed
out.

In comparing the keeping qualities of the beer M and the beer T
(the latter being the brewery beer), we made the following
observations:—[182]

On November 25th we began to detect in the brewery beer an unsound
flavour; a large deposit, too, had formed; the beer had lost its
brilliancy, and frothed enormously. The deposit swarmed with diseased
ferments, especially those represented in Nos. 1 and 7 of Plate I. The
beer M, on the contrary, was in brilliant condition, with an
insignificant deposit, and an ordinary froth, if anything, rather small,
and beautifully bright.

On December 3rd the beer M was still good, very clear, and in excellent
preservation; it was considered by professional brewers as remarkably
sound.

December 22nd, the same beer M was still very bright and good.

January 20th, the beer was still bright; for the first time, however, we
detected in the deposit in the bottles, which was still small, the
filaments of turned beer. This unsoundness was in its earliest stage.
Now, comparing the relative unsoundness of the two beers, we see that M
kept at least two months longer than the corresponding brewery beer.
This example shows us that as far as the keeping powers and the quality
of beer are concerned, the existing process would gain considerably by
the employment of pure wort and pure ferment; and, indeed, it seems
likely that the new process may be introduced into breweries with this
object in view.

In the course of the summer of 1875 we made the following observations
on the keeping qualities of a beer brewed on the new system, all the
details of which had been rigorously carried out. The beer brewed at
Tantonville during the months of June and July, at a temperature of 13°
C. (55·4° F.), in 50-litre and 80-litre casks (11 and 18-gallon), had
been sent by slow trains to Arbois (Jura), where we were staying for a
time. The temperature of the wine cellars in which these barrels were
stored was, on June 1st, 12·5° C. (54·5° F.); this rose gradually until
September 1st, when it attained 18° C. (64·4° F.). In this cellar the
brewery beer, brewed in the ordinary way, underwent change in the course
of fifteen days or three weeks, whilst the beer brewed on the new system
remained sound for several months. It is true that some of the barrels
lost their frothiness, and that the beer in them underwent a peculiar
vinous change, but these effects in no way depend on the conditions
peculiar to the new process.

Comparing the beers K, M, T, of which we have been speaking, we see
that, however useful the aeration and oxidation of the wort may be in
quickening fermentation and facilitating clarification, yet it is by no
means indispensable to the success of our operations that we should
introduce into our worts large quantities of oxygen, whether by solution
or combination. Beyond a certain limit—a limit which is undoubtedly
overstepped in the existing process—oxygen is injurious to the palate
characteristics and aroma of beer.

These comparisons have proved to us that the new process can be applied
to wort aerated to the third of its saturate-capacity for oxygen, and
pitched with a good “low” yeast, taken from the fermentation of a wort
aerated in the same way, and that the beers thus obtained not only
possess vastly superior keeping properties, but are equal in quality and
superior in palate-fulness to beers brewed with the same wort on the
existing system. We should be perfectly justified in forming this
conclusion as to the _strength_[183] of the beer furnished by the new
process, even if on tasting it we found that the new beer M was merely
equal in strength to Tourtel’s beer brewed in the ordinary manner, since
the wort in the new process, other conditions being the same, is weaker
than the same wort treated in the usual way, from not having undergone
that evaporation on the coolers which concentrates it. If we were to
restore to the concentrated wort of ordinary brewing all the water lost
by it through evaporation, the beer that we should obtain would be
sensibly weakened.[184]

One thing, however, is that we must employ good varieties of “low”
yeast. We have seen how the employment of certain forms of yeast renders
the clarification of beers difficult, as well as extremely slow, and
almost prevents their falling bright at the end of fermentation. These
yeasts, moreover, frequently impart to beer a peculiar yeast-bitten
flavour, which does not disappear even after a prolonged stay in cask.
Even repeated growth of these yeasts, whether in closed or in open
vessels, and no matter what quantity of air we may supply them with
before fermentation, seems to have no effect in changing their
character. The only thing we can do with these varieties of yeast is to
get rid of them with all speed, and to replace them with others.

Notwithstanding the comparative success that has attended various trials
of the new process on the commercial scale, that process has not yet
been practically adopted: and here we must bear in mind that we have not
to deal with any casual invention or mechanical improvement that could
be introduced all at once into the working of a brewery; we are dealing
with operations of considerable delicacy, which necessitate the adoption
of a special plant to carry them out. Under such conditions time and
labour are required to effect a change in the established processes of a
great industry. This, however, cannot diminish the confidence that we
have in the future of our process, and it is our hope that the same
confidence will be shared in by all those who may give this work an
attentive perusal.

Footnote 162:

  M. Galland, a brewer in Maxéville, near Nancy, published with his
  name, in November, 1875, a pamphlet, which was reproduced in the
  brewing journals of that date, bearing the title, _It is said, “the
  air being impure, let us exclude it;” I say, “The air being impure,
  let us purify it.”_ These two aphorisms, together or apart, constitute
  the essential novelty of my researches on beer, and M. Galland is
  mistaken in attempting to appropriate the merit of the second
  alternative (see my note in the _Comptes rendus_ of the 17th November,
  1873, and the text of the letters-patent obtained 13th March of that
  year). M. Galland has devised some arrangements for putting the latter
  of these two schemes into practice; but it is possible, of course, to
  effect this in a variety of ways. M. Velten, a brewer in Marseilles,
  had already accomplished this in his efforts to carry out practically
  the procedure advocated in the present work.

Footnote 163:

  [Non-technically, stirred about.—ED.]

Footnote 164:

  As stated in the paragraph on aërobian ferments, in Chapter V., “low”
  yeasts, to be preserved in their state of “lowness,” must be submitted
  to often-repeated growths—every fifteen days in winter and every ten
  days in summer, that is to say, they must be grown afresh after each
  of these intervals. If this is done, there will be no reason to
  apprehend the formation of aërobian ferments, which, as we have stated
  before, may embarrass us by transforming our “low” yeasts into “high”
  yeasts.

Footnote 165:

  It has been observed by brewers that, sometimes, without any apparent
  cause, a yeast suddenly becomes inactive and fermentation ceases.
  Accidents of this kind may probably be explained in the same manner as
  the facts of which we are speaking. If a wort has not been aerated, or
  if it has been deprived of oxygen by a commencing development of
  microscopic organisms, the yeast formed in it will be very inferior,
  and the fermentation may stop at its commencement or soon afterwards.
  In such a case, an aeration of the yeast and wort would be the best
  remedy.

Footnote 166:

  PASTEUR, _Comptes rendus de l’Académie des Sciences_, vol. lii. p.
  1260, and _Études sur le Vin_, 2nd Edition, p. 277.

Footnote 167:

  We may here remark that the system of gutters in the above apparatus
  is much simpler than that described in connection with Figs. 76 and
  77. The water which falls on the cover is carried off, when the gutter
  is full, by a circle of grooves, inclined so that the streams running
  from them meet and form more readily a sheet of water, which flows
  over the exterior surface of the cylindrical vessel.

Footnote 168:

  [It will be well for the reader to bear in mind, that the word
  “strength,” used by Pasteur many times in this chapter, has a
  different meaning to that which attaches to it in the minds of English
  brewers, who in nearly every case use it in reference to _original
  gravity_, while the author employs it, in this chapter, at any rate,
  to denote the _palate characteristic of strength_, in other words
  _palate-fulness_. For this reason we have thought it best in many
  cases to actually substitute the term “palate-fulness,” or “body,” for
  the literal translation of the French word “force.”—F. F.]

Footnote 169:

  [As some confusion has existed in the nomenclature of these salts, it
  may be as well to offer some explanation.

  The salt here used for absorbing oxygen was discovered by
  Schützenberger, and named by him _hydrosulphite of soda_. It no longer
  now goes by that name, being called _hyposulphite of soda_, NaHSO_{2}.

  The salts formerly known as _hyposulphites_ are now called
  _thiosulphates_, as Na_{2}S_{2}O_{3}.

  Thus to put them together we have:—

     Hyposulphite (Hydrosulphite)             NaHSO_{2}
     Bisulphite                               NaHSO_{3}
     Thiosulphate (Hyposulphite)              Na_{2}S_{2}O_{3}

  The thiosulphates were formerly regarded as containing the elements of
  water in their composition, thus:—Na_{2}H_{2}S_{2}O_{4}, which being
  halved would give NaHSO_{2}, isomeric with hyposulphite, as Pasteur
  says. It is further to be observed that Pasteur uses the old notation,
  in which the number of atoms of sulphur and oxygen are the double of
  what they are in the new.—D. C. R.]

Footnote 170:

  SCHÜTZENBERGER, _Comptes rendus de l’Académie des Sciences_, vol.
  lxxv., p. 880.

Footnote 171:

  M. Schützenberger applies the term _saturated_ to a solution of
  hydrosulphite prepared thus, or very nearly so; a current of
  sulphurous acid is passed through a solution of commercial bisulphite
  of soda, to excess; 100 c.c. (3-½ fl. oz.) of this solution and 30
  grammes (46 grains) of zinc filings are put into a small flask, so as
  to completely fill it; the bottle is corked up and the mixture is
  shaken briskly for about a quarter of an hour. Lastly, the contents of
  this flask are poured into a large 2-litre flask, with water and
  containing milk of lime, prepared by mixing 100 grammes (3·2 troy oz.)
  of quicklime in the water just before it is used. The whole is shaken
  briskly for some minutes and then left to settle. The supernatant
  liquid soon becomes bright. This is the hydrosulphite; but in this
  state it is too concentrated; and should be syphoned into another
  2-litre flask half full of water. In the alkaline condition this salt
  absorbs gaseous oxygen much less rapidly than in the acid, so that the
  liquids will retain their strength much longer, if they are kept in
  well-corked bottles.

Footnote 172:

  The numbers _n_ and _n´´_ will vary as the wort, or liquid which we
  have to test, is perfectly neutral or otherwise. Should it be acid
  _n´´ n_, should it be alkaline _n n´´_. This would be a very exact
  method of estimating the acidity or alkalinity of any coloured liquid.

Footnote 173:

  [The Balling saccharometer being almost unknown in England, we may
  explain that its indications are for percentages of sugar in
  saccharine solutions, or of extract in worts; 17·9° Balling,
  therefore, means 17·9 per cent. of sugar or extract in the respective
  liquids.—F. F.]

Footnote 174:

  Experiments made, at our request, by MM. Calmettes and Grenet, at
  Tantonville; in Tourtel’s brewery.

Footnote 175:

  See foot-note, page 367.

Footnote 176:

  [For non-technical readers we may explain the expressions “gathered,”
  here used, and “turning out,” used on page 365. “Turning out”
  describes the operation of emptying the _copper_ contents into the
  _hop-back_, or the _hop-back_ contents on to the _coolers_.
  “Gathering” refers to the time when the worts are finally intermixed
  and _weighed_, prior to the commencement of vinous fermentation.—F.
  F.]

Footnote 177:

  We know also from the direct experiments of M. Schützenberger,
  performed on aerated water with which yeast had been mixed, that yeast
  causes all the oxygen in solution to disappear very quickly, so that
  hydrosulphite gives no evidence of a trace. (See SCHÜTZENBERGER,
  _Revue scientifique_, vol. iii. (2), April, 1874).

Footnote 178:

  [The bottling needle (_foret â aiguille_) is a contrivance for
  permitting a cork to be driven into a bottle completely filled with
  liquid, without bursting the bottle. It consists of a
  slightly-tapering iron pin about 1/8th inch in diameter and 2 inches
  in length, somewhat flattened, and slightly curved throughout its
  entire length, with a groove running down one side from end to end,
  the pin being jointed with a ring, like a common ring cork-screw. In
  using it the pin is driven into the bottle alongside the cork, thus
  allowing the excess of liquid to escape as the cork advances. When the
  cork is completely home, the needle is withdrawn, and the elasticity
  of the cork enables it to fill up the space left, so that we have the
  bottle corked air-tight, and no air left between the cork and
  liquid.—D. C. R.]

Footnote 179:

  We have remarked in our observations on No. 6 of Plate I. (p. 6) that
  amongst the amorphous granular deposits of wort and beer we often find
  minute balls of resinous and colouring matter, perfectly spherical and
  very dense, which if the liquids be shaken up will render them very
  turbid, but which readily and rapidly deposit again, without remaining
  in suspension in the least. Such then is the form in which the
  deposits of wort in course of fermentation are precipitated, when the
  wort has been freely exposed to oxygen. One day in the laboratory we
  were desirous of starting a fermentation in a vessel capable of
  holding 12 hectolitres (264 gallons). But as we only had at our
  disposal a copper capable of holding 2-½ hectolitres, we procured the
  wort from a neighbouring brewery in two barrels of 6 hectolitres each.
  This wort we re-heated, in portions, in our 2-½ hectolitre copper, a
  treatment which had the effect of oxidizing the wort more than it
  would have been in the brewery. In this case the beer fell remarkably
  bright, and the cells of yeast were accompanied by the deposit of
  minute agglomerations sketched in Plate I., No. 6. We have repeated
  this experiment on a smaller scale and have obtained the same result.

Footnote 180:

  It is evident that this arrangement may be modified in many ways. Any
  of the ordinary worms, or, generally speaking, any of the more modern
  refrigerators invented during the last few years, may be adopted. The
  only point that is of importance is the preservation of the purity of
  the wort during cooling.

  The Baudelot refrigerator is extensively adopted in France; for this
  reason we used it in our experiments at Tantonville. We might equally
  well, by enclosing the worm in a casing of sheet iron or tinned
  copper, pass our wort over the exterior of the tubes, the cold water
  passing through them. The wort would cool quicker in this way than
  with the arrangement described in the text, and if we arrange to admit
  only pure air into the case, always under conditions of purity. The
  aeration, moreover, could be made as much as we wished.

Footnote 181:

  This arrangement limits the proportion of oxygen that may be
  introduced into the wort by direct oxidation. But it would be easy to
  increase this at will, by causing the wort as it comes from the copper
  and the hop-back to pass into a cylinder turning horizontally on its
  axis and furnished with blades fixed inside, so as to divide the wort
  and bring it better into contact with the air in the cylinder. Instead
  of a revolving cylinder we might use a fixed vessel, in which the wort
  could be stirred up by some arrangement outside. In either case we
  should have to take care that the air was pure when it came into
  contact with the wort, but this would be a matter of no difficulty; we
  would simply have to make communication with the outer air by means of
  a tube filled with cotton wool. Any air that might be in the vessel at
  the moment when the wort was introduced would be purified by the high
  temperature of the wort coming from the copper. We should, moreover,
  gain the great advantage of being able to bring oxygen to bear on our
  wort in determinate amounts. From this vessel it would pass on to the
  refrigerator. We might again raise the wort oxidized on the coolers to
  a temperature of 75° C. (167° F.), to recool it in this manner and
  aerate it by means of the pure-air pipe.

Footnote 182:

  One of the barrels of the brewery beer was bottled about the end of
  October, at the same time that a barrel of M was.

Footnote 183:

  Refer foot-note, page 354.

Footnote 184:

  The evaporation on the coolers varies according to the arrangements in
  different breweries; but in no case is it less than several hundredths
  of the total volume. One special advantage of the new process is that
  it gives us, _ceteris paribus_, a volume of beer that is 5, 6, or 7
  per cent. greater than that which we should obtain by the old process,
  without in any way affecting the strength of the beer. It is easy to
  ascertain the quantity that evaporates on the coolers, by determining
  the quantity of water that must be added to a known volume of wort
  coming from the coolers to bring its density back exactly to that of
  the original wort, both being calculated to the same temperature.
  Bate’s English saccharometer, which shows differences of nearly
  1/1000th in density, may be employed with advantage in this
  determination.




                               APPENDIX.


Whilst this work was passing through the press there appeared two small
works on the subject of the generation of inferior organisms.

One of them was by M. Fremy. The author’s object seems to have been
merely to give an account, under a new form, of the part which he took
in the discussion on the origin of ferments that was carried on before
the Academy of Sciences in 1871-1872. In the course of that discussion
M. Fremy had announced his intention of publishing an extensive Memoir,
full of facts, bearing on the subject. The perusal of the promised work
gave us much disappointment. Not only were our experiments, and the
conclusions which we drew from them, given there, for the most part in a
manner which we could not possibly accept, but, moreover, M. Fremy had
confined himself to deducing, by the help of his favourite hypothesis, a
series of _à priori_ opinions based on half-finished experiments, not
one of which, in our opinion, had been brought to the state of
demonstration. To tell the truth, his work was the romance of
hemi-organism, just as M. Pouchet’s work of an earlier date was the
romance of heterogenesis. And yet, what could be clearer than the
subject under discussion? We maintain, adducing incontestable
experimental evidence in support of our theory, that living, organized
ferments spring only from similar organisms likewise endowed with life;
and that the germs of these ferments exist in a state of suspension in
the air, or on the exterior surface of objects. M. Fremy asserts that
these ferments are formed by the force of hemi-organism acting on
albuminous substances, in contact with air. We may put the matter more
precisely by two examples:—

Wine is produced by a ferment, that is to say, by minute, vegetative
cells which multiply by budding. According to us, the germs of these
cells abound in autumn on the surface of grapes and the woody parts of
their bunches; and the proofs which we have given of this fact are as
clear as any evidence can be. According to M. Fremy, the cells of
ferment are produced by spontaneous generation, that is to say, by the
transformation of nitrogenous substances contained in the juice of the
grape, as soon as that juice is brought into contact with air.

Again, blood flows from a vein; it putrefies, and in a very short time
swarms with bacteria or vibrios. According to us the germs of these
bacteria and vibrios have been introduced by particles of dust floating
in the air or derived from the surface of objects, possibly the body of
the wounded animal, or the vessels employed, or a variety of other
objects. M. Fremy, on the other hand, asserts that these bacteria or
vibrios are produced spontaneously, because the albumen, and the fibrin
of the blood themselves possess a semi-organization, which causes them,
when in contact with air, to change spontaneously into these
marvellously active minute beings.

Has M. Fremy given any proof of the truth of his theory? By no manner of
means; he confines himself to asserting that things are as he says they
are. He is constantly speaking of hemi-organism and its effects, but we
do not find his affirmations supported by a single experimental proof.
There is, nevertheless, a very simple means of testing the truth of the
theory of hemi-organism; and on this point M. Fremy and ourselves are
quite at one. This means consists in taking a quantity of grape juice,
blood, wine, &c., from the very interior of the organs which contain
those liquids, with the necessary precautions to avoid contact with the
particles of dust in suspension in the air or spread over objects.
According to the hypothesis of M. Fremy, these liquids must of necessity
ferment in the presence of pure air. According to us, the very opposite
of this must be the case. Here, then, is a crucial experiment of the
most decisive kind for determining the merits of the rival theories, a
criterion, moreover, which M. Fremy perfectly admits. In 1863, and again
in 1872, we published the earliest experiments that were made in
accordance with this decisive method. The result was as follows:—The
grape juice did not ferment in vessels full of air, air deprived of its
particles of dust—that is to say, it did not produce any of the ferments
of wine; the blood did not putrefy—that is to say, it yielded neither
bacteria nor vibrios; urine did not become ammoniacal—that is to say, it
did not give rise to any organism; in a word the origin of life
manifested itself in no single instance.

In the presence of arguments so irresistible as these, M. Fremy,
throughout the 250 pages of his work, continues to repeat that these
results, which, he admits, seem subversive of his theory, are,
nevertheless, explicable by the circumstance that the air in our
vessels, although pure at first, underwent a sudden chemical change when
it came in contact with the blood, or urine, or grape juice; that the
oxygen became converted into carbonic acid gas, and that, in
consequence, hemi-organism could no longer exercise its force. We are
astonished at this assertion, for M. Fremy must be aware that, since
1863, we have given analyses of the air in our vessels after they had
remained sterile for several days—for ten, twenty, thirty, or forty
days—at the highest atmospheric temperatures, and that oxygen was still
present, often even in proportions almost identical with those to be
found in atmospheric air.[185] Why has M. Fremy made no allusion to
these analyses? This was the chief, the essential point in question.
Besides, if M. Fremy had wished to test the truth of his explanation,
there was a very simple means of restoring the purity of the air in
contact with the liquids open to him; he might have passed through his
vessels a slow and continuous current of pure air, day and night. We
have done this a hundred times, and we have always found that the
sterility of the putrescible or fermentable liquids remained unaffected.

The hemi-organism hypothesis is, therefore, absolutely untenable, and we
have no doubt that our learned friend will eventually declare as much
before the Academy, since he has more than once publicly expressed his
readiness to do so as soon as our demonstrations appear convincing to
him. How can he resist the evidence of such facts and proofs?
Persistence in such a course can benefit nobody, but it may depreciate
the dignity of science in general esteem. It would gratify us extremely
to find the rigorous exactness of our studies on this subject
acknowledged by M. Fremy, and regarded by that gentleman with the same
favour bestowed upon it everywhere abroad. It may be doubted if there
exists at the present day a single person beyond the Rhine who believes
in the correctness of Liebig’s theory, of which M. Fremy’s hemi-organism
is merely a variation. If M. Fremy still hesitates to accept our
demonstrations, the observations of Mr. Tyndall may effect his
conversion.

The other publication to which we alluded was the work of the celebrated
English physicist, John Tyndall. It was read before the Royal Society of
London, at a meeting held on January 13, 1876.

The following letter explains how the illustrious successor of Faraday
at the Royal Institution came to undertake these researches:—

    “London, February 16, 1876.

    “Dear Mr. Pasteur,—

    “In the course of the last few years a number of works bearing such
    titles as ‘The Beginnings of Life’; ‘Evolution and the Origin of
    Life,’ &c., have been published in England by a young physician, Dr.
    Bastian. The same author has also published a considerable number of
    articles in different reviews and journals. The very circumstantial
    manner in which he describes his experiments, and the tone of
    assurance with which he advances his conclusions, have produced an
    immense impression on the English as well as the American public.
    But what is more serious still, from a practical point of view, is
    the influence that these writings have exercised on the medical
    world. He has attacked your works with great vigour, and, although
    he has made but slight impression on those who know them thoroughly,
    yet he has succeeded in producing a very great and, I may add, a
    very pernicious one on others.

    “The state of confusion and uncertainty had come to be so great
    that, about six months ago, I thought that I should be rendering a
    service to science, and at the same time performing an act of
    justice to yourself, in submitting the question to a fresh
    investigation. Putting into execution an idea which I had
    entertained for some six years, the details of which were set forth
    in an article in the _British Medical Journal_, which I had the
    pleasure of sending you, I have gone over a great deal of the ground
    on which Dr. Bastian had taken his stand, and, I believe, refuted
    many of the errors by which the public had been misled.

    “The change which has taken place since then in the tone of the
    English medical journals is quite remarkable, and I am inclined to
    think that the general confidence of the public in the exactness of
    Dr. Bastian’s experiments has been considerably shaken.

    “In taking up these researches again, I have had occasion to refresh
    my memory by another perusal of your works; they have revived in me
    all the admiration which I experienced when I first read them. It is
    my intention now to pursue these researches until I have dissipated
    any doubts that may be entertained in respect to the unassailable
    exactness of your conclusions.

    “For the first time in the history of science, we are justified in
    cherishing confidently the hope that, as far as epidemic diseases
    are concerned, medicine will soon be delivered from empiricism, and
    placed on a real scientific basis; when that great day shall come,
    humanity will, in my opinion, recognise the fact that the greatest
    part of its gratitude will be due to you.

    “Believe me, ever very faithfully yours,

    “JOHN TYNDALL.”

We need scarcely say that we read this letter with the liveliest
gratification, and were delighted to learn that our studies had received
the support of one renowned in the scientific world alike for the
rigorous accuracy of his experiments as for the lucid and picturesque
clearness of all his writings. The reward as well as the ambition of the
man of science consists in earning the approbation of his
fellow-workers, or that of those whom he esteems as masters.

Mr. Tyndall has observed this remarkable fact, that in a box, the sides
of which are coated with glycerine, and the dimensions of which may be
variable and of considerable size, all the particles of dust floating in
the air inside fall and adhere to the glycerine in the course of a few
days. The air in the case is then as pure as that in our double-necked
flasks. Moreover, a transmitted ray of light will tell us the moment
when this purity is obtained. Mr. Tyndall has proved, in fact, that to
an eye rendered sensitive by remaining in darkness for a little, the
course of the ray is visible as long as there are any floating particles
of dust capable of reflecting or diffusing light, and that, on the other
hand, it becomes quite obscure and invisible to the same eye as soon as
the air has deposited all its solid particles. When it has done this,
which it will do very quickly in two or three days, if we employ one of
the boxes used by Mr. Tyndall—it has been proved that any organic
infusions whatever may be preserved in the case without undergoing the
least putrefactive change, and without producing bacteria.

On the other hand, bacteria will swarm in similar infusions, after an
interval of from two to four days, if the vessels which contain them are
exposed to the air by which the cases are surrounded. Mr. Tyndall can
drop into his boxes, at any time he wishes, some blood from a vein or an
artery of an animal, and show conclusively that such blood will not,
under these circumstances, undergo any putrefactive change.

Mr. Tyndall concludes his work with a consideration of the probable
application of the results given in his paper to the etiology of
contagious diseases. We share his views on this subject entirely, and we
are obliged to him for having recalled to mind the following statement
from our _Studies on the Silkworm Disease_:—“Man has it in his power to
cause parasitic diseases to disappear off the surface of the globe, if,
as we firmly believe, the doctrine of spontaneous generation is a
chimera.”

THE END.

Footnote 185:

  See _Comptes rendus_, vol. lxi., p. 734, 1863.




                                 INDEX.


 A

 Absorption of gases by air-free liquids, 292
   oxygen by blood, 50;
       by urine, 50
     from solutions by _bacteria_, 295

 Acidity, natural, of wine a preservative, 2, and footnote
   of beer heated, 20
   action on ferments, 35

 Acetate of lime from fermentation of tartrate, 288

 Acid, sulphuric, facilitating filtration, 250

 Acid, carbonic, _v._ carbonic acid

 Adaptability of liquids to certain growths, 36, 73, 85
   (supposed) of vibrios to aërobian or anaërobian conditions, 309, 310

 Aeration, reviving influence of, 138
   adoption by brewers of, 253
   tardy, of wort in deep vessels, 348
   on “coolers,” its importance, 348, 349

 Aeration-conditions in ordinary brewing process, 350, 351, 364, 365

 Aeration of wort, apparatus for regulating, 352

 Aeration, influence on clarification of worts, 381
   experiments on its influence on growth, 107, 130

 Aërobian, definition, 116
   ferment, growth of, 208, 209
   ferments, general characteristics, 210;
     origin of, 210 (footnote);
     cultivation of, 211;
     aspects of, 212-217;
     distinguishing features of, 218
   life in ferments overlooked, 260

 “Age,” as applied to a ferment, 169

 Age of cells, 246

 Aged aspect of exhausted cells, 133, 147

 Air, influence on ferment-life, 242
   renewal of, in brewers’ yeast, 246, 247
   mode of expulsion from growing media, 285
   unnecessary to life of _vibrios_, 292
   injurious to life of _vibrios_, 304

 Air, compressed, and ferment-life, 324
   composition unaffected by contact with blood, &c., 398

 Albumen-transformation theory of fermentation, 273

 Albuminous liquids, growth of yeast in, 265

 Alcohol, percentage is heated beer, 20

 Alcoholic ferment, minute species of, 71

 Alcohol, detection in minute quantity, 78, 79 (footnote)
   produced by _penicillium_, 99, and following pages
     by _aspergillus glaucus_, 101, and following pages
     by _mycoderma vini_, 111, 113
       explanation of, 114

 Alcoholic fermentation, general explanation of, 114, 115

 Alcohol, proportion of, to mucor forming it, 134, and following pages

 Alcohol produced by moulds, 258 (footnote)
   production of, within fruits, 267

 Alcoholic fermentation, restricted meaning, 275 (footnote)
   necessary relation with yeast-cells, 275

 _Altenaria tenuis_, 157

 Ammonia, a test for vegetable organisms (Robin), 312

 Ammoniacal urine, 45, 46

 Anaërobian, definition, 116
   growth of yeast, 239, and following pages
   precautions to be observed in, 248
   life of fruit-cells, 272
   growth of _vibrios_, 302

 Animal or vegetable nature of organisms, 312, and following pages

 Anti-ferments, 45

 Apparatus for sterilizing liquids, 27
   for producing pure beer, 340, &c.
   for pure pitching, 344
   for pure aeration, 352
   for cooling beer with regulated supply of pure air, 388, 389

 Appert’s experiment, 62

 Aroma of beer destroyed by excess of air, 353

 Asbestos, useful plug, 27, and footnote, 30

 Ascospores of yeast, 150 (footnote)

 Aspect of yeast variable, 37

 _Aspergillus glaucus_, functioning as ferment, 101, and following pages
   different aspects of, 105

 Atmospheric germs, 6, 26, 38
   variety of, 39, 76, 87 (footnote)

 _Autonomy_ of organisms, 84 (footnote)


 B

 Bacteria, 35, 36;
   medium for growth of, 294;
   absorption of air from solutions by, 295

 Bacteria and butyric vibrios, how related, 296
   influence of oxygen upon, 305

 Bail mentioned, 92, 93, 127


 Balling saccharometer explained, 363 (footnote)

 Barley-wine, 1 (footnote), 230

 Barley decoctions, experiments on development of ferments in, (Fremy)
    273 (footnote)

 Bary, De, mentioned, 92;
   on relations of yeast to other organisms, 180, 181

 Bastian’s experiments, 403

 Baudelot refrigerator, 387 (footnote)

 Bavarian beer, 10

 Béchamp’s _microzyma_ theory, 121
   influence of air on fermentation, 178 (footnote)

 Beer, definition, 1;
     difference between it and wine, 1
   changeable nature of: effects upon brewing purposes, 2, 3
   two kinds only, “high” and “low:” difference, 7
   samples of bottled, examined, 222
   general precautions for pure manufacture of, 338
   improved apparatus for commercial production, 340, and following
      pages

 Beet root preservation in pits, 269 (footnote)

 Berkeley mentioned, 92

 Bellamy’s researches on fermentation in fruits, 270

 Berard on fermentation of fruits, 270, 271

 Berthelot’s mode of isolating inverting constituent of yeast, 322
    (footnote)

 Bert, action of compressed air on ferments, 324

 Birds, experiment upon, described, 309

 Bistournage, 43 (footnote)

 Bisulphite of lime used by bottlers, 15

 Blood, study of sterilized, 49, 50

 Blood-crystals, 50 (footnote)

 Boiling sterilizes liquids, 34

 Bottling needle, 372 (footnote)

 Bottled beer, treatment of, 16

 _Bouche_ influenced by presence of oxygen, 387

 Bouchardat, 323

 Brefeld, strictures on Pasteur’s theory criticised, 280
   convinced of truth of Pasteur’s theory, 315, 316

 Breweries, statistics of, 10

 Brewing, change in processes of, 7
   practices largely empirical, 222

 Brewing processes under conditions of purity, 390

 Budding, rate of, experiment on, 145
   process of, 146

 Buffon’s hypothesis mentioned, 121

 Bulbs, glass, for study of growths, 156 (footnote)
   for vibrios, 298

 Bunsen, tables of solubility of oxygen in water, 360

 Butyric vibrios in must, 65;
   in wort, 70

 Butyric acid from fermentation of lactates, 297
   not a suitable food for vibrios, why?, 301 (footnote)

 Butyric fermentations yield variable products, 308


 C

 Cagniard Latour, on cause of fermentation, 60

 Calmettes, M., 369, 371;
   experiments on the curve of cooling of wort, 377, 378

 Carbolic acid for purifying yeasts, 232

 Carbonate of lime crystals formed in fermentation of lactate, 294


 Carbonic acid, influence on preservation and fermentation of fruits,
    268
   evolution from fermentation of tartrate of lime by vibrios, 287
   amount of evolution, 288
   mode of collection of, 288
   influence on bacteria, 305 (footnote)

 Caseous ferment, occurrence, 200;
   aspect, 201;
   endurance of heat, 203 (footnote);
   meaning of title, 202;
   origin of in brewers’ high yeast, 203, 204;
   origin of in English pale ale, 204, 224;
   aërobian form of, 215

 Cells, power of endurance, 134
   aspect of dead, 139 (footnote)

 Cells, glass, for study of growths, 155 (footnote)

 Cells, probable function in elaborating proteic matter, 335

 Cellulose, not soluble in ammonia (Robin), 312

 Change of yeast, usual remedy for disease, 22

 Chauveau on castration, 43

 Circumstances modifying nature of germs present in atmosphere, 73, 87
    (footnote)

 _Cladosporium_, 55 (footnote)

 Clarification of liquids by fungi, 66 (footnote)
   of wort, 381, and following pages
   of a wort and its beer not always correlated, 382, 383

 Cohn’s medium for growth of vibrios, 294 (footnote)

 Colour darkened by oxidation in pure liquids, 57

 Coloration of vibrio-fermented liquors, 291

 Colpoda, 39, 40

 Composition of medium, influence on life, 296

 Conidia, definition, 137

 Conditions affecting the ferment character of cells, 266

 Consumption of beer in France, statistics, 17 (footnote)

 Contagion and ferments, 41, and following pages

 Continuity, non-, of germs in air, 62

 Continuous vital activity of cells, 278

 Contact-action, theory of, 326

 “Coolers,” importance in aeration of wort, 348, 349
   influence on worts, 364

 Cooling of wort must be rapid in ordinary brewing, 2
   artificial of “low” beers, 12

 Cooling of wort in presence of carbonic acid, 342;
   difficulties of the process, 346, and following pages

 Corpuscles on grapes and stalks, 54

 Corpuscles refractive in bodies of vibrios, 300, _v._ also cysts

 Correlation of special germs with special fruits, 61
   of special ferment and fermentation product, 277

 Cotze and Feltz, 43

 Crushers for the vintage, 268 (footnote)

 Cream of tartar, _v._ tartrate

 Cultivation of yeast under conditions of purity, 29-32
   of pure penicillium, mode of, 88, and following pages
   of aërobian ferments, 211, and following pages


 Cysts of vibrios, 306, 307


 D

 Davainne, on splenic fever, &c., 42

 Daughter-cells, 146

 Dead cells, aspect of, 139 (footnote)

 Declat’s treatment of infectious diseases, 44

 _Dematium_, 167;
   resemblance to _Saccharomyces pastorianus_, 179, 180, 181, 214
   resemblance to “caseous” yeast, 201

 Degrees, Balling, _v._ Balling

 Deposits, amorphous, of wort, 6, 193, 385, and footnote

 Deterioration of beer correlated with presence of foreign organisms,
    26, 32

 Differential vitality, a means of separating ferments, 226

 Difficulty of experiments on growths, 63, 85


 Disease-ferments, what they are, why so called, 4
   classification and account of, 5, 6
   origin of, 6
   inactive at low temperatures, 14
   often found only in deposits, 24
   not everywhere in atmosphere, 31

 Disease-germs usually latent, 220
   development in bottled beer, 222

 Diseases of wort and beer, meaning of, 19
   mode of proving the cause of, 19, 20

 Diseased beer always result of disease ferments, 26

 Distribution of germs limited, 61

 Division, fissiparous, of vibrios, 299

 Dried yeast, 81

 Dryness decreases sensitiveness of moulds to heat, 35

 Dumas, distinction between organized and unorganized ferments, 323

 Dust, atmospheric, contains disease-germs, 6, 26
   on fruits, experiments with, 153, and following pages
   when fertile, 157, and following pages

 Dutch yeast, 200

 Duval, Jules, experiments on transformation of ferments illusory, 37


 E

 _Efflorescence_ of fermented liquors, 108, 117

 Egg-albumen, experiments on, 51

 Egypt, beer first brewed in, 17

 Empiricism in ordinary brewing, 222

 Energy stored by cells, 133, 134

 Endogenous sporulation of yeast, 150 (footnote), 172

 English beers all “high,” 7
   temperatures and yeast employed, 8 (footnote)
   breweries, usages of, 8 (footnote), 14

 Errors, causes of, _v._ experimental errors

 Equations of fermentations variable, 276, 277

 Examination of deposits, mode of, 21 (footnote)

 Exhaustion, definition of, 171 (footnote)

 Exhausted vibrios, 290


 Experimental errors, 63, 85, 92
   avoided by use of double-necked flasks, 120

 Experiments, exactness of Pasteur’s, 95 (footnote)
   to prove connection between quality of ferment and quality of beer,
      26, and following pages
   on living fluids, 47, and following pages
   comparative, on pure must and must with corpuscles boiled and
      unboiled, 54, and following pages
   by Gay-Lussac on must, 62, 63
   by Pasteur after Gay-Lussac, 64
   on distribution of ferments, 65, and following pages
   on distribution of fungus-spores, 68
   in wide shallow dishes, 69, and following pages
   comparative on germs in air, 72, and following pages
   with non-fermentative species of _torula_, 78
   on spontaneous impregnations, 65, 66, 69, 73, 79, 87 (footnote)
   on spontaneous fermentation, 184
   on dried yeast, 81, and following pages
   on influence of aeration on growths, 107
   on aeration and its absence, 130, and following pages
   on function of oxygen on ferment-life, 238, and following pages
   on the capacity of yeast for oxygen, 255
   on influence of carbonic acid on fruits, 268
   on growth of vibrios apart from air, 285
   on fermentation of lactate of lime apart from air, 292, and following
      pages
   on influence of air on vibrio-life, 303, 304
   on influence of air on bacterium-life, 305
   on gradual adaptability of organisms to adverse life-conditions, 309
   on influence of air on fermentation, 349
   on solubility-coefficients of wort for oxygen, 361-3
     of brewers’ worts, 366, and following pages
   on combination of oxygen with worts, 371, and following pages
   on the rapidity of the combination, 376
   on amount of combination, 379
   on non-transformation of _mycoderma vini_, 110, and following pages,
      113 (footnote)
     of _mycoderma aceti_, 124, and following pages
     of _mucor racemosus_, 128, and following pages
   on non-transformation of yeast into penicillium, 333-335
   on cultivating pure _penicillium_, 88, and following pages
   on its transformation into yeast, 91
   transformation, Trécul’s, details of, 98
   with submerged _aspergillus_, 101, and following pages
     _penicillium_, 99
   in disproof of the _hemi-organism_ theory, 273 (footnote)
   on growth of mixed moulds, 112
   on purification of mixed ferments, 226, and following pages
   on growth of _mucor mucedo_, 140, 141
   on proportion between weights of mucor and alcohol formed, 134, and
      following pages
   on the anaërobian cultivation of yeast, 239, and following pages
   on variation of proportion of sugar used to yeast formed, 249
   on growth of yeast in sugar solutions, 318, and following pages,
      331-333
   on dust on fruits, 153, and following pages
   on seasonal influences on fertility of dust-germs, 157, and following
      pages
   on exhaustion of yeast, 169, and following pages
     of “high” yeast, 189, 190
   on revival of yeast, 207, 208
   on cultivation of aërobian ferment, 211, and following pages
   on gradual _senescence_ of yeast, 245
   on production of a pure beer, 338, and following pages
   on clarification of worts and beers, 382, and following pages
   comparative, on the qualities of beers brewed by different processes,
      391
     on rate of budding, 145

 Exportation of “high” beers unsatisfactory, 16


 F

 Ferment, _v._ also yeast

 Ferments of disease, _v._ disease-ferments

 Ferments, special, 14, 15

 Ferments and animal diseases, 41, and following pages
   butyric, lactic, alcoholic, 72
   moulds functioning as, 100, 101, and following pages, 111, 113, 129,
      133
   general character of a, 115
   of grape, varieties, origin, 150, and following pages
   alcoholic, summary of, 196
   intermixture of, 224, 225
   mode of separation of mixed, 226 and following pages
   succession of, in must, 227
   exceptional vital processes of, 236, 237

 Ferment power in relation to time discussed, 252
   character, how related to heat, 270
   and fermentation correlated, 277
   a chemical substance existing in cells (Traube), 283 (footnote)
   of tartrate of lime, 290


 Ferments, two classes, distinctive characteristics, 323

 Fermentation, rapid, inexpedient, 3
   spontaneous, in case of must, 4
   “top” and “bottom,” _v._ “high” and “low”
   masked by moulds in shallow vessels, 75 (footnote)
   by _penicillium_ (Tréoul) 94
   by _mycoderma vini_, 111, 113
   by _mucor racemosus_, 129, 139
   alcoholic, general explanation of, 114, 115
   conditions of, in sweetened mineral liquids, 211
   without air, 242
   with and without air, results compared, 243, 244
   a cell-life without air, 259
   a general phenomenon, 266, 267
   of fruits not truly “alcoholic,” 276
   not definable, according to Brefeld, as life without air, 280
   of lactate of lime, 294

 Fermentative energy, 252
   character dependent on conditions, 266

 Filamentous tissue (Turpin), 123

 Fitz on fermentation, 142

 Fissiparous division of vibrios, 299

 Flask sterilizing, 27, 29

 Flasks with double necks, advantage of, 120

 Fluid, Raulin’s, 88 (footnote)

 Flavour dependent on ferment species, 230

 Foreign organisms correlated with unsound beer, 26, 32
   greatly promoted by adaptability of liquids, 36

 Formula for solubility-coefficient of any wort for oxygen, 364


 Fremy’s statement of _hemi-organism_, 52
   answer to Pasteur’s facts, 58
   explanation of vintage fermentation, 272
   “organic impulse,” 325
   latest assertions, 396-399

 Fruits, ferment organisms on surface of, 153, and following pages
   internal fermentation of, 267, and following pages
   yeast cells not present within, 267 (footnote)
   influence of carbonic acid gas on preservation of, 268
   respiratory processes of, according to Bérard, 270
   fermentation within, Lechartier and Bellamy, 270
   crushed and uncrushed, fermentation of, 274

 Fruit-cells, anaërobian life of, 272

 Fungi, wide distribution of spores, 68
   absorption of oxygen by, 257
   production of alcohol by, 258 (footnote)

 Fungoid manner of growth of well-aerated yeast, 251


 G

 Galland’s claims of priority, 338 (footnote)

 Gay-Lussac’s experiments on grape-juice, 59, 60

 Gayon’s experiments on egg-albumen, 51

 “Gathered,” 367 (footnote)

 Generation, theories of, contrasted, 397


 Germs of ferments in air, &c., 6, 26, 38
   brought by other matters, 38
   absent from fruits, when? 58, 59, 157, and following pages
   not universally distributed, 61, 63, 181 (footnote)
   distribution experiments, 65, and following pages, 87 (footnote)
   and their correlated fruits, 61
   of disease latent, 220

 _Germ_, use of term by Pasteur, 313

 Germ theory of disease discussed, 46, 47

 Globuline tissue (Turpin), 123

 Globulines, punctiform, 121, and following pages

 Globules, 275 (footnote)

 Glycerine, fermentation of, by vibrios, 306, 307

 Gosselin, M., report, 44
   and Robin on ammoniacal urine, 45

 Gramme, value in grains, 135 (footnote)

 Granules in wort, explanation of, 95

 Graham’s, Dr., criticisms of Pasteur, 13 (footnote), 196 (footnote)

 Graham, Dr., on aspect of bottom yeast, 194 (footnote)

 Grape juice, experiments on, 57, 59

 Grape-ferments, _v._ ferments

 Grapes, do they contain cells of yeast? 267

 Greasiness of _mycoderma vini_, 80, and footnote


 H

 Hallier mentioned, 92

 Hard water, influence on aspect of yeast, 194 (footnote)

 _Head_ of vibrio, 292

 Heating sufficient as preventing deterioration of liquids, 20
   influence on beer, 20

 Heat, production of, its relation to ferment-power, 270

 _Hemi-organism_, chimerical, 53, 162, 399, 273 (footnote)
   latest assertions by Fremy on subject of, 396-399
   theory of vintage-fermentation, 272, 273

 Heterogenesis, facts against, 51


 “High” fermentation, meaning of, 8, 9
   beers, disadvantages of, 12, 13
   ferment, aspect of, 188, 189
   characteristics of, summary, 191
   ferment (new), occurrence, 198
     aspect and characteristics of, 199
     aërobian form of, 216

 High yeast, aërobian form, aspect of, 214

 Hoffmann, H., transformation of ferment, 92, 93

 Hop-oil as a beer-antiseptic, 16, and footnote

 Hopping influence on growths _quâ_ temperature, 96

 Hot countries, absence of breweries in 16

 Hydrogen from vibrionic life, 300
   occasional absence in butyric fermentations, 308


 Hydrosulphite of soda, composition, use in determinations of oxygen,
    355, and footnote
   preparation of _saturated_ solution, 357 (footnote)
   alterability of solutions of, 356
   improved method of M. Raulin, 356, and following pages


 I

 Ice, quantities consumed in “low” breweries, 11

 Illusions as to absence of foreign organisms, 36, 85, 92

 Impregnations, spontaneous, 65, 66, 69, 73, 79

 Impregnation, mode of (_penicillium glaucum_), 86

 Impurity of ferments, source of experimental errors, 37
   of yeast masked for a time, 220

 Increase of yeast disproportionate to sugar used, 237

 Infusions, nature of organisms in, 39

 Infusoria, 35

 Insoluble substances in wort, 386

 Inverting constituent of yeast, 321, and footnote

 Isolation of ferment, 77


 L

 Lactic ferments, 5, 36
   transformation from and into other ferments (Duval), 37

 Lactate of lime, fermentation of, 292

 Lechartier and Bellamy, researches on fermentation in fruits, 270

 Leptothrix, 36

 Liebig’s views of fermentation, 317, and following pages
   on fermentation of malate of lime, 321
   definition of a ferment, 324
   modified theory, 326;
     answer to, by Pasteur, 326, 327
   neglect of microscopical observations, 329, 330

 Lime, bisulphite, use of, by bottlers, 15
   carbonate sterilized, use of in growths, 126
   dextro-tartrate, 284
   acetate and metacetate, 288
   lactate, fermentation of, 292

 Lister’s, Prof., letter on germ-theory, 43

 London breweries, usages of, 8 (footnote)
   Pasteur’s visit to, 22-24


 “Low” fermentation, meaning of, 9, 10;
   advantages, 12
   beer breweries, statistics of, 10
     properties of, according to Dr. Graham, 13
   yeast and “high” yeast distinct, 192, 193
   yeast, aspect of, 193;
     characteristics, 195
     aërobian form of, 215

 Low temperatures prejudicial to disease-ferments, 14


 M

 Malignant pustule, 42

 Mashings, 3

 Medium, mineral, for growing lactic vibrios, 293, 297 (footnote)
   Cohn’s formula, 294 (footnote)
   for growth of bacteria, 294

 Medium, composition of, influence on life, 296

 Microscopical study of yeast important, 23
   formerly neglected in English breweries, 22-24

 Microscopical examination of vibrios, 298, 299

 Microzyma, 121;
   source of _mycoderma aceti_ according to Béchamp, 124

 Milk, temperature of sterilization of, 34

 Milk-sugar, growth of yeast in, 265

 _Mother of vinegar_, _v._ mycoderma aceti

 Moulds thrive in acid liquors, 36
   functioning as ferments, 100, 101, and following pages, 111, 113,
      129, 133
   growth of, and production of alcohol, 257, 258 (footnote)
   suggested employment of, industrially, 261

 Mucedines, 36, 40

 _Mucor mucedo_ and _racemosus_ on must, 66

 _Mucor racemosus_, different aspects of, 105
   pure growth of, 128, and following pages

 Mucor normal, growth of, 132
   weight of to alcohol formed, 134, and following pages
   morphology of abnormal growth, 137

 _Mucor mucedo_ distinguished from _racemosus_, 140
   growth in double-necked flasks, 140, 141

 Müntz, 323

 Must, fermentation of, always regular, 3
   pure fermentation of, 54, and following pages
   succession of ferments in, 227, 228

 _Mycelium_ and _mycoderma vini_ on wine, 56, 65

 Mycoderma in wort experiments, 70

 _Mycoderma vini_, arborescent form of, 77
   growth of pure, experiments on, 110, and following pages, 120
   growth with _penicillium_, 112
     with _mucor_, 112
   endogenous sporulation, 151 (footnote)


 _Mycoderma aceti_ transformations (Béchamp), 124
   pure growth of, 124, and following pages


 N

 _Nageurs_ used in low fermentation, 9

 Nature of liquids, influence on growths, 36, 73, 85

 Natural liquids for pure growths, use of, 40, 41
   experiments on, 47, and following pages

 Neutrality, conditions of, as affecting sterilization of liquids, 34;
   explanation of fact, 35

 Neutralization of acidity in pure growths, mode of, 126

 New high ferment, _v._ high

 New process of brewing, 391-393

 Nitrogenous soluble parts of yeast, 319, 320

 Nomenclature used by Pasteur purposely vague, 314

 Normal growth of mucor, 132


 O

 Organic substances, have they any tendency to become organized? 33

 Organic liquids sterilized by boiling, 34

 Organizable globulines (Turpin), 123

 Organisms and animal diseases, 42

 _Ouillage_, 2

 Oxidation of germ-free liquids, 57
   processes of fungi, 261, and footnote
   of wort, excessive, injurious, 353, 354

 Oxygen absorbed by blood, 50
     by urine, 50
   and fermentation, according to Gay-Lussac, 60
   store-energy imparted to cells by, 134
   no influence upon fermentation, (Béchamp), 178 (footnote)
   function in fermentation, experiments on, 238, and following pages
   influence on fermentation (Schützenberger and Pasteur), 253, 254
   amount absorbable by yeast, 255
   deficiency of, function in fermentation, 259
     influence on products, 100, 108, 113
     influence on morphology of moulds and ferments, 105, 106, 133, 137,
        262
   necessity of, to growth of yeast discussed, 280
   unnecessary and adverse to vibrionic life, 284, and following pages
   necessary to bacterial life, 305
   removal from solutions by bacteria, 295
   growth of vibrios apart from, 302
   compressed, influence on ferment life, 324
   determination of, in worts (Schützenberger), 355, and following pages
   solubility-coefficients in water (Bunsen), 360
   usual amounts in solution in brewers’ worts, 366, 367
     changes in amounts during brewing processes, 369, 370
   combination of, with hopped wort, 371, and following pages
   experiments on rapidity of combination, 376
     on amount of, under brewing conditions, 379
   in combination with wort not available for yeast, 380, 381
   clarification of wort by, 385


 P

 Palate-fulness definition, 354, and footnote
   impaired by oxidation, 354

 Parasites and their germs, 40
   influence on animal diseases, 41

 Pasteur’s repetition of Trécul’s experiments, 98, 99
   subject of his inquiries stated, 311
   experiments, exactness of, 95 (footnote)

 Pasteurization, meaning and use, 15 (footnote)

 Patches of froth in growth of pure yeast, 31

 _Penicillium glaucum_ on must, 66
   growth of pure, 86, and following pages
   precaution, 89
   transformed into ferment (Trécul), 94
   spores, varieties of, 97
   production of alcohol by, 99, and following pages
   transformation into mycoderma, 109

 Phenol for purifying yeasts, 232

 Pitching, mode of, for pure beer, 342, and following pages
   flasks, 344
   peculiar in London breweries, explanation, 350, 351

 Plaster of Paris and yeast powder, 81, and following pages

 _Ploussard_ grapes, experiments on, 161

 Polymorphism of organisms, 84 (footnote), also _v._ transformation

 Precautions for pure fermentation of must, 64
   brewers’, to check disease-germs, 220, and following pages
   for pure anaërobian growth of yeast, 248

 Preservation of yeast, 207

 Preoccupation of liquids by organisms, 36, 109, 220

 Products of fermentation variable, 276, 277

 Price of beer as affected by losses from disease, 24

 Proliferous pellicles, 121

 Proportions of alcoholic products variable, 276, 277

 Proportions of products diagnostic of the fermentation, 279

 Proteic matter elaborated by cells, 335

 “Pulling up,” 343

 Pure growth of yeast, precautions for, 29-32
   growths in natural liquids, 40, 41
   wort and ferment, advantages of, 391-393

 Purification of mixed ferments, 226, and following pages
   practical methods, growth in sweetened water, 230
   shallow basins, 231
   in acid and alcoholic liquids, 231
   with aid of carbolic acid, 232

 Putrid wort, ferments of, 5

 Putrefaction prevented by use of sterilizing flask, 27
   of yeast, cause of, 221
   of tartrate of lime, 291


 Q

 Qualities of “high” and “low” beers, 12, 13, 19, 196

 Quality of beer dependent on kind of ferment, 26, and following pages


 R

 Racking, 222
   precautions necessary in, 351

 Raulin’s fluid, 88 (footnote)
   improvement on Schützenberger’s oxygen process, 356, and following
      pages
   experiments on solubility of oxygen in worts, 361-363

 Rayer on splenic fever, &c., 42

 Reducing action of vibrios, 291

 Rees, Dr., 150 (footnote)

 Refrigerator, Baudelot’s, 387 (footnote)

 Revival of mould-cells by aeration, 130, 131 (footnote), 138

 Revival of starved yeast, 148, 208
   vibrios, 301, 302

 Ripening of fruits, 270, 271

 Robin, Ch., mentioned, 93;
   strictures on Pasteur, 310, 311
   recantation of views on fermentation, 314


 S

 _Saccharomyces apiculatus_, 71, and footnote, 150
   _exiguus_, 185,
     _ellipsoideus_, 165
   _pastorianus_ 151;
     mode of growth of, 167
   two aspects, globular and filamentous, 168, 169
   exhaustion and revival of, aspects, 172, and following pages
   occurrence as impurity in most ferments, 225
   most suitable for growth experiments in sugar solutions, 332

 _Saccharomyces pastorianus_, _ellipsoideus_, _apiculatus_ in must, 227,
    and following pages

 _Sang de rate_, 43

 Schützenberger on budding of yeast, 146, and footnote

 Schützenberger’s strictures on Pasteur’s views answered, 252, and
    following pages
   process for determining oxygen in solutions, 355

 Seasons, influence on success in brewing, 25
   at which germs are absent on fruits, 58, 59

 Secondary fermentation in English beers, 224

 _Senescence_ of yeast cells, 208
   gradual of yeast cells, experiments on, 245

 Shallow basins for purification of yeasts, 231

 Sodium hydrosulphite, _v._ hydrosulphite

 Solubility-coefficients of oxygen in water (Bunsen), 360
   in worts (Raulin), 361-363

 Sour beer, ferments of, 5

 Soundness of beer always dependent on purity of yeast, 26, 32

 Specialization of ferment-variations, 197

 Specimens, necessary precautions for taking, 126 (footnote)

 Splenic fever, 42

 Spontaneous fermentation used in must, not in beer, 4
   fermentation or putrefaction prevented by use of sterilizing flask,
      28
   ferment, definition of, 182;
     experiment on, 184
   generation, facts against, 51, 52, 57
     supported by experimental errors, 62, 63
     (Trécul’s theory of), 94, 95
   impregnations, 65, 66, 69, 73, 79
     use in isolating ferments, 77

 Spores on grapes, gooseberries, &c., 54
   of fungi widely distributed, 68

 Statistics of breweries, 10
   of French beer consumption, 17 (footnote)

 Starved yeast, appearance of, 148

 Stability of sterilized liquids, 286

 _Stemphylium_ spores, 55 (footnote)

 Sterilizing apparatus, 27, 29, 285
   flask, 28

 Sterilization-temperature of various liquids, 34

 Stock beer, 223

 Store beer, must be surrounded by ice, 16

 Straw wine, peculiar fermentation of, 166

 _Strength_, Pasteur’s use of word, 354
   saving by the new process, 394

 Submerged _penicillium_, 99
   _aspergillus_, 101, and following pages
   _mycoderma_, 111, 113, and following pages
   _mucor_, 129, and following pages, 133

 Submerging growths, precautions for, 91 (footnote)

 Succession of transformations (Trécul’s scheme), 93, 94

 Sugar decomposition by submerged cells, 114
   different modes of, by different cells, 115
   decomposed disproportionate to yeast formed, 237, and following pages
   variation of disproportion in different cases, 249
   amount decomposed in a given time, as an index of fermentative energy
      (Schützenberger’s views), 252
   solutions pure with mineral salts, growth of yeast in, 317, and
      following pages
   denial of the fact by Liebig and reply by Pasteur, 328, 329

 Surface growth of yeast in pure culture, 31

 Sweetened water for exhausting yeast, 169, 170, 190
   for purification of yeasts, 230


 T


 Tartrate-acid of potash for purifying yeasts, 231
   -dextro of lime, fermentation of, 284, and following pages
   products of, 288
   ferment of, 290

 Temperatures in use in London breweries, 8 (footnote)
   high, prejudicial to quality of “low” beer, 19
   at which disease-ferments perish, 20;
     differs in different liquids, 34, 96
   influence on fermentation, 129

 Temperatures suitable for “high” or “low” yeasts respectively, 192
   influence of on mixed “high” and “caseous” yeasts, 203
   for observing active vibrios, 299

 Theories of generation opposite stated, 397

 Tieghem, Van, on ammoniacal urine, 45


 Torula, sense in which used, 73 (footnote)
   varieties of, 77
   non-fermentative species, 78


 Transformation of ferments, according to Duval, 37
   of non-fermentative to fermentative impracticable, 80
   of _penicillium_ into yeast impracticable, 91
   of ferment into moulds (Hoffmann), 92
   series, Trécul’s scheme of, 93, 94
   of _penicillium_ to _mycoderma_ (Ch. Robin), 109 (footnote)
   of _mycoderma vini_ refuted, 113 (footnote)
   Turpin’s system of, 122, and following pages
   of _mycoderma aceti_ (Béchamp), 124
   historical account of views on, 128 (footnote)
   of _mucor_ (Bail), 127
   of filamentous into globular yeast, 169
   of yeast into _penicillium_, &c., impracticable, 333-335
   mutual of low and high yeast, discussed, 192, 193
   of “high” yeast into “caseous” ferment illusory, 203
   of albumen, theory of the vintage, 272, 273
   theory disproved generally, 273 (footnote)

 Traube, Dr., on ammoniacal urine, 46
   researches on fermentation, 282
   theory of fermentation, 283 (footnote)

 Trécul and Fremy, _v._ Fremy

 Trécul’s theory of successive transformations, 93, 94
   details of transformation experiments, 98
   theory refuted, 99

 _Trousseau_ grapes, experiments on, 162

 “Turned” beer, ferments of, 5;
   filaments of, 23

 “Turning out,” 367 (footnote)

 Turpin, M., mentioned, 92

 Turpin’s system of transformations, 122, and following pages, 113
    (footnote)

 Tyndall, letter to Pasteur, 399-401


 U

 Unsoundness of beer correlated with disease-organisms, 26, 32

 Urea-ferment, the transformation of (Duval), 37

 Urine, ammoniacal, 45, 46

 Urine, sterilized, study of, 49, 50


 V

 Variability of fermentation products, 277

 Variations of ferment strengthened and established, 197

 Varieties of yeast, 149, and following pages

 Vaureal, De, budding of yeast, 146 (footnote)

 Vegetable distinguished from animal organisms by ammonia (Robin), 312

 Vesicular tissue (Turpin), 123

 Vibrio, 36;
   butyric, 65, 70
   also an example of anäerobian life, 282, 284
   active and exhausted, 290
   reducing action of, 291

 Vibrionic ferment of tartrate of lime, 290

 Vibrios, _head_ of, 292;
   supposed reproductive corpuscles, 306
   growth of, in lactate media, 293
   medium for growth of, according to Cohn, 294 (footnote)
   not genetically related to bacteria, 296
   of butyric fermentation, description of, 298, 300
   mode of examining microscopically, 298
   fissiparous division of, 299
   measurements of, 300
   cannot live on butyrates, 301 (footnote)
   revival of, 301, 302;
   anaërobian growth of, 302
   life of, destroyed by oxygen, 303, 304

 Vigour of ordinary brewer’s yeast, 246

 _Vin de paille_, 166

 Vinegar, temperature at which it is sterilized, 34

 Vinous flavour in stock beer, 224

 Vintage, varied conditions of, 268 (footnote)
   fermentation, theory of, according to Fremy, 272, and following pages

 Viscous wort, ferments of, 5

 Visit to London brewery by Pasteur, 22-24

 Vital processes of ferment exceptional, 237
   activity of yeast apart from air, 259
   potential in cells, 278

 Vitiation of experiments, causes of, 63, 85, 92


 W

 Wad-dressing, antiseptic, 44

 Water, hard, influence on aspect of yeast, 194 (footnote)

 Weights of _mucor_ and alcohol, proportion of, 134, and following pages

 Weight of yeast grown, what due to, 257 (footnote)

 Wide dishes, experiments on fermentation in, 69, 70
   favourable to mould developments, 75 (footnote)

 Wine, less liable to deteriorate than beer, 2
   temperature of sterilization, 34

 Wort, definition, 2;
   cooling of, 3, 4
   temperature of sterilization, 34
   solubility of oxygen in, 361, and following pages
   formula for solubility in any wort, 364

 Worts, brewers’, usual amounts of oxygen in solution, 366, 367
   experiments on amounts, 379

 Wort, hopped, its affinity for oxygen, 371, and following pages
   mode of transmitting it free of oxygen, 371, 372
   insoluble substances in, 386


 Y

 Yeast, _v._ also ferment, germs, torulæ nature and properties of, 143,
    and following pages
   starved and well-nourished, appearances contrasted, 147, 148
   varieties of, 149, and following pages
   commercial origin of, where? 187
   relations to other organisms, 180, 181
   commercial mixtures, 224, 225
   practical purification of, 230-233
   impurities in masked for a time, 220
   exceptional characteristics of, 237
   growth of in sterilizing flasks, 29-32
   not transformable into any other organism, 37
   aspect may change under modified circumstances, 37
   non-transformation of _mycoderma vini_ into, 120
   _mucor_ into, 132
   non-fermentative species of, 79, 80, 206, 207
   “high,” characteristic aspect of, 188-192
   well aerated, fungoid mode of growth, 251
   anaërobian growth, cause of fermentation, 259
   growth of, in solutions of sugar, 318, and following pages
   growth in relation to proportion of sugar used, 237, and following
      pages
   difficult propagation in saccharine mineral media, 329, 330
   growth of, without producing alcohol, 265
   capacity of absorbing oxygen, 255
   necessity of oxygen for its growth discussed, 280
   incapable of using oxygen in combination in worts, 380, 381
   soluble nitrogenous part of, 320, 321, 79 (footnote)
   dried into dust still active, 81, and following pages
   does not perish at temperatures at which disease-ferments do, 20
   sudden inactivity of, cause and cure, 347 (footnote)
   change of, a trade custom, 22
   reason of addition of yeast to wort, 3
   proportion commonly added, 3
   reason of the large proportion used, 343.

 Yeast-cells abundant in brewing laboratories, 75
   gradual senescence of, 245

 Yeast-cells, mode of examining fruits for, 267 (footnote)
   necessary relation to “alcoholic fermentation,” 275

 Yeast-water, definition, 79 (footnote)
   exhaustion of yeast by, 171
   use of in pure growths (_penicillium_), 88

 “Youth” of cells, 246


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    ○ Footnotes have been moved to follow the chapters in which they are
      referenced.
    ○ Subscripts are transcribed by an underscore and a value enclosed
      in braces, such as O_{2}. Superscripts are transcribed by an
      up-caret and a value enclosed in braces, such as MC^2, or
      sometimes by an up-caret and a single character, such as 4^e.





End of Project Gutenberg's Studies on Fermentation, by Louis Pasteur