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THE SCIENCE SERIES


  1. +The Study of Man.+--By A. C. HADDON. Illustrated, 8^o, $2.00.

  2. +The Groundwork of Science.+--By ST. GEORGE MIVART. 8^o, $1.75.

  3. +Rivers of North America.+--By ISRAEL C. RUSSELL.
       Illustrated, 8^o, $2. 00.

  4. +Earth Sculpture.+--By JAMES GEIKIE. Illustrated, 8^o, $2.00.

  5. +Volcanoes.+--By T. G. BONNEY. Illustrated, 8^o, $2.00.

  6. +Bacteria.+--By GEORGE NEWMAN. Illustrated, 8^o, $?

G. P. PUTNAM'S SONS, NEW YORK AND LONDON


  The Science Series

  EDITED BY

  Professor J. McKeen Cattell, M.A., Ph.D.

  AND

  F. E. Beddard, M.A., F.R.S.

BACTERIA

[Illustration]




  BACTERIA

  ESPECIALLY AS THEY ARE RELATED
  TO THE ECONOMY OF NATURE
  TO INDUSTRIAL PROCESSES
  AND TO THE PUBLIC HEALTH

  BY

  GEORGE NEWMAN

  M.D., F.R.S. (EDIN.), D.P.H. (CAMB.), ETC.

  DEMONSTRATOR OF BACTERIOLOGY IN KING'S COLLEGE, LONDON

  ILLUSTRATED

  NEW YORK
  G. P. PUTNAM'S SONS

  LONDON
  JOHN MURRAY

  1899

  COPYRIGHT, 1899

  BY

  G. P. PUTNAM'S SONS


  The Knickerbocker Press, New York




PREFACE


The present volume is not a record of original work, nor is it a
text-book for the laboratory. Theoretical and practical text-books of
Bacteriology plentifully exist both in England and America. There are
two large works widely used, one by Professor Crookshank, entitled
_Bacteriology and Infective Diseases_, the other by Dr. Sternberg,
_A Manual of Bacteriology_. There are also, in English, a number of
smaller works by Abbott, Ball, Hewlett, Klein, Macfarland, Muir and
Ritchie, and Sims Woodhead. This book is of a less technical nature. It
is an attempt, in response to the editor of the series, to set forth
a popular scientific statement of our present knowledge of bacteria.
Popular science is a somewhat dangerous quantity with which to deal. On
the one hand it may become too popular, on the other too technical. It
is difficult to escape the Scylla and Charybdis in such a voyage.

I am much indebted to Professor Crookshank, who, in reading the
manuscript, has helped me by many valuable criticisms. My thanks are
also due to Sir C. T. D. Acland, Bart., for many kind suggestions,
and to Mr. E. J. Spitta, M.R.C.S., who has been good enough to take a
number of excellent photo-micrographs for me. Some other illustrations
have been derived from the _Atlas of Bacteriology_, brought out jointly
by Messrs. Slater and Spitta. For these also I am glad to have an
opportunity of expressing my thanks. It should be understood that the
outline drawings are only of a diagrammatic nature.

  GEORGE NEWMAN.

  LONDON, 1899.




CONTENTS


                                                    PAGE

  INTRODUCTION                                        ix


  CHAPTER I

  THE BIOLOGY OF BACTERIA                              1


  CHAPTER II

  BACTERIA IN WATER                                   37


  CHAPTER III

  BACTERIA IN THE AIR                                 96


  CHAPTER IV

  BACTERIA AND FERMENTATION                          111


  CHAPTER V

  BACTERIA IN THE SOIL                               137


  CHAPTER VI

  BACTERIA IN MILK, MILK PRODUCTS, AND OTHER FOODS   178


  CHAPTER VII

  THE QUESTION OF IMMUNITY AND ANTITOXINS            240


  CHAPTER VIII

  BACTERIA AND DISEASE                               264


  CHAPTER IX

  DISINFECTION                                       322


  APPENDIX                                           337




ILLUSTRATIONS


[Illustrations starred (*) are reproduced by permission of the
Scientific Press from Drs. Spitta and Slater's _Atlas of Bacteriology_.]

                                                               PAGE

  VARIOUS FORMS OF BACTERIA                                       9

  SARCINA                                                        10

  NORMAL AND PLEOMORPHIC FORMS OF TUBERCLE                       13

  BACILLI, SHOWING FLAGELLA                                      15

  VARIOUS FORMS OF SPORE FORMATION AND FLAGELLA                  18

  POTATO IN A ROUX TUBE PREPARED FOR CULTIVATION                 22

  STAPHYLOCOCCUS PYOGENES AUREUS INCUBATOR           _to face_   22

  CULTURE MEDIA READY FOR INOCULATION                            23

  INOCULATING NEEDLES                                            24

  PASTEUR'S LARGE INCUBATOR FOR CULTIVATION AT ROOM
    TEMPERATURE                                      _to face_   24

  METHOD OF PRODUCING HYDROGEN BY KIPP'S APPARATUS
   FOR CULTIVATION OF ANAËROBES                                  27

  ANAËROBIC CULTURE                                              28

  KOCH'S STEAM STERILISER                                        31

  LEVELLING APPARATUS FOR KOCH'S PLATE                           40

  MOIST CHAMBER IN WHICH KOCH'S PLATES ARE INCUBATED             41

  HOT-AIR STERILISER                                             42

  THE HANGING DROP                                               44

  DRYING STAGE FOR FIXING FILMS                                  45

  TYPES OF LIQUEFACTION OF GELATINE                              47

  WOLFHÜGEL'S COUNTER                                            49

  PETRI'S DISH                                                   50

  BERKEFELD FILTER                                               52

  APPARATUS FOR FILTERING WATER TO FACILITATE
    ITS BACTERIOLOGICAL EXAMINATION                  _to face_   52

  BACTERIA OF TYPHOID FEVER                                      56

  BACILLUS COLI COMMUNIS                                         60

  THE COMMA-SHAPED BACILLUS OF CHOLERA                           66

  *BACILLUS TYPHOSUS                                 _to face_   66

  *BACILLUS TYPHOSUS                                      "      66

  *BACILLUS COLI COMMUNIS                                 "      66

  *BACILLUS MYCOIDES                                      "      66

  PASTEUR-CHAMBERLAND FILTER                                     80

  PROTEUS VULGARIS                                               86

  BACILLUS ENTERIDITIS SPOROGENES                                86

  A PLAN OF SEPTIC TANK AND FILTER-BEDS                          91

  FILTER-BEDS                                                    94

  MIQUEL'S FLASK                                                 97

  SEDGWICK'S SUGAR-TUBE                                          99

  SEDGWICK'S TUBE                                               100

  SACCHAROMYCES CEREVISIÆ                                       117

  ASCOSPORE FORMATION                                           120

  GYPSUM BLOCK                                                  121

  YEAST                                             _to face_   122

  ASCOSPORE FORMATION IN YEAST                           "      122

  NITROGEN-FIXING BACTERIA FROM ROOTLET NODULES          "      122

  *BACILLUS OF TETANUS                                   "      122

  SACCHAROMYCES ELLIPSOIDEUS                                    126

  SACCHAROMYCES PASTORIANUS                                     126

  BACILLUS ACIDI LACTICI                                        131

  BACILLUS BUTYRICUS                                            133

  KIPP'S APPARATUS                                              140

  FRÄNKEL'S TUBE                                                141

  BUCHNER'S TUBE                                                141

  A METHOD OF GROWING CULTIVATIONS IN A VACUUM OVER PYROGALLIC
    SOLUTION                                                    143

  MICROCOCCUS FROM SOIL                                         151

  NITROUS ORGANISM                                  _to face_   158

  NITRIC ORGANISM                                        "      158

  NITROGEN-FIXING ORGANISM FROM SECRETION
    OF ROOT-NODULES                                      "      158

  ROOTLET OF PEA WITH NODULES                                   163

  NITROGEN-FIXING BACTERIA IN SITU IN NODULE
   ON ROOTLET OF A PEA                              _to face_   164

  NITROGEN-FIXING BACTERIA IN SITU IN ROOTLET-NODULE
    OF A PEA                                             "      164

  NITROGEN-FIXING BACTERIA IN SITU IN ROOT-NODULE
    OF A PEA                                             "      164

  BACILLUS OF TETANUS                                           170

  BACILLUS OF SYMPTOMATIC ANTHRAX                               172

  BACILLUS OF MALIGNANT ŒDEMA                                   172

  A CENTRIFUGE                                                  228

  SUSPENDED SPINAL CORD                                         255

  FLASK USED IN THE PREPARATION OF THE TOXIN OF DIPHTHERIA      262

  *BACILLUS TUBERCULOSIS                            _to face_   280

  *BACILLUS TUBERCULOSIS                                 "      280

  *STREPTOCOCCUS PYOGENES                                "      280

  *BACILLUS ANTHRACIS                                    "      280

  FLASK USED IN THE PREPARATION OF TUBERCULIN                   282

  BACILLUS OF DIPHTHERIA                                        289

  TYPES OF STREPTOCOCCUS                                        298

  MICROCOCCUS TETRAGONUS                                        299

  DIPLOCOCCUS OF NEISSER                                        300

  BACILLUS OF ANTHRAX AND BLOOD CORPUSCLES                      302

  THREADS OF BACILLUS ANTHRACIS, SHOWING SPORES                 302

  BACILLUS OF PLAGUE                                            306

  *BACILLUS OF PLAGUE                               _to face_   310

  *BACILLUS OF LEPROSY                                   "      310

  STREPTOTHRIX ACTINOMYCES                               "      310

  BACILLUS MALLEI                                        "      310

  DIPLOCOCCUS OF PNEUMONIA                                      312

  BACILLUS OF INFLUENZA                                         315




INTRODUCTION


We live in a world that is teeming with life. From the earliest times
of man that life has been studied and the observations recorded. Thus
there has slowly come to be a considerable accumulation of knowledge
concerning the various forms (morphology) and functions (physiology)
of organised life. This we call the science of biology. It has for its
object the study of organic beings, and for its end the knowledge of
the laws of their organisation and activity. Slowly, too, in the midst
of this gradual accumulation of facts, we begin to see incoherence
becoming coherent, chaos becoming cosmos, chance and accident becoming
law. Further, the contemplation and comprehension which built up the
edifice of modern biology is assuming a new relationship to practical
life. Biology can no longer be considered only as an academic
occupation or as a theoretical pabulum upon which the leisured mind
may ruminate. With rapid strides and determined face this giant of
knowledge has marched into the arena of practical politics. The world
is opening its eyes to a reality which it had mistaken for a vision.

This application of biology to life and its problems has in recent
years been nowhere more marked than in the realm of bacteriology.
This comparatively new science, associated with the great names of
Pasteur, Koch, and Lister, furnishes indeed a stock illustration of
the applicability of pure biology. Turn where we will, we shall find
the work of the unseen hosts of bacteria daily claiming more and more
attention from practical people. Thus biology, even when clothed in the
form of microscopic cells, is coming to occupy a new place in the minds
of men. "Its evolution," as Professor Patrick Geddes declares, "forms
part of the general social evolution." Certainly its recent rapid
development forms a remarkable feature in the practical science of our
time. Not only in the diagnosis and treatment of disease, nor even in
the various applications of preventive medicine, but in ever-increasing
degree and sphere, micro-organisms are recognised as agents of utility
or otherwise no longer to be ignored. They occur in our drinking water,
in our milk supply, in the air we breathe. They ripen cream, and
flavour butter. They purify sewage, and remove waste organic products
from the land. They are the active agents in a dozen industrial
fermentations. They assist in the fixation of free nitrogen, and they
build up assimilable compounds. Their activity assumes innumerable
phases and occupies many spheres, more frequently proving themselves
beneficial than injurious. They are both economic and industrious in
the best biological sense of the terms.

Yet bacteriology has its limitations. It is well to recognise this,
for the new science has in some measure suffered in the past from
over-zealous friends. It cannot achieve everything demanded of it,
nor can it furnish a cause for every disease. It is a science fuller
of hope than proved and tested knowledge. We are as yet only upon the
threshold of the matter. As in the neighbouring realm of chemistry, it
is to be feared that bacteriology has not been without its alchemy.
The interpretations and conclusions which have been drawn from time to
time respecting bacteriological work have led to alarmist views which
have not, by later investigation, been fully supported. Again, the
science has had devotees who have fondly believed, like the alchemists,
that the twin secret of transmuting the baser metals into gold and of
indefinitely prolonging human life was at last to be known. But neither
the worst fears of the alarmist nor the most sanguine hopes of the
alchemist have been verified. Science, fortunately, does not progress
at such speed, or with such kindly accommodation. It holds many things
in its hands, but not finally life or death. It has not yet brought to
light either "the philosopher's stone" or "the vital essence."

What has already been said affords ample reason for a wider
dissemination of the elementary facts of bacteriological science. But
there are other reasons of a more practical nature. Municipalities
are expending public moneys in water analysis, in the examination of
milk, in the inspection of cows and dairies, in the bacterial treatment
of sewage, and in disinfection and other branches of public health
administration. Again, the newly formed National Association for
the Prevention of Tuberculosis, our increasing colonial possessions
with their tropical diseases, even medical science itself, which
is year by year becoming more preventive, make an increasing claim
upon public opinion. The successful accomplishment and solution of
these questions depend in a measure upon an educated public opinion
respecting the elements of bacteriology. Recently it was urged that
"the first elements of bacteriology should be shadowed forth in the
primary school."[1] This course was advised owing to such knowledge
being of value to those engaged in dairying. As we shall point out at a
later stage, many of the undesirable changes occurring in milk are due
to bacteria, even as the success of the butter and cheese industries
depends on the use and control of the fermentative processes due to
their action. Much of the uncertainty attending the manufacture of
dairy products can only be abolished by the careful application of
some knowledge of the flora of milk. In Denmark and in Scandinavia
the importance of such knowledge is realised and acted upon. America,
too, has not been slow to respond to these needs; but in England
comparatively little has been done in this direction.[2]

Whilst there can be no doubt as to the advantage of a wider
dissemination of the ascertained facts concerning bacteria, it
should be borne in mind that only patient, skilled observation and
experimental research in well-equipped laboratories can advance this
branch of science, or indeed train bacteriologists. The lives of Darwin
and of Pasteur adequately illustrate this truth. Yet it is observable
that States and public bodies are slow to act upon it, and frequently
in the past the most useful and substantial support for the advancement
of science has been forthcoming only from private sources. As the world
learns its intimate relation to science and the interdependence between
its life and scientific truth, it may be expected more heartily to
support science.




BACTERIA




CHAPTER I

_THE BIOLOGY OF BACTERIA_[3]


The first scientist who demonstrated the existence of micro-organisms
was Antony von Leeuwenhoek. He was born at Delft, in Holland, in 1632,
and enthusiastically pursued microscopy with primitive instruments.
He corroborated Harvey's discovery of the circulation of the blood
in the web of a frog's foot; he defined the red blood corpuscles of
vertebrates, the fibres of the lens of the human eye, the scales of the
skin, and the structure of hair. He was neither educated nor trained in
science, but in the leisure time of his occupation as a linen-draper he
learned the art of grinding lenses, in which he became so proficient
that he was able to construct a microscope of greater power than had
been previously manufactured. The compound microscope dates from 1590,
and when Leeuwenhoek was about forty years old, Holland had already
given to the world both microscope and telescope. Robert Hooke did for
England what Hans Janssen had done for Holland, and established the
same conclusion that Leeuwenhoek arrived at independently, viz.,
that a simple globule of glass mounted between two metal plates and
pierced with a minute aperture to allow rays of light to pass was
a contrivance which would magnify more highly than the recognised
microscopes of that day. It was with some such instrument as this that
the first micro-organisms were observed in a drop of water. It was not
until more than a hundred years later that these "animalcules," as
they were termed, were thought to be anything more than accidental to
any fluid or substance containing them. Plenciz, of Vienna, was one
of the first to conceive the idea that decomposition could only take
place in the presence of some of these "animalcules." This was in the
middle of the eighteenth century. Just about a century later, by a
series of important discoveries, it was established beyond dispute that
these micro-organisms had an intimate causal relation to fermentation,
putrefaction, and infectious diseases. Spallanzani, Pasteur, and
Tyndall are the three who more than others contributed to this
discovery. Spallanzani was an Italian, who studied at Bologna, and was
in 1754 appointed to the chair of logic at Reggio. But his inclinations
led him into the realm of natural history. Amongst other things, his
attention was directed to the doctrine of _spontaneous generation_,
which had been propounded by Needham a few years previously. In 1768
Spallanzani became Professor of Natural History at Pavia, and whilst
there he demonstrated that if infusions of vegetable matter were
placed in flasks and hermetically sealed, and then brought to the
boiling point, no living organisms could thereafter be detected, nor
did the vegetable matter decompose. When, however, the flasks were
very slightly cracked, and air gained admittance, then invariably
both organisms and decomposition appeared. Schwann, the founder of
the cell-theory, and Schulze, both showed that if the air gaining
access to the flask were either passed through highly heated tubes
or drawn through strong acid the result was the same as if no air
entered at all, viz., no organisms and no decomposition. The result
of these investigations was that scientific men began to believe that
no form of life arose _de novo_ (_abiogenesis_), but had its source
in previous life (_biogenesis_). It remained to Pasteur and Tyndall
to demonstrate this beyond dispute, and to put to rout the fresh
arguments for spontaneous generation which Pouchet had advanced as late
as 1859. Pasteur collected the floating dust of the air, and found by
means of the microscope many organised particles, which he sowed on
suitable infusions, and thus obtained rich crops of "animalculæ." He
also demonstrated that these organisms existed in different degrees in
different atmospheres, few in the pure air of the Mer de Glace, more
in the air of the plains, most in the air of towns. He further proved
that it was not necessary to insist upon hermetic sealing or cotton
filters to keep these living organisms in the air from gaining access
to a flask of infusion. If the neck of the flask were drawn out into a
long tube and turned downwards, and then a little upwards, even though
the end be left open, no contamination gained access. Hence, if the
infusion were boiled, no putrefaction would occur. The organisms which
fell into the open end of the tube were arrested in the condensation
water in the angle of the tube; but even if that were not so, the
force of gravity acting upon them prevented them from passing up the
long arm of the tube into the neck of the flask. A few years after
Pasteur's first work on this subject Tyndall conceived a precise method
of determining the absence or presence of dust particles in the air by
passing a beam of sunlight through a glass box before and after its
walls had been coated with glycerine. Into the floor of the box were
fixed the mouths of flasks of infusion. These were boiled, after which
they were allowed to cool, and might then be kept for weeks or months
without putrefying or revealing the presence of germ life. Here all the
conditions of the infusions were natural, except that in the air above
them there was no dust.

The sum-total of result arising from all these investigations was
to the effect that no spontaneous generation was possible, that the
atmosphere contained unseen germs of life, that the smallest of
organisms responded to the law of gravitation and adhered to moist
surfaces, and that micro-organisms were in some way or other the cause
of putrefaction.

The final refutation of the hypothesis of spontaneous generation was
followed by an awakened interest in the unseen world of micro-organic
life. Investigations into fermentation and putrefaction followed each
other rapidly, and in 1863 Davaine claimed that Pollender's bacillus
of anthrax, which was found in the blood and body tissues of animals
dead of anthrax, was the cause of that disease. From that time to this
in every department of biology bacteria have been increasingly found
to play an important part. They cause changes in milk, and flavour
butter; they decompose animal matter, yet build up the broken-down
elements into compounds suitable for use in nature's economy; they
assist in the fixation of free nitrogen; they purify sewage; in certain
well-established cases they are the cause of specific disease, and
in many other cases they are the likely cause. No doubt the disposal
of spontaneous generation did much to arouse interest in this branch
of science. Yet it must not be forgotten that the advance of the
microscope and bacteriological method and technique have played a large
share in this development. The sterilisation of culture fluids by heat,
the use of aniline dyes as staining agents, the introduction of solid
culture media (like gelatine and agar), and Koch's "plate" method have
all contributed not a little to the enormous strides of bacteriology.
Owing to its relation to disease, physicians have entered keenly into
the arena of bacteriological research. Hence, from a variety of causes,
it has come about that the advance has been phenomenal.

We shall now take up a number of points in the biology of bacteria
which call for early attention, and which are mostly the outcome of
comparatively recent work on the subject.

_The Place of Bacteria in Nature._ As we have seen, for a considerable
period of time after their first detection these unicellular organisms
were considered to be members of the animal kingdom. As late as 1838,
when Ehrenberg and Dujardin drew up their classification, bacteria
were placed among the Infusorians. This was in part due to the powers
of motion which these observers detected in bacteria. It is now, of
course, recognised that animals have no monopoly of motion. But what,
after all, are the differences between animals and vegetables so low
down in the scale of life? Chiefly two: there is a difference in
life-history (in structure and development), and there is a difference
in diet. A plant secures its nourishment from much simpler elements
than is the case with animals; for example, it obtains its carbon
from the carbonic acid gas in air and water. This it is able to do,
as regards the carbon, by means of the green colouring matter known
as _chlorophyll_, by the aid of which, with sunlight, carbonic acid
is decomposed in the chlorophyll corpuscles, the oxygen passing back
into the atmosphere, the carbon being stored in the plant in the form
of starch or other organic compound. The supply of carbon in the
chlorophyll-free plants, among which are the bacteria, is obtained
by breaking up different forms of carbohydrates. Besides albumen and
peptone, they use sugar and similar carbohydrates and glycerine as a
source of carbon. Many of them also have the capacity of using organic
matters of complex constitution by converting such into water, carbonic
acid gas, and ammonia. Their hydrogen comes from water, their nitrogen
from the soil, chiefly in the form of nitrates. From the soil, too,
they obtain other necessary salts. Now all these substances are in an
elementary condition, and as such plants can absorb them. Animals, on
the other hand, are only able to utilise compound food products which
have been, so to speak, prepared for them; for example, albuminoids
and proteids. They cannot directly feed upon the elementary substances
forming the diet of vegetables. This distinction, however, did not at
once clear up the difficult matter of the classification of bacteria.
It is true, they possess motion, are free from chlorophyll, and even
feed occasionally upon products of decomposition--three physiological
characters which would ally them to the animal kingdom. Yet by their
structure and capsule of cellulose and by their life-history and mode
of growth they unmistakably proclaim themselves to be of the vegetable
kingdom. In 1853 Cohn arrived at a conclusion to this effect, and since
that date they have become more and more limited in classification and
restricted in definition.

Even yet, however, we are far from a scientific classification for
bacteria. Nor is this matter for surprise. The development in this
branch of biology has been so rapid that it has been impossible
to assimilate the facts collected. The facts themselves by their
remarkable variety have not aided classification. Names which a few
years ago were applied to individual species, like _Bacillus subtilis_,
or _Bacterium termo_, or _Bacillus coli_, are now representative,
not of individuals, but of families and groups of species. Again,
isolated characteristics of certain microbes, such as motility, power
of liquefying gelatine, size, colour, and so forth, which at first
sight might appear as likely to form a basis for classification, are
found to vary not only between similar germs, but in the same germ.
Different physical conditions have so powerful an influence upon these
microscopic cells that their individual characters are constantly
undergoing change. For example, bacteria in old cultures assume a
different size, and often a different shape, from younger members of
precisely the same species; _Bacillus pyocyaneus_ produces a green to
olive colour on gelatine, but a brown colour on potato; the bacillus
of Tetanus is virulently pathogenic, and yet may not act thus unless
in company with certain other micro-organisms. Hence it will at once
appear to the student of bacteriology that, though there is great
need for classification amongst the six or seven hundred species of
microbes, our present knowledge of their life-history is not yet
advanced enough to form more than a provisional arrangement.

We know that bacteria are allied to moulds on the one hand and yeasts
on the other, and that they have no differentiation into root, stem, or
leaf; we know that they are fungi (having no chlorophyll), in which no
sexual reproduction occurs, and that their mode of multiplication is by
division. From such facts as these we may build up a classification as
follows:--

                            VEGETABLE KINGDOM.
                                    |
           +---------------+--------+-------+------------------+
           |               |                |                  |
     Thallophyta.      Muscineæ.      Pteridophyta.      Phanerogamia.
    [= The lowest forms
    of vegetable life. No
    differentiation into
    root, stem, or leaf.]
           |
       Protophyta.
    [= No sexual reproduction.]
           |
   +-------+------------+
   |                    |
  Algæ.               Fungi.
  [= Chlorophyll    [= No chlorophyll.]
     present.]          |
                        |
              +--+--+--+--+--+--+
              |  |  |  |  |  |  |
                                |
                          Schizomycetes        {(1) Coccaceæ[4]--round cells.
                     [= multiplication by cell {
                        division or by spores] {(2) Bacteriaceæ--rods and threads.
                                               {
                                or             {(3) Leptotricheæ. }
                            Bacteria.          {                  } Higher Bacteria.
                                               {(4) Cladotricheæ. }


_Structure and Form._ Having now located micro-organisms in the economy
of nature, we may proceed to describe their subdivisions and form. For
practical convenience rather than academic accuracy, we may accept
the simple division of the family of bacteria into three chief forms,
viz.:--

                 { (1) Round cell form--_coccus_.
                 {
  Lower Bacteria { (2) Rod form--_bacillus_.
                 {
                 { (3) Thread form--_spirillum_.

  Higher Bacteria--Leptothrix, Streptothrix, Cladothrix, etc.

A classification dependent as this is upon the form alone is not by any
means ideal, for it ignores all the higher and complicated functions of
bacteria, but it is, as we have said, practically convenient.

[Illustration: VARIOUS FORMS OF BACTERIA

  1. Micrococcus
  2. Diplococcus
  3. Streptococcus
  4. Staphylococcus
  5. Leuconostoc, showing Arthrospores
  6. Merismopedia
  7. Sarcina
  8. Bacilli
  9. Spirillum]

1. _The Coccus._ This is the group of round cells. They vary in size as
regards species, and as regards the conditions, artificial or natural,
under which they have been grown. Some are less than 1/25000 of an inch
in diameter; others are half as large again, if the word large may be
used to describe such minute objects. No regular standard can be laid
down as reliable with regard to their size. Hence the subdivisions
of the cocci are dependent not upon the individual elements so much
as upon the relation of those elements to each other. A simple round
cell of approximately the size already named is termed a _micrococcus_
(μικρος, small). Certain species of micrococci always or almost always
occur in pairs, and such a combination is termed a _diplococcus_. Some
diplococci are united by a thin capsule, which may be made apparent by
special methods of staining; of others no limiting or uniting membrane
can be seen with the ordinary high powers of the microscope.[5] Again,
one frequently finds a species which is exactly described by saying
that two micrococci are in contact with each other, and move and act
as one individual, but otherwise show no alteration; whilst others
are seen which show a flattening of the side of each micrococcus
which is in relation to its partner. Perhaps the diplococci in an even
greater degree than the micrococci respond to external conditions
both as regards size and shape. It must further be borne in mind that
a dividing micrococcus assumes the exact appearance of a diplococcus
during the transition stage of the fission. Hence, with the exception
of several well-marked species of diplococci, this form is somewhat
arbitrary. The third kind of micrococcus is that formed by a number
of elements in a twisted chain, named _streptococcus_ (στρεπτος,
twisted). This form is produced by cells dividing in one axis, and
remaining in contact with each other. It occurs in a number of
different species, or what are supposed by many authorities to be
different species, owing to their different effects. Morphologically
all the streptococci are similar, though a somewhat abortive attempt
was once made to divide them into two groups, according to whether
they were long chains or short. As a matter of fact, the length of
streptococci depends in some cases upon biological properties, in
others upon external treatment or the medium of cultivation which has
been used. Sometimes they occur as straight chains of only half a dozen
elements; at other times they may contain thirty to forty elements,
and twist in various ways, even forming rosaries. The elements, too,
differ not only in size, but in shape, appearing occasionally as
oval cells united to each other at their sides. The fourth form is
constituted by the micrococci being arranged in masses like grapes, the
_staphylococcus_ (σταφυλις, a bunch of grapes). The elements are often
smaller than in the streptococcus, and the name itself describes the
arrangement. There is no matrix and no capsule. This is the commonest
organism found in abscesses, etc. The _sarcina_ is best classified
amongst the cocci, for it is composed of them, in packets of four or
multiples of four, produced by division vertically in two planes. If
the division occurs in one plane, we have as a result small squares
of round cells known as _merismopedia_. In both these conditions it
frequently happens that the contiguous sides of the elements of packets
become faceted or straightened against each other. It may happen, too,
particularly in the _sarcinæ_, that segmentation is not complete,
and that the elements are larger than in any other class of cocci.
They stain very readily. Nearly all the cocci are non-motile, though
Brownian movement may readily be observed.

[Illustration: SARCINA]

2. _The Bacilli._ These consist of rods, having parallel sides and
being longer than they are broad. They differ in every other respect
according to species, but these two characteristics remain to
distinguish them. Many of them are motile, others not. The ends or
poles of a bacillus may be pointed, round, or almost exactly square and
blocked. They all, or nearly all, possess a capsule. Individuals of the
same species may differ greatly, according to whether they have been
naturally or artificially grown, and pleomorphic forms are abundant.

3. _The Spirilla._ This wavy thread group is divisible into a number
of different forms, to which authorities have given special names. It
is sufficient, however, to state that the two common forms are the
non-septate spiral thread (like the _Spirillum Obermeier_ of relapsing
fever), which takes no other form but a lengthened spirillum; and the
spirillum which breaks up into elements or units, each of which appears
comma-shaped (like the cholera bacillus). The degree of curvature in
the spirilla, of course, varies. They are the least important of the
lower bacteria.

The _Higher Bacteria_ group includes more highly organised members
of the Schizomycetes. They possess filaments, which may be branched,
and almost always have septa and a sheath. Perhaps the most marked
difference from the lower bacteria is in their reproduction. In
the higher bacteria we have what is in fact a flower--terminal
fructification by conidia. In this group of vegetables we have the
Beggiatoa, Leptothrix, Cladothrix, and, at the top, the Streptothrix.
It has been demonstrated that _Streptothrix actinomycotica_ and
_Streptothrix maduræ_ are the organismal cause, respectively, of
Actinomycosis and Madura-foot, two diseases which have hitherto been
obscure.

_Pleomorphism._ This term designates an irregular development of
a species. Different media and external conditions bring about in
protoplasm as susceptible as mycoprotein a variety of morphological
phases. These may occur in succession, and represent different stages
in the life-history of a bacterium, or they may be involution forms
resulting from a change of environment, and occurring as "faults" in
the species. In the _Bacillus coli_, _B. typhosus_, bacillus of Plague,
and _B. tuberculosis_ pleomorphism undoubtedly occurs, and is manifest
in the change of shape. This is particularly marked in old cultures
of the last named. The ordinary well-known bacillus may grow out into
threads, with bulbous endings, granular filaments, drumsticks, and
diplococcal forms. Speaking generally, the older the culture, the more
marked is the variation.

_Polymorphism_ is a term used to define the theory which held that
bacteria were one of the intermediate shapes or forms between
something lower and something higher in the vegetable kingdom.
Neither pleomorphism nor polymorphism is fully understood, and many
bacteriologists find shelter from both in the term _involution form_.
What we do know is that the species already named, for example, take on
divers forms when placed under different conditions.

_Composition._ From what we have seen of the diet of micro-organisms,
we shall conclude that in some form or other they contain the elements
nitrogen, carbon, and hydrogen. All three substances are combined
in the _mycoprotein_ or protoplasm of which the body of the microbe
consists. This is generally homogeneous, and there is no sign of a
nucleus. It possesses a fortunate affinity for aniline dyes, and by
this means organisms are stained for the microscope. Besides the
variable quantity of nitrogen present, mycoprotein may also contain
various mineral salts. The uniformity of the cell protoplasm may
be materially affected by disintegration and _segmentation_ due to
degenerative changes. _Vacuoles_ also may appear from a like cause,
which it is necessary to differentiate from spores. Two other signs
of degeneration are the appearance of granules in the body of the
cell protoplasm known as _metachromatic granules_, owing to their
different staining propensities, and the _polar bodies_ which are seen
in some species of bacteria. Surrounding the mass of mycoprotein, we
find in most organisms a capsule or membrane composed, in part at
least, of _cellulose_. This sheath plays a protective part in several
ways. During the adult stage of life it protects the mycoprotein,
and holds it together. At the time of reproduction or degeneration
it not infrequently swells up, and forms a viscous hilum or matrix,
inside which are formed the new sheaths of the younger generation. It
may be rigid, and so maintain the normal shape of the species, or,
on the other hand, flexible, and so adapted to rapid movement of the
individual.

[Illustration: NORMAL AND PLEON-FORMS OF THE BACILLUS OF TUBASH.]

Here, then, we have the major parts in the constitution of a
bacillus--its body, mycoprotein; its capsule, cellulose. But,
further than this, there are a number of additional distinctive
characteristics as regards the contents inside the capsule which
call for mention. _Sulphur_ occurs in the Beggiatoa which thrive
in sulphur springs. _Starch_ is commoner still. _Iron_ as oxide
or other combination is found in several species. Many are highly
coloured, though these are generally the "innocent" bacteria, in
contradistinction to the disease-producing. A pigment has been found
which is designated _bacterio-purpurin_. According to Zopf, the
colouring agents of bacteria are the same as, or closely allied to, the
colouring matters occurring widely in nature. Migula holds that most
of the bacterial pigments are non-nitrogenous bodies. There are a very
large number of chromogenic bacteria, some of which produce exceedingly
brilliant colours. Among some of the commoner forms possessing this
character are _Bacillus et micrococcus violaceus_ (violet); _B. et M.
aurantiacus_ (orange); _B. et M. luteus_ (yellow); _M. roseus_ (pink);
many of the _Sarcinæ_; _B. aureus_ (golden-yellow); _B. fluorescens
liquefaciens et non-liquefaciens_ (green); _B. pyocyaneus_ (green); _B.
prodigiosus_ (blood-red).

_Motility._ When a drop of water containing bacteria is placed upon
a slide, a clean cover glass superimposed, and the specimen examined
under an oil immersion lens, various rapid movements will generally
be observed. These are of four kinds: (1) A dancing stationary
motion known as _Brownian movement_. This is molecular, and depends
in some degree upon heat and the medium of the moving particles. It
is non-progressive, and is well known in gamboge particles. (2) An
_undulatory_ serpentine movement, with apparently little advance being
made. (3) A _rotatory_ movement, which in some water bacilli is very
marked, and consists of spinning round, with sometimes considerable
velocity, and maintained for some seconds or even minutes. (4) A
_progressive_ darting movement, by which the bacillus passes over some
considerable distance.

The conditions affecting the motion of bacteria are but partly
understood. Heating the slide or medium accelerates all movement.
A fresh supply of oxygen, or indeed the addition of some nutrient
substance, like broth, will have the same effect. There are also the
somewhat mysterious powers by which cells possess inherent attraction
or repulsion for other cells, known as _positive_ and _negative
chemiotaxis_. These powers have been observed in bacteria by Pfeiffer
and Ali-Cohen.

[Illustration: BACILLI, SHOWING FLAGELLA]

The essential condition in the motile bacilli is the presence of
_flagella_.[6] These cilia, or hairy processes, project from the
sides or from the ends of the rod, and are freely motile and elastic.
Sometimes only one or two terminal flagella are present; in other
cases, like the bacillus of typhoid fever, five to twenty may occur all
round the body of the bacillus, varying in length and size, sometimes
being of greater length even than the bacillus itself. It is not yet
established as to whether these vibratile cilia are prolongations of
capsule only, or whether they contain something of the body
protoplasm. Migula holds the former view, and states that the position
of flagella is constant enough for diagnostic purposes. They are but
rarely recognisable except by means of special staining methods.
_Micrococcus agilis_ (Ali-Cohen) is the only coccus which has flagella
and active motion.

_Modes of Reproduction._ Budding, division, and spore formation are the
three chief ways in which Schizomycetes and Saccharomycetes (yeasts)
reproduce their kind. _Budding_ occurs in some kinds of yeast, and
would be classified by some authorities under spore formation, but in
practice it is so obviously a "budding" that it may be so classified.
The capsule of a large or mother cell shows a slight protrusion
outwards which is gradually enlarged into a daughter yeast and later
on becomes constricted at the neck. Eventually it separates as an
individual. The protoplasm of spores of yeasts differs, as Hansen has
pointed out, according to their conditions of culture.

_Division_, or fission, is the commonest method of reproduction. It
occurs transversely. A small indentation occurs in the capsule, which
appears to make its way slowly through the whole body of the bacillus
or micrococcus until the two parts are separate, and each contained in
its own capsule. It has been pointed out already that in the incomplete
division of micrococci we observe a stage precisely similar to a
diplococcus. So also in the division of bacilli an appearance occurs
described as a diplobacillus.

Simple fission requires but a short period of time to be complete.
Hence multiplication is very rapid, for within half an hour a new
adult individual can be produced. It has been estimated that at this
rate one bacillus will in twenty-four hours produce 17,000,000 similar
individuals; or, expressed in another way, Cohn calculated that in
three days, under favourable circumstances, this rate of increase would
form a mass of living organisms weighing 7300 tons, and numbering
about 4772 billions. Favourable conditions do not occur, fortunately,
to allow of such increase, which, of course, can only be roughly
estimated. But the above figures illustrate the enormous fertility of
micro-organic life. When we remember that in some species it requires
10,000 or 15,000 fully grown bacilli placed end to end to stretch
the length of an inch, we see also how exceedingly small are the
individuals composing these unseen hosts.

_Spore formation_ may result in the production of germinating cells
inside the capsule of the bacillus, _endospores_, or of modified
individuals, _arthrospores_. The body of a bacillus, in which
sporulation is about to occur, loses its homogeneous character
and becomes granular, owing to the appearance of globules in the
protoplasm. In the course of three or four hours the globule enlarges
to fill the diameter of the rod, and assumes a more concentrated
condition than the parent cell. At its maturity, and before its
rupture of the bacillary capsule, a spore is observed to be bright and
shining, oval and regular in shape, with concentrated contents, and
frequently causing a local expansion of the bacillus. In a number of
rods lying endwise, these local swellings produce a beaded or varicose
appearance, even simulating a streptococcus. In the meantime the rod
itself has become slightly broader and pale. Eventually it breaks down
by segmentation or by swelling up into a gelatinous mass. The spore
now escapes and commences its individual existence. Under favourable
circumstances it will germinate. The tough capsule gives way at one
point, generally at one of the poles, and the spore sprouts like a
seed. In the space of about one hour's time the oval refractile cell
has become a new bacillus. One spore produces by germination one
bacillus. Spores never multiply by fission, nor reproduce themselves.

Hueppe has stated that there are certain organisms (like leuconostoc,
and some streptococci) which reproduce by the method of _arthrospores_.
Defined shortly, this is simply an enlargement of one or more cell
elements in the chain which thus takes on the function of maternity.
On either side of the large coccus may be seen the smaller ones, which
it is supposed have contributed of their protoplasm to form a mother
cell. An arthrospore is said to be larger, more refractile, and more
resistant than an ordinary endospore. Many bacteriologists of repute
have declined hitherto to definitely accept arthrospore formation as a
proved fact.

[Illustration: VARIOUS FORMS OF SPORE FORMATION AND FLAGELLA

A. Stages in formation of spore and its after development. B. Spirillum
with terminal flagella.]

It is important to note that spore formation in bacteria must not
be considered as a method of multiplication. The general rule
is undoubtedly that one bacillus produces one spore, and one
spore germinates into one bacillus. It is a reproduction, not a
multiplication. Indeed, the whole process is of the nature of a
resting stage, and is due (_a_) to the arrival of the adult bacillus
at its biological zenith, or (_b_) to the conditions in which it
finds itself being unfavourable to its highest vegetative growth,
and so it endeavours to perpetuate its species. Most authorities are
probably of the latter opinion, though there is not a little evidence
for the former. Exactly what conditions are favourable to sporulation
is not known. Nutriment has probably an intimate effect upon it. The
temperature must not be below 16° C., nor much above 40° C. Oxygen, as
we have seen, is favourable, if not necessary, to many species, which
will in cultivation in broth rise to the surface and lodge in the
pellicle to form their seeds. Moisture, too, is considered a necessity.

The position and size of the spore are of considerable use in
differential diagnosis. The terminal spore of _Bacillus tetani_ is
well known. It is rarely seen at both ends of the bacillus, and
hence when poised only at one end causes the "drumstick" appearance.
In the bacillus of Quarter Evil the spore is generally towards one
end of the rod rather than in the middle; in Malignant Œdema the
bacillus in the blood grows out into long threads, and when such a
thread sporulates the spore is also near one end. The latter further
illustrates the fact that in some species the spore is of greater
diameter than the mother cell, and hence dilates the bacillary capsule.
The spores of anthrax are typical oval endospores. When free in the
field of the microscope, spores must be distinguished from fat cells,
micrococci, starch cells, some kinds of ova, yeast cells, and other
like objects. Spores are detected frequently by their resistance to
ordinary stains and the necessity of colouring them by special staining
methods. When, however, a spore has taken on the desired colour, it
retains it with tenacity. In addition to their shape, size, thickened
capsule, and staining characteristics, spores also resist desiccation
and heat in a much higher degree than bacilli not bearing spores. Roux
and some other eminent bacteriologists suggest that bacteria should be
classified according to their method of spore formation.

THE INFLUENCE OF EXTERNAL CONDITIONS ON GROWTH OF BACTERIA

_Nutritive Medium._ In the very earliest days of the study of
micro-organisms it was observed that they mostly congregate where there
is pabulum for their nourishment. The reason why fluids such as milk,
and dead animal matter such as a carcass, and living tissues such as
a man's body contain so many microbes is because each of these three
media is favourable to their growth. Milk affords almost an ideal
food and environment for microbes. Its temperature and constitution
frequently meet their requirements. Dead animal matter, too, yields
a rich diet for some species (saprophytes). In the living tissues
bacteria obtain not only nutriment, but a favourable temperature
and moisture. Outside the human body it has been the endeavour of
bacteriologists to provide media as like the above as possible, and
containing many of the same elements of food. Thus the life-history
may be carried on outside the body and under observation. By means of
cover-glass preparations for the microscope we are able to study the
form, size, motility, flagella, spore formation, and peculiarities
of staining, all of which characters aid us in determining to what
species the organism under examination belongs. By means of artificial
nutrient media we may further learn the characters of the organism in
"pure culture,"[7] its favourable temperature, its power or otherwise
of liquefaction, the curdling milk, or of gas production, its behaviour
towards oxygen, its power of producing indol, pigment, and chemical
bodies, as well as its thermal death point and resistance to light
and disinfectants. It is well known that under artificial cultivation
an organism may be greatly modified in its morphology and physiology,
and yet its conformity to type remains much more marked than any
degeneration which may occur.

The basis of many of these artificial media is _broth_. This is made
from good lean beef, free from fat and gristle, which is finely
minced up and extracted in sterilised water (one pound of lean beef
to every 1000 cc. of water). It is then filtered and sterilised. It
will be understood that such an extract is acid. To provide _peptone
beef-broth_, ten grains of peptone and five grains of common salt
are added to every litre of acid beef-broth. It is rendered slightly
alkaline by the addition of sodium carbonate, and is filtered and
sterilised. _Glycerine-broth_ indicates that 6 to 8 per cent. of
glycerine has been added after filtration, _glucose-broth_ 1 or 2 per
cent. of grape-sugar. This latter is used for anaërobic organisms. The
use of broth as a culture medium is of great value. It is undoubtedly
our best fluid medium, and in it may not only be kept pure cultures of
bacteria which it is desired to retain for a length of time, but in
it also emulsions and mixtures may be placed preparatory to further
operations. _Gelatine_ is broth solidified by the addition of 100 grams
of best French gelatine to the litre. Its advantage is twofold: it is
transparent, and it allows manifestation of the power of liquefaction.
When we speak of a liquefying organism we mean a germ having the power
of producing a peptonising ferment which can at the temperature of the
room break down solid gelatine into a liquid. _Grape-sugar gelatine_ is
made like grape-sugar broth. _Agar_ was introduced as a medium which
would not melt at 25° C., like gelatine, but remain solid at blood-heat
(37·5° C.; 98·5° F.). It is a seaweed generally obtained in dried
strips from the Japanese market. Ten to fifteen grams are added to
every litre of peptone-broth. Filtration is slow and often difficult,
and the result not as transparent as desirable. The former difficulty
is avoided by filtering in the Koch's steamer or with a hot-water
filter, the latter by the addition of the white of an egg. Glycerine
and grape-sugar may be added as elsewhere. _Blood agar_ is ordinary
agar with fresh sterile blood smeared over its surface. _Blood serum_
is drawn from a jar of coagulated horse-blood, in which the serum has
risen to the top. This is collected in sterilised tubes and coagulated
in a special apparatus (the serum inspissator). _Potato_ is prepared
by scraping ordinary potatoes, washing in corrosive sublimate, and
sterilising. They may then be cut into various shapes convenient for
cultivation. Upon any of these forms of solid media the characteristic
growth of the organism can be observed. Of the nutrient elements
required, nitrogen is obtained from albumens and proteids, carbon
from milk-sugar, cane-sugar, or the splitting up of proteids; salts
(particularly phosphates and salts of potassium) are readily obtainable
from those incorporated in the media; and the water which is required
is obtainable from the moisture of the media.

[Illustration: POTATO IN A ROUX TUBE PREPARED FOR CULTIVATION

Glycerine is placed in the bulb of the tube]

There are two common forms of test-tube culture, viz.: on the surface
and in the depth of the medium. In the former the medium is sloped,
and the inoculating needle is drawn along its surface; in the latter
the needle is thrust vertically downwards into the depth of the solid
medium. Plate cultures and anaërobic cultures will be described at a
later stage. When the medium has been inoculated the culture is placed
at a temperature which will be favourable. Two standards of temperature
are in use in bacteriological laboratories. The one is called _room
temperature_, and varies from 18° C.-20° C.; the other is _blood-heat_,
and varies from 35° C.-38° C. It is true, some species will grow below
18° C., and others above 38° C. The pathogenic (disease-producing)
bacteria thrive best at 37° C., and the non-pathogenic at the ordinary
temperature of the room. The different degrees of temperature are
regulated by means of incubators. For the low temperatures gelatine
is chosen; as a medium for the higher temperatures agar.

[Illustration: STAPHYLOCOCCUS PYOGENES AUREUS

× 1000]

[Illustration: INCUBATOR

(Temperature of blood-heat, registered by thermometer, and regulated by
thermo-regulator)]

_Moisture_ has been shown to have a favourable effect upon the growth
of microbes. Drying will of itself kill many species (_e. g._, the
spirillum of cholera), and, other things being equal, the moister a
medium is, the better will be the growth upon it. Thus it is that the
growth in broth is always more luxuriant than that on solid media. Yet
the growth of _Bacillus subtilis_ and other species is an exception to
this rule, for they prefer a dry medium.

[Illustration: CULTURE MEDIA READY FOR INOCULATION]

_Temperature._ Most bacteria grow well at room temperature, but they
will grow more luxuriantly and speedily at blood-heat. The optimum
temperature is generally that of the natural habitat of the organism.
In exceptional cases growth will occur as low as 5° C. or as high as
70° C. Indeed, some have been cooled to-20° C. and-30° C., and yet
retained their vitality,[8] whereas some few can grow at 60-70° C.
These latter are termed _thermophilic_ bacteria. The average thermal
death-point is at or about 50° C.

[Illustration: INOCULATING NEEDLES

Plantinum wire fused into glass handles]

_Light_ acts as an inhibitory or even germicidal agent. This fact
was first established by Downes and Blunt in a memoir to the Royal
Society in 1877. They found by exposing cultures to different degrees
of sunlight that thus the growth of the culture was partially or
entirely prevented, being most damaged by the direct rays of the sun,
although diffuse daylight acted prejudicially. Further, these same
investigators proved that of the rays of the spectrum which acted
inimically the blue and violet rays acted most bactericidally, next
to the blue being the red and orange-red rays. The action of light,
they explain, is due to the gradual oxidation which is induced by the
sun's rays in the presence of oxygen. Duclaux, who worked at this
question at a later date, concluded that the degree of resistance to
the bactericidal influence of light which some bacteria possess might
be due to difference in species, difference in culture media, and
difference in the degrees of intensity of light. Tyndall tested the
growth of organisms in flasks exposed to air and light on the Alps, and
found that sunlight inhibited the growth temporarily. A large number
of experimenters in Europe and England have worked at this fascinating
subject since 1877, and though many of their results appear
contradictory, we may be satisfied to adopt the following conclusions
respecting the matter:

(1) Sunlight has a deleterious effect upon bacteria, and to a less
extent on their spores.

(2) This inimical effect can be produced by light irrespective of rise
in temperature.

(3) The ultra-violet rays are the most bactericidal, and the infra-red
the least so, which indicates that the phenomenon is due to chemical
action.

(4) The presence of oxygen and moisture greatly increases this action.

(5) The sunlight acts prejudicially upon the culture medium, and
thereby complicates the investigation and after-growth.

(6) The time occupied in the bactericidal action depends upon the heat
of the sun and the intrinsic vitality of the organism.

(7) With regard to the action of light upon pathogenic organisms, some
results have recently been obtained with _Bacillus typhosus_. Janowski
maintains that direct sunlight exerts a distinctly depressing effect on
typhoid bacilli. At present more cannot be said than that sunlight and
fresh air are two of the most powerful agents we possess with which to
combat pathogenic germs.

[Illustration: PASTEUR'S LARGE INCUBATOR FOR CULTIVATION AT ROOM
TEMPERATURE]

A very simple method of demonstrating the influence of light is to grow
a pure culture in a favourable medium, either in a test-tube or upon
a glass plate, and then cover the whole with black paper or cloth. A
little window may then be cut in the protective covering, and the whole
exposed to the light. Where it reaches in direct rays it will be found
that little or no growth has occurred; where, on the other hand, the
culture has been in the dark, abundant growth occurs. In diffuse light
the growth is merely somewhat inhibited. It has been found that the
electric light has but little action upon bacteria, though that which
it has is similar to sunlight. Recent experiments with the Röntgen rays
have given negative results.

In 1890 Koch stated that tubercle bacilli were killed after an
exposure to direct sunlight of from a few minutes to several hours.
The influence of diffuse light would obviously be much less. Professor
Marshall Ward has experimented with the resistant spores of _Bacillus
anthracis_ by growing these on agar plates and exposing to sunlight.
From two to six hours' exposure had a germicidal effect.

It should be remembered that several species of sea-water bacteria
themselves possess powers of phosphorescence. Pflüger was the first to
point out that it was such organisms which provided the phosphorescence
upon decomposing wood or decaying fish. To what this light is due,
whether capsule, or protoplasm, or chemical product, is not yet known.
The only facts at present established are to the effect that certain
kinds of media and pabulum favour or deter phosphorescence.

_Desiccation._ A later opportunity will occur for consideration of
the effect of drying upon bacteria. Here it is only necessary to say
that, other things being equal, drying diminishes virulence and lessens
growth.

_Oxygen._ Pasteur was the first to lay emphasis upon the effect which
free air had upon micro-organisms. He classified them according to
whether they grew in air, _aërobic_, or whether they flourished most
without it, _anaërobic_. Some have the faculty of growing with or
without the presence of oxygen, and are designated as _facultative_
aërobes or anaërobes. As regards the cultivation of anaërobic germs, it
is only necessary to say here that hydrogen, nitrogen, or carbonic acid
gas may be used in place of oxygen, or they may be grown in a medium
containing some substance which will absorb the oxygen.

_Modes of Bacterial Action._ In considering the specific action of
micro-organisms, it is desirable, in the first place, to remember
the two great functional divisions of saprophyte and parasite. A
_saprophyte_ is an organism that obtains its nutrition from dead
organic matter. Its services, of whatever nature, lie outside the
tissues of living animals. Its life is spent apart from a "host." A
_parasite_, on the other hand, lives always at the expense of some
other organism which is its host, in which it lives and upon which it
lives. There is a third or intermediate group, known as "facultative,"
owing to their ability to act as parasites or saprophytes, as the
exigencies of their life-history may demand.

[Illustration: METHOD OF PRODUCING HYDROGEN BY KIPP'S APPARATUS FOR
CULTIVATION OF ANAËROBES (See page 139)]

[Illustration: ANAËROBIC CULTURE

(Buckner's Tube) with Pyrogallic Solution in Bulb.]

The saprophytic organisms are, generally speaking, those which
contribute most to the benefit of man, and the parasitic the reverse,
though this statement is only approximately true. In their relation
to the processes of fermentation, decomposition, nitrification, etc.,
we shall see how great and invaluable is the work which saprophytic
microbes perform. Their result depends, in nearly all cases, upon the
organic chemical constitution of the substances upon which they are
exerting their action, as well as upon the varieties of bacteria
themselves. Nor must it be understood that the action of saprophytes is
wholly that of breaking down and decomposition. As a matter of fact,
some of their work is, as we shall see, of a constructive nature; but,
of whichever kind it is, the result depends upon the organism and its
environment. This, too, may be said of the pathogenic species, all
of which are in a greater or less degree parasitic. It is well known
how various are the constitutions of man, how the bodies of some
persons are more resistant than those of others, and how the invading
microbe will find different receptions according to the constitution
and idiosyncrasy of the body which it attacks. Indeed, even after
invasion the infectivity of the special disease, whatever it happens
to be, will be materially modified by the tissues. When we come to
turn to the micro-organisms which are pathogenic parasites we shall
further have to keep clear in our minds that their action is double
and complex, and not single or simple. In the first place, we have
an infection of the body due to the bacteria themselves. It may be a
general and widespread infection, as in anthrax, where the bacilli
pass, in the blood or lymph current, to each and every part of the
body; or it may be a comparatively local one, as in diphtheria, where
the invader remains localised at the site of entrance. But, be that as
it may, the micro-organisms themselves, by their own bodily presence,
set up changes and perform functions which may have far-reaching
effects. It is obvious that the wider the distribution the wider is
the area of tissue change, and _vice versâ_. Yet there is something
of far greater importance than the mere presence of bacteria in human
or animal tissues; for the secondary action of disease-producing
germs--and possibly it is present in all bacteria--is due to their
poisonous products, or _toxins_, as they have been termed. These may
be of the nature of ferments, and they become diffused throughout the
body, whether the bacteria themselves occur locally or generally. They
may bring about very slight and even imperceptible changes during the
course of the disease, or they may kill the patient in a few hours.
Latterly bacteriologists have come to understand that it is not so much
the presence of organisms which is injurious to man and other animals,
as it is their products which cause the mischief; and the amount of
toxic product bears no known proportion to the degree of invasion by
the bacteria. The various and widely differing modes of action in
bacteria are therefore dependent upon these three elements: the tissues
or medium, the bacteria, and the products of the bacteria; and in all
organismal processes these three elements act and react upon each other.

A word may be said here respecting the much-discussed question of
_species_ in bacteria. A species may be defined as "a group of
individuals which, however many characters they share with other
individuals, agree in presenting one or more characters of a peculiar
and hereditary kind with some certain degree of distinctness."[9] Now,
as regards bacteria, there is no doubt that separate species occur
and tend to remain as separate species. It is true, there are many
variations, due in large measure to the medium in which the organisms
are growing,--variations of age, adaptation, nutrition, etc.,--yet
the different species tend to remain distinct. Involution forms occur
frequently, and degeneration invariably modifies the normal appearance.
But because of the occurrence of these morphological and even
pathological differences it must not be argued that the demarcation of
species is wholly arbitrary.

_Means of Sterilisation._ As this term occurs frequently in even a book
of this untechnical nature, and as it is expressive of an idea which
must always be present to the mind of the bacteriologist, it may be
desirable to make some passing allusion to it.

Chemical substances, perfect filtration, and heat are the three means
at our command in order to secure germ-free conditions of apparatus or
medium. The first two, though theoretically admissible, are practically
seldom used, the former of the two because the addition of chemical
substances annuls or modifies the operation, the latter of the two on
account of the great practical difficulties in securing perfection.
Hence in the investigation involved in bacteriological research heat
is the common sterilising agent. A temperature of 70° C. (158° F.)
will kill all bacilli; even 58° C. will kill most kinds. Boiling at
100° C. (212° F.) for three minutes will kill anthrax spores, and
boiling for thirty to sixty minutes will kill all bacilli and all
spores. This difference in the _thermal death-point_ between bacilli
and their spores enables the operator to obtain what are called
"pure cultures" of a desired bacillus from its spores which may be
present. For example, if a culture contains spores of anthrax and is
contaminated with micrococci, heating to 70° C. (158° F.) will kill
all the micrococci, but will not affect the spores of anthrax, which
can then grow into a pure culture of anthrax bacilli. _Fractional or
discontinuous sterilisation_ depends on the principle of heating to
the sterilising point for bacilli (say 70°C.) on one day, which will
kill the bacilli, but leave the spores uninjured. But by the following
day the spores will have germinated into bacilli, and a second heating
to 70°C. will kill them before they in their turn have had time to
sporulate. Thus the whole will be sterilised, though at a temperature
below boiling.

Successful sterilisation, therefore, depends upon killing both bacteria
and their spores, and nothing short of that can be considered as
sterilisation. The following methods are those generally used in the
laboratory. For dry heat (which is never so injurious to organisms
as moist heat)[10]: (_a_) _the Bunsen burner_, in the flame of which
platinum needles, etc., are sterilised; (_b_) _hot-air chamber_, in
which flasks and test-tubes are heated to a temperature of 150-170°
C. for half an hour. For moist heat: (_c_) _boiling_, for knives and
instruments; (_d_) _Koch's steam steriliser_, by means of which a crate
is slung in a metal cylinder, at the bottom of which the water is
boiled; (_e_) _the autoclave_, which is the most rapid and effective
of all the methods. This is in reality a Koch steriliser, but with
apparatus for obtaining high pressure. The last two (_d_, _e_) are
used for sterilising the nutriment media upon which bacteria are
cultivated outside the body. Blood serum would, however, coagulate at a
temperature over 60° C. (124° F.), and hence a special steriliser has
been designed to carry out fractional sterilisation daily for a week at
about 55° C.-58° C.

[Illustration: KOCH'S STEAM STERILISER]

_The Association of Organisms._ At a later stage we shall have an
opportunity of discussing symbiosis and allied conditions. Here it is
only necessary to draw attention to a fact that is rapidly becoming of
the first importance in bacteriology. When species were first isolated
in pure culture it was found that they behaved somewhat differently
under differing circumstances. This modification in function has been
attributed to differences of environment and physical conditions.
Whilst it is true that such external conditions must have a marked
effect upon such sensitive units of protoplasm as bacteria, it has
recently been proved that one great reason why modification occurs in
pure artificial cultures is that the species has been isolated from
amongst its colleagues and doomed to a separate existence. One of
the most abstruse problems in the immediate future of the science of
bacteriology is to learn what intrinsic characters there are in species
or individuals which act as a basis for the association of organisms
for a specific purpose. Some bacteria appear to be unable to perform
their regular function without the aid of others. An example of such
association is well illustrated in the case of tetanus, for it has been
shown that if the bacilli and spores of tetanus alone obtain entrance
to a wound the disease may not follow the same course as when with the
specific organism the lactic-acid bacillus or the common organisms of
suppuration or putrefaction also gain entrance. There is here evidently
something gained by association. Again, the virulence of other bacteria
is also increased by means of association. The _Bacillus coli_ is an
example, for, in conjunction with other organisms, this bacillus,
although normally present in health in the alimentary canal, is able to
set up acute intestinal irritation, and various changes in the body
of an inflammatory nature. It is not yet possible to say in what way
or to what degree the association of bacteria influences their _rôle_.
That is a problem for the future. But whilst we have examples of this
association in streptococcus and the bacillus of diphtheria, _B. coli_
and yeasts, tetanus and putrefactive bacteria, _Diplococcus pneumoniæ_
and streptococcus, and association amongst the various suppurative
organisms, we cannot doubt that there is an explanation to be found
here of many hitherto unsolved results of bacterial action. This is
the place in which mention should also be made of higher organisms
associated for a specific purpose with bacteria. There is some evidence
to support the belief that some of the Leptotricheæ (Crenothrix,
Beggiatoa, Leptothrix, etc.) and the Cladotricheæ (Cladothrix) perform
a preliminary disintegration of organic matter before the decomposing
bacteria commence their labours. This occurs apparently in the
self-purification of rivers, as well as in polluted soils.

_Antagonism of Bacteria._ Study of the life-history of many of the
water bacteria will reveal the fact that they can live and multiply
under conditions which would at once prove fatal to other species. Some
of these water organisms can indeed increase and multiply in distilled
water, whereas it is known that other species cannot even live in
distilled water, owing to the lack of pabulum. Thus we see that what is
favourable for one species may be the reverse for another.

Further, we shall have opportunity of observing, when considering
the bacteriology of water and sewage, that there is in these media
in nature a keen struggle for the survival of the fittest bacteria
for each special medium. In a carcass it is the same. If saprophytic
bacteria are present with pathogenic, there is a struggle for the
survival of the latter. Now whilst this is in part due to a competition
owing to a limited food supply and an unlimited population, as occurs
in other spheres, it is also due in part to the inimical influence of
the chemical products of the one species upon the life of the bacteria
of the other species. Moreover, in one culture medium, as Cast has
pointed out, two species will often not grow. When Pasteur found that
exposure to air attenuated his cultures, he pointed out that it was not
the air _per se_ that hindered his growth, but it was the introduction
of other species which competed with the original. The growth of the
spirillum of cholera is opposed by _Bacillus pyogenes fœtidus_.
_B. anthracis_ is, in the body, opposed by either _B. pyocyaneus_ or
_Streptococcus erysipelatis_, and yet it is aided in its growth by _B.
prodigiosus_. _B. aceti_ is, under certain circumstances, antagonistic
to _B. coli communis_.

In several of the most recent of the admirable reports of Sir Richard
Thorne issued from the Medical Department of the Local Government
Board, we have the record of a series of experiments performed by Dr.
Klein into this question of the antagonism of microbes. From this
work it is clearly demonstrated that whatever opposition one species
affords to another it is able to exercise by means of its poisonous
properties. These are of two kinds. There is, as is now widely known,
the poisonous product named the _toxin_, into which we shall have
to inquire more in detail at a later stage. There is also in many
species, as Dr. Klein has pointed out, a poisonous constituent or
constituents included in the body protoplasm of the bacillus, and which
he therefore terms the _intracellular poison_. Now, whilst the former
is different in every species, the latter may be a property common to
several species. Hence those having a similar intracellular poison are
antagonistic to each other, each member of such a group being unable to
live in an environment of its own intracellular poison. Further, it has
been suggested that there are organisms possessing only one poisonous
property, namely, their toxin--for example, the bacilli of tetanus and
diphtheria--whilst there are other species, as above, possessing a
double poisonous property, an intracellular poison and a toxin. In this
latter class would be included the bacilli of Anthrax and Tubercle.

Reference has been made to the associated work of higher vegetable life
and bacteria. The converse is also true. Just as we have bacterial
diseases affecting man and animals, so also plant life has its
bacterial diseases. Wakker, Prillieux, Erwin Smith, and others have
investigated the pathogenic conditions of plants due to bacteria, and
though this branch of the science is in its very early stages, many
facts have been learned. _Hyacinth disease_ is due to a flagellated
bacillus. _The wilt of cucumbers and pumpkins_ is a common disease in
some districts of the world, and may cause widespread injury. It is
caused by a white microbe which fills the water-ducts. Wilting vines
are full of the same sticky germs. Desiccation and sunlight have a
strongly prejudicial effect upon these organisms. Bacterial _brown-rot_
of potatoes and tomatoes is another plant disease probably due to a
bacillus. The bacillus passes down the interior of the stem into the
tubers, and brown-rots them from within. There is another form of
brown-rot which affects cabbages. It blackens the veins of the leaves,
and a woody ring which is formed in the stem causes the leaves to
fall off. This also is due to a micro-organism, which gains entrance
through the water-pores of the leaf, and subsequently passes into the
vessels of the plants. It multiplies by simple fission, and possesses a
flagellum.

There can be no doubt that these complex biological properties of
association and antagonism, as well as the parasitic growth of bacteria
upon higher vegetables, are as yet little understood, and we may be
glad that any light is being shed upon them. In the biological study
of soil bacteria in particular may we expect in the future to find
examples of association, even as already there are signs that this
is so in certain pathogenic conditions. In the alimentary canal, on
the other hand, and in conditions where organic matter is greatly
predominating, we may expect to see further light on the subject of
antagonism.

_Attenuation of Virulence or Function._ It was pointed out by some of
the pioneer bacteriologists that the function of bacteria suffered
under certain circumstances a marked diminution in power. Later workers
found that such a change might be artificially produced. Pasteur
introduced the first method, which was the simple one of allowing
cultures to grow old before sub-culturing. Obviously a pure culture
cannot last for ever. To maintain the species in characteristic
condition it is necessary frequently to sub-culture upon fresh
media. If this simple operation be postponed as long as possible
consistent with vitality, and then performed, it will be found that
the sub-culture is _attenuated_, _i. e._, weakened. Another mode is
to raise the pure culture to a temperature approaching its thermal
death point. A third way of securing the same end is to place it under
disadvantageous external circumstances, for example a too alkaline or
too acid medium. A fourth, but rarely necessary, method is to pass
it through the tissues of an insusceptible animal. Thus we see that,
whilst the favourable conditions which we have considered afford full
scope for the growth and performance of functions of bacteria, we are
able by a partial withdrawal of these, short of that ending fatally, to
modify the character and strength of bacteria. In future chapters we
shall have opportunity of observing what can be done in this direction.




CHAPTER II

_BACTERIA IN WATER_


In entering upon a consideration of such a common article of use as
water, we shall do well to describe in some detail the process by which
we systematically investigate the bacteriology of a water, or, indeed,
of any similar fluid suspected of bacterial pollution.

The collection of samples, though it appears simple enough, is
sometimes a difficult and responsible undertaking. Complicated
apparatus is rarely necessary, and fallacies will generally be
avoided by observing two directions. In the first place, the sample
should be chosen as representative as possible of the real substance
or conditions we wish to examine. Some authorities advise that it
is necessary to allow the tap to run for some minutes previous
to collecting the sample; but if we desire to examine for lead
chemically or for micro-organisms in the pipes biologically, then
such a proceeding would be injudicious.[11] Hence we must use common
sense in the selection and obtaining of a sample, following this one
guide, namely, to collect as nearly as possible a sample of the exact
water the quality of which it is desired to learn. In the second
place, we must observe strict bacteriological cleanliness in all our
manipulations. This means that we must use only sterilised vessels or
flasks for collecting the sample, and in the manipulation required
we must be extremely careful to avoid any pollution of air or any
addition to the organisms of the water from unsterilised apparatus.
A flask polluted in only the most infinitesimal degree will entirely
vitiate all results.

Accompanying the sample should be a more or less full statement of
its source. There can be no doubt that, in addition to a chemical and
bacteriological report of a water, there should also be made a careful
examination of its source. This may appear to take the bacteriologist
far afield, and in point of fact, as regards distance, this may be so.
But until he has seen for himself what "the gathering-ground" is like,
and from what sources come the feeding streams, he cannot judge the
water as fairly as he should be able to do. The configuration of the
gathering-ground, its subsoil, its geology, its rainfall, its relation
to the slopes which it drains, the nature of its surface, the course of
its feeders, and the absence or presence of cultivated areas, of roads,
of houses, of farms, of human traffic, of cattle and sheep--all these
points must be noted, and their influence, direct or indirect, upon the
water carefully borne in mind.

When the sample has been duly collected, sealed, and a label affixed
bearing the date, time, and conditions of collection and full
address, it should be transmitted with the least possible delay to
the laboratory. Frequently it is desirable to pack the bottles in a
small ice case for transit. On receipt of such a sample of water the
examination must be immediately proceeded with, in order to avoid, as
far as possible, the fallacies arising from the rapid multiplication
of germs. Even in almost pure water, at the ordinary temperature of a
room, Frankland found organisms multiplied as follows:--

            No. of Germs
  Hours.      per cc.

   0           1,073
   6           6,028
  24           7,262
  48          48,100

Another series of observations revealed the same sort of rapid increase
of bacteria. On the date of collection the micro-organisms per cc. in
a deep-well water (in April) were seven. After one day's standing at
room temperature the number had reached twenty-one per cc. After three
days under the same conditions it was 495,000 per cc. At blood-heat the
increase would, of course, be much greater, as a higher temperature
is more favourable to multiplication. But this would depend upon the
degree of impurity in the water, a pure water _decreasing_ in number
on account of the exhaustion of the pabulum, whereas, for the first
few days at all events, an organically polluted water would show an
enormous increase in bacteria.

Furthermore, it is desirable to remember that organisms, in an ordinary
water, do not continue to increase indefinitely. There is a limit
to all things, even to numbers in bacteriology. Cramer, of Zurich,
examined the water of the Lake after it had been standing for different
periods, with the following results:--

  Hours and Days of  No. of Micro-organisms
  Examination.       per cc.[12]

   0 hours               143
  24   "              12,457
   3 days            328,543
   8   "             233,452
  17   "              17,436
  70   "               2,500

The writer's own experience is entirely in agreement with this
cessation of multiplication at or about the end of a week, and the
later decline.

_Method of Examination._ At the outset of a systematic study of a water
it is well to observe its physical characters. The colour, if any,
should be noted. Suspended matter and deposit may indicate organic
or inorganic pollution. If abundant or conspicuous, a microscopic
examination of the sediment may be made. The reaction, whether acid,
neutral, or alkaline, must be tested, and the exact temperature taken.
Any and every fact will help us, perhaps not so much to determine the
contents of the water as to interpret rightly the facts we deduce from
the entire examination.

[Illustration: LEVELLING APPARATUS FOR KOCH'S PLATE]

At the beginning of the bacteriological work the water should be
examined by means of the gelatine plate method. This consists in
drawing up into a fine sterilised pipette a small quantity of the water
and introducing it thereby into a test-tube of melted gelatine at a
temperature below 40° C.[13] It will depend upon the apparent quality
of the water as to the exact quantity introduced into the gelatine;
about .5 or .1 of a cubic centimetre is a common figure. The stopper
is then quickly replaced in the test-tube, and the contents gently
mixed more or less equally to distribute the one-tenth cubic centimetre
throughout the melted gelatine. A sterilised sheet of glass (4 inches
by 3) designated a _Koch's plate_ is now taken and placed upon the
stage of a levelling apparatus, which holds iced water in a glass jar
under the stage. The gelatine is now poured out over the glass plate,
and by means of a sterilised rod stroked into a thin, even film all
over the glass. It is then covered with a bell-jar and left at rest
to set. The level stage prevents the gelatine running over the edge
of the plate; the iced water under the stage expedites the setting
of the gelatine into a fixed film. When it is thus set the plate is
placed upon a small stand in a moist chamber, and the whole apparatus
removed to the room temperature incubator. A _moist chamber_ is a glass
dish, in which some filter paper, soaked with corrosive sublimate, is
inserted, and the dish covered with a bell-jar. By this means the risks
of pollution are minimized, and moisture maintained. In all cases at
least two plates must be prepared of the same sample of water, and it
is often advisable to make several. They may be made with different
media for different purposes, and with different quantities of water,
though the same method of procedure is adopted. In a highly polluted
water extremely small quantities would be taken, and, _vice versâ_, in
pure water a large quantity.

[Illustration: MOIST CHAMBER IN WHICH KOCH'S PLATES ARE INCUBATED]

When we come to discuss the relation of disease organisms to water,
particularly those causing typhoid fever, we shall learn that they
are both scarce and intermittent. This point has been dwelt upon
frequently by Dr. Klein, and it is clear that such a state of things
greatly enhances the difficulties in detecting such bacteria, and he
has proposed a simple procedure by which the difficulty of finding the
_Bacillus typhosus_ in a large body of water may be met.

[Illustration: HOT AIR STERILISER

For the Sterilization of Glass Apparatus, etc.]

One or two thousand cubic centimetres of the water under examination
are passed through a sterilised Berkefeld filter by means of siphon
action or an air-pump. The candle of the filter retains on its outer
surface all, or nearly all, the particulate matter contained in the
water. The matter thus retained on this outer surface is brushed by
means of a sterile brush into 10 or 20 cc. of sterilised water. Thus
we have all the organisms contained in two litres of the water reduced
into 10 cc. of water. From this, so to speak, concentrated emulsion of
the bacteria of the original water, phenol-gelatine plates or Eisner
plates (both acid media) may be readily made. In this way we not only
catch many bacteria which would evade us if we were content with the
examination merely of a few drops of the water, but we eliminate
by means of the acid those common water bacteria, like _Bacillus
fluorescens liquefaciens_, which so greatly confuse the issue.

[Illustration: HOT AIR STERILISER

For the Sterilizatin of Glass Apparatus, etc.]

In the course of two or three days the film of gelatine on the plate
becomes covered with _colonies_ of germs, and the next step is to
examine these quantitatively and qualitatively. We may here insert a
simple scheme by which this may be most fully and easily accomplished:--

1. _Naked-Eye Observation of the Colonies._ By this means at the
very outset certain facts may be obtained, viz., the size, elevation,
configuration, margin, colour, grouping, number, and kinds of colonies,
all of which facts are of importance, and assist in final diagnosis.
Moreover, in the case of gelatine plates (it is otherwise in agar)
one is able to observe whether or not there is present what is termed
_liquefaction of the gelatine_. Some organisms produce in their
development a peptonizing ferment which breaks down gelatine into a
fluid condition. Many have not this power, and hence the characteristic
is used as a diagnostic feature.

2. _Microscopic Examination of Colonies_, which confirms or corrects
that which has been observed by the naked eye. Fortunately some
micro-organisms when growing in colonies produce cultivation features
which are peculiar to themselves (especially is this so when growing
in test-tube cultures), and in the early stages of such growths a low
power of the microscope or magnifying glass facilitates observation.

3. _Make cover-glass preparations_: (_a_) unstained--"the hanging
drop"; (_b_) stained--single stains, like gentian-violet, methyl
blue, fuchsin, carbol fuchsin, etc.; double stains--Gram's method,
Ziehl-Neelsen's method, etc.

[Illustration: THE HANGING DROP]

This third part of the investigation is obviously to prepare specimens
for the microscope. "The hanging drop" is a simple plan for securing
the organisms for microscopic examination in a more or less natural
condition. A hollow ground slide, which is a slide with a shallow
depression in it, is taken, and a small ring of vaseline placed
round the edge of the depression. Upon the under side of a clean
cover-glass is placed a drop of pure water, and this is inoculated
with the smallest possible particle taken from one of the colonies
of the gelatine plate on the end of a sterilised platinum wire. The
cover-glass is then placed upon the ring of vaseline, and the drop
hangs into the space of the depression. Thus is obtained a view of the
organisms in a freely moving condition, if they happen to be motile
bacteria. As a matter of practice the hollow slide may be dispensed
with, and an ordinary slide used.

[Illustration: DRYING STAGE FOR FIXING FILMS]

With regard to staining, it will be undesirable here to dwell at length
upon the large number of methods which have been adopted. The "single
stain" may be shortly mentioned. It is as follows: A clean cover-glass
is taken (cleaned with nitric acid and alcohol, or bichromate of potash
and alcohol), and a drop of pure sterilised water placed upon it. This
is inoculated with the particle of a colony on the end of a platinum
needle, and a scum is produced. The film is now "fixed" by slowly
drying it over a flame. When the scum is thus dried, a drop of the
selected stain (say gentian-violet) is placed over the scum and allowed
to remain for varying periods: _sarcinæ_ about thirty seconds; for
many of the bacilli three or four minutes. It is then washed off with
clean water, dried, and mounted in Canada balsam. The organisms will
now appear under the microscope as violet in colour, and will thus be
clearly seen.

The "double staining" is adopted when we desire to stain the organisms
one colour and the tissue in which they are situated a contrast colour.
Some of the details of these methods are mentioned in the Appendix.

4. _Sub-culture._ The plate method was really introduced by Koch in
order to facilitate isolation of species. In a flask it is impossible
to isolate individual species, but when the growth is spread over a
comparatively large area, like a plate, it is possible to separate the
colonies, and this being done by means of a platinum wire, the colonies
may be replanted in fresh media; that is to say, a _sub-culture_ may be
made, each organism cultivated on its favourite soil, and its manner of
life closely watched. We have already mentioned the chief media which
are used in the laboratory, and in an investigation many of these would
be used, and thus _pure cultures_ would be obtained. Let us suppose
that a water contains six kinds of bacteria. On the plate these six
kinds would show themselves by their own peculiar growth. Each would
then be isolated and placed in a separate tube, on a favourite medium,
and at a suitable temperature. Thus each would be a _pure culture_;
_i. e._, one and only one, species would be present in each of the six
tubes. By this simple means an organism can be, we say, _cultivated_,
in the same sort of way as in floriculture. From day to day we can
observe the habits of each of our six species, and probably at an early
stage of their separated existences we should be able to diagnose
what species of bacteria we had found in the water. If not, further
microscopic examination could be made, and, if necessary, secondary or
tertiary sub-cultures.

5. _Inoculation of Animals._ It may be necessary to observe the action
of supposed pathogenic organisms upon animals. This is obviously a last
resource, and any abuse of such a process is strictly limited by law.
As a matter of fact, an immense amount of bacteriological investigation
can be carried on without inoculating animals; but, strictly speaking,
as regards many of the pathogenic bacteria, this test is the most
reliable of all. Nor would any responsible bacteriologist be justified
in certifying a water as healthy for consumption by a large community
if he was in doubt as to the disease-producing action of certain
contained organisms.

[Illustration: TYPES OF LIQUEFACTION OF GELATINE]

By working through some such scheme as the above we are able to detect
what quantity and species of organisms, saprophytic or parasitic, a
water or similar fluid contains. For, observe what information we
have gained. We have learned the form (whether bacillus, micrococcus,
or spirillum), size, consistence, motility, method of grouping, and
staining reactions of each micro-organism; the characters of its
culture, colour, composition, presence or absence of liquefication or
gas formation, its rate of growth, smell, or reaction; and lastly, when
necessary, the effect that it has upon living tissues. Here, then, are
ample data for arriving at a satisfactory conclusion respecting the
qualitative estimation of the suspected water.

As to to the quantitative examination, that is fulfilled by counting
the number of colonies which appear, say by the third and fourth day,
upon the gelatine plates. Each colony has arisen, it is assumed, from
one individual, so that if we count the colonies, though we do not
thereby know how many organisms we have upon our plate, we do know
approximately how many organisms there were when the plate was first
poured out, which are the figures we require, and which can at once
be multiplied and returned as so many organisms per cubic centimetre.
There is, unfortunately, at present no exact standard to which all
bacteriologists may refer.

Miquel and Crookshank have suggested standards which allow "very pure
water" to contain up to 100 micro-organisms per cc. Pure water must not
contain more than 1000, and water containing up to 100,000 bacteria
per cc. is contaminated with surface water or sewage. Macé gives the
following table:

  Very pure water                   0-    10 bacteria per cc.
  Very good water                  20-   100    "        "
  Good water                      100-   200    "        "
  Passable (mediocre) water       200-   500    "        "
  Bad water                       500- 1,000    "        "
  Very bad water                1,000-10,000 and over    "

Koch first laid emphasis on the quantity of bacteria present as an
index of pollution, and whilst different authorities have all agreed
that there is a necessary quantitative limit, it has been so far
impossible to arrive at one settled standard of permissible impurity.

Besson adopts the standard suggested by Miquel, and, on the whole,
French bacteriologists follow suit. They also agree with him, generally
speaking, in not placing much emphasis upon the numerical estimation of
bacteria in water. In Germany and England it is the custom to adopt a
stricter limit. Koch in 1893 fixed 100 bacteria per cc. as the maximum
number of bacteria which should be present in a properly filtered
water. Hence the following has been recognised more or less as the
standard:

    0- 100  bacteria per cc.  =a good potable water,
  100- 500     "       "      =a suspicious water.
  500-1000 or  more    "      =a water which should have
                               further filtration before
                               being used for drinking
                               purposes.

The personal view of the writer after some experience of water
examination would favour a standard of "under 500" being a potable
water, if the 500 were of a nature indicating neither sewage pollution
nor disease. Miquel holds that not more than ten different species of
bacteria should be present in a drinking water, and such is a useful
standard. The presence of rapidly liquefying bacteria _associated
with sewage or surface pollution_ would, even though present in fewer
numbers than a standard, condemn a water. Thus it will be seen that it
is impossible to judge alone by the numbers unless they are obviously
enormously high.

[Illustration: WOLFHÜGEL'S COUNTER]

When we are counting colonies upon a Koch's plate, _Wolfhügel's
counter_ may be used. This is a thin plate of glass a size larger than
Koch's plates, and upon it are scratched squares, each square being
divided into nine smaller squares. The Wolfhügel plate is superimposed
upon the Koch's plate, and the colonies counted in one little square or
set of squares and multiplied.

[Illustration: PETRI'S DISH]

By using flat, shallow, circular glass dishes, generally known as
_Petri's dishes_, instead of Koch's plates, much manipulation and time
is saved, and, on the whole, less risk of pollution occurs. Moreover,
these are easily carried about and transferred from place to place.
When counting colonies in a Petri's dish it is sufficient to divide the
circle into eight equal divisions, and counting the colonies in the
average divisions, multiply and reduce to the common denominator of one
cc. For example, if the colonies of the plate appear to be distributed
fairly uniformly we count those in one of the divisions. They reach,
we will suppose, the figure of 60; 60 × 8=480 micro-organisms in the
amount taken from the suspected water and added to the melted gelatine
from which the plate was made. This amount was .25 cc. Therefore we
estimate the number of micro-organisms in the suspected water as 60 ×
8=480 × 4= 1920 m.-o. per cc., which is over standard by about 1500.
A water might then be condemned upon its quantitative examination
alone or qualitative alone, or both. If the quantity were even that
of an artesian well, say 4-10 m.-o. per cc., but those four or ten
were all _Bacillus typhosus_, it would clearly be condemned on its
quality, though quantitatively it was an almost pure water. If, on the
contrary, the water contained 10,000 m.-o. per cc., and none of them
disease-producing, it would still be condemned on the ground that so
large a number of organisms indicated some kind of organic pollution
to supply pabulum for so many organisms to live in one cc. of the
water. It is not the number _per se_ which condemns. The large number
condemns because it indicates probable pollution with surface water or
sewage in order to supply pabulum for so many bacteria per cc.

It should always be remembered that a chemical report and a
bacteriological report should assist each other. The former is able
to tell us the quantity of salts and condition of the organic matter
present; the latter the number and quality of micro-organisms. Neither
can take the place of the other and, generally speaking, both are more
or less useless until we can learn, by inspection and investigation
of the source of the water, the origin of the organic matter or
contamination. Hence a water report should contain not only a record
of physical characters, of chemical constituents, and of the presence
or absence of micro-organisms, injurious and otherwise, but it should
also contain information obtained by personal investigation of the
source. Only thus can a reasonable opinion be expected. Moreover, it
is generally only possible to form an accurate judgment of a water
from watching its history, that is, not from one examination only,
but from a series of observations. A water yielding a steady standard
of bacterial contents is a much more satisfactory water, from every
point of view, than one which is unstable, one month possessing 500
bacteria per cc. and another month 5000. It is obvious that rainfall
and drought, soil and trade effluents, will have their influence in
materially affecting the bacterial condition of a water.

[Illustration: BERKEFELD FILTER

In Position for Filtration of Water to be Examined.]

It is perhaps scarcely necessary to add that we have not in the
above account of the examination of water included all, or nearly
all, the various methods adopted for acquiring a knowledge of the
bacterial contents of the water. Many of these are of too detailed
and technical a nature to enter into here. Three points, however, we
may touch upon. In the first place, as we have said, the particulate
matter out of a large body of water should be concentrated in a small
quantity. Accordingly it has become the custom to pass 2000 or 3000
cc. of the suspected water through a Berkefeld filter. When this
has been accomplished, by means of a sterile brush the particulate
matter on the candle of the filter is brushed off into 10 or 15 cc. of
sterilised water. This simple arrangement is analogous to the use of
gravity or centrifugal methods of securing the solid matter in milk.
The smaller quantity of water is then readily examined, and scanty
germs more readily detected. A second point elaborating the scheme
of water examination is the choice of media for sub-culturing. Mere
examination on gelatine is not sufficient. Even in making the primary
plate cultivations it is well to vary the media--agar, carbol-gelatine,
Elsner, etc. But when colonies have appeared upon these plates it is
important to sub-culture with accuracy and good judgment upon all or
any media--gelatine, agar, broth, potato, milk, blood serum, glucose
agar, glycerine agar, etc.--that will reveal the real characters of the
bacteria present. A method proposed by Professor Sheridan Delépine is
to place some of the suspected water in sterilised test-tubes without
further treatment, and incubate at 37° C. for twelve or eighteen hours,
and then plate out and estimate the number of bacteria as in the
ordinary course. "In polluted water, containing an excess of organic
matter," he says, "an extremely rapid multiplication of bacteria is
observed. In unpolluted water, containing only water bacteria and a
very small amount of organic matter, very little or no multiplication
takes place, and the growth of the water bacteria liquefying gelatine
is checked to a remarkable extent." Thirdly, by none of these methods
should we be able to isolate anaërobic bacteria, and to furnish a
complete report these also must receive careful attention.

[Illustration: APPARATUS FOR FILTERING WATER TO FACILITATE ITS
BACTERIOLOGICAL EXAMINATION]

_The Bacteriology of Water._ In many natural waters there will be found
varied contents even in regard to flora alone: _algæ_, _diatoms_,
_spirogyræ_, _desmids_, and all sorts of vegetable detritus. Many of
these organisms are held responsible for divers disagreeable tastes
and odours. The colour of a water may also be due to similar causes.
Dr. Garrett, of Cheltenham, has recorded the occurrence of redness
of water owing to a growth of _Crenothrix polyspora_, and many other
similar cases make it evident that not unfrequently great changes may
be produced in water by contained microscopic vegetation.

With the exception of water from springs and deep wells, all unfiltered
natural waters contain numbers of bacteria. The actual number roughly
depends upon the amount of organic pabulum present, and upon certain
physical conditions of the water. As we have already seen, bacteria
multiply with enormous rapidity. In some species multiplication does
not appear to depend on the presence of much organic matter, and,
indeed, some can live and multiply in sterilised water: _Micrococcus
aquatilis_ and _Bacillus erythrosporus_. Again, others depend not
upon the quantity of organic matter, but upon its quality. And
frequently in a water of a high degree of organic pollution it will
be found that bacteria have been restrained in their development
by the competition of other species monopolising the pabulum.
Probably at least one hundred different species of non-pathogenic
organisms have been isolated from water. Some species are constantly
occurring, and are present in almost all natural waters. Amongst
such are _B. liquefaciens_, _B. fluorescens liq._, _B. fluorescens
non-liquefaciens_, _B. termo_, _B. aquatilis_, _B. ubiquitus_, and
not a few _micrococci_, etc. Percy Frankland[14] collected water from
various quarters at various times and seasons, and some of his results
may here be added:

RIVER THAMES WATER COLLECTED AT HAMPTON

Number of Micro-organisms Obtained from 1 cc. of Water.

  ----------------------+--------------+--------------+--------------
        MONTH.          |   +1886.+    |   +1887.+    |     +1888.+
  ----------------------+--------------+--------------+--------------
                        |              |              |
  January               |    45,000    |    30,800    |    92,000
  February              |    15,800    |     6,700    |    40,000
  March                 |    11,415    |    30,900    |    66,000
  April                 |    12,250    |    52,100    |    13,000
  May                   |     4,800    |     2,100    |     1,900
  June                  |     8,300    |     2,200    |     3,500
  July                  |     3,000    |     2,500    |     1,070
  August                |     6,100    |     7,200    |     3,000
  September             |     8,400    |    16,700    |     1,740
  October               |     8,600    |     6,700    |     1,130
  November              |    56,000    |    81,000    |    11,700
  December              |    63,000    |    19,000    |    10,600
  ----------------------+--------------+--------------+--------------

Again, another example:

RIVER LEA WATER COLLECTED AT CHINGFORD

Number of Micro-organisms Obtained from 1 cc. of Water.

  ----------------------+--------------+--------------+--------------
        MONTH.          |   +1886.+    |   +1887.+    |     +1888.+
  ----------------------+--------------+--------------+--------------
                        |              |              |
  January               |    39,300    |    37,700    |    31,000
  February              |    20,600    |     7,900    |    26,000
  March                 |     9,025    |    24,000    |    63,000
  April                 |     7,300    |     1,330    |    84,000
  May                   |     2,950    |     2,200    |     1,124
  June                  |     4,700    |    12,200    |     7,000
  July                  |     5,400    |    12,300    |     2,190
  August                |     4,300    |     5,300    |     2,000
  September             |     3,700    |     9,200    |     1,670
  October               |     6,400    |     7,600    |     2,310
  November              |    12,700    |    27,000    |    57,500
  December              |   121,000    |    11,000    |     4,400
  ----------------------+--------------+--------------+--------------

"During the summer months these waters are purest as regards
micro-organisms, this being due to the fact that during dry weather
these rivers are mainly composed of spring water, whilst at other
seasons they receive the washings of much cultivated land."--Frankland.

Prausnitz has shown that water differs, as would be expected, according
to the locality in the stream at which examination is made. His
investigations were made from the river Isar before and after it
receives the drainage of Munich:

                                              No. of Colonies
                                                  per cc.

  Above Munich                                        531
  Near entrance of principal sewer                227,369
  13 kilometres from Munich                         9,111
  22     "        "    "                            4,796
  33     "        "    "                            2,378

Professor Percy Frankland also points out how the river Dee affords
another example, even more perfect, of pollution and restoration
repeated several times until the river becomes almost bacterially pure.

We cannot here enter more fully into the many conditions of a water
which affect its bacterial content than to say that it varies
considerably with its source, at different seasons, and under different
climatic conditions. An enormous increase will occur if the sediment is
disturbed, and conversely sedimentation and subsidence during storage
will greatly diminish the numbers of bacteria. Sand filtration, plus a
"nitrifying layer," will remove more than 90 per cent. of the bacteria.
Sea-water contains comparatively few bacteria, and the deeper the water
and the farther it is from shore so much less will be the bacterial
pollution.


THE CHIEF DISEASE ORGANISMS FOUND IN WATER

We will now consider several of the more important disease-producing
bacteria found in water.

+Bacillus Typhosus+ (Eberth-Gaffky). In 1880-81 Eberth announced the
discovery of this bacillus in cases of clinical enteric fever. In 1884
it was first cultivated outside the body by Gaffky. Since then other
organisms have been held responsible for the causation of enteric (or
typhoid) fever. In 1885 the _B. coli communis_ was recognised, and it
has been a matter of great debate amongst bacteriologists as to how far
these two organisms are the same species, and the typhoid germ merely
a higher evolution of the _B. coli_. The differentiating signs between
them will be referred to shortly. Bacteriologists generally regard the
Eberth-Gaffky bacillus as the specific cause of the disease, though
complete proof is still wanting.

[Illustration: BACTERIA OF TYPHOID FEVER]

_Microscopic Characters_ (in pure culture). Rods, 2-4 µ long, .5 µ
broad, having round ends. Sometimes threads are observable, being 10 µ
in length. In the field of the microscope the bacilli differ in length
from each other, but are all the same thickness approximately. Round
and oval cells constantly occur even in pure culture, and many of
these shorter forms of typhoid are identical in morphology with some
of the many forms of _Bacillus coli_. There are no spores. Motility is
marked; indeed, in young culture it is the most active pathogenic germ
we know. The small forms dart about with extreme rapidity; the longer
forms move in a vermicular manner. Its powers of movement are due to
some five to twenty flagella of varying length, some of them being much
longer than the bacillus itself, though, owing to the swelling of the
bacillus under flagellum-staining methods, it is difficult to gauge
this exactly. The flagella are terminal and lateral, and are elastic
and wavy. Their active contraction produces an evident current in the
field of the microscope.

_Cultures._ This organism may be isolated from ulcerated Peyer's
patches in the intestine, from the liver, the spleen, and the
mesenteric glands. Owing to the mixture of bacteria found elsewhere, it
is generally best to isolate it from the spleen. The whole spleen is
removed, and a portion of its capsule seared with a hot iron to destroy
superficial organisms. With a sterilised knife a small cut is made
into the substance of the organ, and by means of a sterilised platinum
wire a little of the pulp is removed and traced over the surface of
agar. _Agar_ reveals a growth in about twenty-four hours at 37° C.,
which is the favourite temperature. A greyish, moist, irregular growth
appears, but it is invariably attached to the track of the inoculating
needle. On _gelatine_ the growth is much the same, but its irregular
edge is, if anything, more apparent. There is no liquefaction and no
gas formation. On plates of gelatine the colonies appear large and
spreading, with jagged edges. The whole colony appears raised and
almost limpet-shaped, with delicate lines passing over its surface.
There is an appearance under a low power of transparent iridescence.
The growth on _potato_ is termed "invisible," and is of the nature of
a potato-coloured pellicle, which looks moist, and may at a late stage
become a light brown in colour, particularly if the potato is alkaline.
_Milk_ is a favourable medium, and is rendered slightly acid. No
coagulation takes place. _Broth_ is rendered turbid.

_Micro-pathology._ Typhoid fever is an infiltration and coagulation,
necrosis, and ulceration of the Peyer's patches in the small intestine
of man. The mesenteric glands show the same features, except that
there is no ulceration. The spleen is enlarged, and contains the germs
of the disease in almost a pure culture. The bacillus is present
in the intestinal contents and excreta, particularly so when the
Peyer's glands have commenced ulceration. In the blood of the general
circulation the bacillus is not demonstrable, except in very rare
instances. Typhoid fever is not, like anthrax, a blood disease.


COMPARATIVE FEATURES OF BACILLUS TYPHOSUS AND B. COLI

            B. TYPHOSUS                           B. COLI

  _Morphology:_ Cylindrical bacillus       Shorter, thicker; filaments rare.
    2.4 µ, unequal lengths; some
    filaments.

  _Flagella:_ Long, wavy, spiral,          Shorter, stiffer, fewer; movement
    and very numerous; movement very         less active.
    active.

  _On Gelatine and Agar:_ Angular,         Even edge, homogeneous; much
    irregular, raised colonies; slow         larger, quicker growth, and less
    growth; translucent; medium              translucent than _B. typhosus_;
    remains clear.                           medium becomes turbid or
                                             coloured.

  _In Gelatine:_ In ordinary               Under the same circumstances
    gelatine and in sugar gelatine           abundant gas is produced.
    no gas is produced.

  _Milk:_ Not curdled by the               Milk is coagulated (within three
    bacillus.                                days).

  _Indol:_ The production of indol         Indol is present.
    in ordinary broth is _nil_.

  _Potato:_ The "invisible growth,"        Thick, yellow growth.
    if potato is acid.

  _Lactose:_ Fermentation very slight.     Fermentation marked.

  _25 per cent. Gelatine at 37° C.:_       Gelatine remains limpid and clear,
    Strongly and uniformly turbid            but possesses thick pellicle.
    (Klein).

  _Elsner's Iodised Potato Gelatine:_      Very fast growth; larger, brown,
    Slow growth; small, very                 less transparent colonies.
    transparent colonies.

  _Widal's Test:_ Bacilli become           Bacilli remain actively motile.
    motionless and clumped together
    when suspended in a drop of blood
    serum from a typhoid patient.

  _Broth containing 0.3 per cent. Phenol   Grows well.
    or Formalin_ (1:7000): No growth.

  _Thermal Death Point:_ 62° C. for five   66° C. for five minutes (Klein).
    minutes (Klein).

  _Vitality in Water and Sewage:_          The _B. coli_ retains for a much longer
    Typhoid bacillus soon ceases to          time its vitality and power of
    multiply and readily dies (Klein).       self-multiplication (Klein).

The two species, _Bacillus typhosus_ and _B. coli_, agree in possessing
the following characters: no spores, no liquefaction of gelatine; both
grow well on phenolated gelatine, and in Parietti's broth; both act
similarly upon animals, though typhoid fever is not a specific disease
of animals.

The _Bacillus typhosus_, though a somewhat susceptible bacillus,
can when dried retain its vitality for weeks. In sewage it is very
difficult indeed to detect, and is soon crowded out. Dr. Andrews and
Mr. Parry Laws, in their bacterial researches into sewage for the
London County Council,[15] found that when they examined specially
infected typhoid sewage it was only with extreme difficulty they
isolated Eberth's bacillus. In ordinary sewage it is clear such
difficulty would be greatly enhanced.

[Illustration: B. COLI COMMUNIS]

We have pointed out elsewhere the relation between soil and typhoid.
In water, even though we know it is a vehicle of the disease,
the _Bacillus typhosus_ has been only very rarely detected. The
difficulties in separating the bacillus from waters (like that at
Maidstone, for example), which appear definitely to have been the
vehicle of the disease, are manifold. To begin with, the enormous
dilution must be borne in mind, a comparatively small amount of
contamination being introduced into large quantities of water.
Secondly, the huge group of the _B. coli_ species considerably
complicates the issues, for it copiously accompanies the typhoid, and
is always able to outgrow it. Further, we must bear in mind a point
that is systematically neglected, namely, that the bacteriological
examination of a water which is suspected of having conveyed the
disease is from a variety of circumstances conducted too late to detect
the causal bacteria. The incubation period of typhoid we may take at
fourteen days. Let us suppose a town water supply is polluted with some
typhoid excreta on the 1st of January. Until the 14th of January there
may be no knowledge whatever of the state of affairs. Two or three
days are required for notification of cases. Several more days elapse
generally before bacteriological evidence is demanded. Hence arises the
anomalous position of the bacteriologist who sets to work to examine a
water suspected of typhoid pollution three weeks previously. There can
be no doubt that these difficulties are very real ones. The solution
to the problem will be found in Dr. Klein's dictum that "a water in
which sewage organisms have been detected in large numbers should be
regarded with suspicion"[16] as the vehicle of typhoid, even though no
typhoid bacilli were discoverable. The chief of these sewage bacteria
are believed to be _Proteus vulgaris_, _B. coli_, _P. zenkeri_, and
_B. enteritidis_, and they are all nearly related to _B. typhosus_.
The presence of the _B. coli_ in limited numbers is not sufficient to
indicate sewage pollution, seeing that it is so widely distributed. But
in large numbers, and in company with the other named species, it is
almost certain evidence of sewage-polluted water.

It may occur to the general reader that, as the typhoid bacillus is not
extremely rare, drinking water may frequently act as a vehicle to carry
the disease to man. But, to appreciate the position, it is desirable
to bear in mind the following facts: the typhoid bacillus is only
found in the human excrement of patients suffering from the disease;
it is short-lived; in ordinary waters there exist organisms which can
exert an influence in diminishing its vitality; exposure to direct
sunlight destroys it; and it has a tendency to be carried down-stream,
or in still waters settle at the bottom by subsidence. Even when all
the conditions are fulfilled, it must not be forgotten that a certain
definite dose of the bacillus is required to be taken, and that by a
susceptible person. Into these latter questions of how bacteria produce
disease we shall have an opportunity of inquiring at a later stage.

We must now mention several of the special media and tests used in the
separation of _Bacillus typhosus_ and _B. coli_.

1. _The Indol Reaction._ Indol and skatol are amongst the final
products of digestion in the lower intestine. They are formed by the
growth, or fermentation set up by the growth, of certain organisms.
Indol may be recognised on account of the fact that with nitrous acid
it produces a dull red colour. The method of testing is as follows. The
suspected organism is grown in pure culture in broth, and incubated
for forty-eight hours at 37° C. Two cc. of a 4 per cent. solution of
potassium nitrite are added to 100 cc. of distilled water, and about
1 cc. of this is added to the test-tube of broth culture. Now a few
drops of concentrated sulphuric acid (unless quite pure, hydrochloric
should be used) are run down the side of the tube. A pale pink to dull
red colour appears almost at once, and may be accentuated by placing
the culture in the blood-heat incubator for half an hour. Much dextrose
(derived from the meat of the broth) inhibits the reaction. _Bacillus
typhosus_ does not produce indol, and therefore does not react to the
test; _B. coli_ and the bacillus of Asiatic cholera do produce indol,
and react accordingly. It should be pointed out, however, that the
bacillus of cholera also produces nitrites. Hence the addition of acid
only to a peptone culture of cholera yields the "red reaction" of indol.

2. _Carbolised Gelatine._ To ordinary gelatine .05 per cent. of phenol
is added. This inhibits many common water bacteria.

3. "_Shake Cultures._" To 10 cc. of melted gelatine a small quantity
of the suspected organism is added. The test-tube is then shaken and
incubated at 22° C. If the organism is _Bacillus coli_, the next day
reveals a large number of gas-bubbles.

4. _Elsner's Medium._ This special potassium-iodide-potato-gelatine
medium is used for the examination of typhoid excreta. It is made as
follows: 500 grams of potato gratings are added to 1000 cc. of water;
stand in cool place for twelve hours, and filter through muslin; add
150 grams of gelatine; sterilise and add enough deci-normal caustic
soda until only faintly acid; add white of egg; sterilise and filter.
Before use add half a gram of potassium iodide to every 50 cc. Upon
this acid medium common water bacteria will not grow, but _Bacillus
typhosus_ and _B. coli_ flourish.

5. _Parietti's Formula_ consists of--phenol, five grams; hydrochloric
acid, four grams; distilled water, 100 cc. To 10 cc. of broth 0.1-0.3
cc. of this solution is added. The tube is then incubated in order to
see if it is sterile. If that is so, a few drops of the suspected water
are added, and the tube reincubated at 37° C. for twenty-four hours. If
the water contains the _B. typhosus_ or _B. coli_, the tube will show a
turbid growth.

6. _Widal's Reaction._ Mix a loopful of blood from a patient suspected
of typhoid fever with a loopful of young typhoid broth culture in a
hanging drop on a hollow ground slide. Cover with a cover glass and
examine under 1/6-inch objective. If the patient is really suffering
from typhoid, there will appear in the hanging drop two marked
characteristics, viz., agglutination and immotility. This aggregation,
together with loss of motility, is believed to be due to the inhibitory
action of certain bacillary products in the blood of patients suffering
from the disease. The test may be applied in various ways, and its
successful issue depends upon one or two small points in technique into
which we cannot enter here, but which the reader will find dealt with
in the appendix.

7. _Flagella-staining._ Special methods must be adopted for staining
the flagella of _Bacillus typhosus_ and _B. coli_. The cover glasses
should be absolutely clean, the cultures young (say eighteen hours
old), and a diluted emulsion with distilled water must be made in a
watch-glass in order to get bacilli discrete and isolated enough. _Van
Ermengem's Method_ is as follows:--Place a loopful of the emulsion on
a clean cover glass and dry it in the air, fixing it lastly by passing
it once or twice through the flame of a Bunsen burner. Place films for
thirty minutes in a solution of one part boric acid (2 per cent.) and
two parts of tannin (15.25 per cent.), which also contains four or five
drops of glacial acetic acid to every 100 cc. of the mixture. Wash in
distilled water and alcohol. Then place for five to ten seconds in a
25.5 per cent. solution of silver nitrate. Immediately thereafter, and
without washing, treat the cover glass to the following solution for
two or three seconds: gallic acid, five grams; tannin, three grams;
fused potassium acetate, ten grams; distilled water, 350 cc. After
this place in a fresh capsule of silver nitrate until the film begins
to turn black. Wash in distilled water, dry, and mount. The process
contracts the bacilli somewhat, but the flagella stain well.

The +Bacillus coli communis+ occupies such an important place in all
bacteriological investigation that a few words descriptive of it
are necessary in this place. The "colon bacillus," as it is termed,
appears to be almost ubiquitous in distribution. The idea once held
that it belonged exclusively to the alimentary canal or sewage is
now discarded. It is one of the most widely distributed organisms in
nature, though, as its name implies, its habitat is in the intestinal
tract of man and animals. It is an aërobic, non-sporulating,
non-liquefying bacillus, about .4 µ in thickness, and twice that
measurement in length; hence it often appears oval or egg-shaped. Its
motility is in varying degree, occasionally being as active as _B.
typhosus_, but generally much less so. It possesses lateral flagella.
On gelatine plates at 20° C. _B. coli_ produces non-liquefying,
greyish-white, round colonies; in a stroke culture on the same medium,
a luxuriant greyish band, much broader and less restricted to the
track of the needle than _B. typhosus_. In depth of medium or "shake"
cultures there is an abundant formation of bubbles of gas (methane or
carbon dioxide) in the medium. On potato it produces a light yellow,
greasy growth, which must be distinguished from the growth of _B.
fluorescens liquefaciens_, _B. pyocyaneus_, and several other species
on the same medium. If the potato is old or alkaline, the yellow
colour may not appear. Milk is curdled solid in from twenty-four to
forty-eight hours, and a large amount of lactic acid produced. In broth
it produces a uniform turbidity, with later on some sediment and a
slight pellicle. It gives the reaction to indol.

It is now the practice to speak of the family of _Bacillus coli_
rather than the individual. The family is a very large one, and shows
throughout but few common characters. The morphology readily changes
in response to medium, temperature, age, etc. Fermentation of sugar,
coagulation of milk, or indeed the indol reaction cannot always be
used as final tests as to whether or not the organism is _B. coli_,
for unfortunately some members of the family do not show each of these
three features. Most varieties, however, appear to show some motility,
a small number of flagella, a typical growth on potato, and develop
more rapidly on all media than _B. typhosus_. These characters, plus
one or more of the three features above named, are diagnostic data upon
which reliance may be placed.

+Cholera.+ This word is used to cover more a group of diseases rather
than one specific well-restricted disease. In recent years it has
become customary to speak of Asiatic cholera and British cholera, as
if indeed they were two quite different diseases. But, as a matter of
fact, we know too little as yet concerning either form to dogmatise on
the matter. Until 1884 practically nothing was known about the etiology
of cholera. In that year, however, Koch greatly added to our knowledge
by isolating a spirillum from the intestine and in the dejecta of
persons suffering from the disease.

Cholera has its home in the delta of the Ganges. From this endemic area
it spreads in epidemics to various parts of the world, often following
lines of communication. It is a disease which is characterised by
acute intestinal irritation, manifesting itself by profuse diarrhœa
and general systemic collapse, with cramps, cardiac depression,
and subnormal temperature. The incubation period varies from only
a few hours to several days. In the intestine, and setting up its
pathological condition, are the specific bacteria; in the general
circulation their toxic products, bringing about the systemic changes.
Cholera is generally conveyed by means of water.

The spirillum of Asiatic cholera (Koch, 1884) generally appears, in the
body and in artificial culture, broken into elements known as "commas."
These are curved rods with round ends, showing an almost equal diameter
throughout, and sometimes united in pairs or even a chain (spirillum).
The latter rarely occur in the intestine, but may be seen in fluid
cultures. The common site for Koch's comma is in the intestinal wall,
crowding the lumina of the intestinal glands, situated between the
epithelium and the basement membrane, abundant in the detached flakes
of mucous membrane, and free in the contents of the intestine. They do
not occur in the blood, nor are they distributed in the organs of the
body.

[Illustration: THE COMMA-SHAPED BACILLI OF CHOLERA]

The bacilli are actively motile, and possess at least one terminal
flagellum. The organism is aërobic, and liquefies gelatine. It
stains readily with the ordinary aniline dyes. It does not produce
spores, though certain refractile bodies inside the protoplasm of the
bacillus in old cultures have been regarded as such. The virulence
of the bacillus is readily attenuated, and both the virulence and
morphology appear to show in different localities and under different
conditions of artificial cultivation a large variety of what are
termed _involution forms_. Unless the organism is constantly being
sub-cultured, it will die. Acid, even the .2 per cent. present in the
gastric juice, readily kills it. Desiccation, 55° C. for ten minutes,
and weak chemicals have the same effect. The bacilli, however, have
comparatively high powers of resistance to cold. Unless examined by
the microscope in a fresh and young stage, it is difficult to
differentiate Koch's comma from many other curved bacilli.

[Illustration: BACILLUS TYPHOSUS (SHOWING FLAGELLA)

× 1000]

[Illustration: BACILLUS TYPHOSUS (WIDAL REACTION)

(Agglutination by serum from typhoid patient)

× 400]

[Illustration: BACILLUS COLI COMMUNIS

(From agar culture, 48 hours growth)

× 1000]

[Illustration: BACILLUS MYCOIDES

(Spore formation. From agar culture)

× 1000

_By permission of the Scientific Press, Limited_]

Its cultivation characters are not always distinctive. Microscopically
the young colonies in gelatine appear as cream-coloured, irregularly
round, and granular. Liquefaction sets in on the second day, producing
a somewhat marked "pitting" of the medium, which soon becomes reduced
to fluid. In the depth of gelatine the growth is very characteristic.
An abundant, white, thick growth exactly follows the track of
the needle, here and there often showing a break in continuity.
Liquefaction sets in on the second day, and produces a distinctive
"bubble" at the surface. The liquefied gelatine does not fall from the
sides of the tube, as in the Finkler-Prior comma of _cholera nostras_,
but occurs inside the border where the gelatine joins the glass. In the
course of a week or two all the gelatine may be reduced to fluid. On
agar Koch's comma produces with rapidity a thick, greyish, irregular
growth. On potato, especially if slightly alkaline, an abundant
brownish layer is formed. Broth and peptone water are excellent media.
In milk it rapidly multiplies, curdling the medium, with production
of acid. Unlike _Bacillus coli_, it does not form gas, but, like
_B. coli_, it produces large quantities of indol and a reduction of
nitrates to nitrites. Hence the indol test may be applied by simply
adding to the peptone culture several drops of strong sulphuric acid,
when in the course of several hours, if not at once, there will be
produced a pink colour, the "cholera red reaction." Although it readily
loses virulence, and its resistance is little, the comma bacillus
retains its vitality for considerable periods in moist cultures, upon
moist linen, or in moist soil. In cholera stools kept at ordinary
room temperature the cholera bacillus will soon be outgrown by the
putrefactive bacteria. The same is true of sewage water.

The lower animals do not suffer from any disease at all similar to
Asiatic cholera, and hence it is impossible to fulfil the postulate
of Koch dealing with animal inoculation. In this respect it is like
typhoid. It is, however, provisionally accepted that Koch's bacillus
is the cause of the disease. The four or five other bacteria which
have from time to time been put forward as the cause of cholera have
comparatively little evidence in their support. It is less from these,
and more from several spirilla occurring in natural waters, that
difficulties of diagnosis arise.

Some hold that, however many comma bacilli be introduced into the
alimentary canal, they will not produce the disease unless there is
some injury or disease of the wall of the intestine. It need hardly
be added that cholera acts, like other pathogenic bacteria, by the
production of toxins. Brieger separated cadaverin and putrescin and
other bodies from cholera cultures, and other workers have separated a
tox-albumen.

_Methods of Diagnosis of Cholera_:

1. The nature of the evacuations and the appearance of the mucous
membrane of the intestine afford striking evidence in favour of a
positive diagnosis. Nevertheless it is upon a minute examination of the
flakes and pieces of detached epithelium that reliance must be placed.
In these flakes will be found in cholera abundance of bacilli having
the size, shape, and distribution of the specific comma of cholera.
The size and shape have been already touched upon. The distribution is
frequently in parallel lines, giving an appearance which Koch described
as the "fish-in-stream arrangement." This distribution of comma bacilli
in the flakes of watery stools is, when present, so characteristic of
Asiatic cholera that it alone is sufficient for a definite diagnosis.
But unfortunately it is not always present, and then search for other
characters must be made.

2. The appearance of cultivation on gelatine, to which reference has
been made, is of diagnostic value.

3. The "cholera red reaction." It is necessary that the culture be pure
for successful reaction.

4. Isolation from water is, according to Dr. Klein, best accomplished
as follows: A large volume of water (100-500 cc.) is placed in a
sterile flask, and to it is added so much of a sterile stock fluid
containing 10 per cent. peptone, 5 per cent. sodium chloride, as will
make the total water in the flask contain 1 per cent. peptone and .5
per cent. salt. Then the flask is incubated at 37° C. If there have
been cholera vibrios in the water, however few, it will be found after
twenty-four hours' incubation that the top layer contains actively
motile vibrios, which can now be isolated readily by gelatine-plate
culture.

5. To demonstrate in a rapid manner the presence of cholera bacilli
in evacuations, even when present in small numbers, a small quantity
must be taken up by means of a platinum wire and placed in a solution
containing 1 per cent. of pure peptone and .5 per cent. sodium chloride
(Dunham). This is incubated as in the case of the water, and in twelve
hours is filled with a turbid growth, which when examined by means of
the hanging drop shows characteristic bacilli.


NATURAL PURIFICATION OF WATER

We have already noticed that rivers purify themselves as they proceed.
There are many excellent examples of this self-purification. The Seine
as it runs through Paris becomes highly polluted with every sort of
filthy contamination. But twenty or thirty miles below the city it
is found to be even purer than above the city before it received the
city sewage. In small rivers it is the same, provided the pollution is
less in amount. Whilst authorities differ with regard to the mode of
self-purification, all agree that in some way rivers receiving crude
sewage are able in a marvellous degree to become pure again.

The conditions influencing this phenomenon are as follows:

(_a_) _The Movement of the Water._ It is probable, however, that
any beneficial result accruing from this cause is due, not to any
mechanical factor in the movement, but to the extra surface of water
available for oxidation processes.

(_b_) _The Pressure of the Water._ It is believed that the volume of
water pressing down upon any given area beneath it weakens the vitality
of certain microbes. In support of this theory, it is urged that the
number of bacteria capable of developing is less the greater the depth
from the surface. Yet it must be remembered that mud at the bottom of a
river, or at the bottom of the sea, is teeming with living organisms.

(_c_) _Light._ We have seen how prejudicial is light to the growth of
organisms in culture media. This is so, though to a less extent, in
water. Arloing held that sunlight could not pierce a layer of water
an inch in thickness and still act inimically on micro-organisms. But
Buchner found that the sun's rays could pass through fifteen or twenty
inches and yet be bactericidal. This evidence appears contradictory.
On the whole, however, authorities agree that the influence of the
sun's rays upon water is distinctly bactericidal and causes a marked
diminution in the quantity of organisms after acting for some hours.
Especially will this be so when the water is spread over a wide area
and is therefore shallow and stationary, or moving but slowly.

(_d_) _Vegetation in Water._ Pettenkofer, in his observations upon the
Iser below Munich, has shown how algæ bring about a marked reduction in
the organic matters present in water.

(_e_) _Dilution._ There can be no doubt in anyone's mind that the
pollutions passing into a flowing river are very soon diluted with the
large quantities of comparatively pure water always forthcoming. But
this, whilst it would lower the percentage of impurity, cannot remove
impurities.

(_f_) _Sedimentation._ Whilst Pettenkofer attributes self-purification
to oxygenation and vegetation, most authorities are now agreed that
it is largely brought about by the subsidence of impure matters,
and by their subsequent disintegration at the bottom of the river.
Sedimentation obviously is greatest in still waters. Hence lake water
contains as a rule very few bacteria. "The improvement in water during
subsidence is the more rapid and pronounced the greater the amount
of suspended matter initially present" (Frankland). Tils has pointed
out that the number of micro-organisms was invariably smaller in the
water collected from the reservoir than in that taken from the source
supplying the latter. Percy Frankland has demonstrated the same effect
of sedimentation by storage as follows:

                                         No. of Colonies in
                                          1 cc. of water.

  1. Intake from Thames, June 25, 1892        1,991
  2. First small storage reservoir            1,703
  3. Second         "       "                 1,156
  4. Large storage reservoir                    464

The large reservoir would of course necessitate a prolonged subsidence,
and hence a greater diminution than in the small reservoirs. Many like
examples might be cited, but a typical one such as the above will
suffice.

(_g_) _Oxidation._ Many experiments and observations have been made to
prove that large quantities of oxygen are used up daily in oxygenising
the Thames water. Oxygenated water will come up with the tide and down
with the fresh water from above London. There will also be oxygen
absorption going on upon the surface of the water, and from these three
sources enough oxygen is obtained to oxidise impurities and produce
what is really an effluent. In many smaller streams the opportunity for
oxidation is afforded by weirs and falls.

Probably all these factors play a part in the self-purification of
rivers, but we may take it that oxidation, dilution, and sedimentation
are three of the principal agencies.

We may here digress to refer in passing to the facts obtainable from
Sir Edward Frankland's report on Metropolitan water supply in 1894,
as they will afford a connecting link between self purification and
artificial purification. Judged by the relatively low proportion of
carbon to nitrogen, the organic matter present in the water was, as
usual, found to be chiefly, if not entirely, of vegetable origin. An
immense destruction of bacteria was found to be effected by storage in
subsidence reservoirs. The bacterial quality of the water might differ
widely from its chemical qualities. These three facts are of primary
importance in the interpretation of water reports, and it will be well
to bear them in mind. Sir E. Frankland also refers to the physical
conditions affecting microbial life in river waters. The importance
of changes of temperature, the effect of sunlight, and rate of flow
had been referred to in previous reports. Respecting the relative
proportion of these factors, he adds:

  "The number of microbes in Thames water is determined mainly
  by the flow of the river, or, in other words, by the rainfall,
  and but slightly, if at all, by either the presence or absence
  of sunshine, or a high or low temperature. With regard to the
  effect of sunshine, the interesting researches of Dr. Marshall
  Ward leave no doubt that this agent is a powerful germicide,
  but it is probable that the germicidal effect is greatly
  diminished, if not entirely prevented, when the solar rays
  have to pass through a comparatively thin stratum of water
  before they reach the living organisms."

From which it is clear that evidence favours the effect of
sedimentation and dilution. These two factors in conjunction with
filtration are, practically speaking, the methods of artificial water
purification, with which we are now in a position to deal.

ARTIFICIAL PURIFICATION OF WATER

_Sedimentation and Precipitation._ Naturally, we see this factor in
action in lakes or reservoirs. For example, the water supply of Glasgow
is the untreated overflow from Loch Katrine. Purification has been
brought about by means of subsidence of impurities. Nothing further is
needed. Artificially, we find it is this factor which is the mechancial
purifier of biological impurity in such methods as _Clark's process_.
By this mode "temporary hardness," or that due to soluble bicarbonate
of lime, is converted into insoluble normal carbonate of lime by the
addition of a suitable quantity of lime-water. Carbonates of lime and
magnesia are soluble in water containing free carbonic acid, but when
fresh lime is added to such water it combines with the free CO_{2} to
form the insoluble carbonate, which falls as a sediment:

  CaCO_{3} + CO_{2} + CaH_{2}O_{2} (lime-water) = 2 CaCO_{3} + H_{2}O.

As the carbonate falls to the bottom of the tank it carries down with
it the organic particles. Hence sedimentation is brought about by means
of chemical precipitation. It is obviously a mechanical process as
regards its action upon bacteria. Nevertheless its action is well-nigh
perfect, and 300 or 400 m.-o. per cc. are reduced to 4 or 5 per cc.
We shall refer to this same action when we come to speak of bacterial
purification of sewage. _Alum_ has been frequently used to purify
waters which contain much suspended matter. Five or six grains of
alum are added to each gallon of water, with some calcium carbonate
by preference. Precipitation occurs, and with it sedimentation of the
bacteria, as before. But, as Babes has pointed out, alum itself acts
inimically on germs; in such treatment, therefore, we get sedimentation
and germicidal action combined.

As a matter of actual practice, however, sedimentation alone is rarely
sufficient to purify water. It is true that the collection of water in
large reservoirs permits subsidence of suspended matters, and affords
time for the action of light and the competitive suicidal behaviour
of the common water bacteria. Yet, after all, filtration is the most
important and most reliable method.

_Sand Filtration_, as a means of purifying water, has been practised
since the early part of the present century. But it was not till
1885 that Percy Frankland first demonstrated the great difference in
bacterial content between a water unfiltered and a water which had
passed through a sand filter. Previous to this time the criterion of
efficiency in water purification had been a chemical one only, and
the presence or absence of bacteria in any appreciable quantity was
described, not in mathematical terms, but in indefinite descriptive
words, like "turbid," "cloudy," etc. It is needless to say that this
difference in estimation was due to the introduction by Koch of the
gelatine-plate method of examination. As a result of Percy Frankland's
work, he formulated the following conclusions as regards the chief
factors influencing the number of microbes passing through the filter.

It depends upon:

(1) _The Storage Capacity for Unfiltered Water._ This, of course, has
reference to the advantages, which we have noticed above, of securing a
large collection of water previous to filtration for subsidence, etc.

(2) _The Thickness of Fine Sand through which Filtration is Carried
on._ An argument needing no further support, for it is clear, other
things being equal, the more sand water passes through the greater the
opportunity of leaving its impurities behind.

(3) _Rate of Filtration._ The slower filtration will be generally the
more complete in its results.

(4) _Renewal of Filter-Beds._ After a certain time the filter-bed
becomes worn out and inefficient; at such times renewal is necessary.
Not only may the age of the filter act prejudicially, but the extra
pressure required will tend to force through it bacteria which ought to
have remained in the filter.

In 1893, Koch brought out his monograph upon _Water Filtration and
Cholera_, and his work had a deservedly great influence upon the whole
question. He shows how the careful filtration of water supplied to
Altona from the Elbe saved the town from the epidemic of cholera which
came upon Hamburg as a result of drinking unfiltered water, although
Altona is situated several miles below Hamburg, and its drinking
water is taken from the river after it has received the sewage of
Hamburg. Now, from his experience of water filtration, Koch arrived at
several important conclusions. In the first place, he maintained that
the _portion of the filter-bed which really removed micro-organisms
effectively was the slimy organic layer upon the surface_. This
layer is produced by a deposit from the still unpurified water lying
immediately above it. The most vital part of the filter-bed is this
organic layer, which, after formation, should not be disturbed until
it requires removal owing to its impermeability. A filter-bed, as is
well known, consists of say three feet of sand and one foot of coarse
gravel. The water to be filtered is collected into large reservoirs,
where subsidence by gravitation occurs. Thence it is led by suitable
channels to the surface of the filter-bed. Having passed through the
three or four feet of the bed, it is collected in a storage reservoir
and awaits distribution. The action of the whole process is both
mechanical and chemical. Mechanically by subsidence, much suspended
matter is left behind in the reservoir. Again, mechanically, much
of that which remained suspended in the water when it reached the
filter-bed is waylaid in the substance of the sand and gravel of the
filter-bed. Chemically also the action is twofold. Oxidation of the
organic matter occurs to some extent as the water passes through the
sand. Until recently this chemical action and the double mechanical
action were believed to be the complete process, and its efficiency
was tested by chemical oxidation and alteration, and absence of the
suspended matter.

Now, however, it is recognised that the second portion of the chemical
action is vastly the more important, indeed, the only vital, part of
the process. This is the chemical effect of the layer of scum and mud
on the surface of the sand at the top of the filter-bed. The mechanical
part of this layer is, of course, the holding back of the particulate
matter which has not subsided in the reservoir; the vital action
consists in what is termed _nitrification_ of unoxidised substance,
which is accomplished in this layer of organic matter. We shall deal
at some length with the principles of nitrification when we come to
speak of soil. But we may say here that by nitrification is understood
a process of oxidation of elementary compounds of nitrogen, by which
these latter are built up into stable bodies which can do little
harm in drinking water. From what has been said it will be seen that
the action of a filter-bed is of a complicated nature. There is (1)
subsidence of the grosser particles of impurity in the water; (2)
mechanical obstruction to impurities in the interstices of the scum,
sand, and gravel in the filter; (3) oxidation of organic matter by the
oxygen held in the pores of the sand and gravel; (4) nitrification
in the vital scum layer, which is accomplished by micro-organisms
themselves. This latter is now considered to be incomparably the most
important part of the filter. That being so, its removal, except when
absolutely necessary, is to be avoided as detrimental to the efficiency
of the filter. New filters have obviously but little of this action.
Hence it is wise to allow a new filter-bed to act for a short period
(say twenty-four to forty-eight hours) before the filtered water is
used for domestic purposes, in order to allow the organic layer to be
formed. This must also be borne in mind after a filter-bed has been
cleaned.

To maintain this nitrifying action of a filter in efficiency, Koch
suggested, in the second place, _that the rate of filtration must not
exceed four inches per hour_. At the Altona water-works this rate of
filtration was maintained, and the number of organisms always remained
below 100 per cc., which, as we have seen, is the standard. Thirdly,
it is important that _periodic bacteriological examinations should
be made_. Koch's emphasis upon this point is well known, and the
cumulative experience of bacteriologists only further supports such a
course being taken. If it be true that efficient sand filtration is a
safeguard against pathogenic germs like typhoid and cholera, then there
can be but one criterion of efficiency, viz., their absence in the
filtered water, which can only be ascertained by regular examination.
But it is not alone for pathogenic germs that filtration is proposed.
Filtered water containing more than 100 micro-organisms of any kind
per cc. is below the standard in purity, and should on no account be
distributed for drinking purposes. In this country chemical analysis,
with a more or less cursory microscopic examination, has been almost
invariably accepted as reliable indication of the condition of the
water. But such an examination is not really any more a fair test of
the working of the filter than it is of the actual condition of the
water. It is true, the quantity of organic matter can be estimated and
the condition in which it exists in combination obtained; but it cannot
tell us what a bacteriological examination can tell us, viz., the
quantity and quality of living micro-organisms present in the water.
Upon this fact, after all, an accurate conclusion depends. There is
abundant evidence to show that no valuable opinion can be passed upon
a water except by both a chemical and a bacteriological examination,
and further by a personal investigation, outside the laboratory, of the
origin of the water and its liabilities to pollution.

So convinced was Koch of the efficiency of sand filtration as
protection against disease-producing germs that he advocated an
adaptation of this plan in places where it was found that a well
yielded infected water. Such pollution in a well may be due to various
causes; surface-polluted water oozing into the well is probably the
commonest, but decaying animal or vegetable matter might also raise the
number of micro-organisms present almost indefinitely. Koch's proposal
for such a polluted well was to fill it up with gravel to its highest
water level, and above that, up to the surface of the ground, with
fine sand. Before the well is filled up in this manner it must, of
course, be fitted with a pipe passing to the bottom and connected with
a pump. This simple procedure of filling up a well with gravel and sand
interposes an effectual filter-bed between the subsoil water and any
foul surface water percolating downwards. Such an arrangement yields as
good, if not better, results than an ordinary filter-bed, on account of
there being practically no disturbance of the bed nor injury done to it
by frost.

The effect of the remedies we have been discussing upon the number of
bacteria is demonstrated in the results which Sir Edward Frankland
arrived at in his investigation of London waters.[17]

MEAN OF MONTHLY EXAMINATIONS FOR THE YEAR

  ------------------+---------+----------------------------+---------------
                    |         |   M.-O. PER CC.            | Average per
                    |         +-------+--------+------- ---+   cent. of
                    |Source of|       |        |           |Micro-organisms
  NAME OF COMPANY.  | Supply. |  At   | After  |   After   |    Removed
                    |         |Source.|Storage.|Filtration.| by Filtration.
  ------------------+---------+-------+--------+-----------+---------------
  The Chelsea Co.   |Thames at|       |        |           |
                    | Hampton |16,138 |  1067  |       34  |      98.96
  West Middlesex Co.|    "    |16,138 |  1788  |       58  |      99.40
  Southwark &       |         |       |        |           |
       Vauxhall Co. |    "    |16,138 |  ....  |       80  |      97.72
                    |         |       |        |    { 623  |    }
  Grand Junction Co.|    "    |16,138 |  2500  |    { 100  |    } 98.46
                    |         |       |        |    {  96  |    }
  Lambeth Co.       |    "    |16,138 |  7820  |       75  |      99.50
  ------------------+---------+-------+--------+-----------+---------------


The teaching of these figures could, with great ease, be reproduced
again and again if such was necessary; but these will suffice to show
that sand filtration, when carefully carried out, offers a more or less
absolute barrier to the passage of bacteria, whether non-pathogenic or
pathogenic.

_Domestic Purification of Water._ Something may here be said, from a
bacteriological point of view, relative to what is called _domestic
purification_. There is but one perfectly reliable method of
sterilising water for household use, viz., _boiling_. As we have seen,
moist heat at the boiling point maintained for five minutes will kill
all bacteria and their spores. The only disadvantages to this process
are the labour entailed and the "flat" taste of the water. Nevertheless
in epidemics due to bad water it is desirable to revert to this simple
and effectual purification.

There are a large number of filters on the market with, in many
cases, but little modification from each other. The materials out of
which they are made are chiefly the following: carbon and charcoal,
iron (spongy iron or magnetic oxide), asbestos, porcelain and other
clays, natural porous stone, and compressed siliceous and diatomaceous
earths. From an extended research in 1894 by Dr. Sims Woodhead and Dr.
Cartwright Wood our knowledge of the quality of these substances as
protectives against bacteria has been largely increased. They concluded
that a filter failed to act in one of two ways. It was either pervious
to micro-organisms, or its power of filtering became modified owing to
(_a_) structural alteration of its composition, or to (_b_) the growing
through of the micro-organisms. The conditions which chiefly influence
the growth of bacteria through a filter appear to be the temperature,
the intermittent use of the filter, and the species of bacteria. The
higher the temperature and the longer the organisms are retained in the
filter the more likely is it that they will grow through, and in the
next usage of the filter appear in the filtrate. As to the species,
those multiplying rapidly and possessing the power of free motility
will naturally appear earlier in a filtrate than others. Woodhead and
Wood, from their searching and most able investigation, concluded that
the Pasteur-Chamberland candle filters (composed of porcelain formed
by a mixture of kaolin and other clays) were the only filters out of
the substances named above which were reliable and protective against
bacteria. They tested over three dozen of the Pasteur filters, and
"in every case these gave a sterile filtrate." Pure cholera bacillus
in suspension (5000 bacilli to every cc.) and typhoid bacillus in
suspension (8000 per cc.) were passed through these filters, and
not a single bacillus was detectable in the filtrate. The Berkefeld
filter (siliceous earth) came second on the list as an effective
filter, and had but the fault of not being a "continuous" steriliser.
A certain Parisian filter ("Porcelaine d'Amiante"), made of unglazed
porcelain, rendered water absolutely free from bacteria. Its action
was, however, very slow. Setting aside these three efficient filters,
we are face to face with the fact that most filters do not produce
germ-free filtrates, even though they are nominally guaranteed to do
so. It is professed for _animal charcoal_, which is widely used, that
it absorbs oxygen, and so fully oxidises whatever passes through it.
This may be so at first, but after a little use it probably does more
harm than good. It appears to add nitrogen and phosphates to water,
which are both nutritive substances on which bacteria grow. Moreover it
readily absorbs impurities from the air. As a matter of experiment and
practice, it has been found by Frankland, Woodhead, and others, that
charcoal actually adds to the number of germs after it has been in use
for some days.

[Illustration: PASTEUR-CHAMBERLAND FILTER

Attached to Water Supply]

_Diseases Conveyed by Water._ There are a few preliminary features
to be noticed before we enter in detail upon the characteristics of
several of the chief pathogenic bacteria in water.

In sterilised water, and in very highly polluted water or sewage,
pathogenic bacteria do not flourish. In the former case they die of
starvation, although there are some experiments on record which appear
not to support this view; in the latter case they are killed by the
enormous competition of common bacteria. Even in ordinary water there
is a wide divergence of behaviour. Some bacteria are destroyed in a few
hours; others appear to flourish for weeks. In all cases the spores are
able to resist whatever injurious properties the water may have much
more persistently than the bacilli themselves. These changes in the
vitality of bacteria in water, partly due to the water and partly to
the other micro-organisms, bring about two characteristics which it is
important to remember, viz., that pathogenic germs in water are, as a
rule, _scanty_ and _intermittent_. It is these features in conjunction
with the enormous quantities of common water bacteria which make the
search for the bacillus of typhoid what Klein has called "searching
for a needle in a rick of hay." Not that it cannot be detected, but
its detection is one of the most difficult of investigations. We
shall refer to this matter again when _Bacillus typhosus_ is under
consideration.

In artificial cultivation water bacteria respond very readily to
external conditions. Increase of alkalinity (.01 grams of sodium
carbonate added to 10 cc. of ordinary gelatine) causes the number of
colonies to be five or six times greater than that revealed by using
ordinary gelatine; on the other hand, very slightly increasing the
acidity of a medium as markedly diminishes the number of bacteria.
Advantage is taken of this in culturing the bacillus of typhoid, which
does not object to an acid medium.

Water may become polluted in a variety of ways, and it is helpful to
classify these as pollutions at the _source_, in the _course_, and at
the _periphery_. Gathering-grounds are frequently the locality of the
pollution. The recent Maidstone epidemic is an example. Here some of
the springs supplying the town with water were contaminated by several
typhoid patients. Frequently on the gathering-ground one may find a
number of houses the waste and refuse of which will furnish ample
surface pollution, which in its turn may readily pass into a collecting
reservoir or a well. Only recently the writer investigated the cause
of typhoid in a large country house, and traced it to pollution of the
private well by surface washings from the stable quarters. Leakage of
house-drains into wells is not an infrequent source of contamination.
The same cause is generally operative in cases of pollution of a water
supply in its _course_ from the source to the cisterns or taps at the
periphery, viz., a sewer or drain leaking into the water supply.

Water companies and those responsible for water supply appear to hold
the opinion that so long as there is sand filtration or subsidence
reservoirs it is unnecessary to consider the gathering-ground or
transit. But, as we have seen, a frost may completely dislocate the
efficient action of a filter, and times of flood may prevent proper
sedimentation; then our dependence for pure water is wholly upon the
gathering-ground and source. Hence we find water contaminated at its
source by polluted wells, by sewage-infected rivers and streams, by
drainage of manured fields, by innumerable excremental pollutions over
the areas of the gathering-grounds, and in transit by careless laying,
poor construction, bad jointing, and close proximity of water-and
drain-pipes. In the third place, we may get a water infected at the
_periphery_, in the house itself. Such cases are generally due to one
of two causes: filthy cisterns or suction. Cisterns _per se_ are more
or less indispensable where a constant service does not exist, but they
should be inspected from time to time and maintained in a cleanly
condition. Suction into the tap has been recently emphasised by Dr.
Vivian Poore as a cause of pollution. It is liable to occur whenever a
tap is left turned on, and a vacuum is produced in the supply-pipe by
intermission of the water supply, so that foul gas or liquid is sucked
back into the house-pipe.

One more point requires our attention. It has relation to bacterially
polluted water when it has gained entrance to the body. It has been
known for some time past that not all waters polluted with disease
germs produce disease. As we have before said, this may depend upon the
infective agent, its quantity and quality; the body being able in many
cases to resist a small dose of poison. It is, however, necessary to
infection, especially in water-borne disease, that the tissues shall
be in some degree disordered. The perverted action of the stomach
influences the acid secretion of the gastric juice, through which
bacilli might then pass uninjured. Particularly must this be so in
the bacillus of cholera, which is readily killed by the normal acid
reaction of the stomach. Hence, in this disease at least, it is the
opinion of bacteriologists that the condition of the mucous membrane
of the stomach is of primary importance. Metschnikoff has indeed
demonstrated the presence of the bacillus of cholera in the intestinal
excretion of apparently healthy persons, which shows that they were
protected by the resistance of their tissues to the bacilli. Further
light has been thrown on this question by the researches of MacFadyen,
who has pointed out that suspensions of cholera bacilli in water passed
through the stomach untouched, and were thus able to exert their evil
influence in other parts of the alimentary canal. When, however,
cholera bacilli were suspended in milk, none appeared to escape the
germicidal action of the gastric juice. The explanation of this is
probably the simple one that the stomach reacted with its secretion of
gastric juice only to food (milk), but simply passed the water on into
the lower and more absorptive parts of the alimentary canal. Such a
condition of affairs clearly increases the danger of water-borne germs.


THE BACTERIOLOGY OF SEWAGE AND SEWAGE-POLLUTED WATERS

It will not be needful to insist upon the obvious fact that bacteria
abound in sewage. Such a large quantity of organic matter, in which
decomposition is constantly taking place, will afford an almost ideal
nidus for micro-organic life. There is indeed but one reason why such
a medium is not absolutely ideal from the microbe's point of view, and
that reason is, that in sewage the vast number of bacteria present make
the struggle for existence exceptionally keen. Not only are the numbers
incredibly large, but we also find a very extensive representation
of species, including both saprophytes and parasites, non-pathogenic
and pathogenic. Not infrequently it is from pollution by sewage that
drinking water is contaminated with disease. A patient, we will say,
suffers from typhoid fever. The specific organism has its habitat
largely, though not exclusively, in the alimentary canal. It passes out
in the excreta, and though sometimes partially disinfected, may escape
without hindrance into the drains, and thus to the sewer or cesspool.
How often, by means of direct connection or by percolation, sewage,
from sewers or cesspools, gains access to drinking water, the history
of typhoid outbreaks in this country only too fully records.

It is impossible to lay down any exact standard of the chemical and
bacteriological quality of sewage. The quality will differ according to
the size of the community, the inclusion or otherwise of trade-waste
effluents, the addition of rain-water, and other like physical
conditions. Moreover, sewage itself when, so to speak, fully formed
is liable to undergo rapid changes owing to fermentation and the
competition of micro-organisms. It is clear that these latter are the
chief agents in bringing about the change, because, if sewage be placed
in hermetically sealed flasks and sterilised by heat, it is found that
no change occurs. From facts such as the above it will be apparent
that no exact standard of chemical or bacterial contents is possible.
Respecting the chemical condition we may shortly say that the chief
characteristic of sewage is its enormous amount of contained organic
matter in suspension or solution; respecting the bacterial content we
may say that the chief species of the very numerous organisms are those
commonly concerned in fermentative putrefaction. London crude sewage
contains on an average about four millions of micro-organisms per cc.
Many of these are "liquefying" bacteria; that is to say, they have the
power of liquefying gelatine, which is generally one of the features
of putrefactive species. In considering the quality of the bacteria
present in sewage, a still wider field of research opens before us.
For though we can say that, roughly, all sewage will contain probably
between four and eight millions of bacteria, we cannot even lay down a
rough standard respecting the kinds of bacteria present more than we
have done already in stating that a very large number indeed out of the
total will belong to putrefactive species.

We may, however, make a provisional list of normal sewage bacteria[18]
as follows:

1. _Bacillus coli communis_ and all its varieties and allies.

2. _Proteus vulgaris_ and the various protean species.

3. _B. enteritidis sporogenes_ (Klein).

4. Liquefying bacteria, _e. g._, _Bacillus fluorescens liquefaciens_,
_B. subtilis_, _B. mesentericus_.

5. Non-liquefying bacteria.

6. Sarcinæ, yeasts, and moulds.

[Illustration: PROTEUS VULGARIS]

[Illustration: B. ENTERITIDIS SPOROGENES]

We have not included in the above inventory any pathogenic bacteria.
Doubtless such species (_e. g._, typhoid[19]) not infrequently find
their way into sewage. But they are not normal habitants, and though
they struggle for survival, the keenness of competition among the dense
crowds of saprophytes makes existence almost impossible for them. Nor
can they expect much sympathy from us in the difficulties of life which
fortunately confront them in sewage.

Of those we have named as normally present it is unnecessary to speak
in detail, with the exception of the newly discovered anaërobe,
_Bacillus enteritidis sporogenes_ of Klein.[20] This bacillus is
credited to be a causal agent in diarrhœa, and has been isolated
by Dr. Klein from the intestinal contents of children suffering from
severe diarrhœa, and from adults having cholera nostras. It has
been readily detected in sewage from various localities, and also in
sewage effluents, after sedimentation, precipitation, and filtration.
Its biological characters are shortly as follows: It is in thickness
somewhat like the bacillus of symptomatic anthrax, thicker and shorter
than the bacillus of malignant œdema, and standing therefore between
the latter and anthrax itself. It is motile and possesses flagella,
but has no threads. It readily forms spores, which develop as a rule
near the ends of the rods and are thicker than the bacilli. It is
stained by Gram's method. In various media (particularly milk) it
produces gas rapidly. It is an anaërobe, and is cultivated in Buchner's
tubes. A recent epidemic of diarrhœa affecting 144 patients in St.
Bartholomew's Hospital was traced to milk in which _B. enteritidis_ was
present.

_Sewer Air._ Though not of material importance as regards bacterial
treatment of sewage, this subject calls for some remark. For long it
has been known that air polluted by sewage emanations is capable of
giving rise to various degrees of ill-health. These chiefly affect two
parts of the body; one is the throat, and the other is the alimentary
canal. Irritation and inflammation may be set up in both by sewer air.
Such conditions are in all probability produced by a lowering of the
resistance and vitality of the tissues, and not by either a conveyance
of bacteria in sewer air or any stimulating effect upon bacteria
exercised by sewer air. What evidence we have is against such factors.
(See p. 105.)

Several series of investigations have been made into the bacteriology
of sewer air, amongst others by Uffelmann, Haldane, Laws, and Andrewes.
From their labours we may formulate four simple conclusions:

1. The air of sewers contains very few micro-organisms indeed,
sometimes not more than two organisms per litre (Haldane), and
generally fewer than the outside air (Laws and Andrewes).

2. There is no relationship between the microbes contained in sewer air
and those contained in sewage. Indeed, there is a marked difference
which forms a contrast as striking as it is at first sight unexpected.
The organisms isolated from sewer air are those commonly present in
the open air. Micrococci and moulds predominate, whereas in sewage
bacilli are most numerous. Liquefying bacteria, too, which are common
in sewage, are extremely rare in sewer air. _Bacillus coli communis_,
which occurs in sewage from 20,000 to 200,000 per cc., is altogether
absent from sewer air.

3. Pathogenic organisms and those nearly allied to them are found in
sewage, but absent in sewer air. Uffelmann isolated the _Staphylococcus
pyogenes aureus_ (one of the organisms of suppuration), but such
a species is exceptional in sewer air. Hence, though sewer air is
popularly held responsible for conveying diphtheria and all sorts of
other virulent bacteria, there is up to the present no evidence of a
substantial nature in support of such views. Sewer air neither conducts
pathogenic organisms nor stimulates the virulence of such.

4. Lastly, only when there is splashing in the sewage, or when bubbles
are bursting (Frankland), is it possible for sewage to part with its
contained bacteria to the air of the sewer.

Whilst we cannot here enter more fully into an account of the bacteria
found in sewage or of their functions, it is necessary to remark upon
one distinguishing feature. A very large number of sewage bacteria
are _decomposing_ and _denitrifying_, that is to say, breakers down,
by means of putrefaction, of organic compounds. The knowledge of this
fact has recently been applied, in conjunction with oxidation, to the
biological treatment of sewage. As this illustrates in a marked degree
some of the facts we have dwelt upon in considering the bacteriology of
soil, and as it is likely that the future will witness a still wider
application of these same facts, it will be necessary to refer in some
detail to the matter.

Hitherto there has been adopted one of four methods of treatment of
sewage. In the first place, in towns situated on the coast the sewage
has, by means of a conduit, been _carried out to sea_. It is clear that
such a course, which is in itself open to criticism, is applicable to
but few towns. In the second place, methods of _chemical treatment_
have been practised. This has generally been of the nature of a
"precipitation" process. Six to twelve grains of quicklime have been
added to each gallon of sewage. The process is simple and cheap, but
it does not remove the organic matter in solution. On the one hand, it
does not produce a valuable manure; on the other, it fails to purify
the effluent. A dozen other methods have been tried, but all based
on the addition of chemical substances to precipitate or change the
organic matter of the sewage. Electrolysis, too, has been proposed. The
third mode adopted in the past has been that known as _intermittent
downward filtration_. This may be defined as "the concentration of
sewage at short intervals on an area of specially chosen porous ground,
as small as will absorb and cleanse it, not excluding vegetation,
but making the product of secondary importance" (Metropolitan Sewage
Commission). The action is mechanical and biological, that is to say,
due in part to nitrification by bacteria in the upper layers of soil.
The fourth plan is that of _irrigation_, or "the distribution of sewage
over a large surface of ordinary agricultural ground, having in view a
maximum growth of vegetation (consistently with due purification) for
the amount of sewage supplied." Like the former, there is biological
influence at work here, though in a less degree. About one acre is
required for every hundred persons in the population. These two latter
modes are much to be preferred to chemical treatment, yet on account of
space and management, as well as on account of the non-removal of the
"sludge," their success has not been all that could be desired. Until
comparatively recent times the above methods of treating sewage were
the only ones available.

In 1881 it appears that M. Louis Mouras, of Vesoul (Haute Saône),
published an account of a hermetically sealed, inodorous, and
automatically discharging cesspool, in which sewage was anaërobically
broken down by "the mysterious agents of fermentation." This is the
first record we have of the newly applied treatment of sewage by simply
allowing Nature to fulfil her function by means of bacteria. We shall
most easily arrive at an appreciation of the recent developments of
the process in England by describing the so called septic tank and
cultivation beds.

[Illustration: A PLAN OF SEPTIC TANK AND FILTER-BEDS

As Used at Exeter]

The _septic tank_ is a large underground vault of cemented brick,
having a capacity of thousands of gallons, according to the population.
That at Exeter has a capacity of 53,800 gallons, and takes the average
sewage of 1500 inhabitants in twenty-four hours. Near the entrance is
a submerged wall, seven feet from the entrance and twelve inches below
the surface of the liquid in the full tank. Within this are caught, by
gravity, gravel and such-like deposits. The remaining solid matter of
the sewage becomes deposited in the tank itself. Both in the sediment
at the bottom of the tank and in the thick scum on the surface the
organic compounds are broken down and made soluble. In the former
position this is accomplished by anaërobic bacteria, in the latter on
the surface by aërobic bacteria. It need hardly be added that these are
denitrifying and putrefactive bacteria, and that those at the bottom
of the tank perform greater service than those at the top. When the
liquid sewage passes out of the tank it differs from the crude sewage
which enters the tank in the following particulars: (_a_) The gravel
and particulate débris have been removed; (_b_) the organic solids in
suspension are so greatly diminished that they are almost absent; (_c_)
there is an increase of organic matter in solution; (_d_) the sewage is
darker in colour and more opalescent; (_e_) compounds like albuminoid
ammonia, urea, etc., have been more or less completely broken down,
and reappear in elementary conditions, like ammonia, methane, carbon
dioxide, and sulphuretted hydrogen. These latter bodies may be in
solution or may have escaped as gas.

The _cultivation beds_ are four or five filters, to which the sewage
from the tank flows in such a manner as to produce a weir. By an
automatic arrangement the fluid is distributed to each filter in turn.
When the second filter is full the first is discharged, and remains
empty during the time that the third and fourth are being filled.
Each filter is thus full, say, about six hours, and has from ten to
twelve hours' rest. These filter-beds (at Exeter) have an area of
eighty square yards and a depth of five feet; collecting drains are
laid on the bottom of the filters, joining main collectors, the latter
terminating in discharging wells. The filtrant is broken furnace
clinker or broken coke.

The changes occurring in these filters are of the nature of oxidation,
with the result that the proportion of the oxidised nitrogen increases
(as nitrites and nitrates), the ammonia becomes less, and the total
solids and organic nitrogen almost disappear. It will thus be seen
that the work of these filters is not merely a straining action. It is
true that particulate matter in the effluent from the tank is caught
on the surface by the film (resulting from previous effluents), but
the real work of the bed is _nitrification_, an oxidation of ammonia
into nitrites and nitrates. This change obviously begins when the tank
effluent flows over the "weir" on to the filter-beds, and the oxygen
thus obtained by the effluent is carried down in solution into the
coke-breeze. Upon the surface of the filtrant are oxidising bacteria.
When the effluent is on the bed they oxidise its contained products;
when the bed is empty and "resting" they oxidise carbon. An advantage
arising from the periodical emptying and filling of the filter is that
the products of decomposition which would eventually inhibit the action
of the aërobic bacteria are washed away, and pass into the nearest
stream, where they become absolutely innocuous.

The "filter" is more correctly termed a _cultivation bed_, for its
purpose is to furnish a very large surface upon which the nitrifying
organisms present, as we have seen, in all soils, may flourish, and
thus feeding upon the organic matter of the sewage, may perform their
function of oxidation.

It is not possible to lay down exact limits as to where denitrification
ends and oxidation begins. To a certain extent, and in varying degree,
they overlap each other. But roughly we may say that in the tank
there is a breaking down (denitrification and decomposition) and in
the filter-beds a building up (nitrification). The case is precisely
parallel to similar changes occurring in soil, and which we have dealt
with elsewhere. The advantage indeed of this biological treatment
of sewage is that it exactly follows the processes of nature, in
contradistinction to the mechanical and chemical methods hitherto
adopted.

At Sutton and some other places the same principles are applied,--that
is to say, bacterial filtration,--but there is no tank. A metal screen
in some measure takes its place, and holds back solid matter from being
carried on to the beds. The filtrant is burnt clay, and it is forked
over occasionally to let in oxygen. The crude sewage is run over the
top of the burnt ballast, where it is left for two or three hours. It
is then slowly run off on to a finer filter, where it also stays two
hours. Thence the effluent is run into the stream.

[Illustration: FILTER-BEDS

As Used at Sutton]

It must be admitted that the bacterial treatment of sewage, though
exhibiting such excellent results where it has been given a fair trial,
is still in a probationary stage. It appears to stand on reason. The
sludge of previous methods is avoided. The sewage is entirely broken
down, and the effluent is a comparatively pure one, yet taking back
nitrogen, as nitrate, to the soil. The whole change, indeed, in the
opinion of Dr. Dupré, is more effective and radical than in chemical
treatment. Further, it has been tested as regards its action upon the
pathogenic bacilli--those of tubercle and typhoid--with the result that
these infective bacteria have been completely destroyed. It appears
that such destruction of infective germs occurs in the tank, and
depends in degree upon the rapidity with which sewage is passed through
the tank. The cultivation beds also have an inimical effect upon
infective bacteria. Hence the final effluent is practically germ-free
as regards pathogenic organisms.




CHAPTER III

_BACTERIA IN THE AIR_


METHODS OF EXAMINING AIR FOR BACTERIA

The basis of the usual methods in practice is to pass air over or
through some nutrient medium. By this means the contained organisms
are waylaid, and finding themselves under favourable conditions of
pabulum, temperature, and moisture, commence active growth, and thus
reveal themselves in characteristic colonies. These are examined, as
directed on page 43, by the microscope and sub-culture. Quantitative
estimation is not generally made, as a fixed standard is even less a
possibility than in milk and soil. Returns of the number of bacteria in
the sample taken may be made for the sake of information, but little
or no conclusion of value can be drawn from such data. The standard
recognised in Europe is the cubic metre, and one may speak, for
example, of the air of a room containing 500, 1000, or 3000 germs per
cubic metre.

The following are the chief methods:

1. _Pouchet's Aëroscope._ This apparatus was in use some time ago in
France, and by its means all the solid matter of a given quantity of
air was drawn through an air-tight glass tube by aspiration and made
to impinge upon a small plate of glycerine. The air escaped to the
aspirator at the sides, leaving upon the glycerine plate only its
particulate matter. This remnant could then be examined.

2. _Koch_ adopted the simplest of all the culture methods, viz.,
exposing a plate of gelatine or agar for a longer or shorter time to
the air of which examination is desired. By gravity the suspended
bacteria fall on the plate and start growth. As a matter of
quantitative exactitude, this method is not to be recommended, but it
frequently proves an excellent method for qualitative estimation.

3. _The Method of Miquel._ Pasteur was the first to analyse air by the
culture method, and he adopted a plan which in principle is _washing_
the air in some fluid culture medium which will retain all the
particulate matter, which may then be cultured directly or sub-cultured
into any favourable medium.

[Illustration: MIQUEL'S FLASK]

Miquel has contrived a simple piece of apparatus for the carrying out
of this principle. It consists of a flask with a central tube through
its own neck for the entrance of the air. On one side of the flask is
a tube to be connected with the aspirator, on the other side of the
flask a tube through which to pour off the contained fluid at the end
of the process. In the flask are placed 30 cc. of sterilised water (or,
indeed, if it be preferred, sterilised broth). The entrance tube is now
unplugged, and the aspirator draws through a fair sample of the air in
the room (say ten litres). This air perforce passes through the water
and by the exit tube to the aspirator, and is thereby washed, leaving
behind in the water all its bacteria. The aspiration is then stopped,
and the entrance tube closed. The water (plus bacteria) is now poured
out into test-tubes of media or plated out on Petri's dishes. Provided
the apparatus has been absolutely sterilised, and that the water was
also sterile, any colonies developing upon the Petri dish are composed
of micro-organisms from the air examined.

4. _The Method of Hesse._ This method is somewhat akin to Pouchet's
aëroscope, but is in addition a culture method. Hesse's tube is
about 2 feet long and 1-1/2 inches bore throughout. At one end is an
india-rubber stopper bored for a glass tube to the aspirator. The other
end is open. Before using, the tube is sterilised, and 40 or 50 cc. of
sterilised gelatine replaced in it. The tube is now rapidly rotated
in a groove on a block of ice or under a cold-water tap, and by this
simple means the gelatine becomes fixed and forms a layer inside the
tube throughout. We have therefore, so to speak, a tube of glass with
a tube of gelatine inside it. The apparatus is now ready for use. It
is fixed on the tripod, and fifteen litres of air are drawn through,
and the tube is properly plugged and incubated at room temperature. In
a day or two days the colonies appear upon the gelatine. They are most
numerous generally in the first part of the tube, and might be roughly
estimated as follows:

  15 litres of air, 6 colonies.
  ⁂ 6/15 × 10,000 = 4000 aërobic bacteria in the cubic metre.

The disadvantages of this process are that dried gelatine does not
catch germs like the broth cultures of Pasteur or Miquel, and that many
organisms are able to go straight through the tube, and failing to be
deposited, pass out at the aspirator exit, and thus are neither caught
nor counted. The Hesse tube is generally used in practice with a pump
consisting of two flasks and a double-way india-rubber tube. The flasks
have a capacity for one litre of water. By a simple adaptation it is
possible to secure siphon action, and hence measure with considerable
exactitude the amount of air passing through the tube.

5. _Methods of Filtration._ To-day most of the above methods have been
discarded, with the exception, perhaps, of Miquel's and modifications
thereof.

[Illustration: SEDGWICK'S SUGAR-TUBE]

Frankland, Petri, Pasteur, Sedgwick, and others have suggested the
adoption of methods of filtration. These depend upon catching the
organisms contained in the air by filtering them through sterilised
sand or sugar, and then examining these media in the ordinary way.
Many different kinds of apparatus have been invented. Petri aspirates
through a glass tube containing sterilised sand, which after use is
distributed in Petri dishes and covered with gelatine. The principal
objection to this method is the presence of the opaque particles of
sand in and under the gelatine. Probably it was this which suggested
the use of soluble filters like sugar. Pasteur introduced the
principle, and Frankland and others have followed it out. The apparatus
most largely used is that known as Sedgwick's Tube. This consists of
a comparatively small glass tube, about a foot long. Half of it has a
bore of 2.5 cm., and the other half a bore of .5 cm. It is sterilised
at 150° C., after which the dry, finely granulated cane-sugar is
inserted in such a way as to occupy an inch or more of the narrow part
of the tube next the wide part. Next to it is placed a wool plug, and
the whole is again sterilised at 130° C. for two hours, care being
taken that the sugar does not melt. After sterilisation an india-rubber
tube is fixed to the end of the narrow portion, and thus it is attached
to the aspirator. The measured quantity (5-20 litres) of air is drawn
through, and any particulate matter is caught in the sugar. Warm,
nutrient gelatine (10-15 cc.) is now poured into the broad end of
the tube, and by means of a sterilised stilette the sugar is pushed
down into the gelatine, where it quickly dissolves. We have now in
the gelatine all the micro-organisms in the air which has been drawn
through the tube. After plugging with wool at both ends, the tube is
_rolled_ on ice or under a cold-water tap in order to fix the gelatine
all round the inner wall of the tube, which is incubated at room
temperature. In a day or two the colonies appear, and may be examined.

[Illustration: SEDGWICK'S TUBE

Fixed upon Tripod for Air Examination]

_Micro-organisms in the Air._ Schwann was one of the first to point
out that when a decoction of meat is effectually screened from the
air, or supplied solely with calcined air, putrefaction does not set
in. Helmholtz and Pasteur confirmed this, but it may be said with some
truth that Schwann originated the germ theory, and Lister applied it
in the treatment of wounds. Lister believed that if he could surround
wounds with filtered air the results would be as good as if they were
shut off from the air altogether.

It was Tyndall[21] who first laid down the general principles upon
which our knowledge of organisms in the air is based. That the dust in
the air was mainly organic matter, living or dead, was a comparatively
new truth; that epidemic disease was not due to "bad air" and "foul
drains," but to germs conveyed in the air, was a prophecy as daring
as it was correct. From these and other like investigations it came
to be recognised that putrefaction begins as soon as bacteria gain an
entrance to the putrefiable substance, that it progresses in direct
proportion to the multiplication of bacteria, and that it is retarded
when they diminish or lose vitality.

Tyndall made it clear that both as regards quantity and quality of
micro-organisms in the air there neither is nor can be any uniformity.
They may be conducted on particles of dust--"the raft theory"--but
being themselves endowed with a power of flotation commensurate
with their extreme smallness and the specific lightness of their
composition, dust as a vehicle is not really requisite. Nevertheless
the estimation of the amount of dust present in a sample of air is a
very good index of danger. It is to Dr. Aitken that we are indebted for
devising a method by which we can measure dust particles in the air,
even though they be invisible. His ingenious experiments, reported
in the _Transactions of the Royal Society of Edinburgh_ (vol. xxxv.),
have demonstrated that by supersaturation of air the invisible dust
particles may become visible. As is now well known, Dr. Aitken has been
able to prove that fogs, mists, and the like do not occur in dust-free
air, and are due to condensation of moisture upon dust particles. But
it should be remembered that, though dust forms a vehicle for bacteria,
dusty air is often comparatively free from bacteria. Hence, after all,
the necessary conditions for dissemination of bacteria in air are two,
namely, some degree of air-current and dry surfaces.

This latter condition is one of essential importance. Bacteria
cannot leave a moist surface either under evaporation or by means
of air-currents.[22] Only when there is considerable molecular
disturbance, such as splashing, can there possibly be microbes
transmitted to the surrounding air. This fact, coupled with the
influence of gravitation, is the reason why sewer gas and all air
contained within moist perimeters is almost germ-free; whereas from dry
surfaces the least air-current is able to raise countless numbers of
organisms. Quite recently this principle has been admirably illustrated
in two series of investigations made upon expired and inspired air. In
a report to the Smithsonian Institution of Washington (1895) upon the
composition of expired air, it is concluded that "in ordinary quiet
respiration no bacteria, epithelial scabs, or particles of dead tissue
are contained in the expired air. In the act of coughing or sneezing
such organisms or particles may probably be thrown out." The interior
of the cavity of the mouth and external respiratory tract is a moist
perimeter, from the walls of which no organisms can rise except under
molecular disturbance. The position is precisely analogous to the
germ-free sewer air as established by Messrs. Laws and Andrewes for the
London County Council. The popular idea that infection can be "given
off by the breath" is contrary to the laws of organismal pollution
of air. The required conditions are not fulfilled, and such breath
infection must be of extremely rare occurrence. The air can only be
infective when filled with organisms arising from dried surfaces.

The other series of investigations were conducted by Drs. Hewlett and
St. Clair Thompson, and dealt with the fate of micro-organisms in
inspired air and micro-organisms in the healthy nose. They estimated
that from 1500 to 14,000 bacteria were inspired every hour. Yet, as we
have pointed out, expired air contains practically none at all. It is
clear, then, that the inspired bacteria are detained somewhere. Lister
has pointed out, from observation on a pneumo-thorax caused by a wound
of the lung by a fractured rib, that bacteria are arrested before they
reach the air-cells of the lung; hence it is at some intermediate stage
that they are detained. Hewlett and Thomson examined the mucus from
the wall of the trachea, and found it germ-free. It was only when they
reached the mucous membrane and moist vestibules and vibrissæ of the
nose that they found bacteria. Here they were present in abundance. The
ciliated epithelium, the moist mucus, and the bactericidal influence
of the wandering or "phagocyte" cells probably all contribute to their
final removal.[23]

There can be no doubt that the large number of bacteria present in the
moist surfaces of the mouth is the cause of a variety of ailments,
and under certain conditions of ill-health organisms may through
this channel infect the whole body. _Dental caries_ will occur to
everyone's mind as a disease possibly due to bacteria. As a matter of
fact, probably acids (due to acid secretion and acid fermentation) and
micro-organisms are two of the chief causes of decay of teeth. Defects
in the enamel, inherent or due to injury, retention of débris on and
around the teeth, and certain pathological conditions of the secretion
of the mouth are predisposing causes, which afford a suitable nidus for
putrefactive bacteria. The large quantity of bacteria which a decayed
tooth contains is easily demonstrated.

From the two series of experiments which we have now considered we may
gather the following facts:

(_a_) That air may contain great numbers of bacteria which may be
readily inspired.

(_b_) That _in health_ those inspired do not pass beyond the moist
surface of the nasal and buccal cavities.

(_c_) That here there are various influences of a bactericidal nature
at work in defence of the individual.

(_d_) That expired air contains, as a rule, no bacteria whatever.

The practical application of these things is a simple one. To keep air
free from bacteria, the surroundings must be moist. Strong acids and
disinfectants are not required. Moisture alone will be effectual. Two
or three examples at once occur to the mind.

_Anthrax_ spores are conveyed from time to time from dried infected
hides and skins to the hands or bodies of workers in warehouses in
Bradford and other places. If the surroundings were moist, and the
hides moist, anthrax spores and all other bacteria would not remain
free in the air.

The bacilli or spores of _tubercle_ present in sputum in great
abundance cannot, by any chance whatever, infect the air until, and
unless, the sputum dries. So long as the expectorated matter remains on
the pavement or handkerchief _wet_, the surrounding air will contain
no bacilli of tubercle. But when in the course of time the sputum
dries, then the least current of air will at once infect itself with
the dried spores and bacilli.

_Typhoid Fever_, too, occupies the same position. Only when the
excrement dries can the contained bacteria infect the air. It is of
course well known that the common channel of infection in typhoid fever
is not the air, whereas the reverse holds true of tuberculosis. The
writer recently obtained some virulent typhoid excrement, and placed
it in a shallow glass vessel under a bell-jar, with similar vessels
of sterilised milk and of water, all at blood-heat. So long as the
excrement remained moist, even though it soon lost its more or less
fluid consistence, the milk and water remained uninfected. But when the
excrement was completely dried it required but a few hours to reveal
typhoid bacilli in the more absorptive fluid, milk, and at a later
stage the water also showed clear signs of pollution. This evidence
points in the same direction as that which has gone before. If the
excrement of patients suffering from typhoid dries, the air will become
infected; if, on the other hand, it passes in a moist state into the
sewer, even though untreated with disinfectants, all will be well as
regards the surrounding air.

Before passing on to consider other matters concerning organisms
in the air, we may draw attention to some interesting observations
recorded by Mr. S. G. Shattock[24] on the negative action of sewer air
in raising the toxicity of lowly virulent bacilli of diphtheria. Some
direct relationship, it has been surmised, exists between breathing
sewer air and "catching" diphtheria. Clearly it cannot be that the
sewer air contains the bacillus. But some have supposed that the sewer
air has had a detrimental effect by increasing the virulent properties
of bacilli already in the human tissues. Two cultivations of lowly
virulent bacilli were therefore grown by Mr. Shattock in flasks upon a
favourable medium over which was drawn sewer air. This was continued
for two weeks or five weeks respectively. Yet no increased virulence
was secured. Such experiments require ample confirmation, but even
from this it will be seen that sewer air does not necessarily have a
favouring influence upon the virulence of the bacilli of diphtheria.

It should be noted that the bacilli of diphtheria are capable of
lengthened survival outside the body, and are readily disseminated by
very feeble air-currents. The condition necessary for their existence
outside the body for any period above two or three days is moisture.
Dried diphtheria bacilli soon lose their vitality. It is probably owing
to this fact that the disease is not as commonly conveyed by air as,
for example, tubercle.[25]

_The influence of gravity_ upon bacteria in the air may be observed
in various ways, in addition to its action within a limited area
like a sewer or a room. Miquel found in some investigations in Paris
that, whereas on the Rue de Rivoli 750 germs were present in a cubic
metre, yet at the summit of the Pantheon only 28 were found in the
same quantity of air. At the tops of mountains air is germ-free, and
bacteria increase in proportion to descent. As Tyndall has pointed
out, even ultra-microscopic cells obey the law of gravitation. This is
equally true in the limited areas of a laboratory or warehouse and in
the open air.

The conditions which affect the number of bacteria in the air
are various. After a fall of rain or snow they are very markedly
diminished; during a dry wind they are increased. In open fields, free
from habitations, they are fewer, as would be expected, than in the
vicinity of manufactories, houses, or towns. A dry, sandy soil or a dry
surface of any kind will obviously favour the presence of organisms in
the air. Frankland found that fewer germs were present in the air in
winter than in summer, and that when the earth was covered with snow
the number was greatly reduced. Miquel and Freudenreich have declared
that the number of atmospheric bacteria is greater in the morning and
evening between the hours of six and eight than during the rest of the
day. But we venture to express the hope that such coincidental facts
may not be exalted into principles.

There is no numerical standard for bacteria in the air as there is in
water. The open air possibly averages about 250 per cubic metre. On
the seacoast this number would fall to less than half; in houses and
towns it would rise according to circumstances, and frequently in dry
weather reach thousands per cubic metre. When it is remembered _that
air possesses no pabulum_ for bacteria as do water and milk, it will be
understood that bacteria do not live in the air. They are only driven
by air-currents from one dry surface to another. Hence the quality
and quantity of air organisms depend entirely upon environment and
physical conditions. In some researches which the writer made into
the air of workshops in Soho in 1896, it was instructive to observe
that fewer bacteria were isolated by Sedgwick's sugar-tube in premises
which appeared to the naked eye polluted in a large degree than in
other premises apparently less contaminated. In the workroom of a
certain skin-curer the air was densely impregnated with particles
from the skin, yet scarcely a single bacterium was isolated. In the
polishing-room of a well-known hat firm, in which the air appeared to
the naked eye to be pure, and in which there was ample ventilation,
there were found four or five species of saprophytic bacteria. Quite
recently Mr. S. R. Trotman, public analyst for the city of Nottingham,
estimated the bacterial quality of the air of the streets of that town
during "the goose fair" held in the autumn. He used a modification of
Hesse's apparatus in which the gelatine is replaced by glycerine. The
air was slowly drawn through and measured in the usual way. Sterilised
water was then added to bring the glycerine to a known volume, the
liquid thoroughly mixed, and a series of gelatine and agar plates made
with quantities varying from 0.1 to 2 cc. By this method a large number
of bacteria were detected in this particular investigation, including
_Staphylococcus pyogenes aureus et albus_, the common _Bacillus
subtilis_, and _B. coli communis_.[26]

During a six years' investigation the air of the Montsouris Park
yielded, according to Miquel, an average of 455 bacteria per cubic
metre. In the middle of Paris the average per cubic metre was nearly
4000. Flügge accepts 100 bacteria per cubic metre as a fair average.
From this fact he estimates that "a man during a lifetime of seventy
years inspires about 25,000,000 bacteria, the same number contained in
a quarter of a litre of fresh milk."[27] Many authorities would place
the average much below 100 per cubic metre, but even if we accept that
figure it is at once clear how relatively small it is. This is due, as
we have mentioned, to sunlight, rain, desiccation, dilution of air,
moist surfaces, etc. So essentially does the bacterial content of air
depend upon the facility with which certain bacteria withstand drying
that Dr. Eduardo Germano[28] has addressed himself first to drying
various pathogenic species and then to mixing the dried residue with
sterilised dust and observing to what degree the air becomes infected.
Typhoid appears to withstand comparatively little dessication, without
losing its virulence. Nevertheless, it is able to retain vitality in
a semi-dried condition, and it is owing to this circumstance in all
probability that it possesses such power of infection. Diphtheria,
on the other hand, is, as we have pointed out, capable of lengthened
survival outside the body, particularly when surrounded with dust. The
question of their power of resisting long drying is an unsettled point.
The power of surviving a drying process is, according to Germano,
possessed by the streptococcus. This is not the case with cholera or
plague. Dr. Germano classifies bacteria, as a result of his researches,
into three groups: first, those like plague, typhoid, and cholera,
which cannot survive drying for more than a few hours; second, those
like the bacilli of diphtheria, and streptococci, which can withstand
it for a longer period; thirdly, those like tubercle, which can very
readily resist drying for months and yet retain their virulence. It
will be obvious that from these data it is inferred that Groups 1 and
2 are rarely conveyed by the air, whereas Group 3 is frequently so
conveyed. Miquel has recently demonstrated that soil bacteria or their
spores can remain alive in hermetically sealed tubes for as long a time
as sixteen years. Even at the end of that period the soil inoculated
into a guinea-pig produced tetanus.[29]

The presence of pathogenic bacteria in the air is, of course, a much
rarer contamination than the ordinary saprophytes. Tubercle has been
not infrequently isolated from dry dust in consumption hospitals, and
in exit ventilating shafts at Brompton the bacillus has been found.
From dried sputum it has, of course, been many times isolated, even
after months of desiccation. M. Lalesque failed to isolate it from the
dry soil surrounding some garden seats in a locality frequented by
phthisical patients. The writer also failed to isolate it from the same
soil. But a very large mass of experimental evidence attests the fact
that the air in proximity to dried tubercular sputum or discharges may
contain the specific bacillus of the disease. Diphtheria in the same
way, but in a lesser degree, may be isolated from the air, and from the
nasal mucous membrane of nurses, attendants, and patients in a ward
set apart for the treatment of the disease. Delalivesse, examining
the air of wards at Lille, found that the contained bacteria varied
more or less directly with the amount of floating matter, and depended
also upon the vibration set up by persons passing through the ward and
the heavy traffic in granite-paved streets adjoining. _Bacillus coli_,
staphylococci, and streptococci, as well as _B. tuberculosis_, were
isolated by this observer.

Some new light has been thrown upon the subject of pathogenic organisms
in air by Neisser in his investigations concerning the amount and
rate of air-currents necessary to convey certain species through the
atmosphere. He states that the bacteria causing diphtheria, typhoid
fever, plague, cholera, and pneumonia, and possibly the common
_Streptococcus pyogenes_, are incapable of being carried by the
molecules of atmospheric dust which the ordinary insensible currents of
air can support, whilst _Bacillus anthracis_, _B. pyocyaneus_, and the
bacillus of tubercle are capable of being aërially conveyed. This work
will require further confirmation, but if its truth be established,
it proves that attempted aërial disinfection of the first group of
diseases is useless.




CHAPTER IV

_BACTERIA AND FERMENTATION_


It was Pasteur who in 1857 first propounded the true cause and process
of fermentation. The breaking down of sugar into alcohol and carbonic
acid gas had been known, of course, for a long period. Since the time
of Spallanzani (1776) the putrefactive changes in liquids and organic
matter had been prevented by boiling and subsequently sealing the flask
or vessel containing the fluid. Moreover, this successful preventive
practice had been in some measure correctly interpreted as due to the
exclusion of the atmosphere, but wrongly credited to the exclusion of
the oxygen of the air. It was not until the beginning of the present
century that authorities modified their view and declared in favour
of yeast cells as the agents in the production of fermentation. That
this process was due to oxygen _per se_ was disproved by Schwann, who
showed that so long as the oxygen admitted to the flask of fermentative
fluid was sterilised no fermentation occurred. It was thus obvious that
it was not the atmosphere or the oxygen of the atmosphere, but some
fermenting agent borne into the flask by the admission of unsterilised
air. It was but a step to further establish this hypothesis by adding
unsterilised air plus some antiseptic substance which would destroy the
fermenting agent. Arsenic was found by Schwann to have this germicidal
faculty. Hence Schwann supported Latour's theory that fermentation was
due to something borne in by the air, and that this something was
yeast. Passing over a number of counter-experiments of Helmholtz and
others, we come to the work of Liebig. He viewed the transformation
of sugar into alcohol and carbonic acid gas simply and solely as a
non-vital chemical process, depending upon the dead yeast communicating
its own decomposition to surrounding elements in contact with it.

Liebig insisted that all albuminoid bodies were unstable, and if left
to themselves would fall to pieces--_i. e._, ferment--without the aid
of living organisms, or any initiative force greater than dead yeast
cells. It was at this juncture that Pasteur intervened to dispel the
obscurities and contradictory theories which had been propounded.

As in all the conclusions arrived at by Pasteur, so in those relating
to fermentation, there were a number of different experiments which
were performed by him to elucidate the same point. We will choose one
of many in relation to fermentation. If a sugary solution of carbonate
of lime is left to itself, after a time it begins to effervesce,
carbonic acid is evolved, and lactic acid is formed; and this latter
decomposes the carbonate of lime to form lactate of lime. This lactic
acid is formed, so to speak, at the expense of the sugar, which
little by little disappears. Pasteur demonstrated the cause of this
transformation of sugar into lactic acid to be a thin layer of organic
matter consisting of extremely small moving organisms. If these be
withheld or destroyed in the fermenting fluid, fermentation will cease.
If a trace of this grey material be introduced into sterile milk or
sterile solution of sugar, the same process is set up, and lactic acid
fermentation occurs.

Pasteur examined the elements of this organic layer by aid of the
microscope, and found it to consist of small short rods of protoplasm
quite distinct from the yeast cells which previous investigators had
detected in alcoholic fermentation. One series of experiments was
accomplished with yeast cells and these bacteria, a second series with
living yeast cells only, a third series with bacteria only, and the
conclusions which Pasteur arrived at as the result of these labours
were as follows:

  "As for the interpretation of the group of new facts which
  I have met with in the course of these researches, I am
  confident that whoever shall judge them with impartiality will
  recognise that the alcoholic fermentation is an act correlated
  to the life and to the organisation of these corpuscles,
  and not to their death or their putrefaction, any more than
  it will appear as a case of contact action in which the
  transformation of the sugar is accomplished in the presence of
  the ferment without the latter giving or taking anything from
  it."

Pasteur occupied six years (1857-1863) with further elucidation of
his wonderful discovery of the potency of these hitherto unrecognised
agents, and the establishment of the fact that "organic liquids do not
alter until a living germ is introduced into them, and living germs
exist everywhere."

It must not be supposed that to Pasteur is due the whole credit of the
knowledge acquired respecting the cause of fermentation. He did not
first discover these living organisms; he did not first study them and
describe them; he was not even the first to suggest that they were the
cause of the processes of fermentation or disease. But, nevertheless,
it was Pasteur who "first placed the subject upon a firm foundation by
proving with rigid experiment some of the suggestions made by others."
Thus it has ever been in the times of new learning and discovery: many
contributors have added their quota to the mass of knowledge, even
though one man appearing at the right moment has drawn the conclusions
and proved the theory to be fact.

In order that no confusion may arise in the mind of the reader, we
may here say that, although fermentation is always due to a living
agent, as proved by Pasteur, the process is conveniently divided into
two kinds.[30] (1) When the action is direct, and the chemical changes
involved in the process occur only in the presence of the cell, the
latter is spoken of as an _organised ferment_; (2) when the action is
indirect, and the changes are the result of the presence of a soluble
material secreted by the cell, acting apart from the cell, this soluble
substance is termed an _unorganised soluble ferment_, or _enzyme_.
The organised ferments are bacteria or vegetable cells allied to the
bacteria; the unorganised ferments, or enzymes, are ferments found in
the secretions of specialised cells of the higher plants and animals.
With the former this book deals in an elementary fashion; with the
latter we have little concern. It will be sufficient to illustrate the
enzymes by a few of the more familiar examples. They form, for example,
the digestive agents in human assimilation. This function is performed,
in some cases, by the enzyme combining with the substance on which
it is acting and then by decomposition yielding the new "digested"
substance and regenerating the enzyme; in other cases, the enzyme, by
its molecular movement, sets up molecular movement in the substance it
is digesting, and thus changes its condition. These digestive enzymes
are as follows: in the saliva, _ptyalin_, which changes starch into
sugar; in the gastric juice of the stomach, _pepsin_, which digests the
proteids of the food and changes them into absorptive peptones; the
pancreatic ferments, _amylopsin_, _trypsin_, and _steapsin_, capable
of attacking all three classes of food stuffs; and the intestinal
ferments, which have not yet been separated in purer condition. In
addition to these, there are ferments in bitter almonds, mustard, etc.
Concerning these unorganised ferments we have nothing further to say.
Perhaps the commonest of them all is _diastase_, which occurs in malt,
and to which some reference will be made later.

Its function is to convert the starch which occurs in barley into
sugar. These unorganised ferments act most rapidly at about 75° C.
(167° F.).[31]

We may now return to the work of Pasteur and the question of _organised
ferments_. Let us preface further remark with an axiom with which
Professor Frankland sums up the vitalistic theory of fermentation,
which was supported by the researches of Pasteur: "_No fermentation
without organisms, in every fermentation a particular organism_." From
these words we gather that there is no one particular organism or
vegetable cell to be designated the micro-organism of fermentation,
but that there are a number of fermentations each started by some
specific form of agent. It is true that the chemical changes induced
by organised ferments depend on the life processes of micro-organisms
which feed upon the sugar or other substance in solution, and excrete
the product of the fermentation. Fermentation nearly always consists
of a process of breaking down of complex bodies, like sugar, into
simpler ones, like alcohol and carbonic acid. Of such fermentation
we may mention at least five: the _alcoholic_, by which alcohol is
produced; the _acetous_, by which wine absorbs oxygen from the air and
becomes vinegar; the _lactic_, which sours milk; the _butyric_, which
out of various sugars and organic acids produces butyric acid; and
_ammoniacal_, which is the putrefactive breaking down of compounds of
nitrogen into ammonia. We have already referred at some length to this
process when considering denitrifying organisms in the soil.

There are four chief conditions common to all these five kinds of
organised fermentation. They are as follows:--

1. The presence of the special living agent or organism of the
particular fermentation under consideration. This, as Pasteur pointed
out, differs in each case.

2. A sufficiency of pabulum (nutriment) and moisture to favour the
growth of the micro-organism.

3. A temperature at or about blood-heat (35-38° C., 98.5° F.).

4. The absence from the solution or substance of any obnoxious or
inimical substances which would destroy or retard the action of the
living organism and agent. Many of the products of fermentation are
themselves antiseptics, as in the case of alcohol; hence alcoholic
fermentation always arrests itself at a certain point.

We are now in a position to consider particular fermentations and
their causal micro-organisms. These latter are of various kinds,
belonging, according to botanical classification, to various different
subdivisions of the non-flowering portion of the vegetable kingdom.
A large part of fermentation is based upon the growth of a class of
microscopic plants termed _yeasts_. These differ from the bacteria in
but few particulars, mainly in their method of reproduction by budding
(instead of dividing or sporulating, like the bacteria). Their chemical
action is closely allied to that of the bacteria. Secondly, there are
special fermentations and modifications of yeast fermentation due
to _bacteria_. Thirdly, a group of somewhat more highly specialised
vegetable cells, known as _moulds_, make a perceptible contribution in
this direction. According to Hansen, these latter, so far as they are
really alcoholic ferments, induce fermentation, not only in solutions
of dextrose and invert sugar, but also in solutions of maltose. _Mucor
racemosus_ is the only member that is capable of inverting a cane-sugar
solution; _M. erectus_ is the most active fermenter, yielding eight
per cent. by volume of alcohol in ordinary beer wort. Each of these
will be referred to as they occur in considering the five important
fermentations already mentioned.

[Illustration: SACCHAROMYCES CEREVISIÆ]

The general microscopic appearance of yeast cells may be shortly
stated as follows: they are round or oval cells, and by budding become
daughter yeasts. Each consists of a membrane and clear homogeneous
contents. As they perform their function of fermentation, vacuoles,
fat-globules, and other granules make their appearance in the enclosed
plasma. As in many vegetable cells a _nucleus_ was detected by Schmitz
by means of special methods of staining, Hansen has found the nucleus
in old yeast cells from "films" without any special staining.


1. _Alcoholic Fermentation._

  Cause, yeast; medium, sugar solutions; result, alcohol and
  carbonic acid.

It was Caignard-Latour who first demonstrated that yeast cells, by
their growth and multiplication, set up a chemical change in sugar
solutions which resulted in the transference of the oxygen from the
hydrogen in the sugar compound to the carbon atoms, that is to say, in
the evolution of carbonic acid gas and the production, as a result, of
alcohol. If we were to express this in a chemical formula, it would
read as follows:

  C_{6}H_{12}O_{6} (plus the yeast) = 2 C_{2}H_{6}O + 2 CO_{2}.

A natural sugar, like grape-sugar, present in the fruit of the vine,
is thus fermented. The alcohol remains in the liquid; the carbonic
acid escapes as bubbles of gas into the surrounding air. It is thus
that brandy and wines are made. If we go a step further back, to
cane-sugar (which possesses the same elements as grape-sugar, but in
different proportions), dissolve it in water, and mix it with yeast,
we get exactly the same result, except that the first stage of the
fermentation would be the changing of the cane-sugar into grape-sugar,
which is accomplished by a soluble ferment secreted by the yeast cells
themselves. If now we go yet one step further back, to starch, the same
sort of action occurs. When starch is boiled with a dilute acid it
is changed into a gum-like substance named dextrin, and subsequently
into a sugar named maltose, which latter, when mixed with these living
yeast cells, is fermented, and results in the evolution of carbonic
acid gas and the production of alcohol. In the manufacture of fermented
drinks from cereal grains containing starch there is therefore a double
chemical process: first the change of starch into sugar by means of
_conversion_,[32] and secondly the change of the sugar into alcohol and
carbonic acid gas by the process of _fermentation_, an organic change
brought about by the living yeast cells.

In all these three forms of alcoholic fermentation the principal
features are the same, viz., the sugar disappears; the carbonic acid
gas escapes into the air; the alcohol remains behind. Though it is
true that the sugar disappears, it would be truer still to say that it
reappears as alcohol. Sugar and alcohol are built up of precisely the
same elements: carbon, hydrogen, and oxygen. They differ from each
other in the proportion of these elements. It is obvious, therefore,
that fermentation is really only a change of _position_, a breaking
down of one compound into two simpler compounds. This redistribution of
the molecules of the compound results in the production of some heat.
Thus we must add heat to the results of the work of the yeasts.

When alcohol is pure and contains no water it is termed _absolute
alcohol_. If, however, it is mixed with 16 per cent. of water, it is
called _rectified spirit_, and when mixed with more than half its
volume of water (56.8 per cent.) it is known as _proof spirit_.

We shall have to consider elsewhere a remarkable faculty which some
bacteria possess of producing products inimical to their own growth.
In some degree this is true of the yeasts, for when they have set up
fermentation in a saccharine fluid there comes a time when the presence
of the resulting alcohol is injurious to further action on their part.
It has become indeed a poison, and, as we have already mentioned, a
necessary condition for the action of a ferment is the absence of
poisonous substances. This limit of fermentation is reached when the
fermenting fluid contains 13 or 14 per cent. of alcohol.

Having discussed shortly the "medium" and the results, we may now turn
to the bacteriology of the matter, and enumerate some of the chief
forms of the yeast plant. Professor Crookshank[33] gives more than a
score of different members of this family of _Saccharomycetes_. Before
dwelling upon some of the chief of these, it will be desirable to
consider a number of properties common to the genus.

The yeast cell is a round or oval body of the nature of a fungus,
composed of granular protoplasm surrounded by a definite envelope, or
_capsule_. It reproduces itself by budding, or, as it is sometimes
termed, _gemmation_. At one end of the cell a slight swelling or
protuberance appears, which slowly enlarges. Ultimately there is
a constriction, and the bud becomes partly and at last completely
separated from the parent cell. In many cases the capsules of the
daughter cell and the parent cell adhere, thus forming a chain of
budding cells. The character of the cell and its method of reproduction
do not depend merely upon the particular species alone, but are also
dependent upon external circumstances. There are differences in the
behaviour of species towards different media at various temperatures,
towards the carbohydrates (especially maltose), and in the chemical
changes which they bring about in nutrient liquids. In connection with
this Professor Hansen has pointed out that, whilst some species can be
made use of in fermentation industries, others cannot, and some even
produce diseases in beer.[34]

[Illustration: ASCOSPORE FORMATION]

One of the most remarkable evidences of the adaptability of the yeasts
to their surroundings and a specific characteristic occurs in what
is sometimes called _ascospore_ formation. If a yeast cell finds
itself lacking nourishment or in an unfavourable medium, it reproduces
itself not by budding, but by forming spores out of its own intrinsic
substance, and within its own capsule. To obtain this kind of spore
formation Hansen used some gypsum blocks as medium on which to grow
his yeast cells. Well-baked plaster of Paris is mixed with distilled
water, and made into a liquid paste. Small moulds are made by pouring
this paste into cardboard dishes, where it hardens again. The mould is
sterilised by heat, and a small portion of yeast is placed on its upper
surface, and then the whole is floated in a small vessel of water and
covered with a bell-jar. Under these conditions of limited pabulum the
cell undergoes the following changes: it increases in size, loses much
of its granularity, and becomes homogeneous, and about thirty hours
after being sown on the gypsum there appear several refractile cells
inside the parent cell. These are the ascospores. In addition to the
gypsum, it is necessary to have a plentiful supply of oxygen, some
moisture (gained from the vessel of water in which the gypsum floats),
a certain temperature, and a young condition of the protoplasm of the
parent yeast cells. Hansen found that the lowest temperature at which
these ascospores were produced was .5-3° C., and at the other extreme
up to 37° C., which is blood-heat. The rapidity of formation also
varies with the temperature, the favourable degree of warmth being
about 22-25° C.

[Illustration: GYPSUM BLOCK]

Hansen pointed out that it was possible by means of sporulation to
differentiate species of yeasts. For it happens that different species
show slight differences in spore formation, _e. g._:

(_a_) The spores of _Saccharomyces cerevisiæ_ expand during the first
stage of germination, and produce partition walls, making a compound
cell with several chambers. Budding can occur at any point on the
surface of the swollen spores. To this group belong _S. pastorianus_
and _S. ellipsoideus_.

(_b_) The spores of _Saccharomyces Ludwigii_ fuse in the first stage,
and afterwards grow out into a promycelium, which produces yeast cells.

(_c_) The spores of _Saccharomyces anomalus_ are different in shape
from the others in that they possess a projecting rim round the base.

Another point in the cultivation of yeasts has been elucidated by a
number of workers, chief among whom perhaps is Hansen, namely, methods
of obtaining _pure cultures_. We know, generally speaking, what this
term means, and there is no difference in its meaning here to what is
understood as its meaning with regard to bacteria. There is, however,
some difference in the mode of securing it. It is only by starting
with one individual cell that we can hope to secure a pure culture of
yeasts. For the study of the morphology of yeasts under the microscope
the problem was not a difficult one. It was comparatively easy to keep
out foreign germs from a cover-glass preparation enough to perceive
germination of spores and growth of mycelium. But when we require pure
cultures for various physiological purposes, then a different standard
and method are necessary.

[Illustration: YEAST (SACCHAROMYCES CEREVISIÆ)

× 1000]

[Illustration: ASCOSPORE FORMATION IN YEAST

(The capsule of the parent cell around the spores is invisible)

× 1000]

[Illustration: NITROGEN FIXING BACTERIA FROM ROOTLET-NODULES
(SUBCULTURE)

× 1000]

[Illustration: BACILLUS OF TETANUS

(From broth culture, showing spore formation)

× 1000

  _By permission of the Scientific Press, Limited_]

Pasteur and Cohn adopted a practice based upon the fact that when
organisms find themselves in a favourable medium they multiply to the
exclusion of others to which the medium is less favourable. Hence if
an impure mixture be placed under such circumstances there comes a
time when those organisms for which the circumstances are favourable
multiply to such an extent that they form an almost pure culture. The
method is open to fallacy, and will rarely result in a really pure
culture; and even if that be secured, it is quite possible that it
will be to the exclusion of the desired culture. Hansen has devised
a much improved process for securing a pure culture of yeast which
depends upon dilution. We believe Lister was one of the first who,
in the seventies, introduced some such plan as this. Hansen employed
dilution with water in the following manner:

Yeast is diluted with a certain amount of sterilised water. A drop is
carefully examined under the microscope, a single cell of yeast is
taken, and a cultivation made upon wort. When it has grown abundantly
a quantity of sterilised water is added. From this, again, a single
drop is taken and added, to, say 20 cc. of sterilised water in a fresh
flask. This flask will contain we will suppose ten cells. It is now
vigorously shaken, and the contents are divided into twenty portions
of 1 cc. each, and added to twenty tubes of sterilised water. It is
highly probable that half of those tubes have received one cell each.
In the course of a few days it can be seen how far a culture is pure.
If only one colony is present, the culture is a pure one, and as this
grows we obtain an absolutely pure culture in necessary quantity.
Even when the gelatine-plate method is used it is desirable to start
with a single cell (Hansen). The advantage of Hansen's yeast method
over Koch's bacterial-plate method is that it has a certain definite
starting-point. This is obviously impossible when dealing with such
microscopic particles as the bacteria proper.

A third matter in the differentiation of yeast species is the question
of _films_. Hansen set to work, after having obtained pure cultures
and ascospores, to examine films appearing on the surface of liquids
undergoing fermentation. The object of this was to ascertain whether
all yeasts produced the same mycelial growth on the surface of the
fermenting fluid. To produce these films the process is as follows:
Drop on to the surface of sterilised wort in a flask a very small
quantity of a pure culture of yeast; secure the flask from movement,
and protect it, not from air, which is necessary, but from falling
particles in the air. In a short time small colonies appear, which
coalesce and form patches, then a film or membrane which covers
the liquid and attaches itself to the sides of the flask. By the
differences in the films and the temperatures at which they form it is
possible to obtain something of a basis for classification. The further
advances in a yeast culture and in our knowledge of the agencies of
fermentation have, however, tended to show that no strict dividing
lines can be drawn. Hansen's researches have, notwithstanding, been
of the greatest moment to the whole industry of fermentation. What
has been found true in bacteriology has also been demonstrated in
fermentation, namely, that though many yeasts differ but little in
structure and behaviour, they may produce very different products
and possess very different properties. Industrial cultivation of
these finer differences in fermentative action has to a large extent
revolutionised the brewing industry.

The formation of films is not a peculiarity of certain species, but
must be regarded as a phenomenon occurring somewhat commonly amongst
yeasts. The requisites are a suitable medium, a yeast cell, a free,
still surface, direct access of air, and a favourable temperature. The
wort loses colour, and becomes pale yellow. Microscopic differences
soon appear between the sedimentary yeast and the film yeast of the
same species, the latter growing out into long mycelial forms, the
character of which depends in part upon the temperature. This often
varies from 3° to 38° C.

A fourth point helpful in diagnosis is the temperature which proves to
be the thermal death-point. _Saccharomyces cerevisiæ_ is killed by an
exposure to 54° C. for five minutes, and 62° C. kills the spores. As a
rule, yeasts can resist a considerably higher temperature when in a dry
state than in the presence of moisture.

Lastly, yeasts may be cultivated on solid media. Hansen employed
wort-gelatine (5 per cent. gelatine), and found that at 25° C. in a
fortnight the growths which develop show such microscopic differences
as to aid materially in diagnosis. _Saccharomyces ellipsoideus I._
exhibits a characteristic network which readily distinguishes it.

There is one other point to which reference must be made. The process
of fermentation may be set up by a "high" or a "low" yeast. These
terms apply to the temperature at which the process commences. "High"
yeasts rise to the surface as the action proceeds, accomplish their
work rapidly, and at a comparatively high temperature, say about 16°
C.; "low" yeasts, on the contrary, sink in the fermenting fluid,
act slowly, and only at the low temperature of 4° or 5° C. This is
maintainable by floating ice in the fluid. Formerly all beer was
made by the "high" mode, but on the continent of Europe "low" yeast
is mostly used, while the "high" is in vogue in England. This latter
method is more conducive to the development of extraneous organisms,
and therefore risky in all but well-ordered brewing establishments.
Whether high and low yeasts consist of one or several species is not
known.

Before proceeding to mention shortly some of the commoner forms of
yeast we must again emphasise Hansen's method of analysis in separating
a species. The shape, size, and appearance of cells are not sufficient
for differentiation, because it is found that the same species when
exposed to different external conditions can occur in very different
forms. Hence Hansen established the analytical method of observing
(1) the microscopic appearance, (2) the formation of ascospores, and
(3) the formation of films. In addition, the temperature limits,
cultivation on solid media, and behaviour towards carbohydrates,
are characters which aid in the separation of yeasts. By basing
differentiation of species upon these features, the following can be
distinguished:

_Saccharomyces Cerevisiæ._ Oval or ellipsoidal cells; reproduction by
budding; ascospores, rapidly at 30° C., slowly at 12° C., not formed
at all at lower temperatures; film formation, seven to ten days at 22°
C.; an active alcoholic ferment, producing in a fortnight in beer wort
from 4 to 6 per cent. by volume of alcohol (Jörgensen). This species
is a typical English "high" yeast, possessing the power of "inverting"
cane-sugar previous to producing alcohol and carbonic acid. It is said
to have no action on milk-sugar.

[Illustration: S. ELLIPSOIDEUS]

[Illustration: S. PASTORIANUS]

_Saccharomyces Ellipsoideus I._ Round, oval, or sausage-shaped cells,
single or in chains; ascospores in twenty-four hours at 25° C. (not
above 30° C., not below 4° C.). Grown on the surface of wort-gelatine,
a network is produced by which they can be recognised (in eight to
twelve days at 33° C.). At 13-15° C. a characteristic branching mass
is produced. It is an alcoholic ferment as active as _S. cerevisiæ_.
_S. Ellipsoideus II._ Round and oval, rarely elongated, a widely
distributed yeast, causing "muddiness" in beer and a bitter taste. It
is essentially a "low" yeast.

_Saccharomyces Conglomeratus_ is a round cell, often united in
clusters, and occurring in rotting grapes, and at the commencement of
fermentation.

_Saccharomyces Pastorianus I._ Oval or club-shaped cells, occurring
in after-fermentation of wine, etc., and producing a bitter taste,
unpleasant odour, and turbidity. The spores frequently occur in the air
of breweries.

_S. Pastor. II._ Elongated cells, possessing an invertose ferment. They
do not, like _S. pastor I._, produce disease in beer.

_S. Pastor. III._ Oval or elongated cells, producing turbidity in beer.
Grown on yeast-water gelatine, the colonies show after sixteen days
crenated hairy edges.

_Saccharomyces Apiculatus._ Lemon-shaped cells. They give rise to a
feeble alcoholic fermentation, and produce two kinds of spores--round
and oval; they appear at the onset of vinous fermentation, but give way
later on to _S. cerevisiæ_.

_Saccharomyces Mycoderma._ Oval or elliptical cells, often in branching
chains. They form the so-called "mould" on fermented liquids, and
develop on the surface without exciting fermentation. When forced to
grow submerged they produce a little alcohol.

_Saccharomyces Exiguus._ Conical cells, appearing in the
after-fermentation of beer.

_Saccharomyces Pyriformis._ Oval cells, converting sugary solutions
containing ginger into ginger-beer.

_Saccharomyces Illicis_, _Hansenii_, _et Aquifolii_ produce a small
percentage of alcohol.


2. _Acetous Fermentation._

  Cause, Mycoderma aceti; medium, wine and other alcoholic
  liquids; result, the formation of vinegar.

If alcohol be diluted with water, and the specific ferment mixed
with it and exposed to the air at 22° C., it is rapidly converted
into vinegar. The change is accompanied by the absorption of oxygen,
one atom of which combines with two of hydrogen to form water, and
a substance remains called _aldehyde_, further oxidation of which
produces the acetic acid. We may express it chemically thus:

  Alcohol.                                  Aldehyde.    Water.

  C_{2}H_{6}O (+ oxygen and the ferment) = C_{2}H_{4}O + H_{2}O.

The aldehyde becomes further oxidised:

  C_{2}H_{4}O + O = C_{2}H_{4}O_{2} (acetic acid).

Now this method of simply oxidising alcohol to obtain acetic acid may
be carried out chemically without any ferment. If slightly diluted
alcohol be dropped upon _platinum black_, the oxygen condensed in
that substance acts with energy upon the spirit, and union readily
occurring, acetic acid results. Here the whole business of the platinum
sponge is to persuade the oxygen of the air and the hydrogen of the
alcohol to unite. In the ordinary manufacture this is accomplished by
the vegetable cells of _Mycoderma aceti_.

There are two chief methods adopted in the commercial manufacture of
vinegar, both of which depend upon the presence of the _Mycoderma_.
The method in vogue at Orleans when Pasteur (about 1862) commenced
his studies of the vinegar organism was to fill vats nearly to the
brim with a weak mixture of vinegar and wine. Where the process is
proceeding the surface is covered with a fragile pellicle, "the
mother of vinegar," which is produced by and consists of certain
micro-organisms whose function is to convey the oxygen of the air to
the liquor in the vats, thus oxidising the alcohol into vinegar. This
oxidation may be carried on even beyond the stage of acetic acid (when
no more alcohol remains to be oxidised), resulting in carbonic acid
gas, which escapes into the air. But as in the alcoholic, so in the
acetic, fermentation, there comes a time when the presence of an excess
of the acid inhibits the further growth of the organism. This point is
approximately when the acetic acid has reached a percentage as high as
14. But if the acid be removed, and fresh alcohol added, the process
recommences.

The second method, sometimes called by the Germans the "quick vinegar
process," is to pour the weakened alcohol through a tall cylinder
filled with wood-shavings, having first added some warm vinegar to the
shavings. After a number of hours the resulting fluid is charged with
acetic acid. What has occurred? Liebig maintained that a chemical and
mechanical change had brought about the change from the alcohol put
into the cylinder and the vinegar drawn off at the exit tube. It was
reserved for Pasteur to demonstrate by experiment that the addition
of the warm vinegar to the shavings was in reality an addition of a
living micro-organism, which, forming a film upon the shavings, became
"the mother of vinegar," and oxidised the alcohol which passed over it,
inducing it to become aldehyde and then acetic acid.

_Mycoderma Aceti_ (described by Persoon 1822, Kützing 1837, and Pasteur
1864). It must be understood that this term is the name rather of
a family than an individual. Pasteur believed it to be a specific
individual, but Hansen pointed out that it was composed of two
distinctly different species (_Bacterium aceti_ and _B. pasteurianum_),
and subsequently other investigators have added members to the acetic
fermentation group of which _M. aceti_ is the type.

This bacterium is made up of small, slightly elongated cells, with a
transverse diameter of 2 or 3 µ, sometimes united in short chains of
curved rods. They frequently show a central constriction, are motile,
and produce in old cultures involution forms. The way in which the
cells act and are made to perform their function is as follows: A
small quantity, taken from a previous pellicle, is sown on the surface
of an aqueous liquid, containing 2 per cent. of alcohol, 1 per cent.
of vinegar, and traces of alkaline phosphates. Very rapidly indeed
the little isolated colonies spread, and, becoming confluent, form a
membrane or pellicle over the whole area of fluid. When the surface is
covered the alcohol acidifies to vinegar. After this it is necessary
to add each day small quantities of alcohol. When the oxidation is
completed the vinegar is drawn off, and the membrane is collected and
washed, and is then again ready for use. It ought not to remain long
out of fermenting liquid, nor ought it to be allowed to over-perform
its function, for thus having oxidised all the alcohol it will commence
oxidation of the vinegar.

In wort-gelatine _Bacterium pasteurianum_ develops round colonies with
a smooth or wavy border, whilst _B. aceti_ has a tendency towards
stellate arrangement. Spores have not been observed, and from a
morphological point of view the two species behave alike. Neither
produces any turbidity in the liquid containing them. In order to
flourish, _B. aceti_ requires a temperature of about 33° C. and a
plentiful supply of oxygen. In a cool store or cellar there is,
therefore, nothing to fear from _B. aceti_. Frankland has isolated a
_Bacillus ethaceticus_, which is a fermentative organism producing
ethyl-alcohol and acetic acid. By oxidation the ethyl-alcohol may be
converted into acetic acid.


3. _Lactic Acid Fermentation._

  Cause, Bacillus acidi lactici; medium, milk-sugar, cane-sugar,
  glucose, dextrose, etc.; result, lactic acid.

The process set up by the lactic ferment is simply a decomposition, an
exact division of one molecule of sugar into two molecules of lactic
acid, there being neither oxidation nor hydration. The conditions
under which the ferment acts are very similar to those we have already
considered. There is frequently carbonic acid gas formed; there is a
cessation of fermentation when the medium becomes too acid; there
is the same method of starting the process by inoculation of sour
milk or cheese or any substance containing the specific bacillus. It
is probable that such inoculated matter will contain a mixture of
micro-organisms, but if the lactic bacillus is present, it will grow so
vigorously and abundantly that the fermentation will be readily set up.

[Illustration: B. ACIDI LACTICI]

_The Bacillus Acidi Lactici._ Rods about 2 µ long and 4 µ wide,
occurring singly or in chains and threads. It is non-motile. Spore
formation is present, the spores appearing irregularly or at one end of
the rod.

On the surface of gelatine a delicate growth appears along the track of
the needle, with round colonies appearing at the edges of the growth.
It does not liquefy gelatine. It grows best at blood-heat; but much
above that it fails to produce its fermentation, and it ceases to
grow under 10° C. It inverts milk-sugar and changes it to dextrose,
from which it then produces lactic acid. Sugars do, however, differ
considerably in the degrees to which they respond to the influence of
the lactic ferment, and some which are readily changed by the alcoholic
ferment are untouched by the _Bacillus acidi lactici_. It will be
necessary to refer again to this micro-organism when we come to speak
of milk and other dairy products.

Van Laer has described a saccharobacillus which produces lactic acid
amongst other products, and brings about a characteristic disease
in beer, named _tourne_. The liquid gradually loses its brightness
and assumes a bad odour and disagreeable taste. The bacillus is a
facultative anaërobe. A number of workers have separated organisms,
having a lactic acid effect, which diverge considerably from the
orthodox type of lactic acid bacillus. This is but further evidence of
a fact to which reference has been made: that nomenclature restricted
to one individual has now become adapted to a family.


4. _Butyric Acid Fermentation._

  Cause, Bacillus butyricus and B. amylobacter; medium, milk,
  butter, sugar and starch solutions, glycerine; result, butyric
  acid.

When sugars are broken down by the _Bacillus acidi lactici_ the lactic
acid resulting may, under the influence of the butyric ferment,
become converted into butyric acid, carbonic acid, and hydrogen.
Neither butyric acid nor lactic acid is as commonly used as alcohol
or vinegar. Both, like vinegar, can be manufactured chemically, but
this is rarely practised. Butyric acid is a common ingredient in old
milk and butter, and its production by bacteria is historically one
of the first bacterial fermentations understood. Moreover, in its
investigation Pasteur first brought to light the fact that certain
organisms acted only in the absence of oxygen. In studying a drop of
butyric fermenting fluid, it was observed that the organisms at the
edge of the drop were motionless and apparently dead, whilst in the
central portion of the drop the bacilli were executing those active
movements which are characteristic of their vitality. To Pasteur's
mind this at once suggested what he was able later to demonstrate,
namely, that these bacilli were paralysed by contact with oxygen.
When he passed a stream of air through a flask containing a liquid in
butyric fermentation, he observed the process slacken and eventually
cease. So were discovered the _anaërobic_ micro-organisms. The aërobic
ferments give rise to oxidation of certain products of decomposition;
the anaërobic organisms, on the other hand, only commence to grow
when the aërobic have used up all the available oxygen. Thus in such
fermentations certain bodies (carbohydrates, fatty acids, etc.) undergo
decomposition, and by oxidation become carbonic acid gas, and the
remainder is left as a "reduced" product of the whole process. Hence
sometimes this is termed fermentation by reduction. The chemical
formula of this butyric reaction may be expressed thus:--

  C_{6}H_{12}O_{6} (by simple decomposition) = 2 C_{3}H_{6}O_{3}.
  Glucose,                                      Lactic acid.

which is followed by the fermentation of the lactic acid:--

  2 C_{3}H_{6}O_{3} = C_{4}H_{8}O_{2} + 2 CO_{2} + 2 H_{2}.
  Lactic acid.        Butyric acid.    Carbonic   Free hydrogen.
                                       acid gas.

[Illustration: B. BUTYRICUS]

_Bacillus Butyricus._ Long and short rods, generally straight, with
rounded ends, single or in chains, reproducing themselves both by
fission and spores, and sometimes growing out into long threads,
actively motile, anaërobic, and liquefying. The spores are widely
distributed in nature, and grow readily on fleshy roots, old cheese,
etc. The favourable temperature is blood-heat, and on liquid media
they produce a pellicle. The resistant spores are irregularly placed
in the rod, and may cause considerable variations in morphology. The
culture gives off a strong butyric acid odour. It grows most readily at
a temperature of about 40° C.

Although, according to Pasteur's researches, the butyric acid ferment
performs its functions anaërobically, many butyric organisms can act in
the presence of oxygen, and yield somewhat different products.

All of them, however, ferment most actively at a temperature at or
about blood-heat, and the spores are able to withstand boiling for from
three to twenty minutes (Fitz). It will be observed that as in lactic
acid fermentation so in butyric, the results are not due to one species
only.


5. _Ammoniacal Fermentation_ (see under Soil).

_Diseases in Beer._ We have seen how a knowledge of fermentation has
been compiled by a large number of workers. Spallanzani, Schwann,
Pasteur, and Hansen all made epoch-making contributions. In the same
way the investigations of diseases in beers and wines were carried out
by many observers, and were closely connected with those relating to
spontaneous generation and mixed cultures of bacteria in fermentation.
These so-called "diseases" are analogous to the taints occurring in
milk and due to fermentations. Turning (_tourne_), turbidity, ropiness,
bitterness, acidity, mouldiness, are all terms used to describe these
diseases. They are chiefly brought about by four agencies:--

  1. Bacteria.
  2. Mixed yeasts.
  3. "Wild" yeasts.
  4. Moulds.

To each species of wild yeast there belongs some taint-producing power
in the fermentations for which it is responsible. _Saccharomyces
ellipsoideus II._ and _S. pastorianus I._, _III._, are such yeasts;
they only produce their diseases when introduced at the commencement of
the fermentation.

_Saccharomyces pastorianus I._ is a low fermentative yeast in elongated
cells, producing a _bitter_ taste to beer and an unpleasant odour. It
can also produce turbidity. _S. pastorianus III._ produces turbidity,
and _S. ellipsoideus II._ has a similar effect.

In 1883 Hansen demonstrated that the much-dreaded turbidity and
disagreeable tastes and smells in beer may be due to mixture of two
yeasts, each of which by itself gives a faultless product.

_Industrial Application of Bacterial Ferments._ From what has been
said we trust it has been made evident that bacteriology has a place
of ever-increasing importance in regard to fermentative processes. Not
only have the causal agents of various fermentations been isolated and
studied, but from their study practical results follow. The question of
pure cultures alone is one of practical importance; the recognition of
the causes of "diseases" of beer is another.

We cannot enter into a full discussion of the rôle of bacteria in
industrial processes, but several of the chief directions may be
pointed out. Without exception, bacteria have a part in them on account
of their powers of fermentation. In securing their food, bacteria
break down material, and bring about chemical and physical change.
The power which organisms have of chemically destroying compounds is
in itself of little importance, but the products which arise as a
result are of an importance in the world which has not hitherto been
recognised. We have used bacteria abundantly in the past, but we have
not perceived that we were doing so. _The maceration industries_ may
be mentioned as illustrative of this use without acknowledgment. The
flax stem is made up of cellular substance, flax fibres, and wood
fibres; the later are of no service in the making of linen, but the
whole is bound together by a gummy, resinous substance. Now this
connective element is got rid of in the process of _retting_. There
is dew-retting and water-retting. The former is practised in Russia,
and consists in spreading the flax on the grass and exposing it to the
influence of dew, rain, air, and light. The result is a soft and silky
fibre. Water-retting is accomplished by means of steeping the flax in
bundles, roots downwards, in tanks or ponds. In ten to fourteen days,
according to the weather, fermentation sets in, and breaks the "shore"
or "shive" from the fibre, and the process is complete. This is always
done by the aid of bacteria, which, under the favourable circumstances,
multiply rapidly, and cause decomposition of the pectin resinous
matter. The same operation occurs in _jute_ and _hemp_. _Sponges_, too,
are cleared in this manner by the rotting of the organic matter in
their interstices. The preparation of _indigo_ from the indigo plant
is brought about by a special bacterium found on the leaves. If the
leaves are sterilised, no fermentation occurs, and no indigo is formed.
_Tobacco-curing_ is also in part due to decomposition bacteria, and
several bacteriologists have experimented independently in fermenting
tobacco leaves by the action of pure cultures obtained from tobacco of
the finest quality.

In all these applications we have advanced only the first stage of
the journey. Nevertheless, here, as in nature on a big scale in the
formation of fertile soils and coal-measures, we find bacteria silently
at work, achieving great ends by co-operating in countless hordes.




CHAPTER V

_BACTERIA IN THE SOIL_


Surface soils and those rich in organic matter supply a varied field
for the bacteriologist. Indeed, it may be said that the introduction
of the plate method of culture and the improved facilities for growing
anaërobic micro-organisms have opened up possibilities of research into
soil micro-biology unknown to previous generations of workers.

From the nature of bacteria it will be readily understood that their
presence is affected by geological and physical conditions of the soil,
and in all soils only within a few feet of the surface. As we go down
below two feet, bacteria become less, and below a depth of five or six
feet we find only a few anaërobes. At a depth of ten feet, and in the
"ground water region," bacteria are scarce or absent. This is held
to be due to the porosity of the soil acting as a filtering medium.
Regarding the numbers of micro-organisms present in soil, no very
accurate standard can be obtained. Ordinary earth may yield anything
from 10,000 to 5,000,000 per gram, whilst from polluted soil even
100,000,000 per gram have been estimated. These figures are obviously
only approximate, nor is an exact standard of any great value.
Nevertheless, Fränkel, Beumer, Miquel, and Maggiora have, as the result
of experiments, arrived at a number of conclusions respecting bacteria
in soil which are of much more practical use. From these results it
appears that, in addition to the "ground water region" being free, or
nearly so, virgin soils contain much fewer than cultivated lands, and
these latter, again, fewer than made soils and inhabited localities. In
cultivated lands the number of organisms augments with the activity of
cultivation and the strength of the fertilisers used. In all soils the
maximum occurs in July and August.

But the condition which more than all others controls the quantity and
quality of the contained bacteria is the degree and quality of the
organic matter in the soil. The quantity of organic matter present in
soil having a direct effect upon bacteria will be materially increased
by placing in soil the bodies of men and animals after death. Dr.
Buchanan Young two or three years ago performed some experiments to
discover to what degree the soil bacteria were affected by these means.
"The number of micro-organisms present in soil which has been used for
burial purposes," he concludes, "exceeds that present in undisturbed
soil at similar level, and this excess, though apparent at all depths,
is most marked in the lower reaches of the soil."[35] The numbers were
as follows:--

  Virgin soil, 4 ft. 6 in. = 53,436 m.o. per gram of soil.
  Burial soil (8 years), 4 ft. 6 in. = 363,411 m.o. per gram of soil.
        "     (3   "  ), 6 ft. 6 in. = 722,751       "        "

_Methods of Examination of Soil._ Two simple methods are generally
adopted. The first is to obtain a qualitative estimation of the
organisms contained in the soil. It consists simply in adding to
test-tubes of liquefied gelatine or broth a small quantity of the
sample, finely broken up with a sterile rod. The test-tubes are now
incubated at 37° C. and 22° C., and the growth of the contained
bacteria observed in the test-tube, or after a plate culture has been
made. The second plan is adopted in order to secure more accurate
quantitative results. One gram or half-gram of the sample is weighed
on the balance, and then added to 1000 cc. of distilled sterilised
water in a sterilised flask, in which it is thoroughly mixed and
washed. From either of these two different sources it is now possible
to make sub-cultures and plate cultures. The procedure is, of course,
that described under the examination of water (p. 41 _et seq._), and
Petri's dishes, Koch's plates, or Esmarch's roll cultures are used.
Many of the commoner bacteria in soil will thus be detected and
cultivated. But it is obvious that this by no means covers the required
ground. It will be necessary for us here to consider the methods
generally adopted for growing anaërobic bacteria, that is to say those
species which will not grow in the presence of oxygen. This anaërobic
difficulty may be overcome in a variety of ways.

1. The air contained in the culture tube may be removed by _ebullition_
and rapid cooling. And whilst this may accurately produce a vacuum,
it is far from easy to introduce the virus without also reintroducing
oxygen.

2. The oxygen may be displaced by some other gas, and though coal-gas,
nitrogen, and carbon dioxide may all be used for this purpose, it has
become the almost universal practice to grow anaërobes in _hydrogen_.
The production of the hydrogen is readily obtained by Kipp's or some
other suitable apparatus for the generation of hydrogen from zinc and
sulphuric acid. The free gas is passed through various wash-bottles
to purify it of any contaminations. Lead acetate (1-10 per cent.
water) removes any traces of sulphuretted hydrogen, silver nitrate
(1-10) doing the same for arseniated hydrogen; whilst a flask of
pyrogallic acid will remove any oxygen. It is not always necessary to
have these three purifiers if the zinc used in the Kipp's apparatus
is pure. Occasionally a fourth flask is added of distilled water,
and this or a dry cotton wool pledget in the exit tube will ensure
germ-free gas. From the further end of the exit tube of the Kipp's
apparatus an india-rubber tube will carry the hydrogen to its desired
destination. With some it is the custom to place anaërobic cultures
in test-tubes, and the test-tubes in a large flask having a two-way
tube for entrance and exit of the hydrogen; others prefer to pass the
hydrogen immediately into a large test-tube containing the culture
(Fränkel's method). Either method ends practically the same, and the
growth of the culture in hydrogen is readily observed. Yet another plan
is to use a yeast flask, and after having passed the hydrogen through
for about half an hour, the lateral exit tube is dipped into a small
flask containing mercury. The entrance tube is now sealed, and the
whole apparatus placed in the incubator. The interior containing the
culture is filled with an atmosphere of hydrogen. No oxygen can obtain
entrance through the sealed entrance tube, or through the exit tube
immersed in mercury. Yet through this latter channel any gases produced
by the culture could escape if able to produce sufficient pressure.

[Illustration: KIPP'S APPARATUS

For the Production of Hydrogen]

[Illustration: FRÄNKEL'S TUBE

For Cultivation of Anaërobes]

3. _The Absorption Method._ Instead of adding hydrogen to the tube or
flask containing the anaërobic culture, it is feasible to add to the
medium some substances, like glucose or pyrogallic acid, which will
absorb the oxygen which is present, and thus enable the anaërobic
requirement to be fulfilled. To various media--gelatine, agar, or
broth (the latter used for obtaining the toxins of anaërobes)--2 per
cent. of glucose may be added. Pyrogallic acid, or pyrogallic acid one
part and 20 per cent. caustic potash one part, is also readily used
for absorptive purposes. A large glass tube of 25 cc. height, named a
Buchner's cylinder, having a constriction near the bottom, is taken;
and about two drachms of the pyrogallic solution are placed in the
bottom of it. A test-tube containing the culture is now lodged in the
upper part above the constriction. The apparatus is now placed in the
incubator at the desired temperature, and the contained culture grows
under anaërobic conditions. As the pyrogallic solution absorbs the
oxygen it assumes a darker tint.

[Illustration: BUCHNER'S TUBE

For Cultivation of Anaërobes]

4. _Mechanical Methods._ These include various ingenious tricks for
preventing an admittance of oxygen to the culture. An old-fashioned
one was to plate out the culture and protect it from the air by
covering it with a plate of mica. A more serviceable mode is to
inoculate, say, a tube of agar with the anaërobic organism, and then
pour over the culture a small quantity of melted agar, which will
readily set, and so protect the culture itself from the air. Oil may
be used instead of melted agar. Another mechanical method is to make
a deep inoculation and then melt the top of the medium over a bunsen
burner, and thus close the entrance puncture and seal it from the air.

5. _Absorption of Oxygen by an Aërobic Culture._ This method takes
advantage of the power of absorption of certain aërobic bacteria,
which are planted over the culture of the anaërobic species. It is not
practically satisfactory, though occasionally good results have been
obtained.

6. _Lastly, there is the Air-pump Method._ By this means it is
obviously intended to extract air from the culture and seal of it
_in vacuo_. The culture tubes are connected with the air-pump, and
exhausted as much as possible.

Of these various methods it is on the whole best to choose either the
hydrogen method, the vacuum, or the plan of absorption by grape-sugar
or pyrogallic. In anaërobic plate cultures grape-sugar agar plus 0.5
per cent. of formate of soda may be used. The poured inoculated plate
should be placed over pyrogallic solution under a sealed bell-glass
and incubated at 37° C. Pasteur, Roux, Joubert, Chamberland, Esmarch,
Kitasato, and others have introduced special apparatus to facilitate
anaërobic cultivation, but the principles adopted are those which have
been mentioned.


THE QUALITATIVE ESTIMATION OF BACTERIA IN THE SOIL

We may now turn to consider the species of bacteria found in the
interstices of soil. They may be classified in five main groups. The
division is somewhat artificial, but convenient:

1. _The Denitrifying Bacteria._ A group whose function has been
elucidated in recent years (largely by the investigations of Professor
Warington) are held responsible for the breaking down of nitrates. With
these may be associated the _Decomposition or Putrefactive Bacteria_,
which break down complex organic products other than nitrates into
simpler bodies.

[Illustration: A METHOD OF GROWING CULTIVATIONS IN A VACUUM OVER
PYROGALLIC SOLUTION]

2. _The Organisms of Nitrification._ To this group belong the two
chief types of nitrifying bacteria, viz., those which oxidise ammonia
into nitrites, and those which change nitrites into nitrates.

3. _The Nitrogen-fixing Bacteria_, found mainly in the nodules on the
rootlets of certain plants.

4. _The Common Saprophytic Bacteria_, whose function is at present but
imperfectly known. Many are putrefactive germs.

5. _The Pathogenic Bacteria._ This division includes the three types,
tetanus, malignant œdema, and quarter evil. Under this heading
we shall also have to consider in some detail the intimate relation
between the soil and such important bacterial diseases as tubercle and
typhoid.

To enable us to appreciate the work which the "economic bacteria"
perform, it will be necessary to consider shortly the place they occupy
in the economy of nature. This may be perhaps most readily accomplished
by studying the accompanying table (p. 145).

A SCHEME SHOWING THE PLACE AND FUNCTION OF THE ECONOMIC MICRO-ORGANISMS
FOUND IN SOIL

                   _Water_   _Chemical Substances_    _Gases_
                      \        [Nitrates, etc.]    [CO_{2}, H, N, O]
                       \            |                    /
                        \           |                   /
                         _|        \|/                |_
                                PLANT LIFE
                                    |
        +------------+----------+---+--------+-------------+----------+
        |            |          |            |             |          |
  Carbohydrates     Fats     Proteids     Vegetable     Mineral     Water
  [albumoses,                [bodies        Acids        Salts
   sugar,                     containing
   starch,                    Nitrogen]
   etc.]                           |
                                   |
                               ANIMAL LIFE
                                   |
    +-----------------+------------+---+-----------------------+
    |                 |                |                       |
  Gases             Water      Urea, Albuminoids,        Nitrogen in many
  [CO_{2}, etc.]             Ammonia compounds, etc.     forms locked up
                                                         in the body

                               \_______________________________________/
                                                 |
                                 PUTREFACTIVE AND DENITRIFYING BACTERIA
                                                 |
        +--------------+----------+-------------+-------------------+
        |              |          |             |                   |
  Free Nitrogen      Gases      Water        Ammonia            [Nitrites]
        |           [CO_{2}]            and other elements
        |                                 of broken-down
        |                                 complex bodies.
        |
        |                                   \_______________________/
        |                                               |
        |                                       NITRIFYING BACTERIA
        |                                         |
        |                                     Nitrites[=Nitrous organism
        |                                         |      (Nitrosomans)]
  NITROGEN-FIXING                             Nitrates[=Nitric organism
    BACTERIA                                             (Nitrosomonas)]
  [In soil and in the nodules                [In soil and available for
   on the rootlets of _Leguminosæ_]             plant life]

The threefold function of plant life is nutrition, assimilation, and
reproduction: the food of plants, the digestive and storage power of
plants, and the various means they adopt for multiplying and increasing
their species. With the two latter we have little concern in this
place. Respecting the nutrition of plant life, it is obvious that,
like animals, they must feed and breathe to maintain life. Plant food
is of three kinds, viz., _water_, _chemical substances_, and _gas_.
Water is an actual necessity to the plant not only as a direct food
and food-solvent, but as the vehicle of important inorganic materials.
The hydrogen, too, of the organic compounds is obtained from the
decomposition of the water which permeates every part of the plant,
and is derived by it from the soil and from the aqueous vapour in
the atmosphere. The chief _chemical substances_ of which vegetable
protoplasm is constituted are six, viz, potassium, magnesium, calcium,
iron, phosphorous, and sulphur. These inorganic elements do not enter
the plant as such, but combined with other substances or dissolved in
water. _Potassium_ occurs in salt form combined with various organic
acids (tartaric, oxalic, etc.), _calcium_ and _magnesium_ as salts of
lime and magnesia in combination both with organic and inorganic acids.
_Iron_ contributes largely to the formation of the green colouring
matter of plants, and is also derived from the soil. _Phosphorus_,
one of the chief constituents of seeds, generally occurs as phosphate
of lime. _Sulphur_, which is an important constituent of albumen, is
derived from the sulphates of the soil. In addition to the above, there
are other elements, sometimes described as non-essential constituents
of plants. Amongst these are _silica_ (to give stiffness), _sodium_,
_chlorine_, _iodine_, _bromine_, etc. All these elements contribute to
the formation or quality of the protoplasm of plants.

The _gases_ essential to plants are four: Carbon dioxide (carbonic
acid), Hydrogen, Oxygen, and Nitrogen. By the aid of the green
chlorophyll corpuscles, and under the influence of sunlight, we know
that leaves absorb the carbon dioxide of the atmosphere, and effect
certain changes in it. The hydrogen, as we have seen, is obtained from
the water. Oxygen is absorbed through the root from the interstices
of the soil. Each of these contributes vitally to the existence of
the plant. The fourth gas, _nitrogen_, which constitutes more than
two thirds of the air we breathe (79 per cent. of the total volume
and 77 per cent. of the total weight of the atmosphere), is, perhaps,
the most important food required by plants. Yet, although this is so,
the plant cannot absorb or obtain its nitrogen in the same manner
in which it acquires its carbon--viz., by absorption through the
leaves--nor can the plant take nitrogen into its own substance by any
means as _nitrogen_, with the exception of the flesh-feeding plants
(insectivorus). Hence, although this gas is present in the atmosphere
surrounding the plant, the plant will perish if nitrogen does not
exist in some combined form in the soil. Nitrates and compounds of
ammonia are widely distributed in nature, and it is from these bodies
that the plant obtains, by means of its roots, the necessary nitrogen.

Until comparatively recently it was held that plant life could not
be maintained in a soil devoid of nitrogen or compounds thereof. But
it has been found that certain classes of plants (the _Leguminosæ_,
for example), when they are grown in a soil which is practically free
from nitrogen at the commencement, do take up this gas into their
tissues. One explanation of this fact is that free nitrogen becomes
converted into nitrogen compounds in the soil through the influence
of micro-organisms present there. Another explanation attributes
this fixation of free nitrogen to microorganisms existing in the
rootlets of the plant. These two classes of organisms, known as the
nitrogen-fixing organisms, will require our consideration at a later
stage. Here we merely desire to make it clear that the main supply
of this gas, absolutely necessary to the existence of vegetable life
upon the earth, is drawn not from the nitrogen of the atmosphere,
but from that contained in nitrogen compounds in the soil. The most
important of these are the _nitrates_. Here then we have the necessary
food of plants expressed in a sentence: _water, gases, salts, the most
important and essential gas and some of the salts being combined in
nitrates_.

Plant life seizes upon its required constituents, and by means of the
energy furnished by the sun's rays builds these materials up into
its own complex forms. Its many and varied forms fulfil a place in
beautifying the world. But their contribution to the economy of nature
is, by means of their products, to supply food for animal life. The
products of plant life are chiefly sugar, starch, fat, and proteids.
Animal life is not capable of extracting its nutriment from soil, but
it must take the more complex foods which have already been built up
by vegetable life. Again, the complementary functions of animal and
vegetable life are seen in the absorption by plants of one of the waste
materials of animals, viz., carbonic acid gas. Plants abstract from
this gas carbon for their own use, and return the oxygen to the air,
which in its turn is of service to animal life.

By animal activity some of these foods supplied by the vegetable
kingdom are at once decomposed into carbonic acid gas and water, which
goes back to nature. Much, however, is built up still further into
higher and higher compounds. The proteids are converted by digestion
into albumoses and peptones, ultimately entirely into peptones; these
in their turn are reconverted into proteids, and become assimilated as
part of the living organism. In time they become further changed into
carbonic acid, sulphuric acid, water, and certain not fully oxidised
products,[36] which contain the nitrogen of the original proteid. In
the table these bodies have been represented by one of their chief
members, viz., _urea_.

It is clear that there is in all animal life a double process
continually going on; there is a building up (anabolism, assimilation),
and there is a breaking down (katabolism, dissimilation). These
processes will not balance each other throughout the whole period
of animal life. We have, as possibilities, elaboration, balance,
degeneration; and the products of animal life will differ in degree and
in substance according to which period is in the predominance. These
products we may subdivide simply into excretions during life and final
materials of dissolution after death, both of which may be used more
or less immediately by other forms of animal or vegetable life, or
mediately after having passed to the soil. We may shortly summarise the
final products of animal life as carbonic acid, water, and nitrogenous
remnants. These latter will occur as urea, new albumens, compounds of
ammonia, and nitrogen compounds of great complexity stored up in the
tissues and body of the animal. The carbonic acid, water, and other
simple substances like them will return to nature and be of immediate
use to vegetable life. But otherwise the cycle cannot be completed, for
the more complex bodies are of no service as such to plants or animals.

1. In order that this complex material should be of service in the
economy of nature, and its constituents not lost, it is necessary that
it should be broken down again into simpler conditions. This prodigious
task is accomplished by the agency of two groups of organisms,
_the decomposition and denitrifying_[37] _bacteria_. The organisms
associated with decomposition processes are numerous; some denitrify
as well as break down organic compounds. This group will be referred
to under "Saprophytic Bacteria." The reduction by the denitrifying
bacteria may be simply from nitrate to nitrite, or from nitrate to
nitric or nitrous oxide gas, or indeed to nitrogen itself. In all these
processes of reduction the rule is that a loss of nitrogen is involved.
How that free nitrogen is brought back again and made subservient to
plants and animals we shall understand at a later stage.

Professor Warington has again recently set forth the chief facts known
of this decomposition process.[38] That the action in question only
occurs in the presence of living organisms was first established by
Mensel in 1875 in natural waters, and by Macquenne in 1882 in soils.
If all living organisms are destroyed by sterilisation of the soil,
denitrification cannot take place, nor can vegetable life exist.
"Bacteria reduce nitrates," says Professor Warington, "by bringing
about the combustion of organic matter by the oxygen of the nitrate,
the temperature distinctly rising during the operation." The reduction
to a nitrite is a common property of bacteria. But only a few species
have the power of reducing a nitrate to gas. These few species are,
however, widely distributed. In 1886 Gayon and Dupetit first isolated
the bacteria capable of reducing nitrates to the simplest element,
nitrogen. They obtained their species from sewage, but ten years later
denitrifying bacteria were isolated from manure. That soil contains a
number of these reducing organisms is known by introducing a particle
of surface soil into some broth, to which has been added one per cent.
of nitre. During incubation of such a tube gas is produced, and the
nitrate entirely disappears.

Whenever decomposition occurs in organic substances there is a
reduction of compound bodies, and in such cases the putrefying
substances obtain their decomposing and denitrifying bacteria from
the air. The chief conditions requisite for bringing about a loss of
nitrogen by denitrification are enumerated by Professor Warington as
follows: (1) the specific micro-organism; (2) the presence of a nitrate
and suitable organic matter; (3) such a condition as to aëration that
the supply of atmospheric oxygen shall not be in excess relatively
to the supply of organic matter; (4) the usual essential conditions
of bacterial growth. "Of these," he says, "the supply of organic
matter is by far the most important in determining the extent to which
denitrification will take place." The necessarily somewhat unstable
condition facilitates its being split up by means of bacteria. The
bacteria in their turn are ready to seize upon any products of animal
life which will serve as their food. Thus, by reducing complex bodies
to simple ones, these denitrifying organisms act as the necessary link
to connect again the excretions of the animal body, or after death the
animal body itself, with the soil.

In a book of this nature it has been deemed advisable not to enter into
minute description of all the species of bacteria mentioned. Some of
the chief are described more or less fully. We cannot, however, do
more than name several of the chief organisms concerned in reducing
and breaking down compounds. As we shall find in the bacteria of
nitrification, so also here, the _entire_ process is rarely, if ever,
performed by one species. There is indeed a remarkable division of
labour, not only between decomposition bacteria and denitrification
bacteria, but between different species of the same group. _Bacillus
fluorescens non-liquefaciens_, _Mycoderma ureæ_, and some of the
staphylococci break down nitrates (denitrification), and also decompose
other compound bodies. Amongst the group of putrefactive bacteria found
in soil may be named _B. coli_, _B. mycoides_, _B. mesentericus_, _B.
liquidus_, _B. prodigiosus_, _B. ramosus_, _B. vermicularis_, _B.
liquefaciens_, and many members in the great family of _Proteus_. Some
perform their function in soil, others in water, and others, again, in
dead animal bodies. Dr. Buchanan Young, to whose researches in soil
we have referred, has pointed out that in the upper reaches of burial
soil, where these bacteria are most largely present, there is as a
result no excess of organic carbon and nitrogen. Even in the lower
layers of such soil it is rapidly broken down.

[Illustration: MICROCOCCUS FROM SOIL]

It will be observed, from a glance at the table, that the chief results
of decomposition and denitrification are as follows: free nitrogen,
carbonic acid, gas and water, ammonia bodies, and sometimes nitrites.
The nitrogen passes into the atmosphere, and is "lost"; the carbonic
acid and water return to nature and are at once used by vegetation.
The ammonia and nitrites await further changes. These further changes
become necessary on account of the fact, already discussed, that plants
require their nitrogen to be in the form of nitrates in order to use
it. Nitrates obviously contain a considerable amount of oxygen, but
ammonia contains no oxygen, and nitrites very much less than nitrates.
Hence a process of oxidation is required to change the ammonia into
nitrites and the nitrites into nitrates.

2. This oxidation is performed by the _nitrifying microorganisms_,
and the process is known as _nitrification_. It should be clearly
understood that the process of nitrifaction may, so to speak, dovetail
with the process of denitrification. No exact dividing line can be
drawn between the two, although they are definite and different
processes. In a carcass, for example, both processes may be going on
concomitantly; so also in manure. There is no hard and fast line to be
drawn in the present state of our knowledge. Other organisms beside the
true nitrification bacteria may be playing a part, and it is impossible
exactly to measure the action of the latter, where they began and where
the preliminary attack upon the nitrogenous compounds terminated. In
all cases, however, according to Professor Warington, the formation of
ammonia has been found to precede the formation of nitrous or nitric
acid.

It was Pasteur who (in 1862) first suggested that the production of
nitric acid in soil might be due to the agency of germs, and it is to
Schlösing and Müntz that the credit belongs for first demonstrating (in
1877) that the true nature of nitrification depended upon the activity
of a living micro-organism. Partly by Schlösing and Müntz and partly
by Warington (who was then engaged in similar work at Rothamsted),
it was later established (1) that the power of nitrification could be
communicated to substances which did not hitherto nitrify by simply
seeding them with a nitrified substance, and (2) that the process of
nitrification in garden soil was entirely suspended by the vapour of
chloroform or carbon disulphide. The conditions for nitrification, the
limit of temperature, and the necessity of plant food, have furnished
additional proof that the process is due to a living organism. These
conditions are briefly as follows:

1. Food (of which phosphates are essential constituents). "The
nitrifying organism can apparently feed upon organic matter, but it
can also, apparently with equal ease, develop and exercise all its
functions with purely inorganic food" (Warington).

Winogradsky prepared vessels and solutions carefully purified from
organic matter, and these solutions he sowed with the nitrifying
organism, and found that they flourished. Professor Warington has
employed the acid carbonates of sodium and calcium with distinct
success as ingredients of an ammoniacal solution undergoing
nitrification.

2. The next condition of nitrification is the presence of oxygen.
Without it the reverse process, denitrification, occurs, and instead
of a building up we get a breaking down, with an evolution of nitrogen
gas. The amount of oxygen present has an intimate proportion to the
amount of nitrification, and with 16 to 21 per cent. of oxygen present
the nitrates are more than four times as much as when the smallest
quantity of oxygen is supplied. The use of tillage in promoting
nitrification is doubtless in part due to the aëration of the soil thus
obtained.

3. A third condition is the presence of a base with which nitric acid
when formed may combine. Nitrification can take place only in a feebly
alkaline medium, but an excess of alkalinity will retard the process.

4. The last essential requirement is a favourable temperature. The
nitrifying organism can act at a temperature as low as 37° or 39° F.
(3-4° C.), but at a higher temperature it becomes much more active.
According to Schlösing and Müntz, at 54° F. (12° C.) nitrification
becomes really active, and it increases as the temperature rises to 99°
F. (37° C.), after which it falls. A high temperature or a strong light
are prejudicial to the process.

We are now in a position to consider shortly some of the characters
of these nitrification bacteria. They may readily be divided into
two chief groups, not in consideration of their form or biological
characteristics, but on account of the duties which they perform. Just
as we observed that there were few denitrifying organisms which could
break down ammonia compounds to nitrogen gas, so is it also true that
there are few nitrifying bacteria which can build up from ammonia to
the nitrates. Nature has provided that this shall be accomplished in
two stages, viz., a first stage from ammonia bodies to nitrites, and
a second stage from nitrites to nitrates. The agent of the former is
termed the _nitrous_ organism, the latter the _nitric_ organism. Both
are contributing to the final production of nitrates which can be used
by plant life.[39]

_The Nitrous Organism_ (Nitrosomonas). Prior to Koch's gelatine method
the isolation of this bacterium proved an exceedingly difficult task.
But even the adoption of this isolating method seemed to give no better
results, and for an excellent reason: the nitrifying organisms will
not grow on gelatine. To Winogradsky and Percy Frankland belongs the
credit of separately isolating the nitrous organism on the surface of
gelatinous silica containing the necessary inorganic food. Professor
Warington, in his lectures under the Lawes Agricultural Trust, has
described this important germ as follows:

  "The organism as found in suspension in a freshly nitrified
  solution consists largely of nearly spherical corpuscles,
  varying extremely in size. The largest of these corpuscles
  barely reaches a diameter of one-thousandth of a millimetre,
  and some are so minute as to be hardly discernible in
  photographs. The larger ones are frequently not strictly
  circular, and are sometimes seen in the act of dividing.

  "Besides the form just described, there is another, not
  universally present in solutions, in which the length is
  considerably greater than its breadth. The shape varies, being
  occasionally a regular oval, but sometimes largest at one end,
  and sometimes with the ends truncated. The circular organisms
  are probably the youngest.

  "This organism grows in broth, diluted milk, and other
  solutions without producing turbidity. When acting on ammonia
  it produces only nitrites. It is without action on potassium
  nitrite. It is, in fact, the nitrous organism which, as we
  have previously seen, may be separated from soil by successive
  cultivations in ammonium carbonate solution."

The elongated forms appear to be a sign of arrested growth. Normally
the organ is about 1.8 µ long, or nearly three times as long as the
nitric organism. It possesses a gelatinous capsule. "The motile cells,
stained by Löffler's method, are seen to have a flagellum in the form
of a spiral." When grown on silica the nitrous organism appears in
the same two forms--zooglea and free cells--as when cultivated in a
fluid. It commences to show growth in about four days, and is at its
maximum on about the tenth day. Winogradsky found that there were
considerable differences in the morphology of the organism according to
the soil from which it was taken. One of the Java soils he investigated
contained a nitrous organism having a spiral flagellum of thirty
micromillimetres; but its movement was slow.

As we have already seen, the most astonishing property of this organism
is its ability to grow and perform its specific function in solutions
absolutely devoid of organic matter. Some authorities hold that it
acquires its necessary carbon from carbonic acid. The mode of culturing
it is as follows:

To sterilised flasks add 100 cc. of a solution made of one gram of
ammonium sulphate, one gram of potassium sulphate, and 1000 cc. of
pure water. To this add one gram of basic magnesium carbonate which
has been previously sterilised by boiling. Now inoculate the flask
with a small portion of the soil under investigation, and after four
or five days sub-culture on the same medium in fresh flasks, and let
this be repeated half a dozen times. Now, as this inorganic medium
is unfavourable to ordinary bacteria of soil, it is clear that after
several sub-cultures the nitrous organism will be isolated in pure
culture.

Winogradsky employs for culturing upon solid media a mineral gelatine.
A solution of from 3 to 4 per cent. of silicic acid in distilled water
is placed in flasks. By the addition of the following salts to such a
solution gelatinisation occurs:

        { Ammonium sulphate        0.4 gram
  (_a_) { Magnesium sulphate       0.05 "
        { Calcium chloride         A trace

        { Potassium phosphate      0.1     gram
  (_b_) { Sodium carbonate         0.6, 0.9 "
        { Distilled water          100 cc.

The sulphates and chloride are mixed in 50 cc. of distilled water,
and the latter substance in the remaining 50 cc. in separate flasks.
After sterilisation and cooling these are all mixed and added in small
quantities to the silicic acid.

Upon this medium it is possible to sub-culture a pure growth from the
film at the bottom of the flasks in which the nitrous organism is first
isolated.

_The Nitric Organism._ It was soon learned that the nitrous organism,
even when obtainable in large quantities and in pure culture, was not
able entirely to complete the nitrifying process. As early as 1881
Professor Warington had observed that some of his cultures, though
capable of changing nitrites into nitrates, had no power of oxidising
ammonia. These he had obtained from advanced sub-cultures of the
nitrous organism, and somewhat later Winogradsky isolated and described
this companion of the nitrous organism. It develops freely in solutions
to which no organic matter has been added; indeed, much organic matter
will prevent its growing. He isolated it from soils from various parts
of the world on the following media:

  Water                     1000.0
  Potassium phosphate          1.0
  Magnesium sulphate           0.5
  Calcium chloride         A trace
  Sodium chloride              2.0

About 20 cc. of this solution is placed in a flat-bottom flask, and a
little freshly washed magnesium carbonate is added. The flask is closed
with cotton wool, and the whole is sterilised. To each flask 2 cc. of
a 2 per cent. solution of ammonium sulphate is subsequently added.
The temperature for incubation is 30° C. Winogradsky concluded that
the oxidation of nitrites to nitrates was brought about by a specific
organism independently of the nitrous organism. He successfully
isolated it in silica jelly. He believes the organism, like its
companion, derives its nutriment solely from inorganic matter, but this
is not finally established.

The form of the nitric organism (or _nitromonas_, as it was once
termed) is allied to the nitrous organism. The cells are elongated,
rarely oval, but sometimes pear-shaped. They are more than half a
micromillimetre in length, and somewhat less in thickness. The cells
have a gelatinous membrane. Like the other nitrifying bacteria, its
development and action are favoured by the presence of the acid
carbonates of calcium and sodium. Of the latter, six grams per litre
or even a smaller quantity gives good results. The sulphate of calcium
can be used, but the organism prefers the carbonates. The differences
between these two bacteria are small, with the exception of their
chemical action. The nitric organism has no action upon ammonia, and
the presence of any considerable amount of ammonium carbonate hinders
its development and prevents its action on a nitrite.[40]

We may here summarise the general facts respecting nitrification.
Winogradsky proposes to term the group _nitro-bacteria_, and to
classify thus:

                      { Nitrosomonas, containing at least two species,
  Nitrous organisms = {   viz., the European and the Java.
                      { Nitrosococcus.
  Nitric organism   =   Nitrobacter.

Nitrification occurs in two stages, each stage performed by a distinct
organism. By one (_nitrosomonas_) ammonia is converted into nitrite; by
the other (_nitrobacter_) the nitrite is converted into nitrate.[41]
Both organisms are widely and abundantly distributed in the
superficial soils. They act together and in conjunction, and for one
common purpose. They are separable by employing favourable media.

  "If we employ a suitable inorganic solution containing
  potassium nitrite, but no ammonia, we shall presently obtain
  the nitric organism alone, the nitrous organism feeding on
  ammonia being excluded. If, on the other hand, we employ an
  ammonium carbonate solution of sufficient strength, we have
  selected conditions very unfavourable to the growth of the
  nitric organism, and a few cultivations leave the nitrous
  organism alone in possession of the field" (Warington).

[Illustration: NITROUS ORGANISM

× 1000]

[Illustration: NITRIC ORGANISM

× 1000]

[Illustration: NITROGEN FIXING ORGANISM FROM SECRETION OF ROOT-NODULES

× 1000]

A word upon the natural distribution of these nitrifying bacteria
before we leave them. They belong to the soil, river water, and sewage.
They are also said to be frequently present in well water. From some
experiments at Rothamsted it appears that the organisms occur mostly
in the first twelve inches, and in subsoils of clay down to three or
four feet. In sandy soils nitrification may probably occur at a greater
depth. These facts should be borne in mind when arranging for the
purification of sewage by intermittent filtration.

We have now given some consideration to the chief events in the
life-cycle of nature depicted in the table. There is but one further
process in which bacteria play a part, and which requires some mention.
It will have been noticed that at certain stages in the cycle there
is more or less appreciable "loss" of free nitrogen. In the process
of decomposition brought about by the denitrifying bacteria, a very
considerable portion of the nitrogen is dissipated into the air
in the form of a free gas. This is the last stage of all proteid
decomposition, so that wherever putrefaction is going on there is a
continual "loss" of an element essential to life. Thus it would appear
at first sight that the sum-total of nitrogen food must be diminishing.

But there are other ways also in which nitrogen is being set free.
In the ordinary processes of vegetation there is a gradual draining
of the soil and a passing of nitrogen into the sea; the products of
decomposition pass from the soil by this drainage, and are "lost" as
far as the soil is concerned. Many of the methods of sewage disposal
are in reality depriving the land of the return of nitrogen which is
its necessity. Again, nitrogen is freed in explosions of gunpowder,
nitroglycerine, and dynamite, for whatever purpose they are used. Hence
the great putrefactive "loss" of nitrogen, with its subsidiary losses,
contributes to reduce this essential element of all life, and if there
were no method of bringing it back again to the soil, it would seem
that plant life, and therefore animal life, would speedily terminate.

It is at this juncture, and to perform this vital function, that the
_nitrogen-fixing bacteria_ play their wonderful part: they bring back
the free nitrogen and fix it in the soil. Excepting a small quantity
of combined nitrogen coming down in rain and in minor aqueous deposits
from the atmosphere, the great source of the nitrogen of vegetation
is the store in the soil and subsoil, whether derived from previous
accumulations or from recent supplies by manure.

Sir William Crookes has recently[42] pointed out the vast importance of
using all the available nitrogen in the service of wheat production.
The distillation of coal in the process of gas-making yields a certain
amount of its nitrogen in the form of sulphate of ammonia, and this,
like other nitrogenous manures, might be used to give back to the soil
some of the nitrogen drained from it. But such manuring cannot keep
pace, according to Sir W. Crookes, with the present loss of fixed
nitrogen from the soil. We have already referred to several ways
in which "loss" of nitrogen occurs. To these may well be added the
enormous loss occurring in the waste of sewage when it is passed into
the sea. As the President of the British Association pointed out,[43]
the more widely this wasteful system is extended, recklessly returning
to the sea what we have taken from the land, the more surely and
quickly will the finite stocks of nitrogen, locked up in the soils of
the world, become exhausted. Let us remember that the plant creates
nothing in this direction; there is nothing in wheat which is not
absorbed from the soil, and unless the abstracted nitrogen is returned
to the soil, its fertility must be ultimately exhausted. When we apply
to the land sodium nitrate, sulphate of ammonia, guano, and similar
manurial substances, we are drawing on the earth's capital, and our
drafts will not be perpetually responded to.[44] We know that a virgin
soil cropped for several years loses its productive powers, and without
artificial aid becomes unfertile. For example, through this exhaustion
forty bushels of wheat per acre have dwindled to seven. Rotation of
crops is an attempt to meet the problem, and the four-course rotation
of turnips, barley, clover, and wheat witnesses to the fact that
practice has been ahead of science in this matter.

The store of nitrogen in the atmosphere is practically unlimited,
but it is fixed and rendered assimilable only by cosmic processes of
extreme slowness. We may shortly glance at these, for it is upon these
processes, plus a return to the soil of sewage, that we must depend in
the future for storing nitrogen as nitrates.

1. Some combined nitrogen is absorbed by the soil or plant from the
air, for example, fungi, lichens, and some algæ, and the absorption
is in the form of ammonia and nitric acid. This is admittedly a small
quantity.

2. Some free nitrogen is fixed within the soil by the agency of porous
and alkaline bodies.

3. Some, again, may be assimilated by the higher chlorophyllous plants
themselves, independently of bacteria (Frank).

4. Electricity fixes, and may in the future be made to fix more,
nitrogen. If a strong inductive current be passed between terminals,
the nitrogen from the air enters into combination with the oxygen,
producing nitrous and nitric acids.

5. Abundant evidence has now been produced in support of the fact that
there is considerable fixation by means of bacteria.

Bacterial life in several ways is able to reclaim from the atmosphere
this free nitrogen, which would otherwise be lost. The first method
to which reference may be made is that involving _symbiosis_. This
term signifies "a living together" of two different forms of life,
generally for a specific purpose. It may be to mutual advantage, a
living for one another, or it may be, by means of an interchange of
metabolism or products, finally to produce or obtain some remote
chemical result. It is convenient to restrict the term symbiosis to
complementary partnerships such as exist between algoid and fungoid
elements in lichens, or between unicellular algæ and Radiolarians,[45]
or between bacteria and higher plants. The partnerships between
hermit crabs and sea-anemones and the like are sometimes defined by
the term _commensalism_ (joint diet). Symbiosis and commensalism must
be distinguished from _parasitism_, which indicates that all the
advantage is on the side of the parasite, and nothing but loss on the
side of the host. The distinction between symbiosis and commensalism
cannot be rigid, but between these conditions which are advantageous
to the partners and parasitism, there is an obvious and radical
difference. Association of organisms together for increase of virulence
and function should be distinguished from symbiosis, and mere existence
of two or more species of bacteria in one medium is not, of course,
symbiosis. Most frequently such a condition would result in injury
and the subsequent death of the weaker partner, an effect precisely
opposite to that defined by this term.

The example of bacteriological symbiosis with which we are concerned
here is that partnership between bacteria and some of the higher plants
(_Leguminosæ_) for the purpose of fixing nitrogen in the plant and in
the surrounding soil.

[Illustration: ROOTLET OF PEA WITH NODULES]

_The Nitrogen-fixing Bacteria_, the third group of micro-organisms
connected with the soil, exist in groups and colonies situated inside
the nodules appearing, under certain circumstances, on the rootlets of
the pea, bean, and other _Leguminosæ_. It was Hellriegel and Wilfarth
who first pointed out that, although the higher chlorophyllous plants
could not directly obtain or utilise free nitrogen, some of them
at any rate could acquire nitrogen brought into combination under
the influence of bacteria. Hellriegel found that the gramineous,
polygonaceous, cruciferous, and other orders depended upon combined
nitrogen supplied within the soil, but that the _Leguminosæ_ did not
depend entirely upon such supplies.

It was observed that in a series of pots of peas to which no nitrogen
was added most of the plants were apparently limited in their growth by
the amount of nitrogen locked up in the seed. Here and there, however,
a plant, under apparently the same circumstances, grew luxuriantly
and possessed on its rootlets abundant nodules. The experiments of
Sir John Lawes and Sir Henry Gilbert at Rothamsted[46] demonstrated
further that under the influence of suitable microbe-seeding of the
soil in which _Leguminosæ_ were planted there is nodule formation on
the roots, and coincidentally increased growth and gain of nitrogen
beyond that supplied either in the soil or in the seed as combined
nitrogen. Presumably this is due to the fixation, in some way, of free
nitrogen. Nobbe proved the gain of nitrogen by non-leguminous plants
(Elœagnus, etc.) when these grow root nodules containing bacteria,
but to all appearances, bacteria differing morphologically from the
_Bacillus radicicola_ of the leguminous plants.

[Illustration: NITROGEN FIXING BACTERIA _IN SITU_ IN NODULE ON ROOTLET
OF PEA

× 400

  --Cellular sheath of Rootlet forming capsule of nodule.

  --Colonies of bacteria _in situ_.]

[Illustration: NITROGEN FIXING BACTERIA _IN SITU_ IN ROOT-NODULE OF PEA

(Section of Nodule)

× 500]

[Illustration: NITROGEN FIXING BACTERIA _IN SITU_ IN ROOT-NODULE OF PEA

(Section of Nodule)

× 600]

These facts being established, the question naturally arises, How
is the fixation of nitrogen to be explained, and by what species
of bacteria is it performed? In the first place, these matters are
simplified by the fact that there is very little fixation indeed by
bacteria in the soil apart from symbiosis with higher plants. Hence we
have to deal mainly with the work of bacteria in the higher plant. Sir
Henry Gilbert concludes[47] that the alternative explanations of the
fixation of free nitrogen in the growth of _Leguminosæ_ seem to be:

  "1. That under the conditions of symbiosis the plant is
  enabled to fix the free nitrogen of the atmosphere by its
  leaves;

  "2. That the nodule organisms become distributed within the
  soil and there fix free nitrogen, the resulting nitrogenous
  compounds becoming available as a source of nitrogen to the
  roots of the higher plant;

  "3. That free nitrogen is fixed in the course of the
  development of the organisms within the nodules, and that the
  resulting nitrogenous compounds are absorbed and utilized by
  the host." "Certainly," he adds, "the balance of evidence at
  present at command is much in favour of the third mode of
  explanation."

If this is finally proved to be the case, it will furnish another
excellent example of the power existing in bacteria of assimilating an
elementary substance.

Most authorities would agree that all absorption of free nitrogen, if
by means of bacteria, must be through the roots. As a matter of fact,
legumes, especially when young, use nitrogen, like all other plants,
derived from the soil. It has been pointed out that, unless the soil is
somewhat poor in nitrogen, there appears to be but little assimilation
of free nitrogen and but a poor development of root nodules.[48] The
free nitrogen made use of by the micro-organism is in the air contained
in the interstices of the soil. For in all soils, but especially
in well-drained and light soils, there is a large quantity of air.
Although it is not known how the micro-organisms in legumes utilise
free nitrogen and convert it into organic compounds in the tissues
of the rootlet or plant, it is known that such nitrogen compounds
migrate into the stem and leaves, and so make the roots really poorer
in nitrogen than the foliage. But the ratio is a fluctuating one,
depending chiefly on the stage of growth or maturity of the plant.

If the nodules from the rootlets of _Leguminosæ_ be examined, the
nitrogen-fixing bacteria can be readily seen. The writer has isolated
these and grown them in pure culture as follows: The nodules are
removed, if possible at an early stage in their growth, and placed for
a few minutes in a steam steriliser. This is advisable in order to
remove the various extraneous organisms attached to the outer covering
of the nodule. They may then be washed in antiseptic solution, and
their capsules softened by soaking. When opened with a sterile knife,
thick creamy matter exudes. On microscopic examination this is found
to be densely crowded with small round-ended bacilli or oval bodies,
known as _bacteroids_. By a simple process of hardening and using the
microtome, excellent sections of the nodules can be obtained which
show these bacteria _in situ_. In the central parts of the section may
be seen densely crowded colonies of the bacteria, which in some cases
invade the cellular capsule of the nodule derived from the rootlet.
Aërobic and anaërobic pure cultures of these bacteria were made. In
some cases these cultures very closely resembled the feathery growth of
the bacillus of anthrax.

4. _The Saprophytic Bacteria in Soil._ This group of micro-organisms
is by far the most abundant as regards number. They live on the dead
organic matter of the soil, and their function appears to be to
break it down into simpler constitution. Specialisation is probably
progressing among them, for their name is legion, and the struggle for
existence keen. After we have eliminated the economic bacteria, most
of which are obviously saprophytes, the group is greatly reduced. It
is also needless to add that of the remnant little beyond morphology
is known, for as their function is learned they are classified
otherwise. It is probable, as suggested, that many of the species of
common saprophytes normally existent in the soil act as _auxiliary
agents_ to denitrification and putrefaction. At present we fear they
are disregarded in equal measure, and for the same reasons, as the
common water bacteria. An excess of either, in soil or water, is not
of itself injurious as far as we know; indeed, it is probably just
the reverse. It is, however, frequently an index of value as to the
amount and sometimes condition of the contained organic matter. The
remarks made when considering water bacteria apply here also, viz.,
that an excess of saprophytes acts not only as index of increase of
organic matter, but as at first auxiliary, and then detrimental, to
pathogenic organisms. It will require accurate knowledge of soil
bacteria generally to be able to say which saprophytic germs, if
any, have no definite function beyond their own existence. It may be
doubted whether the stern behests of nature permit of such organisms.
However that may be, we may feel confident, though at present there
are many common bacteria in soil, as also in water, the life object of
which is not ascertained, that as knowledge increases and becomes more
accurate this special provisional group will become gradually absorbed
into other groups having a part in the economy of nature, or in the
production of disease. At present the decomposition, denitrifying,
nitrifying,[49] and nitrogen-fixing organisms are the only saprophytes
which have been rescued from the oblivion of ages, and brought more or
less into daylight. It is but our lack of knowledge which requires the
present division of saprophytes whose business and place in the world
is unknown.

5. _The Pathogenic Organisms found in Soil._ In addition to these
saprophytes and the economic bacteria, there are, as is now well known,
some disease-producing bacteria finding their nidus in ordinary soil.
The three chief members of this group are the bacillus of Tetanus
(lockjaw), the bacillus of Quarter Evil, and the bacillus of Malignant
Œdema.

_Tetanus._ The pathology of this terrible disease has during recent
years been considerably elucidated. It was the custom to look upon it
as "spontaneous," and arising no one knew how; now, however, after the
experiments of Sternberg and Nicolaier, the disease is known to be due
to a micro-organism common in the soil of certain localities, existing
there either as a bacillus or in a resting stage of spores. Fortunately
Tetanus is comparatively rare, and one of the peculiar biological
characteristics of the bacillus is that it grows only in the absence of
oxygen. This fact contributed not a little to the difficulties which
were met with in securing its isolation.

Tetanus occurs in man and horses most commonly, though it may affect
other animals. There is usually a wound, often an insignificant one,
which may occur in any part of the body. The popular idea that a severe
cut between the thumb and the index finger leads to tetanus is without
scientific foundation. As a matter of fact, the wound is nearly always
on one or other of the limbs, and is infected simply because they come
more into contact with soil and dust than does the trunk. It is not the
locality of the wound nor its size that affects the disease. A cut with
a dirty knife, a gash in the foot from the prong of a gardener's fork,
the bite of an insect, or even the prick of a thorn have before now
set up tetanus. Wounds which are jagged, and occurring in absorptive
tissues, are those most fitted to allow the entrance of the bacillus.
The wound forms a local manufactory, so to speak, of the bacillus and
its secreted poisons; the bacillus always remains in the wound, but
the toxins may pass throughout the body, and are especially absorbed
by the cells of the central nervous system, and thus give rise to the
spasms which characterise the disease. Suppuration generally occurs in
the wound, and in the pus thus produced may be found a great variety
of bacteria, as well as the specific agent itself. After a few days
or, it may be, as much as a fortnight, when the primary wound may be
almost forgotten, general symptoms occur. Their appearance is often the
first sign of the disease. Stiffness of the neck and facial muscles,
including the muscles of the jaw, is the most prominent sign. This is
rapidly followed by spasms and local convulsions, which, when affecting
the respiratory or alimentary tract, may cause a fatal result. Fever
and increased rate of pulse and respiration are further signs of the
disease becoming general. After death, which results in the majority of
cases, there is very little to show the cause of fatality. The wound
is observable, and patches of congestion may be found on different
parts of the nervous system, particularly the medulla (grey matter),
pons, and even cerebellum. Evidence has recently been forthcoming
at the Pasteur Institute to support the theory that tetanus is a
nervous disease, more or less allied to rabies, and is best treated by
intra-cerebral injection of antitoxin, which then has an opportunity of
opposing the toxins at their favourite site. (Roux and Borrel.)

In the wound the bacillus is present in large numbers, but mixed
up with a great variety of suppurative bacteria and extraneous
organisms. It is in the form of a straight short rod with rounded
ends, occurring singly or in pairs of threads, and slightly motile.
It has been pointed out that by special methods of staining, flagella
may be demonstrated.[50] These are both lateral and terminal, thin
and thick, and are shed previously to sporulation. Branching also has
been described. Indeed, it would appear that, like the bacillus of
tubercle, this organism has various pleomorphic forms. Next to the
ordinary bacillus, filamentous forms predominate, particularly so in
old cultures. Clubbed forms, not unlike the bacillus of diphtheria,
may often be seen from agar cultures. Without doubt the most peculiar
characteristic of this bacillus is its sporulation. The well-formed
round spores occur readily at incubation temperature. They occupy a
position at one or other pole of the bacillus, and have a diameter
considerably greater than the rod. Thus the well-known "drumstick" form
is produced. In practice the spores occur freely in the medium and
in microscopical preparation. Like other spores, they are extremely
resistant to heat, desiccation, and antiseptics. They can resist
boiling for several minutes.

[Illustration: BACILLUS OF TETANUS]

As we have seen, this bacillus is a strict anaërobe, growing only in
the absence of oxygen. The favourable temperature is 37° C., and it
will only grow very slowly at or below room temperature.

An excellent culture is generally obtainable in glucose gelatine. The
growth occurs, of course, only in the depth of the medium, and appears
as fine threads passing horizontally outwards from the track of the
needle. At the top and bottom of the growth these fibrils are shorter
than at the middle or somewhat below the middle. For extraction of the
soluble products of the bacillus glucose broth may be used.

In some countries, and in certain localities, the bacillus of tetanus
is a very common habitant of the soil, and when one thinks how
frequently wounds must be more or less contaminated with such soil, the
question naturally arises, How is it that the disease is, fortunately,
so rare? Probably we must look to the advance of bacteriological
science to answer this and similar questions at all adequately. Much
has recently been done in Paris and elsewhere to emphasise the relation
which other organisms have to such bacteria as those of typhoid and
tetanus. When considering typhoid, we saw that in addition to the
presence of the specific germ other conditions were requisite before
the disease actually occurred. So in tetanus, Kitasato and others have
pointed out that the presence of certain other bacteria, or of some
foreign body, is necessary to the production of the disease. The common
organisms of suppuration are particularly accused of increasing the
virulence of the bacillus of tetanus. How these auxiliary organisms
perform this function has not been fully elucidated. Probably, however,
it is by damaging the tissues and weakening their resistance to such a
degree as to afford a favourable multiplying ground for the tetanus. It
is right to state that some authorities hold that they act by using up
the surrounding oxygen, and so favouring the growth of tetanus.

_Quarter Evil_ (or symptomatic anthrax) is a disease of animals,
produced in a manner analogous to tetanus. It is characterised by a
rapidly increasing swelling of the upper parts of the thigh, sacrum,
etc., which, beginning locally, may attain to extraordinary size and
extent. It assumes a dark colour, and crackles on being touched. There
is high temperature, and secondary motor and functional disturbances.
The disease ends fatally in two or three days.

Slight injuries to the surface of the skin or mucous membrane are
sufficient for the introduction of the causal bacillus. This organism
is, like tetanus, an anaërobe, existing in the superficial layers
of the soil. From its habitat it readily gains entrance to animal
tissues. It has spores, but though they are of greater diameter than
the bacillus itself they are not absolutely terminal. Hence they merely
swell out the capsule of the bacillus, and produce a club-shaped rod.
They form gas while growing in the tissues and in artificial culture.
External physical conditions have little effect upon this bacillus,
and the dried and even buried flesh retains infection for a very long
period of time.

[Illustration: B. OF SYMPTOMATIC ANTHRAX]

[Illustration: B. OF MALIGNANT ŒDEMA]

The third disease-producing microbe found naturally in soil is that
which produces the disease known as Malignant Œdema. Pasteur called
this _gangrenous septicœmia_. Unlike quarter evil, malignant
œdema may occur in man in cases where wounds have become septic.
Animals become inoculated with this bacillus from the surface of soil,
straw-dust, upper layers of garden-earth, or decomposing animal and
vegetable matter.

The bacillus occurs in the blood and tissues as a long thread, composed
of slender segments of irregular length. It is motile and anaërobic.
The spores are larger than the diameter of the bacillus, and the
organism produces gas; so much is this the case in artificial culture,
that the medium itself is frequently split up.

Both malignant œdema and symptomatic anthrax are similar in some
respects to anthrax itself. There are, however, a number of points for
differential diagnosis. The enlargement of the spleen, the non-motility
of the bacillus, the enormous numbers of bacilli throughout the body,
the square ends, equal inter-bacillary spaces, aërobic growth, and
characteristic staining afford ample evidence of anthrax.

_The Relation of Soil generally to certain Bacterial Diseases._ Recent
investigations have, in effect, considerably added to our knowledge of
pathogenic germs in soil; and whilst the three species enumerated above
are still considered as types normally present in soil, it must not be
forgotten that other virulent disease producers either live in the soil
or are greatly influenced by its conditions.

Fränkel and Pasteur have both demonstrated the possible presence of
anthrax. Fränkel maintained that it could not live there long, and at
ten feet below the surface no growth occurred. This may have been due
to the low temperature of such a depth. Pasteur held that earthworms
are responsible for conveying the spores of anthrax from buried
carcasses to the surface, and thus bringing about reinfection. Cholera,
too, has been successfully grown in soil, except during winter. The
presence of common saprophytes in the soil is prejudicial to the
development of the cholera spirillum, and under ordinary circumstances
it succumbs in the struggle for existence. From experiments recently
conducted for the Local Government Board by Dr. Sidney Martin, evidence
is forthcoming in support of the view that the bacillus of typhoid can
live in certain soils. Samples of soil polluted with organic matter
formed a favourable environment for living bacilli of typhoid for
456 days, whereas in sterilised soil, without organic matter, these
organisms lived only twenty-three days. Tubercle also has been kept
alive for several weeks in soil.

In passing, a single remark may be made in relation to the long
periods during which bacteria can retain vitality in soil. Farm soils
have, as is well known, been contaminated with anthrax in the late
summer or autumn, and have retained the infectious virus till the
following spring, and it has even then cropped up again in the hay of
the next season. In 1881 Miquel took some samples of soil at a depth of
ten inches, containing six and a half million bacteria per gram. After
drying for two days at 30° C., the dust was placed in hermetically
sealed tubes, which were put aside in a dark corner of the laboratory
for sixteen years. Upon re-examination it is reported that more than
three million germs per gram were still found, amongst them the
specific bacillus of tetanus. Whether or not there is any fallacy in
these actual figures, there is abundant evidence in support of the fact
that bacteria, non-pathogenic and pathogenic, can and do retain their
vitality, and sometimes even their virulence, for almost incredibly
long periods of time.

It is now some years since Sir George Buchanan, for the English Local
Government Board, and Dr. Bowditch, for the United States, formulated
the view that there is an intimate relationship between dampness
of soil and the bacterial disease of Consumption (tuberculosis of
the lungs). The matter was left at that time _sub judice_, but the
conclusion has been drawn, and surely a legitimate one, that the
dampness of the soil acted injuriously in one of two ways. It either
lowered the vitality of the tissues of the individual, and so increased
his susceptibility to the disease, or in some way unknown favoured
the life and virulence of the bacillus. That is one fact. Secondly,
Pettenkofer traced a definite relationship between the rise and fall
of the ground water with pollution of the soil and enteric (typhoid)
fever.[51] A third series of investigations concluded in the same
direction, viz., the researches of Dr. Ballard respecting summer
diarrhœa. This, it is generally held, is a bacterial disease,
although no single specific germ has been isolated as its cause.
Ballard demonstrated that the summer rise of diarrhœa mortality does
not commence until the mean temperature of the soil, recorded by the
four-foot thermometer, has attained 56.4° F., and the decline of such
diarrhœa coincides more or less precisely with the fall in soil
temperature. This temperature (56.4° F.) is, therefore, considered
as the "critical" four-foot earth temperature, that is to say, the
temperature at which certain changes (putrefactive, bacterial, etc.)
take place in the pores of the earth, with the consequent development
of the diarrhœal poison.

After a very elaborate and prolonged investigation on behalf of
the Local Government Board, Dr. Ballard formulates the causes of
diarrhœa in the following conclusions:[52]

(_a_) The essential cause of diarrhœa resides ordinarily in the
superficial layers of the earth, where it is intimately associated with
the life processes of some micro-organism not yet detected or isolated.

(_b_) That the vital manifestations of such organism are dependent,
among other things, perhaps principally upon conditions of season and
the presence of dead organic matter, which is its pabulum.

(_c_) That on occasion such micro-organism is capable of getting abroad
from its primary habitat, the earth, and having become air-borne,
obtains opportunity for fastening on non-living organic material,
and of using such organic matter both as nidus and as pabulum in
undergoing various phases of its life history.

(_d_) That from food, as also from contained organic matter of
particular soils, such micro-organism can manufacture, by the chemical
changes wrought therein through certain of its life processes, a
substance which is a virulent chemical poison.

Here, then, we have a large mass of evidence from the data collected
by Buchanan, Bowditch, Pettenkofer, and Ballard. But much of this work
was done anterior to the time of the application of bacteriology to
soil constitution. Recently the matter has received increased attention
from various workers abroad, and in England from Dr. Sidney Martin,
Professor Hunter Stewart, Dr. Robertson, and others. The greater part
of this work we cannot here consider. But some reference must be
made to Dr. Robertson's admirable researches into the growth of the
bacillus of typhoid in soil. By experimental inoculation of soil with
broth cultures, he was able to isolate the bacillus twelve months
after, alive and virulent. He concludes that the typhoid organism is
capable of growing very rapidly in certain soils, and under certain
circumstances can survive from one summer to another. The rains of
spring and autumn or the frosts and snows of winter do not kill them
off so long as there is sufficient organic pabulum. Sunlight, the
bactericidal power of which is well known, had, as would be expected,
no effect except upon the bacteria directly exposed to its rays. The
_bacillus typhosus_ quickly dies out in the soil of grass-covered
areas. Dr. Robertson holds that the chief channel of infection between
typhoid-infected soil and man is dust. As in tubercle and anthrax,
so in typhoid, dried dust or excreta containing the bacillus is the
vehicle of disease.

Hitherto we have addressed ourselves to those diseases the known
causal organisms of which reside, normally or abnormally, in the
soil. But closely allied to these matters connected with the rôle of
pathogenic bacteria in soil is the question of what has been termed the
_miasmatic_ influence of soil. The term "miasm" has had an extensive
and somewhat diffuse application in medical science. It may happen in
the future that typhoid will be classified strictly as a miasmatic
disease. But at present, in the transition state of the science, it
would hardly be justifiable to classify typhoid with a typically
miasmatic disease like malaria. Yet it is clear that mention should
here be made of a group of diseases of which malaria is the type, and
of which the tropics generally are the native land. The bacterial
etiology of the group is by no means worked out. The cause of malaria
alone is not yet a closed subject. However the details of the etiology
of this group finally arrange themselves, there is little doubt of two
facts, viz., the diseases are probably produced by bacteria or allied
protozoa, and soil plays an important part in their production.

From what has been said, it will be seen that though a considerable
amount of knowledge has been obtained respecting bacteria in the soil,
it may be conjectured that actually there is still a great deal to
ascertain before the micro-biology of soil is in any measure complete
or even intelligent. The mere mention of tetanus and typhoid in the
soil, and their habits, nutriment, and products therein, not to mention
the work of the economic bacteria, is to open up to the scientific
mind a vast realm of possibility. It is scarcely too much to say that
a fuller knowledge of the part which soil plays in the culture and
propagation of bacteria may suffice to revolutionise the practice of
preventive medicine. Truly, our knowledge at the moment is rather a
heterogeneous collection of isolated facts and theories, some of which,
at all events, require ample confirmation; still, there is a basis for
the future which promises much constructive work.




CHAPTER VI

_BACTERIA IN MILK, MILK PRODUCTS, AND OTHER FOODS_


Injurious micro-organisms in foods are, fortunately for the consumers,
usually killed by cooking. Vast numbers are, as far as we know, of
no harm whatever. Alarming reports of the large numbers of bacteria
which are contained in this or that food are generally as irrelevant
as they are incorrect. Bacteria, as we have seen, are ubiquitous. In
food we have abundance of the chief thing necessary to their life and
multiplication--favourable nutriment. Hence we should expect to find in
uncooked or stale food an ample supply of saprophytic bacteria. There
was much wholesome truth in the assertions made some two years ago by
the late Professor Kanthack, to the effect that good food as well as
bad frequently contained large numbers of bacteria, and often of the
same species. It is well that we should become familiarised with this
idea, for its accuracy cannot be doubted, and its usefulness at the
present time may not be without its beneficial effect.

Nevertheless, it is well we should know the bacterial flora of good and
bad foods for at least two reasons. First, there is no doubt whatever
that a considerable number of cases of poisoning can be traced every
year to food containing harmful bacteria or their products. To several
of the more notorious cases we shall have occasion to refer in passing.
Secondly, we may approach the study of the bacteriology of foods with
some hope that therein light will be found upon some important habits
and effects of microbes. There can be little doubt that food-bacteria
afford an example of association and antagonism of organisms to which
reference has already been made. Any information that can be gleaned to
illumine these abstruse questions would be very welcome at the present
time. But there is a still further, and possibly an equally important,
point to bear in mind, namely, the economic value of microbes in food.
In a short account like the present it will be impossible to enter into
hypotheses of pathology, but we shall at least be able to consider
some of these interesting experiments which have been conducted in the
sphere of beneficial bacteria.

The injurious effects of organisms contained in foods has been
elucidated by the excellent work of the late Dr. Ballard. From the
careful study of a number of epidemics due to food poisoning, this
patient observer was able, without the aid of modern bacteriology,
to arrive at a simple principle which must not be forgotten. Food
poisoning is due either to bacteria themselves or to their products,
which are contained in the substance of the food. In cases of the
first kind, bacteria gaining entrance to the human alimentary canal,
set up their specific changes and produce their toxins, and by so
doing in course of time bring about a diseased condition, with its
consequent symptoms. On the other hand, if the products, sometimes
called _ptomaines_, are ingested as such, the symptoms set up by their
action in the body tissues appear earlier. From these facts Dr. Ballard
deduced the simple principle that if there is no incubation period or,
at all events, a comparatively short space of time between eating the
poisoned food and the advent of disease, the agents of the disease are
products of bacteria. If, on the other hand, there is an incubation
period, the agents are probably bacteria.

It is necessary to mention two other facts. Dr. Cautley[53] has
recently been engaged in isolating from poisoned foods the different
species of bacteria present. It would appear that these are limited,
as a rule, to two or three kinds. As regards disease, the organisms of
suppuration are the most common. Liquefying or fermentative bacteria
are frequently present, the _Proteus_ family being well represented.
In addition there are, according to circumstances, a number of common
saprophytes. Now, as we have pointed out, these organisms may act
injuriously by some kind of cooperation, or they may by themselves
be harmless, and pathological conditions be due to the occasional
introduction of pathogenic species.

The other fact, requiring recognition from anyone who proposes to study
the bacteriology of foods, is that a certain appreciable amount of
the responsibility for food poisoning rests with the tissues of the
individual ingesting the food. There is ample evidence in support of
the fact that not all the persons partaking of infected food suffer
equally, and occasionally some escape altogether. We know little or
nothing of the causes of such modification in the effect produced.
It may be due to other organisms, or chemical substances already in
the alimentary canal of the individual, or it may be due to some
insusceptibility or resistance of the tissues. Be that as it may, it is
a matter which must not be neglected in estimating the effects of food
contaminated with bacteria or their products.

_Milk._ There are few liquids in general use which contain such
enormous numbers of germs as milk. To begin with, milk is in every
physical way admirably adapted to be a favourable medium for bacteria.
It is constituted of all the chief elements of the food upon which
bacteria live. It is frequently at a temperature favourable to their
growth. It is _par excellence_ an absorptive fluid. A dish of ordinary
water and a dish of newly drawn milk laid side by side, and under
similar conditions of temperature, will rapidly demonstrate the
difference in degree of absorptivity between the two fluids. Yet,
whilst this general fact is true, we must emphasise at the outset the
possibility and practicability of securing absolutely pure sterile
milk. Recently some milking was carried out under strict antiseptic
precautions, with the above sterile result. The udder was thoroughly
cleansed, the hands of the milker washed with corrosive sublimate and
then pure water, the vessels which were to receive the milk had been
carefully sterilised, and the whole process was carried out in strict
cleanliness. The result was that the sample of milk remained sweet and
good and contained no germs. It should be stated that the first flow of
milk, washing out the milk-ducts of the udder, was rejected. This fact
of the sterility of cleanly drawn milk is not a new one, and has been
established by many bacteriologists. Milk, then, is normally a sterile
secretion. How does it gain its enormously rich flora of bacteria?

_Sources of Pollution of Milk._ These are various, and depend upon many
minor circumstances and conditions. For all practical purposes there
are three chief opportunities between the cow and the consumer when
milk may become contaminated with bacteria:

1. At the time of milking.

2. During transit to the town, or dairy, or consumer.

3. After its arrival.

_Pollution at the Time of Milking_ arises from the animal, the
milker, or unclean methods of milking. It is now well known that in
tuberculosis of the cow affecting the udder the milk itself shows the
presence of the bacillus of tubercle. In a precisely similar manner all
bacterial diseases of the cow which affect the milk-secreting apparatus
must inevitably add their quota of bacteria to the milk. To this matter
we shall have occasion to refer again. There is a further contamination
from the animal when it is kept unclean, for it happens that the
unclean coat of a cow will more materially influence the number of
micro-organisms in the milk than the popularly supposed fermenting food
which the animal may eat. It is from this external source rather than
from the diet that organisms occur in the milk. The hairy coat offers
many facilities for harbouring dust and dirt. The mud and filth of
every kind that may be habitually seen on the hinder quarters of cattle
all contribute largely to polluted milk. Nor is this surprising. Such
filth at or near the temperature of the blood is an almost perfect
environment for many of the putrefactive bacteria.

The milker is also a source of risk. His hands, as well as the clothes
he is wearing, can and do readily convey both innocent and pathogenic
germs to the milk. Clothed in dust-laden garments, and frequently
characterised by dirty hands, the milker may easily act as an excellent
purveyor of germs. Not a few cases are also on record where it
appears that milkers have conveyed germs of disease from some case of
infectious disease, such as scarlet fever, in their homes. But under
the more efficient registration of such disease which has recently
characterised many dairy companies, the danger of infection from this
source has been reduced to a minimum. The habit of moistening the hands
with a few drops of milk previous to milking is one to be strongly
deprecated.

Professor Russell recounts a simple experiment which clearly
demonstrates these simple but effective sources of pollution:

  "A cow that had been pastured in a meadow was taken for the
  experiment, and the milking done out of doors, to eliminate
  as much as possible the influence of germs in the barn air.
  Without any special precaution being taken the cow was
  partially milked, and during the operation a covered glass
  dish, containing a thin layer of sterile gelatine, was exposed
  for sixty seconds underneath the belly of the cow in close
  proximity to the milk-pail. The udder, flank, and legs of
  the cow were then thoroughly cleaned with water, and all of
  the precautions referred to before were carried out, and the
  milking then resumed. A second plate was then exposed in the
  same place for an equal length of time, a control also being
  exposed at the same time at a distance of ten feet from the
  animal and six feet from the ground to ascertain the germ
  contents of the surrounding air. From this experiment the
  following instructive data were gathered. Where the animal was
  milked without any special precautions being taken there were
  3250 bacterial germs _per minute_ deposited on an area equal
  to the exposed top of a ten-inch milk-pail. Where the cow
  received the precautionary treatment as suggested above, there
  were only 115 germs per minute deposited on the same area.
  In the plate that was exposed to the surrounding air at some
  distance from the cow there were 65 bacteria. This indicates
  that a large number of organisms from the dry coat of the
  animal can be kept out of milk if such simple precautions as
  these are carried out."[54]

The influence of the barn air, and the cleanliness or otherwise of
the barn, is obviously great in this matter. As we have seen, moist
surfaces retain any bacteria lodged upon them; but in a dry barn,
where molecular disturbance is the rule rather than the exception, it
is not surprising that the air is heavily laden with microbic life.
Here again many improvements have been made by sanitary cleanliness
in various well-known dairies. Still there is much more to be done
in this direction to ensure that the drawn milk is not polluted by a
microbe-impregnated atmosphere.

The risks in _transit_ differ according to many circumstances. Probably
the commonest source of contamination is in the use of unclean utensils
and milk-cans. Any unnecessary delay in transit affords increased
opportunity for multiplication; particularly is this the case in the
summer months, for at such times all the conditions are favourable to
an enormous increase of any extraneous germs which may have gained
admittance at the time of milking. Thus we have (1) the milk itself
affording an excellent medium and supplying ideal pabulum for bacteria,
(2) a more or less lengthened railway journey or period of transit
giving ample time for multiplication, (3) the favourable temperature
of summer heat. We shall refer again to the rate of multiplication of
germs in milk.

Lastly, many are the advantages given to bacteria when milk has reached
its commercial destination. In milk-shops and in the home there are not
a few risks to be added on to the already imposing category. Water is
occasionally, if not frequently, added to milk to increase its volume.
Such water of itself will make its own contribution to the flora of the
milk, unless indeed, which is unlikely, the water has been recently and
thoroughly boiled before addition to the milk. Again, it is impossible
to suppose that in small homes--perhaps of only one room--where the
milk stands for several hours, pollution is avoidable. From a hundred
different sources such milk runs the risk of being polluted.

Before proceeding, a word must be said respecting the first milk
which flows from the udder in the process of milking, and which is
known as the _fore-milk_. This portion of the milk is always rich in
bacterial life on account of the fact that it has remained in the
milk-ducts since the last milking. However thorough the manipulation,
there will always be a residue remaining in the ducts, which will,
and does, afford a suitable nidus and incubator for organisms. The
latter obtain their entrance through the imperfectly closed teat
of the udder, and pass readily into the milk-duct, sometimes even
reaching the udder itself and setting up inflammation (_mastitis_).
Professor Russell states that he has found 2800 germs in the fore-milk
in a sample of which the average was only 330 per cc. Schultz found
83,000 micro-organisms per cc. in the fore-milk, and only 9000 in
the mid-milk. As a matter of fact, most of this large number belong
to the _lactic-acid fermentation_ group, and the fore-milk rarely
contains more than two or three species, and still more rarely any
disease-producing bacteria. Still, they occur in such enormous numbers
that their addition to the ordinary milk very materially alters its
quality. Bolley and Hall, of North Dakota, report sixteen species of
bacteria in the fore-milk, twelve of which produced an acid reaction.
Dr. Veranus Moore, of the United States Department of Agriculture,[55]
concludes from a large mass of data that freshly drawn fore-milk
contains a variable but generally enormous number of bacteria, but
only several species, the last milk containing, as compared with
the fore-milk, very few micro-organisms. The bacteria which become
localised in the milk-ducts, and which are necessarily carried into the
milk, are for the greater part rapidly acid-producing organisms, _i.
e._, they ferment milk-sugar, forming acids. They do not produce gas.
Still their presence renders it necessary to "pasteurise" as soon as
possible. Dr. Moore holds that much of the intestinal trouble occurring
in infants fed with ordinarily "pasteurised" milk arises from acids
produced by these bacteria between the drawing of the milk and the
pasteurisation.

_The Number of Bacteria in Milk._ From all that has been said
respecting the sources of pollution and the favourable nidus which milk
affords for bacteria, it is not surprising that a very large number of
germs are almost always present in milk. The quantitative estimation
of milk appears more alarming than the qualitative. It is true some
diseases are conveyed by bacteria in milk, but on the whole most of the
species are non-pathogenic. Nor need the numbers, though serious, too
greatly alarm us, for, as we shall see at a later stage, disease is a
complicated condition, and due to other agencies and conditions than
merely the bacteria, which may be the _vera causa_. In addition to the
fact that the high numbers have but a limited significance, we must
also remember that there is no uniformity whatever in these numbers.
The conditions which chiefly control them are (1) temperature, (2) time.

_The Influence of Temperature._ We have already noticed, when
considering the general conditions affecting bacteria, how potent
an agent in their growth is the surrounding temperature. Generally
speaking, temperature at or about blood-heat favours bacterial growth.
Freudenreich has drawn up the following table which graphically sets
forth the effect of temperature upon bacteria in milk:


          3 hours.  6 hours.  9 hours.  24 hours.

  59° F.     1 +       2.5         5        163
  77° F.     2        18.5       107     62,100
  95° F.     4       1,290     3,800      5,370

This instructive table claims some observations. It will be noticed
that at 59° F. there is very little multiplication. That may be
accepted as a rule. At 77° F. the multiplication, though not
particularly rapid at the outset, results finally, at the end of
the twenty-four hours, in the maximum quantity. These were probably
common species of saprophytic bacteria, which increase readily at a
comparatively low temperature. During the subsequent hours, after the
twenty-four, we should expect a decline rather than an increase in
62,000, owing to the keen competition consequent upon the limitation
of the pabulum. From a consideration of these figures we conclude that
a warm temperature, somewhat below blood-heat, is most favourable
to multiplication of bacteria in milk; that the common saprophytic
organisms multiply the most rapidly; that, in the course of time,
competition kills off a large number.

Let us take another example, from Professor Conn:

                                77° F.             95° F.
   2 hours after milking    (liquefied the
                          plate of gelatine)       1,275,000
   6   "     "      "          14,620,000         45,900,000
   9   "     "      "          36,550,000         57,800,000
  24   "     "      "      13,702,000,000     13,812,500,000

  [Bacteria per cub. inch.]

These almost incredibly large figures illustrate much the same points,
particularly the rapid multiplication at blood-heat, and the later rise
at 77° C.

_The Influence of Time_ is not less marked than that of temperature, as
the following table will show:

  Milk drawn at 59° C. =     153,000  m.o.  per  cub.  in.
  After 1 hour         =     616,000   "     "    "     "
    "   2 hours        =     539,000   "     "    "     "
    "   4   "          =     680,000   "     "    "     "
    "   7   "          =   1,020,000   "     "    "     "
    "   9   "          =   2,040,000   "     "    "     "
    "  24   "          =  85,000,000   "     "    "     "

        (Conn.)

Freudenreich gives another example, as follows:

  Milk drawn at 15.5° C. =     27,000  m.o.  per  cc.
    After  4 hours       =     34,000   "     "    "
      "    9   "         =    100,000   "     "    "
      "   24   "         =  4,000,000   "     "    "

Concerning these figures little comment is necessary. But here again,
we may remember that this rapid multiplication continues only up to a
certain point, after which competition brings about a marked reduction.

The effect of temperature and time has been illustrated by Dr. Buchanan
Young's recent researches, laid before the Royal Society of Edinburgh.
He estimated that in the Edinburgh milk supply three hours after
milking there were 24,000 micro-organisms per cc. _in winter_; 44,000
_in spring_; 173,000 _in late summer and autumn_. Again, he found that
five hours after milking there were 41,000 micro-organisms per cc. in
country milk, and more than 350,000 micro-organisms per cc. in town
milk. Many London milks would exceed 500,000 per cc.[56]

There is no standard or uniformity in the numerical estimation of
bacteria in milk. A host of observers have recorded widely varying
returns due to the widely varying circumstances under which the milk
has been collected, removed, stored, and examined. Nor is it possible
to establish any standard which may be accepted as a _normal_ or
healthy number of bacteria, as is done in water examination. Bitter has
suggested 50,000 micro-organisms per cc. as a maximum limit for milk
intended for human consumption.

But owing to differences of nomenclature and classification, in
addition to differences in mode of examination at present existing
in various countries, it is impossible to state even approximately
how many bacteria and how many species of bacteria have been isolated
from milk. Until some common international standard is established
mathematical computations are practically worthless. They are
needlessly alarming and sensational. And it should be remembered that
great reliance cannot be placed upon these numerical estimations.
They vary from day to day, and even hour to hour. Furthermore, vast
numbers of bacteria are economic in the best sense of the term, and
the bacteria of milk are chiefly those of a fermentative kind, and not
disease-producers.

_Kinds of Bacteria in Milk._ It is clear from the foregoing that the
only valuable estimation of bacteria in milk is a qualitative one. The
kinds commonly found may be classified thus:

1. Non-pathogenic; fermenting and various unclassified micro-organisms.

2. Pathogenic; tuberculosis, typhoid, cholera, scarlet fever,
diphtheria, and suppurative diseases have all been spread by the agency
of milk.


1. _The Fermentation Bacteria_

At the most we can make a merely provisional classification of these
processes. Many of them are intimately related. Of others, again, our
knowledge is at present very limited. It may be advisable, before
proceeding, to consider shortly what are the constituents of milk upon
which living ferments of various kinds exert their action. A tabulation
of the chief constituents would be as follows:

                { (1) Water                     87.5 per cent.
    Ordinary    { (2) Milk-sugar                 4.9  "   "
  fresh milk =  { (3) Fat                        3.6  "   "
  100 per cent. { (4) Proteids (casein, etc.)    3.3  "   "
                { (5) Mineral matter             0.7  "   "
                                               -----
                                               100.0

Another mode of expressing average milk constitution would be thus:

  Fat              4.1  per  cent.
  Solids not fat   8.8   "    "
                  ----
                  12.9   "    "

It is probably too obvious to need remark that milks vary in standard,
but the above figures may be taken as authentic averages.

_Milk-sugar, or Lactose_ (C_{12}H_{24}O_{12}). This is an important and
constant constituent of milk. It forms the chief substance in solution
in whey or serum. Milk-sugar approximates to dextrose in its action on
polarised light. By boiling with sulphuric acid it is converted into
dextrose and galactose.

_Fat_ occurs in milk as suspended globules, and by churning may be made
into butter.

_The Proteids_ include casein, albumen, lactoprotein, and a small
quantity of globulin. These are the nitrogenous bodies.

_Mineral Matter._ The ash of milk, obtained by careful ignition of the
solids, contains calcium, magnesium, potassium, sodium, phosphoric
acid, sulphuric acid, chlorine, and iron, phosphoric acid and lime
being present in the largest amounts.

(1) _Lactic Acid Fermentation._ If milk is left undisturbed, it is well
known that eventually it becomes sour. The casein is coagulated, and
falls to the bottom of the vessel; the whey or serum rises to the top.
In fact, a coagulation analogous to the clotting of blood has taken
place. In addition to this, the whole has acquired an acid taste. Now,
this double change is not due to any one of the constituents we have
named above. It is, in short, a fermentation set up by a living ferment
introduced from without. The constituent most affected by the ferment
is the milk-sugar, which is broken down into lactic acid, carbonic acid
gas, and other products.

For many years it has been known that sour milk contained bacteria.
Pasteur first described the _Bacillus acidi lactici_, which Lister
isolated and obtained in pure culture. Hueppe contributed still further
to what was known of this bacillus, and pointed out that there were
a large number of varieties, rather than one species, to be included
under the term _B. acidi lactici_. We have already seen that these
bacilli do not as a rule liquefy gelatine, form spores, are non-motile,
and are easily killed by heat.

When a certain quantity of lactic acid has been formed the fermentation
ceases. It will recommence if the liquid be neutralised with carbonate
of lime, or pepsine added. Since Pasteur's discovery of a causal
bacillus for this fermentation, other investigators have added a
number of bacteria to the lactic acid family. Some of these in pure
culture have been used in dairy industry to add to the butter a pure
sour taste, a more or less aromatic odour, and a higher degree of
preservation.

(2) _Butyric Acid Fermentation._ This form of fermentation is also one
which we have previously considered.

Both in lactic and butyric fermentation we must recognise that in the
decomposition of milk-sugar there are almost always a number of minor
products occurring. Some of the chief of these are gases. Hydrogen,
carbonic acid, nitrogen, and methane occur, and cause a characteristic
effect which is frequently deleterious to the flavour of the milk
and its products. Most of the gas-producing ferments are members
of the lactic acid group, and are sometimes classified in a group
by themselves. In cheese-making the gases create the pin-holes and
air-spaces occasionally seen.

(3) _Curdling Fermentations without Acid Production._ Of these there
are several, caused by different bacteria. What happens is that the
milk coagulates, as we have described, but no acid is produced, the
whey being sweet to the taste rather than otherwise. Digestion of
casein may or may not take place.

We must now mention several fermentations about which little is known.
They are designated by terms denoting the outward condition of the
milk, without giving any information respecting the real physiological
alteration which has occurred.

(4) _Bitter Fermentation._ Some bitter conditions of milk are due to
irregularity of diet in the cow. Similar changes occur in conjunction
with some of the acid fermentations. Weigmann and Conn have, however,
shown that there is a specific bitterness in milk due to bacteria which
appear to produce no other change. Hueppe suggests that it may be
due in part to a proteid decomposition resulting in bitter peptones.
There seems to be some evidence for supposing that the bitter bacteria
produce very resistant spores, which enable them to withstand treatment
under which the lactic acid succumbs.

(5) _Slimy Fermentation._ This graphic but inelegant word is used to
denote an increased viscosity in milk, and its tendency when being
poured to become ropy and fall in strings. Such a condition deprives
the milk of its use in the making of certain cheeses, whilst in
other cases it favours the process. In Holland, for example, in the
manufacture of Edam cheese, this "slimy" fermentation is desired.
_Tættemœlk_, a popular beverage in Norway, is made from milk
that has been infected with the leaves of the common butter wort,
_Pinguicula vulgaris_, from which Weigmann separated a bacillus
possessing the power of setting up slimy fermentation. There are,
perhaps, as many as a dozen species of bacteria which have in a greater
or less degree the power of setting up this kind of fermentation. In
1882 Schmidt isolated the _Micrococcus viscosus_, which occurs in
chains and rosaries, affecting the milk-sugar. It grows at blood-heat,
and is not easily destroyed by cold. Its effect on various sugars is
the same. The _M. Freudenreichii_, the specific micro-organism of
"ropiness" in milk, is a large, non-motile, liquefying coccus, which
can produce its result in milk within five hours. On account of its
resistance to drying, it is difficult to eradicate when once it makes
its appearance in a dairy. The organism used in making Edam cheese
is the _Streptococcus Hollandicus_, and in hot milk it can produce
ropiness in one day. A number of bacilli have been detected by several
observers and classified as slime fermentation bacteria. The _Bacillus
lactis pituitosi_, a slightly curved, non-liquefying rod, which is said
to produce a characteristic odour, in addition to causing ropiness,
brings about some acidity. _B. lactis viscosus_ is slow in starting its
fermentation, but maintains its action for as long as a month. Many
of the above organisms, with others, produce "slimy" fermentation in
alcoholic beverages as well as in milk.

(6) _Soapy Milk._ This is still another form of fermentation, the
etiology of which has been elucidated by Weigmann. The _Bacillus
saponacei_ imparts to milk a peculiar soapy flavour. It was detected in
the straw of the bedding and hay of the fodder, and from such sources
may infect the milk. There is little or no coagulation.

(7) _Chromogenic Changes._ We have already remarked that colour is the
natural and apparently only product of many of the innocent bacteria.
They put out their strength, so to speak, in the production of bright
colours. The chief colours produced by germs in milk are as follows:

_Red Milk._ _Bacillus prodigiosus_, in the presence of oxygen, causes
a redness, particularly on the surface of milk. It was the work of
this bacillus that caused "the bleeding host," which was one of the
superstitions of the Middle Ages. _B. lactis erythrogenes_ produces
a red colour only in the dark, and in milk that is not strongly acid
in reaction. When grown in the light this organism produces a yellow
colour. There is a red sarcina (_Sarcina rosea_) which also has the
faculty of producing red pigment. One of the yeasts is another example.

It must not be forgotten that redness in milk may actually be due to
the presence of blood from the udder of the cow. In such a case the
blood and milk will be inextricably mixed together, and not in patches
or a pellicle.

_Blue Milk_ is due to the growth of _Bacillus cyanogenus_. This is an
actively motile rod, the presence of which does not materially affect
the milk, but causes the milk products to be of poor quality.

_Yellow Milk._ _Bacillus synxanthus_ is held responsible for curdling
the milk, and then at a later stage, in redissolving the curd, produces
a yellow pigment.

_Violet and Green Pigments_ in milk are also the work of various
bacteria.


2. _Various Unclassified Bacteria_

In milk this is a comparatively small group, for it happens that
those bacteria in milk which cannot be classified as fermentative or
pathogenic are few. The almost ubiquitous _Bacillus coli communis_
occurs here as elsewhere, and might be grouped with the gaseous
fermentative organisms on account of its extraordinary power of
producing gas and breaking up the medium (whether agar or cheese)
in which it is growing. What its exact rôle is in milk it would be
difficult to say. It may act, as it frequently does elsewhere, by
_association_ in various fermentations. Some authorities hold that its
presence in excessive numbers may cause epidemic diarrhœa in infants.

Several years ago a commission was appointed by the _British Medical
Journal_ to inquire into the quality of the milk sold in some of the
poorer districts of London. Every sample was found to contain _Bacillus
coli_, and it was declared that this particular microbe constituted 90
per cent. of all the organisms found in the milk.[57] We record this
statement, but accept it with some misgiving. The diagnosis of _B.
coli_ four or five years ago was not such a strict matter as to-day.
Still, undoubtedly, this particular organism is not uncommonly found
in milk, and its source is unclean dairying. In the same investigation
_Proteus vulgaris_, _B. fluorescens_, and many liquefying bacteria
were frequently found. Their presence in milk means contamination with
putrefying matter, surface water, or a foul atmosphere.

A number of water bacteria find their way into milk in the practice
of adulteration, and foul byres afford ample opportunity for aërial
pollution.

Another unclassified group occasionally present in milk is represented
by moulds, particularly _Oidium lactis_, the mould which causes a white
fur, possessing a sour odour. It is allied to the _Mycoderma albicans_
(_O. albicans_), which also occurs in milk, and causes the whitish-grey
patches on the mucous membrane of the mouths of infants (_thrush_).
These and many more are occasionally present in milk.


3. _The Disease-Producing Power of Milk_

The general use of milk as an article of diet, especially by the
younger and least resistant portion of mankind, very much increases
the importance of the question as to how far it acts as a vehicle
of disease. Recently considerable attention has been drawn to the
matter, though it is now a number of years since milk was proved to
be a channel for the conveyance of infectious diseases. During the
last twenty years particular and conclusive evidence has been deduced
to show that milch cows may themselves afford a large measure of
infection. The recent extensive work in tuberculosis by the Royal
Commission has done much to obtain new light on the conveyance of
that disease by milk and meat. The enormous strides in the knowledge
of diphtheria and other germ diseases have also placed us in a better
position respecting their conveyance by milk. Generally speaking, for
reasons already given, milk affords an ideal medium for bacteria, and
its adaptibility therefore for conveying disease is undoubted. We may
now suitably turn to speak shortly of the outstanding facts of the
chief diseases carried by milk.

_Tuberculosis._ It is well known that this disease is not a rare one
amongst cattle. The problem of infective milk is, however, simplified
at the outset by recognising the now well-established fact that the
milk of tuberculous cows is only certainly able to produce tuberculosis
in the consumers when the _tuberculous disease affects the udder_.
This is not necessarily a condition of advanced tuberculosis. The
udder may become affected at a comparatively early stage. But to make
the milk infective the udder must be tubercular, and milk from such
an udder possesses a most extraordinary degree of virulence. When
the udder itself is thus the seat of disease, not only the derived
milk, but the skimmed milk, butter-milk, and even butter, all contain
tuberculous material actively injurious if consumed. Furthermore,
tubercular disease of the udder spreads in extent and degree with
extreme rapidity. From these facts it will be obvious that it is of
first-rate importance to be able to diagnose udder disease. This is not
always possible in the early stage. The signs upon which most reliance
may be placed are the enlargement of the lymph-glands lying above the
posterior region of the udder; the serous, yellowish milk which later
on discharges small coagula; the partial or total lack of milk from one
quarter of the udder (following upon excessive secretion); the hard,
diffuse nodular swelling and induration of a part or the whole wall of
the udder; and the detection in the milk of tubercle bacilli. The whole
organ may increase in weight as well as size, and on _post-mortem_
examination show an increase of connective tissue, a number of large
nodules of tubercle, and a scattering of small granular bodies, known
as "miliary" tubercles. _Tuberculin_ may be used as an additional test.
The udder is affected in about two per cent. of tuberculous cows.

There are a variety of causes in addition to the _vera causa_,
the presence of the bacillus of tubercle, which make the disease
common amongst cattle. Constitution, temperament, age, work, food,
and prolonged lactation are the individual features which act as
predisposing conditions; they may act by favouring the propagation of
the bacillus or by weakening the resistance of the tissues. To this
category must further be added conditions of environment. Bad stabling,
dark, ill-ventilated stalls, high temperature, prolonged and close
contact with other cows, all tend in the same direction.

Though there can be no doubt as to the virulence of tuberculous milk,
it may be remembered with satisfaction that only about two per cent.
of tuberculous cows have unmistakably tubercular milk. Even of this
tubercular milk, unless it is very rich in bacilli and is ingested
in large quantities, the risks are practically small or even absent.
Practically the danger from drinking raw milk exists only for persons
who use it as their sole or principal food, that is to say, young
children and certain invalids. With adults in normal health the danger
is greatly minimised, as the healthy digestive tract is relatively
insusceptible. Moreover, dairy milk is almost invariably _mixed_
milk; that is to say, if there is a tubercular cow in a herd yielding
tubercle bacilli in her milk, the addition of the milk of the rest of
the herd so effectually _dilutes_ the whole as to render it almost
innocuous.

But if for practical purposes we look upon all milk derived from
tubercular udders as highly infective, we may adopt a comparatively
simple and efficient remedy. To avoid all danger it is sufficient to
bring the milk to a boil for a few minutes before it is consumed; in
fact, the temperature of 85° C. (160° F.) prolonged for five minutes
kills all bacilli. The common idea that boiled milk is indigestible,
and that the boiling causes it to lose much of its nutritive value, is
largely groundless.

Milk may become tubercular through the carelessness or dirty habits of
the milker. Such a common practice as moistening the hands with saliva
previously to milking may, in cases of tubercular milkers, effectually
contaminate the milk. Again, it may become polluted by dried tubercular
excreta getting into it. Such conveyances must be of rare occurrence,
yet their possibility should not be forgotten.

An infant suckled by a tuberculous mother would run similarly serious
risks of becoming infected with the disease.

In Liverpool, Dr. E. W. Hope, the Medical Officer of Health, has
organised an admirable system of examination by skilled bacteriologists
to find to what degree the Liverpool milk supply is contaminated
with tubercle. The final result of this pioneer work, which ought
really to be undertaken by every great corporation responsible to the
citizens for a pure water and pure milk supply, is to the effect that
in Liverpool 5.2 per cent. of the samples of milk taken from the city
shippons contains tubercle bacilli. As regards the milk sent in from
the country, the return is that 13.4 per cent. is contaminated with the
bacillus of tubercle.

  -------------------------------+--------------------------------+--------
           TOWN SHIPPONS.        |        COUNTRY SHIPPONS.       | TOTAL.
  -------+-----------+-----------+--------+-----------+-----------+--------
  Total. | Infected. | Per cent. | Total. | Infected. | Per cent. |
   228   |    12     |    5.2    |   67   |     9     |   13.4    |  295
  -------+-----------+-----------+--------+-----------+-----------+--------

Such results are very significant, and indicate the importance of all
large corporations obtaining the service of systematic and periodic
bacteriological examination of the milk supply. Nor are the results
surprising, for when we remember the habits of the tubercle bacillus
we cannot conceive a more favourable nurture ground than the typical
byre. "Nothing worse than the insanitary conditions of the life of
the average dairy cow," says Sir George Brown, late of the Board of
Agriculture, "can be imagined." It will be obvious that the above facts
make it incumbent upon responsible authorities to see that not a stone
is left unturned to enforce cleanliness in all dairy work, isolation of
diseased cows, and strict treatment of all infected milk.

_Typhoid Fever._ Jaccoud in France and Hart in England have shown that
enteric fever (typhoid) is not infrequently spread by milk. An epidemic
affecting 386 persons in Stamford, Conn., U.S.A., was traced to milk,
97 per cent. of the cases coming from one single milk supply. Dr.
McNail recently recorded an outbreak of twenty-two cases of enteric,
due to a polluted milk supply.

Within the last twelve months much attention has been drawn to a milk
source of typhoid infection by the epidemic of typhoid at Bristol.
Dr. D. S. Davies has pointed out that a brook received the sewage of
thirty-seven houses, the overflow of a cesspool serving twenty-two
more, the washings from fields over which the drainage of several
others was distributed, and the direct sewage from at least one other,
and then flowed directly through a certain farm. The water of this
stream supplied the farm pump, and the water itself, it is scarcely
necessary to add, was highly charged with putrescent organic matter and
micro-organisms. This water was used for washing the milk-cans from
this particular farm, otherwise the dairy arrangements were efficient.
Part of the milk was distributed to fifty-seven houses in Clifton;
in forty-one of them cases of typhoid occurred. Another part of the
milk was sold over the counter; twenty households so obtaining it were
attacked with typhoid fever, and a number of further infections and
complications arose. This evidence would appear to support the fact
that milk may act in the same way, though not in such a high degree, as
water in the conveyance of typhoid fever.

It may be pointed out that specific typhoid is not a disease of
animals; consequently no danger need be apprehended from milk if it
is properly cared for _after_ it comes from the cow. Typhoid milk is
almost invariably due to the addition of typhoid-infected water,
either by way of adulteration or in the process of washing out the
milk-cans. Cases have, however, been recorded in which there has been
direct transmission to the milk from a person convalescing from the
disease, and also indirect transmission by a milker serving also in the
capacity of nurse to a patient in his own family.

Though the typhoid bacillus appears not to have the power of
multiplying in milk, it has the faculty of existing and thriving in
milk, even when it has curdled or soured, for a considerable time, and
may thus infect milk products like butter and cheese. But infection by
milk products may be eliminated as of too rare occurrence to deserve
attention. The bacillus does not coagulate the milk like its ally
the _Bacillus coli communis_, which is a much more frequent and less
injurious inhabitant of milk.

_Cholera._ The cholera bacillus, as we have already pointed out, is
unable to live in an acid medium. Hence its life in milk is a limited
one, and generally depends on some alkaline change in the milk. Heim
found that cholera bacilli would live in raw milk from one to four
days, depending upon the temperature. D. D. Cunningham, from the
results of a large number of investigations in India, concludes that
the rapidly developing acid fermentations normally or usually setting
in, connected with the rapid multiplication of other common bacteria
and moulds, tend to arrest the multiplication of cholera bacilli, and
eventually to destroy their vitality. Boiling milk appears, on the
contrary, to increase the suitability of milk as a nidus for cholera
bacilli, partly by its germicidal effect upon the acid-producing
microbes, and partly because it removes from the milk the enormous
numbers of common bacteria, which in raw milk cause such keen
competition that the cholera bacillus finds existence impossible.

Professor W. J. Simpson, lately the Medical Officer of Health for
Calcutta, has placed on record an interesting series of cholera cases
on board the _Ardenclutha_, in the port of Calcutta, which arose from
drinking milk which had been polluted with one quarter of its volume
of cholera-infected water. This water came from a tank into which some
cholera dejecta had passed. Of the ten men who drank the milk four
died, five were severely ill, and one, who drank but very little of
the milk, was only slightly ill. There was no illness whatever amongst
those who did not drink the milk.

_Diphtheria._ Recent observations on the infectivity of diphtheria
in milk by Schottelius have established the fact that milk is a good
medium for the bacillus of diphtheria, but that it rarely acts as
a vehicle for transmitting the disease. Klein has emphasised the
possibility of this means of infection. In the first place, it is
obvious that the milk may become infected from a human source--from
pollution with diphtheritic discharges or dried "fomites." Secondly,
from a variety of different quarters evidence has been forthcoming
to throw some suspicion upon the cow itself as the agent. Klein
states that "a new eruptive disease on the teats and udder of the
cow," consisting of papules, vesicles, and induration, may be set up
by the subcutaneous inoculation of a pure culture of the _Bacillus
diphtheriæ_. In these eruptions a bacillus similar to the _B.
diphtheriæ_ was demonstrated. On _a priori_ grounds this evidence
substantiates a belief that diphtheria, in some form or other, may be
a disease of cows. Other observers have not been able to confirm these
observations, and the whole matter of cow diphtheria must remain for
the present _sub judice_.

As long ago as 1879 W. H. Power traced an epidemic of diphtheria in
North London to the milk supply. In 1887 the same authority studied
another outbreak, and other observers have produced further evidence
in favour of the conveyance of this disease by milk. Air infection of
milk by the _Bacillus diphtheriæ_ probably occurs only very rarely, on
account of the fact that the organism is readily killed by desiccation,
and yet such is necessary before it can be airborne. The most frequent
mode of infection of milk with this disease is from the throats, hands,
bodies, or clothing of dairy workers suffering from a mild or acute
form of the disease.

The specific and proved cases in which milk has acted as the vehicle of
diphtheria are, it is true, comparatively few. Yet, nevertheless, the
possibility of milk infection in this disease is not one which we can
afford to neglect.

_Scarlet Fever._ Here again the evidence is not complete, chiefly
owing to the fact that no specific organism of scarlet fever has yet
been discovered. Many cases have, however, illustrated the undeniable
conveyance of the disease by milk. Even before 1881 a number of milk
epidemics of scarlet fever had been traced out. In 1882 these were
further added to by Mr. W. H. Power's report concerning a series
of cases in Central London. That report was remarkable for the
introduction of a new feature, viz., the evidence produced in favour
of the infection of milk from some disease of the cow. The Medical
Department of the Local Government Board from that time took up a
position of suspended judgment concerning the belief hitherto credited
that milk could only be infected by _human_ scarlet fever. In 1886
there was a remarkable epidemic in Marylebone, and the theory was
suggested by Dr. Klein and Mr. Power that the cow from which the milk
was derived suffered from scarlet fever.

Into the extensive controversy which raged round "the Hendon disease,"
as it was called, affecting the cows supplying the Marylebone milk,
we cannot here enter. It will be sufficient to say that a long
discussion took place as to whether or not this Hendon disease was or
was not scarlet fever. The difficulty of course largely arose from the
fact before mentioned that we do not at present know the specific
micro-organism of scarlet fever. The Agricultural Department supported
the view of Professor Crookshank that the cow disease at Hendon was
_cow-pox_, and Professor Axe further pointed out that there was evidence
of the Hendon milk having been contaminated with human scarlet fever.
Whichever conclusion was adopted, all were agreed upon one point, viz.,
that the disease had been conveyed from Hendon to persons in Marylebone
by means of the milk.

Mr. Ernest Hart in 1897 published a very large number of records of
scarlatinal milk infection from all parts of the country, and though
the cause of the disease is obscure, there is now no doubt that it may
be and is conveyed by means of milk.

_Other Diseases Conveyed by Milk._ In addition to the above, there are
other diseases spread by means of polluted milk. From time to time
exceptional cases have occurred in which a disease like anthrax has
been spread by this means. But it is not to such rare cases that we
refer. There are two very common diseases in which milk has been proved
to play a not inconsiderable part, viz., _thrush_ and _diarrhœa_.

The mould which gives rise to the curd-like patches in the throats of
children, and which is known as _Oidium albicans_, frequently occurs
in milk. Soft white specks are seen on the tongue and mucous membrane
of the cheeks and lips, looking not unlike particles of milk curd.
If a scraping be placed upon a glass slide with a drop of glycerine
and examined by means of the microscope, the spores and mycelial
threads of this mould will be seen. The spores are oval, and possess
a definite capsule. The threads are branched and jointed at somewhat
long intervals. Milk affords an excellent medium for the growth of this
parasite. Thus undoubtedly we must hold milk partly responsible for
spreading this complaint. _Penicillium_, _Aspergillus_, and _Mucor_ are
also frequent moulds in milk.

Professor MacFadyen[58] has given a full account of the ways in which
milk becomes pathogenic, and his views have received further support
from Professor Sheridan Delépine, who has examined more than one
hundred samples of milk from Liverpool and Manchester. The result of
this investigation has been that milk must be held to be one of the
most potent causes of the summer diarrhœa of children. Indeed, a
bacillus has been isolated identical with one which was apparently
the cause of this complaint, which carries off such a large number
of infants every summer. It resembles closely the _Bacillus coli
communis_, which is an almost constant inhabitant of the alimentary
canal, and is held by many bacteriologists to play, especially in
conjunction with yeasts and other saprophytic organisms, an active rôle
in the intestine of man.

In a recent official report[59] Dr. Hope, of Liverpool, states that
"the method of feeding plays a most important part in the causation
of diarrhœa; when artificial feeding becomes necessary, the most
scrupulous attention should be paid to feeding-bottles." Careless
feeding, in conjunction with a warm, dry summer, invariably results
in a high death-rate from this cause. These two causes interact upon
each other. A warm temperature is a favourable temperature for the
growth of the poisonous micro-organism; a dry season affords ample
opportunity for its conveyance through the air. Unclean feeding-bottles
are obviously an admirable nidus for these injurious bacteria, for in
such a resting-place the three main conditions necessary for bacterial
life are well fulfilled, viz., heat, moisture, and pabulum. The heat is
supplied by the warm temperature, the moisture and food by the dregs of
milk left in the bottle; and the dry air assists in transit.

Before passing on to other matters, reference must be made to
poisonous products other than bacteria which occur in milk and set up
ill-health. Vaughan, of Michigan, pointed out at the London Congress
of Hygiene in 1891 that he had separated a poisonous alkaloid, which
he called _tyrotoxicon_. This, as its name denotes, was a toxic or
poisonous substance, probably produced by some form of microbe. It
may be taken as a type of the organic chemical substances frequently
occurring in milk.


METHODS OF PRESERVING MILK

From the somewhat extensive category of diseases which may be
milk-borne, it will be suitable now to speak of some of the means at
our disposal for obtaining and preserving good, pure milk.

We considered at the commencement of this chapter the most frequent
channels of contamination. If these be avoided or prevented, and if
the milk be derived from cows in good health and well kept, the risk
of infection is reduced to a minimum. But we have seen that much, if
not most, of the pollution of milk arises after the milking process
and during transit and storage preparatory to use. Bacteria are so
ubiquitous that to prevent the entrance of any at all is almost beyond
hope. Can anything be done to prevent their multiplication or to kill
them in the milk? Fortunately the answer is in the affirmative.

There are two means at hand to secure these results. First, we may
add to the milk various chemical or physical preservatives. Borax or
boric acid, formaldehyde, salicylic acid, and other chemical bodies
are used for this purpose. The commonest of these is that named first.
The Food and Drugs Act (Section VI., 1875) permits the addition of
an ingredient not injurious to health if the same is required for
protection or preparation of the article in question. It is, however,
a difficult matter to determine what amount of boric acid is injurious
to health, for this differs widely in different persons. It has been
laid down by one authority that even so small an amount as one-tenth
per cent. might have inconvenient results, owing to its cumulative
effect. _Formaldehyde_ is without doubt an excellent antiseptic, and
the more its efficacy becomes known so much the more probably will
it be used. The _salicylates_, which are mild antiseptics, have long
been used as preservatives. These substances, then, can be added to
milk in quantities not recognisable to the taste (salicylic acid
about .75 grain, and boracic acid .4 grain, to the litre of milk).
They will materially increase the time that milk will remain sweet,
they will prevent a number of micro-organisms living in the milk, and
will inhibit multiplication of others.[60] Secondly, it is possible
very perceptibly to remove the infectivity of milk by filtration and
temperature variations.

_Filtration_ has been practised for some time by the Copenhagen Dairy
Company and by Bolle, of Berlin. The filters used consist of large
cylindrical vessels divided by horizontal perforated diaphragms into
five superposed compartments, of which the middle three are filled
with fine sand of three sizes. At the bottom is the coarsest sand,
and at the top the finest. The milk enters the lowest compartment by
a pipe under gravitation pressure, and is forced upwards, and finally
is run off into an iced cooler, and from that into the distribution
cans. By this means the number of bacteria is reduced to one-third.
The difficulty of drying and sterilising enough sand to admit a
large turnover of milk is a serious one. This, in conjunction with
the belief that filtration removes some of the essential nutritive
elements of milk, has caused the process to be but little adopted.
Dr. Seibert states that if milk be filtered through half an inch of
compressed absorbent cotton, seven-eighths of the contained bacteria
will be removed, and a second filtration will further reduce the number
to one-twentieth. One quart of milk may thus be filtered in fifteen
minutes.

The common methods now in vogue for the protection of milk are based
upon _germicidal temperatures_. Low temperatures, it is true, do
not easily destroy life, but they have a most beneficial effect
upon the keeping quality of milk. At the outset of the process of
cooling, strong currents of air are started in the milk-can, which
act mechanically as deodorisers. But if the temperature be lowered
sufficiently, the contained bacteria become inactive and torpid, and
eventually are unable to multiply or produce their characteristic
fermentations. At about 50° F. (10° C.) the activity ceases, and
at temperatures of 45° F. (7° C.) and 39° F. (4° C.) organisms are
deprived of their injurious powers. If it happens that the milk is to
be conveyed long distances, then even a lower temperature is desirable.
The most important point with regard to the cooling of milk is that it
should take place quickly. Various kinds of apparatus are effective in
accomplishing this. Perhaps those best known are Lawrence's cooler and
Pfeiffer's cooler, the advantage of the latter being that during the
process the milk is not exposed to the air. It must not be forgotten
that cooling processes are not sterilising processes. They do not
necessarily kill bacteria; they only inhibit activity, and under
favourable circumstances the torpid bacteria may again acquire their
injurious faculties. Hence during the cooling of milk greater care must
be taken to prevent aërial contamination than is necessary during the
process of sterilising milk. No cooling whatever should be attempted in
the stable; but, on the other hand, there should be no delay. Climate
makes little or no difference to the practical desirability of cooling
milk, yet it is obvious that less cooling will be required in the cold
season.

We now come to the protective processes known as _sterilisation_ and
_pasteurisation_. As we have already seen, sterilisation indicates
a complete and final destruction of bacteria and their spores. As
applied to methods of preserving milk, sterilisation means the use of
heat at, or above, boiling-point, or boiling under pressure. This may
be applied in one application of one to two hours at 250° F., or it
may be applied at stated intervals at a lower temperature. The milk
is sterilised--that is to say, contains no living germs--is altered
in chemical composition, and is also boiled or "cooked," and hence
possesses a flavour which to many people is unpalatable.

Now, such a radical alteration is not necessary in order to secure
non-infectious milk. The bacteria causing the diseases conveyable
by milk succumb at much lower temperatures than the boiling-point.
Advantage is taken of this in the process known as "pasteurisation."
By this method the milk is heated to 167-185° F. (75-85° C.). Such a
temperature kills harmful microbes, because 75° C. is decidedly above
their average thermal death-point, and yet the physical changes in the
milk are practically _nil_, because 85° C. does not relatively approach
the boiling-point. There is no fixed standard for pasteurisation,
except that it must be above the thermal death-point of pathogenic
bacteria, and yet below the boiling-point. As a matter of fact, 158° F.
(70° C.) will kill all souring bacteria as well as disease-producing
organisms found in milk. If the milk is kept at that temperature for
ten or fifteen minutes, we say it has been "pasteurised." If it has
been boiled, with or without pressure, for half an hour, we say it
has been "sterilised." The only practical difference in the result is
that sterilised milks have a better keeping quality than pasteurised,
for the simple reason that in the latter some living germs have been
unaffected.

Sterilisation may of course be carried out in a variety of
modifications of the two chief ways above named. When the process
is to be completed in one event an _autoclave_ is used, in order to
obtain increased pressure and a higher temperature. Milk so treated
is physically changed in greater degree than in the slower process.
The slow or _intermittent_ method is, of course, based on Tyndall's
discovery that actively growing bacteria are more easily killed than
their spores. The first sterilisation kills the bacteria, but leaves
their spores. By the time of the second application the spores have
developed into bacteria, which in turn are killed before they can
sporulate.

The methods of pasteurisation are continually being modified and
improved, especially in Germany and America. Most of the variations in
apparatus may be classed under two headings. There are, first, those
in which a sheet of milk is allowed to flow over a surface heated
by steam or hot water. This may be a flat, corrugated surface or a
revolving cylinder. The milk is then passed into coolers. Secondly,
milk is pasteurised by being placed in reservoirs surrounded by an
external shell containing hot water or steam. Dr. A. L. Russell[61] has
described one apparatus consisting of a pasteuriser, a water-cooler,
and an ice-cooler. The pasteuriser is heated by hot water in the
outside casement. To equalise rapidly the temperature of the water
and milk a series of agitators must be used. These are suspended on
movable rods, and hang vertically in the milk and water chambers. By
this ingenious arrangement the heat is diffused rapidly throughout the
whole mass, and as the temperature of the milk reaches the proper point
the steam is shut off, and the heat of the whole body of water and milk
will remain constant for the proper length of time.

The somewhat difficult problem of drawing off the pasteurised milk from
the vat without reinfecting it by contact with the air is solved by
placing a valve inside the chamber, and by means of a pipe leading the
pasteurised milk directly and rapidly into the coolers. These are of
two kinds, which may be used separately or conjointly. In one set of
cylinders there is cold circulating water, in the other finely crushed
ice.

Domestic pasteurisation can be accomplished readily by heating the milk
in vessels in a water-bath raised to the required temperature for half
an hour.

Without entering into a long discussion upon the various methods
adopted, we may summarise some of the chief essential conditions. It
need scarcely be said that the operation must be efficiently conducted,
and in such a way as to maintain absolute control over the time and
temperature. The apparatus should be simple enough to be easily
cleansed, sterilised, and economical in use. Arrangements must always
be made to protect the milk from reinfection during and after the
process. The entire preparation of the milk for market may be summed up
in four items:

  1. Pasteurisation in heat reservoir.

  2. Rapid cooling in water-or ice-coolers.

  3. All cans, pails, bottles, and other utensils to be
  thoroughly sterilised in steam.

  4. The prepared milk must be placed in sterilised bottles and
  sealed up.

The quality of the milk to be pasteurised is an important point. All
milks are not equally suited for this purpose, and those containing a
large quantity of contamination, especially of spores, are distinctly
unsuitable. Such milks, to be purified, _must_ be sterilised. Dr.
Russell has laid down a standard test for the degree of contamination
which may be corrected by pasteurisation by estimating the degree of
acidity, a low acidity (_e. g._, 0.2 per cent.) usually indicating a
smaller number of spore-bearing germs than that which contains a high
percentage of acid.

Lastly, while the heating process is of course the essential feature
of efficient pasteurisation, it must not be forgotten that rapid
and thorough cooling is almost equally important. As we have seen,
pasteurisation differs from complete sterilisation in that it leaves
behind a certain number of microbes or their spores. Cooling inhibits
the germination and growth of this organismal residue. If after the
heating process the milk is cooled and kept in a refrigerator, it will
probably keep sweet from three to six days, and may do so for three
weeks.

Before leaving this subject we may glance for a moment at the bacterial
results of pasteurisation and sterilisation. The chief two of these
are the enhanced keeping quality and the removal of disease-producing
germs. The former is due in part to the latter, and also to the
removal of the lactic acid and other fermentative bacteria. As a
general rule these bacteria do not produce spores, and hence they are
easily annihilated by pasteurisation. True, a number of indifferent
bacteria are untouched, and also some of the peptonising species. The
cooling itself contributes to the increased keeping power of the milk,
especially in transit to the consumer.

Pasteurised milks have the following three economical and commercial
advantages over sterilised milks, namely, they are more digestible, the
flavour is not altered, and the fat and lact-albumen are unchanged.
Professor Hunter Stewart, of Edinburgh, about two years ago,
compiled from a number of experiments the following instructive and
comprehensive table (page 212).

It will be admitted that this table exhibits much in favour of
pasteurisation; yet the crucial test must ever be the effect upon
pathogenic bacteria. Flügge has conducted a series of experiments upon
the destruction of bacteria in milk, and he states that a temperature
of 158° F. (70° C.) maintained for thirty minutes will kill the
specific organisms of tubercle, diphtheria, typhoid, and cholera.
MacFadyen and Hewlett have demonstrated,[62] by sudden alternate
heating and cooling, that 70° C. maintained for half a minute is
generally sufficient to kill suppurative organisms and such virulent
types of pathogenic bacteria as _Bacillus diphtheriæ_, _B. typhosus_,
and _B. tuberculosis_.

  ------------+-----------+--------------+----------------+---------+-----------+-----------
              |Average No.|  Temperature |    No. of      | Soluble |  Soluble  |
              |of Microbes| and Duration |   Microbes     | Albumen |  Albumen  |  Taste
    No. of    |per cc. in |      of      |  per cc. in    |   in    |    in     |    of
  Experiments.|Milk before|Pasteurisation|  Pasteurised   |  Fresh  |Pasteurised|Pasteurised
              |Treatment. |      in      |   Milk after   |  Milk,  |   Milk,   |   Milk.
              |           |    Minutes.  |    24 Hours.   |per cent.| per cent. |
  ------------+-----------+--------------+----------------+---------+-----------+-----------
       5      |   136,262 |  10' 60° C.  | 1722 average   |  0.423  |   0.418   |Unaffected
       4      |    53,656 |  30' 60° C.  | 1 sterile      |  0.435  |   0.427   |     "
              |           |              | 3 averaged 955 |         |           |
      12      |    78,562 |  10' 65° C.  | 6 sterile      |  0.395  |   0.362   |    Not
              |           |              | 6 averaged 686 |         |           |appreciably
              |           |              |                |         |           | affected
      12      |   132,833 |  30' 65° C.  | 9 sterile      |  0.395  |   0.333   |     "
              |           |              | 3 averaged 233 |         |           |
      13      |    49,867 |  10' 70° C.  |   sterile      |  0.422  |   0.269   | Slightly
              |           |              |                |         |           |  boiled
       9      |    38,320 |  30' 70° C.  |      "         |  0.421  |   0.253   |    "
       2      |    77,062 |  10' 75° C.  |      "         |  0.38   |   0.07    |  Boiled
       3      |    48,250 |  30' 75° C.  |      "         |  0.38   |   0.05    |    "
       1      | 1,107,000 |  10' 80° C.  |      "         |  0.375  |   0.00    |    "
       1      | 1,107,000 |  30' 80° C.  |      "         |  0.375  |   0.00    |    "
  ------------+-----------+--------------+----------------+---------+-----------+-----------

Respecting the numerical diminution of microbes brought about by
pasteurisation and sterilisation, respectively, we may take the
following two sets of experiments. Dr. N. L. Russell[63] tabulates the
immediate results of pasteurisation as follows:

  ----------------------------------------------+---------------------------
                  UNPASTEURISED.                |      PASTEURISED.
  -----------+---------+------------+-----------+--------+---------+--------
             | Minimum.|  Maximum.  |  Average. |Minimum.| Maximum.|Average.
  Full cream |         |            |           |        |         |
    milk.    |  25,300 | 18,827,000 | 3,674,000 |    0   |  37,500 |  6,140
  Cream, 25%.| 425,000 | 32,800,000 | 8,700,000 |    0   |  57,000 | 24,250
  -----------+---------+------------+-----------+--------+---------+--------

As regards the later effect of the process, he states that in fifteen
samples of pasteurised milk examined from November to December nine
of them revealed no organisms, or so few that they might almost be
regarded as sterile; in those samples examined after January the lowest
number was 100 germs per cc., while the average was nearly 5,000. With
the pasteurised cream a similar condition was to be observed.

Dr. Hewlett[64] defines pasteurisation briefly as heating the milk to
68° C. for twenty or thirty minutes, and this treatment he quotes as
destroying 99.75 per cent. of the total number of organisms. Bitter's
table of results at 158° F. bears out the same:

    BEFORE PASTEURISATION.        AFTER PASTEURISATION.
    No. of Bacteria in 10 Drops.  No. of Bacteria in 10 Drops.

  1.      102,600                        2-3
  2.      251,600                       30-40
  3.       25,000                        3-5
  4.       37,500                        2-5
  5.       94,000                        2


BACTERIA IN MILK PRODUCTS

Cream is generally richer in bacteria than milk. _Set_ cream contains
more bacteria than _separated_ cream, but germs are abundant in both.
Yet whilst it is true that cream contains a large number of bacteria,
it must be pointed out that the butter fat in cream is a less suitable
food for organisms than is the case with milk. Hence the fermentative
changes set up in cream are of less degree than in milk, particularly
so if separated from the milk. Butter-milk and whey vary much in their
bacterial content. Butter necessarily follows the standard of the
cream. But as the butter fat is not well adapted for bacterial food,
the number of bacteria in butter is usually less than in cream.[65]
Moreover, they are soon reduced both in quality and quantity. Butter
examined after it is several months old is often found to be almost
free from germs; yet in the intervening period a variety of conditions
are set up directly or indirectly through bacterial action.

_Rancid_ butter is partly due to organisms. _Putrid_ butter is caused,
according to Jensen, by various putrefactive bacteria, one form of
which is named _Bacillus fœtidus lactis_. This organism is killed at
a comparatively low temperature, and is therefore completely removed
by pasteurisation. _Ill-flavoured_ butter may be due to germs or an
unsuitable diet of the cow and a retention of the bad quality of the
resulting milk. _Lardy_ and _oily_ butters have been investigated by
Storch and Jensen and traced to bacteria. Lastly, _bitter_ butter
occasionally occurs, and is due to fermentative changes in the milk.
Butter may also contain pathogenic bacteria, like tubercle. The _B.
coli_ can live for one month in butter.

Cheese suffers from very much the same kind of "diseases" as butter,
except that chromogenic conditions occur more frequently. The latter
are, under certain circumstances, more the result of chemical than
bacterial action. Most of the troubles in cheese originate in the milk.

_Method of Examination of Butter._ Several grams of the butter should
be placed in a large test-tube, which is then two-thirds filled with
sterilised water and placed in a water-bath at about 45° C. until the
butter is completely melted. A small quantity may then be added to
gelatine or agar and plated out on Petri dishes or in flat-bottomed
flasks in the usual way. After which the tube may be well shaken
and returned to the bath inverted. In the space of twenty or thirty
minutes the butter has separated from the water with which it has been
emulsified. It is then placed in the cold to set. The water may be
now either centrifugalised or placed in sedimentation flasks, and the
deposit examined for bacteria.

_The Uses of Bacteria in Dairy Produce._ In considering the relation
of bacteria to milk we found that many of the species present were
injurious rather than otherwise, and when we come to consider bacteria
in dairy products, like butter and cheese, we find that the dairyman
possesses in them very powerful _allies_. Within recent years almost
a new industry has arisen owing to the scientific application of
bacteriology to dairy work.

As a preliminary to butter-making the general custom in most countries
is to subject the cream to a process of "ripening." As we have seen,
cream in ordinary dairies and creameries invariably contains some
bacteria, a large number of which are in no sense injurious. Indeed,
it is to these bacteria that the ripening and flavouring processes are
due. They are perfectly consistent with the production of the best
quality of butter. The aroma of butter, as we know, controls in a large
measure its price in the market. This aroma is due to the decomposing
effect upon the constituents of the butter of the bacteria contained
in the cream. In the months of May and June the variety and number
of these types of bacteria are decidedly greater than in the winter
months, and this explains in part the better quality of the butter at
these seasons. As a result of these ripening bacteria the milk becomes
changed and soured, and slightly curdled. Thus it is rendered more
fit for butter-making, and acquires its pleasant taste and aroma. It
is then churned, after which bacterial action is reduced to a minimum
or is absent altogether. Sweet-cream butter lacks the flavour of
ripened or sour-cream butter. The process is really a fermentation,
the ripening bacteria acting on each and all of the constituents of
the milk, resulting in the production of various bye-products. This
fermentation is a decomposition, and just as we found when discussing
fermentation, so here also the action is beneficial only if it is
stopped at the right moment. If, for example, instead of being stopped
on the second day, it is allowed to continue for a week, the cream will
degenerate and become offensive, and the pleasant ripening aroma will
be changed to the contrary.

Bacteriologists have demonstrated that butters possessing different
flavours have been ripened by different species of bacteria.
Occasionally one comes across a dairy which seems to be impregnated
with bacteria that improve cream and flavour well. In other cases
the contrary happens, and a dairy becomes impregnated with a species
having deleterious effects upon its butter. This species may arise
from unclean utensils and dairying, from disease of the cow, or from
a change in the cow's diet. Thus it comes about that the butter-maker
is not always able to depend upon good ripening for his cream. At
other times he gets ripening to occur, but the flavour is an evil one,
and the results correspond. It may be bitter or tainted, and just as
certainly as these flavours develop in the cream, so is it certain
that the butter will suffer. Fortunately the bacterial content of the
cream is generally either favourable or indifferent in its action. Thus
it comes about that the custom is to allow the cream simply to ripen,
so to speak, of its own accord, in a vat exposed to the influence of
any bacteria which may happen to be around. This generally proves
satisfactory, but it has the great disadvantage of being indefinite and
uncertain. Occasionally it turns out wholly unsatisfactory, and results
in financial loss.

There are various means at our command for improving the ripening
process. Perfect cleanliness in the entire manipulation necessary
in milking and dairying, combined with freedom from disease in the
milch cows, will carry us a long way on the road towards a good
cream-ripening. Recently, however, a new method has been introduced,
largely through the work and influence of Professor Storch in Denmark,
which is based upon our new knowledge respecting bacterial action in
cream-ripening. We refer to the artificial processes of ripening set
up by the addition of _pure cultures of favourable germs_.[66] If a
culture of organisms possessing the faculty of producing in cream a
good flavour be added to the sweet cream, it is clear that advantage
will accrue. This simple plan of _starting_ any special or desired
flavour by introducing the specific micro-organism of that flavour
may be adopted in two or three different ways. If cream be inoculated
with a large, pure culture of some particular kind of bacteria, this
species will frequently grow so well and so rapidly that it will check
the growth of the other bacteria which were present in the cream at
the commencement and before the starter was added. That is, perhaps,
the simplest method of adding an artificial culture. But secondly, it
will be apparent to those who have followed us thus far, that if the
cream is previously _pasteurised_ at 70° C. these competing bacteria
will have been mostly or entirely destroyed, and the pure culture, or
starter, will have the field to itself. There is a third modification,
which is sometimes termed ripening by _natural starters_. A natural
starter is a certain small quantity of cream taken from a favourable
ripening--from a clean dairy or a good herd--and placed aside to
sour for two days until it is heavily impregnated with the specific
organism which was present in the whole favourable stock of which the
natural starter is but a part. It is then added to the new cream the
favourable ripening of which is desired. Of the species which produce
good flavours in butter the majority are found to be members of
the acid-producing class; but probably the flavour is not dependent
upon the acid. Moreover, the aroma of good ripening is also probably
independent of the acid production.

Of all the methods of ripening--natural ripening, the addition of
natural starters, the addition of pure cultures with or without
pasteurisation--there can be no doubt that pure culture after
pasteurisation is the most accurate and dependable. The use of natural
starters is a method in the right direction; yet it is, after all,
a _mixed_ culture, and therefore not uniform in action. In order to
obtain the best results with the addition of pure cultures, Professor
Russell has made the following recommendations:

1. The dry powder of the pure culture must be added to a small amount
of milk that has been first pasteurised, in order to develop an active
growth from the dried material.

2. The cream to be ripened must first be pasteurised, in order to
destroy the developing organisms already in it, and thus be prepared
for the addition of the pure culture.

3. The addition of the developing starter to the pasteurised cream and
the holding of the cream at such a temperature as will readily induce
the best development of flavour.

4. The propagation of the starter from day to day. A fresh lot of
pasteurised milk should be inoculated daily with some of the pure
culture of the previous day, not the ripening cream containing the
culture. In this way the purity of the starter is maintained for a
considerable length of time. Those starters are best which grow rapidly
at a comparatively low temperature (60-75° F.), which produce a good
flavour, and which increase the keeping qualities of the butter. Now,
whilst it is true that the practice of using pure cultures in this way
is becoming more general, very few species have been isolated which
fulfil all the desirable qualities above mentioned. In America starters
are preferred which yield a "high" flavour, whereas in Danish butter
a mild aroma is commoner. In England as yet very little has been done,
and that on an experimental scale rather than a commercial one.[67]
In 1891 it appears that only 4 per cent. of the butter exhibited at
the Danish butter exhibitions was made from pasteurised cream plus a
culture starter; but in 1895, 86 per cent. of the butter was so made.
Moreover, such butter obtained the prizes awarded for first-class
butter with preferable flavour. Different cultures will, of course,
yield different flavoured butter. If we desire, say, a Danish butter,
then some species like "Hansen's Danish Starter" would be added; if we
desire an American butter, we should use a species like that known as
"Conn's Bacillus, No. 41." But whilst these are two common types, they
are not the only suitable and effective starters. On certain farms in
England there are equally good cultures, which, placed under favourable
temperatures in new cream, would immediately commence active ripening.

Professor H. W. Conn, who, with Professor Russell, has done so much
in America for the advancement of dairy bacteriology, reports[68] a
year's experience with the bacillus to which reference has been made,
and which is termed No. 41. It was originally obtained from a specimen
of milk from Uruguay, South America, which was exhibited at the World's
Fair in Chicago, and proved the most successful flavouring and ripening
agent among a number of cultures that were tried. The conclusions
arrived at after a considerable period of testing and experimentation
appear to be on the whole satisfactory. A frequent method of testing
has been to divide a certain quantity of cream into two parts, one
part inoculated with the culture and the other part left uninoculated.
Both have then been ripened under similar conditions, and churned in
the same way; the differences have then been noted. It is interesting
to know that, as a result of the year's experience, creameries have
been able to command a price varying from half a cent to two cents
a pound _more_ for the "culture" butters than for the uninoculated
butters. The method advised in using this pure culture is to pasteurise
(by heating at 155° F.) six quarts of cream, and after cooling to
dissolve in this cream the pellet containing bacillus No. 41. The cream
is then set in a warm place (70° F.), and the bacillus is allowed to
grow for two days, and is then inoculated into twenty-five gallons of
ordinary cream. This is allowed to ripen as usual, and is then used
as an infecting culture, or "starter," in the large cream vats in the
proportion of one gallon of infecting culture to twenty-five gallons of
cream, and the whole is ripened at a temperature of about 68° F. for
one day. The cream ripened by this organism needs to be churned at a
little lower temperature (say 52°-54° F.) but to be ripened at a little
higher temperature than ordinary cream to produce the best results.
Cream ripened with No. 41 has its keeping power much increased, and
the body or grain of the butter is not affected. More than two hundred
creameries in America used this culture during 1895, and Professor Conn
reports that this has proved that its use for the production of flavour
in butter is feasible in ordinary creameries and in the hands of
ordinary butter-makers provided they will use proper methods and proper
discretion.

_Bacteria in Cheese-making._ The cases where it has been possible to
trace bacterial disease to the consumption of butter and cheese have
been rare. Notwithstanding this fact, it must not be supposed that
therefore cheese contains few or no bacteria. On the contrary, for
the making of cheese bacteria are not only favourable, but actually
essential, for in its manufacture the casein of the milk has to be
separated from the other products by the use of _rennet_, and is then
collected in large masses and pressed, forming the fresh cheese. In the
course of time this undergoes ripening, which develops the peculiar
flavours characteristic of cheese, and upon which its whole value
depends.

We have said that the casein is separated by the addition of rennet,
which has the power of coagulating the casein. But this precipitation
may also be accomplished by allowing acid to develop in the milk
until the casein is precipitated, as in some sour-milk or cottage
cheeses. The former method is of course the usual one in practice. It
has been suggested that the bacteria contained in the rennet exert
a considerable influence on the cheese, but this, although rennet
contains bacteria, is hardly established. It is not here, however, that
bacteria really play their rôle. After this physical separation, when
the cheese is pressed and set aside, is the period for the commencement
of the ripening process.

That bacteria perform the major part of this ripening process, and
are essential to it, is proved by the fact that when they are either
removed or opposed the curing changes immediately cease. If the milk be
first sterilised, or if antiseptics, like thymol, be added, the results
are negative. It is not yet known whether this peptonising process is
due to the influence of a single organism or not. The probability,
however, is that it is to be ascribed to the action of that group of
bacteria known as the _lactic-acid_ organisms. Nor is it yet known
whether the peptonisation of the casein and the production of the
flavour are the results of one or more species. Freudenreich believes
them to be due to two different forms.

However that may be, we meet with at least four common groups of
bacteria more or less constantly present in cheese-ripening, either
in the early or late stages. First, there are the _lactic-acid
bacteria_, by far the largest group, and the one common feature of
which is the production by fermentation of lactic acid; secondly,
there are the _casein-digesting bacteria_, present in relatively small
numbers; thirdly, the _gas-producing bacteria_, which give to cheese
its honeycombed appearance; lastly, an indifferent or _miscellaneous_
group of extraneous bacteria, which were in the milk at the outset of
cheese-making, or are intruders from the air or rennet. All these four
groups may bring about a variety of changes, beneficial and otherwise,
in the cheese-making.

In order that the relation of bacteria to cheese may be more fully
understood, we may draw attention to some experiments conducted by
Professor H. L. Russell as to the numbers of bacteria present during
different stages of the ripening, excluding those already referred to
as present in the rennet. It appears that there is always at first
a marked increase in the number of micro-organisms, which is soon
followed by a more gradual decline. While the casein-digesting and
gas-producing classes suffer a general and more or less rapid decline,
the lactic-acid bacteria develop to an enormous extent, from which fact
it would appear that cheese offers ideal conditions for the development
of the latter. In some most interesting records Professor Russell has
divided the ripening process into three divisions:

1. _Period of Initial Bacterial Decline in Cheese._ Where the green
cheeses were examined immediately after removing from the press, it was
usually found that a diminution in numbers of bacteria had taken place.
This period of decline lasts but a short time, not beyond the second
day. Lower temperature and expulsion of the whey would account for this
general decline in all species of bacteria.

2. _Period of Bacterial Increase._ Soon after the cheese is removed
from the press a most noteworthy change takes place in green cheese.
A very rapid increase of bacteria occurs, confined almost exclusively
to the lactic-acid group. This commences in green cheese about the
eighth day, and continues more or less for twenty days. In Cheddar
cheese it commences about the fifth day, reaches its maximum about the
twentieth day, declines rapidly to the thirtieth day, and gradually for
a hundred following days. During the first forty days of this period
the casein-digesting and gas-producing organisms are present, and at
first increasing, but relatively to only a very slight degree. With
this rapid increase in organisms the curd begins to lose its elastic
texture, and before the maximum number of bacteria is reached the
curing is far advanced. Freudenreich has shown that acid inhibits the
growth of the casein-digesting microbes and _vice versâ_.

3. _Period of Final Bacterial Decline._ The cause of this decline
can only be conjectured, but it is highly probable that it is due
to a general principle to which reference has frequently been made,
viz., that after a certain time the further growth of any species of
bacteria is prevented by its own products. We may observe that the
gas-producing bacteria in Cheddar cheese last much longer than the
peptonising organisms, for they are still present up to eighty days.
Professor Russell aptly compares the bacterial vegetation of cheese
with its analogue in a freshly seeded field. "At first multitudes of
weeds appear with the grass. These are the casein-digesting organisms,
while the grass is comparable to the more native lactic-acid flora. In
course of time, however, grass, which is the natural covering of soil,
'drives out' the weeds, and in cheese a similar condition occurs." In
milk the lactic-acid bacteria and peptonising organisms grow together;
in ripening cheese the former eliminate the latter.

We have seen that the conclusion generally held respecting these
lactic-acid bacteria is that they are the main agents in curing the
cheese. Upon this basis a system of pure starters has been adopted,
the characteristics of which must be as follows: (_a_) The organism
shall be a pure lactic-acid-producing germ, incapable of producing
gaseous products; (_b_) it should be free from any undesirable aroma;
(_c_) it should be especially adapted for vigorous development in
milk. The starter may be propagated in pasteurised or sterilised milk
from a pure culture from the laboratory. The advantages accruing from
the uses of this lactic-acid culture, as compared with cheese made
without a culture, are that with sweet milk it saves time in the
process of manufacture; that with tainted milk, in which acid develops
imperfectly, it is an aid to the development of a proper amount of
acid for a typical Cheddar cheese; and that the flavour and quality of
such cheese is preferable to cheese which has not been thus produced.
Professor Russell is of opinion that the lactic-acid organisms are to
be credited with greater ripening powers than the casein-digesting
organisms, but it must not be forgotten that these two great families
of bacteria are still more or less on trial, and it is not yet possible
finally to dispose of either of them. Mr. F. J. Lloyd holds that though
"the greater the number of lactic-acid bacilli in the milk the greater
the chance of a good curd," still "this organism alone will not produce
that nutty flavour which is so sought after as being the essential
characteristic of an excellent Cheddar cheese."[69]

There are several difficulties to be encountered by dairymen starting a
ripening by the addition of a pure culture. To begin with, there is the
initial difficulty of not being able to pasteurise milk intended for
cheese, as rennet will not coagulate pasteurised milk (Lloyd). Hence
it is impossible to avoid some contamination of the milk previous to
the addition of the culture. The continual uncontaminated supply of
pure culture is by no means an easy matter. The maintenance of a low
temperature to prevent the rapid multiplication of extraneous bacteria
will, in some localities, be a serious difficulty. These difficulties
have, however, not proved insurmountable, and by various workers in
various localities and countries culture-ripening is being carried on.

_Abnormal Ripening._ Unfortunately, from one cause or another, faulty
fermentations and changes are not infrequently set up. Many of these
may be prevented, being due to lack of cleanliness in the process or
in the milking; others are due to the gas-producing bacteria being
present in abnormally large numbers. When this occurs we obtain what
is known as "gassy" cheese, on account of its substance being split
up by innumerable cavities and holes containing carbonic acid gas,
or sometimes ammonia or free nitrogen. Some twenty-five species of
micro-organisms have been shown by Adamety to cause this abnormal
swelling. In severe cases of this gaseous fermentation the product is
rendered worthless, and even when less marked the flavour and value are
much impaired. Winter cheese contains more of this species of bacteria
than summer. Acid and salt are both used to inhibit the action of these
gas-producing bacteria and yeasts, and with excellent results.

We may remark that the character of the gas holes in cheese is not of
import in the differentiation of species. If a few gas bacteria are
present, the holes will be large and less frequent; if many, the holes
will be small, but numerous. (Swiss cheese having this characteristic
is known as Nissler cheese.)

Many of these gas germs belong to the lactic-acid group, and are
susceptible to heat. A temperature of 140° F. maintained for fifteen
minutes is fatal to most of them, largely because they do not form
spores. The sources of the extensive list of bacteria found in cheese
are of course varied, more varied indeed than is the case with milk.
For there are, in addition to the organisms contained in the milk
brought to the cheese factory, the following prolific sources, viz.,
the vats and additional apparatus; the rennet (which itself contains a
great number); the water that is used in the manufacture.

In addition to the abnormalities due to gas, there are also other
faulty types. The following chromogenic conditions occur: _red cheese_,
due to a micrococcus; _blue cheese_, produced, according to Vries,
by a bacillus; and _black cheese_, caused by a copious growth of
low fungi. _Bitter cheese_ is the result of the _Micrococcus casei
amari_ of Freudenreich, a closely allied form of Conn's micrococcus of
bitter milk. Sometimes cheese undergoes a putrefactive decomposition,
and becomes more or less putrid. These latter conditions, like the
gassy cheeses, are due to the intrusion of bacteria from without,
or from udder disease of the cow. Healthy cows, clean milking, and
the introduction of pure cultures are the methods to be adopted for
avoiding "diseases" of cheese and obtaining a well-flavoured article
which will keep.

Finally, we may quote five conclusions from the prolonged researches
of Mr. Lloyd[70] which cannot but prove helpful to the Cheddar cheese
industry in England:

1. To make Cheddar cheese of excellent quality, the _Bacillus acidi
lactici_ alone is necessary; other germs will tend to make the work
more rather than less difficult. Hence scrupulous cleanliness should be
a primary consideration of the cheese-maker.

2. No matter what system of manufacture be adopted, two things are
necessary. One is that the whey be separated from the curd, so that
when the curd is ground it shall contain not less than 40 per cent. of
water, and not more than 43 per cent.; the other point is that the
whey left in the curd shall contain, developed in it before the curd is
put in the press, at least 1 per cent. of lactic acid if the cheese is
required for sale within four months, and not less than 8 per cent. of
lactic acid if the cheese is to be kept ripening for a longer period.

3. The quality of the cheeses will vary with the quality of the milk
from which they have been made, and proportionately to the amount of
fat present in that milk.

4. "Spongy curd" is produced by at least five organisms, and one of
these is responsible for a disagreeable taint found in curd. They
occur in water. Hence the desirability of securing clean water for all
manipulative purposes, and also for the drinking purposes of the milch
cow.

5. The fact that certain bacteria are found in certain localities and
dairies is due more to local conditions than to climatic causes.

It is needless to remark that these conclusions once more emphasise the
fact that strict and continual cleanliness is the one desideratum for
bacteriologically good dairying. That being secured in the cow at the
milking, in the transit, and at the dairy, it is a comparatively simple
step, by means of pasteurisation and the use of good pure cultures of
flavouring bacteria, to the successful application of bacteriology to
dairy produce.

_Methods of Examination of Milk_:

1. _Preparation of Microscopic Slides._ This course might at once occur
to the mind as the first to adopt in searching for bacteria in milk.
Devices have accordingly been proposed for saponification previous to
staining. Some recommend the addition of a few drops of a solution
of sodium carbonate; others use methylene blue and chloroform. But,
whatever plan of staining is adopted, this method of examination in its
simplest form is in no degree a criterion of the bacterial content of a
large quantity of milk.

Hence it has come to be recognised that one of two manipulations must
precede such microscopic examination. These simple processes are known
by the terms of _sedimentation_ and _centrifugalisation_. Sedimentation
means merely placing the milk in conical glasses in a cool place for
twenty-four hours. The introduction of improved forms of the centrifuge
has brought the second method of securing a sediment into preference.
Five cubic centimetres of the milk are introduced into the graduated
bottle, which is then placed in the centrifuge, and whirled for one
or two minutes. Thus a deposit of particulate matter is ensured.
Cover-glass specimens of the sediment or deposit are then prepared and
stained in the ordinary way.

[Illustration: A CENTRIFUGE

Used in the Examination of Milk]

In testing for tubercle something more is generally necessary. To the
50 cc. of the milk set aside for sedimentation 10 cc. of liquefied,
colourless carbolic acid are added. The mixture is shaken and poured
into the conical glass. After standing for twenty-four hours a little
of the sediment is taken by means of a pipette and examined by ordinary
methods, though after "fixing" the films with heat they are some times
passed through equal parts of alcohol and ether. The stain is of course
that usually adopted in tubercle, namely, the Ziehl-Neelsen. Scheurlen
suggested a method for demonstrating the tubercle bacillus in milk by
steeping the cover glasses first in alcohol and then ether, after which
they were stained with Ziehl-Neelsen.

2. _Plate Culture._ The milk is to be diluted a thousand or more times
with sterile water, and ordinary plate cultures made in Petri dishes or
flat-bottomed conical flasks. The colonies should be counted as late as
possible; but even then the isolation of pathogenic germs is uncertain.
As regards further procedure, the ordinary methods of sub-culturing
adopted in water examination must be strictly followed, and the special
tests for _Bacillus typhosus_ and _B. coli_ applied. As we have already
seen, the quantitative estimation of organisms in milk is not of the
same value as in water.

3. _Inoculation._ To test the capacity of the milk for causing disease,
before or after centrifugalisation, preferably the latter, a certain
quantity of the sediment may be inoculated into guinea-pigs. In
suspected tubercle 2 cc. may be taken; in diphtheria a little less
will suffice. The inoculation should be either intraperitoneal or
subcutaneous. Many authorities hold that this test is the only safe one
to protect the public from milk containing germs of disease.


BACTERIA IN OTHER FOODS

_Shell-fish_ have recently claimed the attention of bacteriologists,
owing to the outbreak of typhoid and other epidemics apparently
traceable to _oysters_.

It is four or five years since Professor Conn startled the medical
world by tracing an epidemic of typhoid fever to the consumption
of some uncooked oysters.[71] Almost at the same time Sir William
Broadbent published in the _British Medical Journal_ a series of
cases occurring in his practice which illustrated the same channel of
infection. Since then a number of similar items of evidence to the same
effect have cropped up. Hence there is little wonder that a number of
investigators concentrated their attention upon this matter. Professors
Herdman and Boyce, of Liverpool, Dr. Cartwright Wood, Dr. Klein, and
Dr. Timbrell Bulstrode are some of the chief contributors to the
elucidation of this problem.

The mode of infection of oysters by pathogenic bacteria is briefly
as follows: The sewage of certain coast towns is passed untreated
out to sea. At or near the outfall, oyster-beds are laid down for
the purpose of fattening oysters. Thus they become contaminated with
saprophytic and pathogenic germs contained in the sewage. It will be
at once apparent that several preliminary questions require attention
before any deductions can be drawn as to whether or not oysters convey
virulent disease to consumers. To the solution of these Dr. Cartwright
Wood was one of the first to address himself.

The precise conditions which render one locality more favourable than
another in respect to oyster culture are not fully known. But it has
been observed that they do not flourish in water containing less than
three per cent. of salt. Hence they are absent from the Baltic Sea,
which, owing to the fresh water flowing into it in rivers, contains a
smaller percentage of salt than three. Oysters appear in addition, to
favour a locality where they find their chosen food of small animalculæ
and particles of organic matter. Such a favourable locality is the
mouth of a river, where tides and currents also assist in bringing
food to the oyster. Unfortunately, however, in a crowded country like
England such localities round her coasts are frequently contaminated
by sewage from outfalls. Thus the oysters and the sewage come into
intimate relation with each other.

Professor Giaxa carried out some experiments in 1889 at Naples which
appeared to show that the bacilli of cholera and typhoid rapidly
disappeared in ordinary sea-water. Other observers at about the same
time, notably Foster and Freitag, arrived at an opposite conclusion.
In 1894 Professor Percy Frankland, in a report to the Royal Society,
declared "that common salt, whilst enormously stimulating the
multiplication of many forms of water bacteria, exerts a directly
and highly prejudicial effect on the typhoid bacilli, causing their
rapid disappearance from the water, whether water bacteria are
present or not." It was at this time, when the matter was admittedly
in an unsatisfactory stage, that Dr. Cartwright Wood made his
experiments.[72] We have not space here to enter into this work. But
his conclusions seem to have been amply established, and were to the
effect that typhoid and cholera bacilli could, as a matter of fact,
exist over very lengthened periods in ordinary sea-water. The next step
was to demonstrate the length of time the bacilli of cholera remained
alive in the pallial cavity and body of the oyster. Dr. Wood found they
did so for eighteen days after infection, though in greatly diminished
numbers. This diminution was due to one or all of three reasons: (_a_)
the effect of the sea-water already referred to as finally prejudicial
to bacilli of typhoid; (_b_) the vital action of the body-cells of the
oyster; (_c_) the washing away of bacilli by the water circulating
through the pallial cavity.

It will have been noticed that up to the present we have learned that
typhoid bacilli can and do live in sea-water, and also inside oysters
up to eighteen days, but in ever-diminishing quantities. The question
now arises: What is the influence of the oyster upon the contained
bacilli? Under certain conditions of temperature organisms may multiply
with great rapidity inside the shell of the oyster. Yet, on the other
hand, the amœboid cells of the oyster, the acid secretion of its
digestive glands, or the water circulating through its pallial cavity,
may act inimically on the germs. Proof can be produced in favour of
the third and last-named mode by which an oyster can cleanse itself of
germs. So far, then, we have met with no facts which make it impossible
for oysters to contain for a lengthened period the specific bacteria
of disease. Let us now turn to their opportunity for acquiring such
disease germs. It is afforded them during the process of what is
termed "fattening." By this process the body of the oyster acquires a
plumpness and weight which enhances its commercial value. This desired
condition is obtained by growing the oyster in "brackish" water, for
thus it becomes filled out and mechanically distended with water.
But if this water contains germs of disease, what better opportunity
could such germs have for multiplication than within the body-cavity
of an oyster? "The contamination of sea-water, therefore, in the
neighbourhood of oyster-beds may undoubtedly lead to the molluscs
becoming infected with pathogenic organisms" (Wood). Yet we have
seen that, apart altogether from the individual susceptibilities or
otherwise of the consumer, there are in the series of events necessary
to infection many occasions when circumstances would practically free
the oysters from infection.

The sources of pollution of oysters are not the fattening beds alone.
The native beds also may afford opportunity for contamination.
Thirdly, in packing and transit, and fourthly, in storage in shops and
warehouses, there is frequently abundant facility for putrefactive
bacteria to gain entrance to the shells of oysters.

Dr. Klein's researches[73] into this question have been wholly
confirmatory of the facts elicited by Dr. Cartwright Wood. Despite
the tendency of the bacilli of cholera and typhoid to die out quickly
in crude sewage, the sewage is sufficiently altered or diluted at the
outfall for these organisms to exist there in a virulent state. We may
give Dr. Klein's conclusions:

1. That the cholera and typhoid bacilli are difficult of demonstration
in sewage known to have received them.

2. Both organisms may persist in sea-water tanks for two or three
weeks, the typhoid bacillus retaining its characteristics unimpaired,
the cholera bacillus tending to lose them.

3. Oysters from sources free of sewage contained no bacteria of sewage.

4. Oysters from sources exposed to risk of sewage contamination did
contain colon bacilli and other sewage bacteria.

5. In one case Eberth's typhoid bacillus was found in the mingled body
and liquor of the oyster.

Nor do typhoid bacilli lose activity or virulence by passing through an
oyster.

These researches once and for all established the fact that oysters
ordinarily grown on oyster-beds contaminated with bacteria may, and
do on occasion, contain the virulent specific bacillus of typhoid,
which can live both in sea-water and within the shell of the oyster.
This being so, it will probably appear to the reader that the risk of
infection of typhoid by oysters is very serious indeed. Yet in actual
practice many conditions have to be fulfilled. For, in addition to the
fact that the oysters must be consumed, as is usual, uncooked, the
following conditions must also be present.

(_a_) Each infective oyster must contain infected sewage, which
presupposes that typhoid excreta from patients suffering from the
disease have passed into that particular sewage untreated and not
disinfected.

(_b_) The infective oyster must be fed upon infected sewage, and still
contain the virus in its substance.

(_c_) It has to be eaten by a susceptible person.

(_d_) There must have been no period of natural cleansing after
"fattening."

Even to this formidable list of conditions we must add the further
remark that, owing to the vitality of the body-cells of the oyster, or
to the lessened vitality of the bacilli of cholera and typhoid, it is
generally the case that the tendency of these organisms is rather to
decrease and die out than live and multiply.

We shall probably maintain a satisfactory balance of truth if we place
alongside these facts the summary of the Local Government Board Report.

  "There can be no doubt," wrote Sir Richard Thorne, "that
  oysters which have been brought into sustained relation with
  the typhoid bacillus are liable to exhibit that microbe
  within the shell contents and to retain it for a while under
  circumstances not only permitting its rapid multiplication
  when transferred again to appropriate media, but conserving at
  the same time its ability to manifest its hurtful properties."

From what has been said the preventive treatment is obvious.
All oyster-layings and shell-fish beds round the coast should
be superintended and inspected by the sanitary authority of the
Government. The importation of foreign oysters, grown on uncontrolled
beds, should, if possible, be _restricted_ or _supervised_. Further,
as a protective measure of the first importance, oysters should be
cleansed, after fattening on a contaminated bed, by being deposited
for several weeks at some point along the coast which is washed by
pure sea-water. Retention in dirty water-tanks, in uncleanly shops and
warehouses, is also to be greatly deprecated.

In order to examine oysters bacteriologically, it is necessary to pay
particular attention to the water in the pallial cavity, the contents
of the alimentary canal, and the washings of the shell itself. Ordinary
media may be used for obtaining a growth of the contained organisms.

_Other shell-fish_ than oysters do, from time to time, cause epidemics
or individual cases of gastro-intestinal irritation, and probably
contain various germs. These they acquire in all probability from their
food, which by their own choice is frequently of a doubtful character.

_Meat._ Parasites are occasionally found in meat, but bacteria are
comparatively rare. Not that they do not occur in the bodies of
animals used for human consumption, for in the glands, mesenteries,
and other organs they are common. But in those portions of the carcass
which are used by man, namely the muscles, bacteria are rare. The
reasons alleged for this are the acid reaction (sarcolactic acid) and
the more or less constant movement during life. A bacterial disease
which, perhaps more than any other, might be expected to be conveyed by
meat is tubercle. Yet the recent Royal Commission on Tuberculosis has
again emphasised the absence of bacilli in the meat substance:

  "In tissues which go to form the butcher's joint, the material
  of tubercle is not often found even where the organs (lungs,
  liver, spleen, membranes, etc.) exhibit very advanced or
  generalised tuberculosis; indeed, in muscle and muscle juice
  it is very seldom that tubercle bacilli are to be met with;
  perhaps they are somewhat more often to be discovered in bone,
  or in some small lymphatic gland embedded in intermuscular
  fat."[74]

The only way in which such meat substance becomes infected with
tubercle appears to be through carelessness in the butcher, who
perchance smears the meat substance with a knife that has been used
in cutting the organs, and so has become contaminated with infected
material. Very instructive also are the results at which Dr. Sims
Woodhead arrived in compiling evidence for the same Commission on the
effect of cooking upon tuberculous meat:

  "Ordinary cooking, such as boiling and more especially
  roasting, though quite sufficient to sterilise the surface,
  and even the substance for a short distance from the surface
  of a joint, cannot be relied upon to sterilise tubercular
  material included in the centre of rolls of meat, especially
  when these are more than three pounds or four pounds weight.
  The least reliable method of cooking for this purpose is
  roasting before a fire; next comes roasting in an oven, and
  then boiling."[75]

From this statement it will be understood that rolled meat may be a
source of infection to a greater degree than the ordinary joint.

Notwithstanding this negative evidence, more than twenty species
of bacteria have been isolated from canned meats and hams, and a
considerable number of poisoning cases have occurred from meat
contaminated with bacteria or their products. The general symptoms
of such meat poisoning are vomiting, diarrhœa, fever, and more or
less prostration. Ballard and Klein isolated a specific microbe from
samples of bacon which appear to have caused an epidemic of infectious
pneumonia at Middlesborough. In 1880 occurred the well-known "Welbeck
disease" epidemic. A public luncheon was followed by severe and even
fatal illness. Seventy-two persons were affected, and four died. A
specific bacillus was isolated by Klein. In 1881 much the same thing
happened at Nottingham, in which fifteen persons were attacked, and one
died. The same bacillus was isolated from the pernicious pork. Again in
1889 an outbreak of diarrhœa at Carlisle was traced to bacterially
diseased pork. But taking these and similar cases at their worst, there
can be no doubt that under no circumstances is meat as infective as
milk.

_Ice-cream._ In 1894 Dr. Klein had occasion to bacteriologically
examine ice-creams sold in the streets of London. In all six samples
were analysed, and in each sample the conclusions resulting were of
a nature sufficiently serious to support the view that the bacterial
flora was not inferior to ordinary sewage. The water in which the
ice-cream glasses were washed was also examined, and found to contain
large numbers of bacteria.

Since that date many investigations have been made into ice-creams. It
appears that they are often made under extremely foul circumstances,
and with anything but sterilised appliances. Little wonder, then,
that the numbers of bacteria present run into millions. In nearly
all recorded cases the quality of the germs as well as the quantity
has been of a nature to cause some concern. _Bacillus coli communis_,
which, though not now considered absolutely indicative of alimentary
pollution, is looked upon as a highly unsatisfactory inhabitant of
water, has been found in considerable abundance. The _Proteus_ family,
which also possesses a putrefactive function, is common in ice-creams.
The _common water bacteria_ are nearly always present.

_Bacillus typhosus_ itself, it is said, has been isolated from some
ice-cream which was held responsible for an outbreak of enteric fever.
The material had become infected during process of manufacture in the
house of a person suffering from unnotified typhoid fever.

Now, whilst reports of the above nature appear very alarming, the
fact is that hundreds of weakly children devour ice-cream with
apparent impunity, and when evil follows it is not infrequently due
to other than bacterial conditions. The cold mass itself may inhibit
the resistance of the gastric tissues. _Tyrotoxicon_, the alkaloid
separated from cheese and cream by Vaughan, may be responsible for
some alimentary irritation. On the whole, the practical effect upon
the community is not in proportion to the bacterial content of the
ice-cream. Yet, nevertheless, we ought to be much more watchful than in
the past to preserve ice-cream from pollution with harmful bacteria.

The two chief constituents which contribute their quota of germ life
to ice-cream are _ice_ and _cream_. In addition, the uncleanly methods
of manufacture render the material likely to contain the six or
seven millions of micro-organisms per cc. which have been on several
occasions estimated. To cleanly methods of dairying we have already
fully referred; to the bacterial content of milk and cream we have
also paid some attention; but we have not had an opportunity of saying
anything of germs in ice.

_Ice_ contains bacteria in varying quantities from 20 per cc. to 10,000
or more. Nor is variation in number affected alone by the condition of
the water, for samples collected from one and the same place differ
widely. The quality follows in large measure the standard of the water.

Water bacteria, _Bacillus coli_, putrefactive bacteria, and even
pathogenic have been found in ice. Many of the latter can live without
much difficulty and are most numerous in ice containing air-bubbles.

Dr. Prudden, of New York, performed a series of experiments in 1887
to show the relative behaviour of bacteria in ice. Taking half a
dozen species, he inoculated sterilised water and reduced it to a
very low temperature for a hundred and three days, with the following
results:--_Bacillus prodigiosus_ diminished from 6,300 per cc. to
3,000 within the first four days, to 22 in thirty-seven days, and
vanished altogether in fifty-one days; a liquefying water bacillus,
numbering 800,000 per cc. at the commencement, had disappeared in four
days; _Staphylococcus pyogenes aureus_ and _B. fluorescens_ showed
large numbers present at the end of sixty-six and seventy-seven days
respectively; _B. typhosus_, which was present 1,000,000 per cc. after
eleven days, fell to 72,000 after 77 days, and 7,000 at the end of 103
days. Anthrax bacilli are susceptible to freezing, but their spores are
practically unaffected (Frankland).

From these facts it will be seen that bacteria live, but do not
multiply, in ice.

In making a bacterial investigation into the flora of ice-cream, it is
necessary to remember that considerable dilution with sterilised water
is required. The usual methods of examining water and milk are adopted.

_Bread_ forms an excellent medium for moulds, but unless specially
exposed the bacteria in it are few. Waldo and Walsh have, however,
demonstrated that baking does not sterilise the interior of bread.
These observers cultivated numerous bacteria from the centre of newly
baked London loaves.[76] The writer has recently made a series of
examinations of the air of several underground bakehouses in Central
London; but, though the air was highly impregnated with flour-dust, few
bacteria were present.

Other foods and beverages may be, and are, from time to time
contaminated in some small degree with bacteria or their spores.
Such contaminations are generally due to uncleanly manufacture or
unprotected storage. The principles of examination or of the prevention
of pollution are similar to those already described.




CHAPTER VII

_THE QUESTION OF IMMUNITY AND ANTITOXINS_


The term _natural immunity_ is used to denote natural resistance to
some particular specific disease. It may refer to race, or age, or
individual idiosyncrasies. We not infrequently meet with examples of
this freedom from disease. Certain races of men do not, as a rule, take
certain diseases. For example, plague and leprosy, though endemic in
some countries, fail to get a footing in England. This, of course, is
due in great measure to the sanitary organisation and cleanly customs
of the English people. Still, it is also due to the fact that the
English appear in some degree to be immune. Some authorities hold that
the immunity against leprosy is due to the fact that the disease has
exhausted itself in the English race. However that may be, we know that
immunity, entire or partial, exists. Children, again, are susceptible
to certain diseases and insusceptible to certain others to which older
people are susceptible. We know, too, that some individuals have a
marked protection against some diseases. Some people coming into the
way of infection at once fall victims to the disease, whilst others
appear to be proof against it. It is only in recent times that any
very intelligent explanations have been offered to account for this
phenomenon. The most recent of these, and that which appears to have
most to substantiate it, is known as immunity due to _antitoxins_.

_The products of bacteria_ are chiefly six:

1. _Pigment._ We have already seen how many organisms exhibit their
energy in the formation of many coloured pigments. They are, as a rule,
"innocent" microbes. Oxygen is required for some, darkness for others,
and they all vary according to the medium upon which they are growing.
Red milk, yellow milk, and green pus afford examples of pigment
produced by bacteria.

2. _Gas._ Quite a number of the common bacteria, like _Bacillus
coli_, produce gas in their growth; hydrogen (H), carbonic acid
(CO_{2}), methane (CH_{4}), and even nitrogen (N) being formed by
different bacteria. Many gases produced during fermentative processes
are the result, not directly of the growth of the bacillus causing
the fermentation, but indirectly owing to the splitting up of the
fermenting fluids.

3. _Acids._ Lactic, acetic, butyric, etc., are common types of acids
resulting from the growth of bacteria.

4. _Liquefying Ferment._ As we have seen, bacteria may be classified
with regard to their behaviour in gelatine medium, whether or not they
produce a peptonising ferment which liquefies the gelatine.

5. _Phosphorescence._ Some species of bacteria in sea-water possess the
power of producing light.

6. _Organic Chemical Products._ When a pathogenic bacillus grows
either in the body or in a test-tube, it produces as a result of its
metabolism certain poisonous substances called _toxins_. These may
occur in the blood as a direct result of the life of the bacillus, or
they may occur as the result of a ferment produced by the bacillus.
They are of various kinds according to the various diseases, and
by their effect upon the blood and body tissues they cause the
symptoms of the disease in question. We know, for instance, that a
characteristic symptom common to many diseases is fever. Now, fever
is produced by the action of the albumoses (bodies allied to the
proteids) upon the heat-regulating centres in the brain. Whenever we
get a bacillus growing in the body which has the power of producing a
toxin albumose, we get fever as a result of that product acting upon
the brain. Albumoses, as a matter of fact, cause a number of symptoms
and poisonous effects, but the mention of one as an illustration will
suffice. Toxins act, roughly speaking, in two ways:

(1) They have a local action, as, for example, in the formation of an
abscess. The presence of the causal bacteria in the tissue brings about
very marked changes. There is a multiplication of connective-tissue
corpuscles, an emigration of leucocytic cells, a congestion of blood
corpuscles. All these elements assist in creating a swelling and
redness, and pain by the subsequent pressure upon the delicate nerve
endings. These, as we all know, are the symptoms of a "gathering"
or abscess. It is a "gathering" in a strict pathological sense--a
gathering of cells to oust the intruder or build around it a wall or
capsule as a protective measure. Now the toxin will commence its local
action. The oldest cells in the mass of congestion will be caused
to break down into liquid; what is called a _necrosis_, or death,
will rapidly set in; and we shall have the connective-tissue cells,
leucocytes, blood corpuscles, etc., losing their form and function, and
"coming to a point" as matter, or _pus_. The local breaking down of
these gatherings of cells into fluid matter is believed to be the work,
not of the bacteria themselves, but of their toxins.

(2) Toxins may be absorbed and distributed generally throughout the
body. They produce degenerative changes in muscles, in organs, and in
the blood itself. Let us take diphtheria as an example. The bacillus
occurs in a false membrane in the throat and occasionally other parts.
It causes first the inflammatory condition giving rise to the membrane,
and then it breaks it down. In the body of the membrane the bacillus
appears to secrete a ferment which by its action and interaction with
the body cells and proteids, chiefly those of the spleen, produces
_albumoses_ and an _organic acid_. These latter bodies are the toxins.
They are absorbed, and pass throughout the body. There are albumoses,
therefore we get the frequent pulse and high temperature of fever; the
toxins irritate the mucous membrane of the intestine, and cause various
fermentative changes in the contents of the intestine, therefore we
get the symptoms of diarrhœa; they penetrate the liver, spleen, and
kidney, therefore we get fatty degeneration and its results in these
organs; they finally affect many of the motor and sensory nerves,
breaking up their axis cylinders into globules, and therefore we get
the characteristic paralysis. Loss of weight naturally follows many of
these degenerative or wasting changes. Here, then, we have some of the
chief changes set up by the toxins, and these changes constitute the
leading symptoms in the disease as it is known clinically.

In addition to the presence of the specific bacillus in the membrane,
we also have a number of other organisms, like the _Bacillus coli_,
_Coccus Brissou_, _Streptococcus pyogenes_, and various staphylococci,
diplococci, etc. Each of these produces or endeavours in the midst
of keen competition and strife to produce, its own specific effect.
Thus we obtain the complications of diphtheria, for example various
suppurative and septic conditions. The whole of this compound process
we may tabulate roughly as follows[77]:

                       | BACILLUS OF DIPHTHERIA = primary infective
  _Bacillus coli._     |     |                      agent.
  _Coccus Brissou._    | Inflammatory changes and fibrinous exudation.
  Staphylococci.       |     |
  Diplococci.          | FERMENT IN MEMBRANE = secondary infective
  Streptococci.        |     |                   agent.
      |                | Passes through body, and
  Toxins.              | by digestion of proteids
      |                | produces              { ALBUMOSES:
  Suppurative glands,  |                       { AN ORGANIC ACID.
    septic poisoning,  |                               |
            etc.       |                     1. Fever.
                       |                     2. Diarrhœa.
                       |                     3. Loss of body weight.
                       |                     4. Fatty degeneration.
                       |                     5. Degeneration of
                       |                          peripheral nerves, and
                       |                          resulting paralysis.

Such is the general effect of toxins in diphtheria. The same principles
apply with equal force in tetanus, typhoid, etc., the only differences
being in degree of virulence, mode of onset, and portions of the body
chiefly affected.

Sidney Martin has recently[78] elaborated the views announced by
him in 1892, and it is right that reference should be made to his
new classification of bacterial poisons. This may be represented as
follows:--

  1. The poisons secreted by the bacterium itself  }
      = (ferment? toxin?)                          }
  2. Products of digestive action of bacterium =   }  = Extracellular
      albumoses:                                   }  bacterial poisons.
  3. Final non-proteid products = animal alkaloid; }
  4. Poisons present in the bodies of the bacillus    = Intracellular
                                                      bacterial poisons.

The poisons of bacteria are, according to Sidney Martin, of a kind
which cannot be fully expressed chemically, but only pathologically.
They may be of a ferment nature in diphtheria and tetanus. The
arguments in support of that view are--(1) that they act in
infinitesimal doses, (2) that they may act slowly and produce death
after many days by profoundly affecting the general nutrition, and (3)
that they are sensitive to the action of heat in a way that no chemical
poisons are known to be. If they are considered as ferments, they must
be substances which have a peculiar affinity for certain tissues of
the body on which they produce their special toxic effect. As for the
products of digestion, they are formed either by the bacillus ingesting
the proteid and discharging it as albumose, or the digestion occurs
by means of a ferment secreted by the bacillus in the body of an
individual or animal suffering from the disease.

Sidney Martin suggests that _anthrax_ produces albumoses and an
alkaloidal substance, the former producing fever, the latter stupor.
In _tetanus_ the bacillus produces a secretion of the bacillus which
causes the convulsions. The albumoses present in this disease are
probably due to the secretory toxin. In _diphtheria_, too, we have a
secretory poison in the membrane and in the tissues, and an albumose
which is possibly the result of the secretion. It will be seen that
these views differ in some particulars from those to which we have
already referred.

However the details of the _modus operandi_ of the formation of toxins
are finally settled, we know that there comes a time when the disease
symptoms vanish, the disease declines, and the patient recovers.
Many of the older schools of medicine explained this satisfactory
phenomenon by saying that this disease exhausted itself after having
"gone through" the body. In a sense that idea is probably true; but
recently a large number of investigators have applied themselves to
this problem, and with some promising results.

Various protective inoculations against anthrax were practised as early
as 1881, and the protected animals remained healthy. In 1887 Wooldridge
succeeded in protecting rabbits from anthrax by a new method, by which
he showed that the growth of the anthrax bacillus in special culture
fluids gave rise to a substance which, when inoculated, conferred
immunity. In 1889 and 1890 Hankin and Ogata worked at the subject,
and announced the discovery in the blood of animals which had died of
anthrax of some substances which appeared to have an antagonistic and
neutralising effect upon the toxins of anthrax and upon the anthrax
bacilli themselves. These substances, they afterwards found, were
products of the anthrax bacillus. Behring and Kitasato arrived at
much the same results for tetanus and diphtheria. The next step was
to isolate these substances, and, separating them from the blood,
investigate still further their constitution. A number of workers
were soon occupied at this task, and since 1891 Buchner, Hankin, the
Klemperers, Roux, Sidney Martin, and others have added to our knowledge
respecting these toxin-opposing bodies known as _antitoxins_. In
diphtheria, as we have seen, the toxins turned out to be soluble bodies
allied to the proteids, albumoses, and an organic acid. Then arose the
question of the source of antitoxins. Some believed they were a kind
of ultratoxin--bodies of which an early form was a toxin; others held
that, as the toxins were products of the bacteria invading the tissues,
the antitoxins were of the nature of ferments produced by the resisting
tissues. Finally, they came to be looked upon as protective substances
produced _in the body cells_ as a result of toxin action, and held in
solution in the blood, and there and elsewhere exerting their influence
in opposition to the toxins.[79] The progress of disease is therefore a
struggle between the toxins and the antitoxins: when the toxins are in
the ascendency we get an increase of the disease; when the antitoxins
are in the ascendency we get a diminution of disease. If the toxins
triumph, the result is death; if the antitoxins and resistance of the
tissues triumph, the result is recovery.

We may now consider shortly how these new facts were received and
what theories of explanation were put forward to explain continued
insusceptibility to disease. It had of course been known for a
long time past that one attack of small-pox, for example, in some
degree protected the individual from a subsequent attack of the same
disease. To that experience it was now necessary to add a large mass
of experimental evidence with regard to toxins and antitoxins. The
theories of immunity were as follows:

1. _The Exhaustion Theory._ The supporters of this idea argued that
bacteria of disease circulating in the body exhausted the body of the
supply of some substance or condition necessary for the growth and
development of their own species.

2. _The Retention Theory._ It was surmised that there were certain
products of micro-organisms of disease retained in the body after an
attack which acted antagonistically to the further growth in the body
of that same species.

3. _The Acquired Tolerance Theory._ Some have advanced the theory
that, after a certain time, the human tissues acquired such a degree
of tolerance to the specific bacteria or their specific products
that no result followed their action in the body. The tissues become
acclimatised to the disease.

4. _The Phagocyte Theory._ This theory, which gained so many adherents
when first promulgated by Metschnikoff, attributes to certain cells
in the tissues the powers of "scavenging," overtaking germs of
disease, and absorbing them into their own protoplasm. This, indeed,
may be actually witnessed, and had been observed before the time
of Metschnikoff. But it was he who applied it to disease. He came
to the conclusion that the successful resistance which an animal
offered to bacteria depended upon the activity of these scavenging
cells, or _phagocytes_. These cells are derived from various cellular
elements normally present in the body: leucocytes, endothelial cells,
connective-tissue corpuscles, and any and all cells in the body which
possess the power of ingesting bacteria. If they are present in large
numbers and active, the animal is insusceptible to certain diseases; if
they are few and inactive, the animal is susceptible.

It appears that the bacteria or other foreign bodies in the blood
which are attacked by the phagocyte become assimilated until they are
a part of the phagocyte itself. Metschnikoff explained also how it
comes to pass that the phagocyte is able to encounter bacteria when
both are circulating through the blood. It is guided in this attack
upon the organisms by a power termed _chemiotaxis_. The bacteria
elaborate a chemical substance which attracts the phagocyte, and
this is termed "positive chemiotaxis."[80] But it may occur that the
chemical substance produced by the bacteria may have an opposite, or
repellent, effect upon the leucocytes, in which case we have "negative
chemiotaxis."[81] It is not to be wondered at that such a theory of
immunity based upon microscopical observations, should at first have
been widely accepted, and there can be no doubt that Metschnikoff
has collected a considerable mass of evidence in support of a theory
of phagocytosis. But when it came to be known that blood serum, from
which all leucocytes (phagocytes) had been removed, possessed the
same immunising effect as before, it was clear that such effect was
a property of the serum _per se_, and not only or wholly due to the
scavenging power of certain cells in it. Even the phagocyte theory
depends largely for its validity upon chemiotaxis, which latter was a
property of the products of the bacteria contained in the blood serum.

5. _The Antitoxin Theory._ We have gathered, then, that whenever
bacteria, introduced into the blood and tissues, fail to multiply or
produce infection (as in saprophytic bacteria, or in immunity of a
particular animal from a specific microbe), this inability to perform
their rôle is brought about by some property in the living and normal
blood serum which opposes their life and action; and further we have
learned that this protective property is exhaustible according to the
number of bacteria, and differs with various species of bacteria, and
in different animals. Buchner designates these protective bodies, held
in solution in the blood, _alexines_, and regards them as belonging
to the albuminous bodies of the lymph and plasma. Where the blood
and tissues do not possess this power, the animal is susceptible.
Now, as we have already seen from the experiments of Ogata, Kitasato,
and others, the blood of an animal dead of anthrax is protective
against anthrax, from which and the foregoing it appears that microbes
produce by their growth in the tissues poisonous substances we term
_toxins_, which have the power of producing in the blood and body
cells substances inimical to themselves, named _antitoxins_, and so
long as these latter substances remain in the tissues the body remains
insusceptible to further attacks of the same disease. Alexines are
_naturally_ produced antitoxins; antitoxins are _acquired_ alexines.
Hence we have the well-known terms "natural" and "acquired immunity."
Of the former we have already spoken. _Acquired immunity_ is a
protection not belonging to the tissues of individuals naturally and as
part of their constitution, but it is acquired during their lives as a
further accomplishment, so to speak, of their tissues. This may happen
in one or both of two ways. Either it may be an involuntary acquired
immunity, or a voluntary acquired immunity. For example, the former is
at once illustrated by an attack of the disease.

Small-pox, typhoid fever, even scarlet fever, are diseases which very
rarely attack the same individual twice. That is because each of these
diseases leaves behind it, on its first appearance, its antitoxic
influence. Hence the individual has _involuntarily_ acquired immunity
against these diseases. An example of voluntary acquired immunity
is also at hand in the old method of preventive inoculation for
small-pox, or _variolation_. This was clearly an inoculation setting
up an artificial and mild attack of small-pox, by which the antitoxins
of that disease were produced, and protected the individual against
further infection of small-pox; that is to say, it was a voluntary
acquired immunity. This form of artificial production of protection is
generally called _artificial immunity_. Let us now marshal together
these various terms in a table as follows:

  _Immunity_ in man {= a condition of protection of insusceptibility to
                    {    certain diseases.

  1. _Natural immunity_ = constitutional protection produced by
       _alexines_.

  2. _Acquired immunity_

    { _Acquired naturally_ (involuntarily) produced by antitoxins formed
    {   byan attack of the disease.
    {
    { _Acquired artificially_ (voluntary)=
    {
    {  (_a_) _Active_ immunity, produced by direct inoculation of the
    {           weakened bacteria or weakened toxins of the disease,
  = {           _e. g._, vaccination, or Pasteur's treatment of rabies,
    {           or Haffkine's inoculation for cholera.
    {
    {  (_b_) _Passive_ immunity, produced by inoculation, not of the
    {           disease or of its toxins, but of the _antitoxins_
    {           produced in the body of an animal suffering from the
    {           specific disease.


It is hoped that previous remarks will have explained the meaning of
the terms used in the above table, with the exception of the last two
phrases of active and passive immunity. We propose now to consider in
some detail the four illustrations quoted under these two headings,
viz., vaccination, Pasteur's treatment of rabies, anti-cholera
inoculation, and antitoxin inoculation. From all accounts, it is to be
feared that these four phases of artificial immunity are hopelessly
confused in the educated public mind. Nor is this to be wondered at
when we reflect upon the rapid growth of the whole science of immunity,
and upon the ever-varying forms of nomenclature through which it has
passed.

_Vaccination for Small-pox._ In 1717 Lady Mary Wortley Montagu[82]
described the inoculation of small-pox as she had seen it practised
in Constantinople. So greatly was she impressed with the efficacy of
this process that she had her own son inoculated there, and in 1721 Mr.
Maitland, a surgeon, inoculated her daughter in London. This was the
first time inoculation was openly practised in England.[83] For one
hundred and twenty years small-pox inoculation (or _variolation_, as
it is more correctly termed) was practised in England, until by Act of
Parliament in 1840 it was prohibited.

There were different ways of performing variolation, but the most
approved method was similar to the modern system of arm-to-arm
vaccination, the arm being inoculated with a lancet in one or more
places with small-pox lymph instead of, as now, with vaccine lymph.
As a rule, only local results or a mild attack of small-pox followed,
which prevented an attack of natural small-pox. Its disadvantage is
apparent on the surface. It was a means of breeding small-pox, for the
inoculated cases were liable to create fresh centres of infection. In
1796 Edward Jenner, who was a country practitioner in Gloucestershire,
observed that those persons affected with cow-pox, contracted in the
discharge of their duty as milkers, did not contract small-pox, even
when placed in risk of infection. Hence he inferred that inoculation of
this mild and non-infectious disease would be preferable to the process
of variolation then so widely adopted in England. Jenner therefore
suggested the substitution of cow-pox lymph (vaccine) in place of
small-pox lymph, as in ordinary variolation.

It should not be forgotten that variolation was thus the first work
done in this country in producing artificial immunity, and was followed
by vaccination, which was only partly understood. Even to-day there is
probably much to learn respecting it. Both variolation and vaccination
may be described as _active immunisation by means of an attenuated form
of the specific virus causing the disease_. The nature of the specific
virus of both small-pox and cow-pox awaits discovery. Burdon Sanderson,
Crookshank, Klein, and Copeman have all demonstrated bacteria in
cow-pox or vaccine lymph, and in 1898 Copeman announced that he had
isolated a specific bacillus and grown it upon artificial media.[84]
Numerous statements have been made to the effect that a specific
bacillus has been found in small-pox also. But neither in small-pox nor
cow-pox is the nature of the contagion really known.[85]

These facts, however, do not remove the suspicion which has hitherto
rested upon vaccine lymph as a vehicle for bacteria of other diseases
which by its inoculation may thus be contracted. A few remarks
are therefore called for at this juncture upon the recent work of
Dr. Monckton Copeman and Dr. Frank Blaxall in respect to what is
known as _glycerinated calf lymph_. Evidence has been forthcoming
to substantiate in some measure the distrust which many of the
public have from time to time felt in the vaccine commonly used in
vaccination, hence the new form as above designated. This retains the
toxic qualities required for immunity, but is so produced that it
possesses in addition three very important advantages; namely, it is
entirely free from extraneous organisms, it is available for a large
number of vaccinations, and it retains full activity for eight months.
It is prepared as follows:

A calf, aged three to six months, is kept in quarantine for a week. If
then found upon examination to be quite healthy, it is removed to the
vaccination station, and the lower part of its abdomen antiseptically
cleaned. The animal is now vaccinated upon this sterilised area with
glycerinated calf lymph. After five days the part is again thoroughly
washed, and the contents of the vesicle, which have of course
appeared in the interval, are removed with a sterilised sharp spoon,
and transferred to a sterilised bottle. This is now removed to the
laboratory, and the exact weight of the material ascertained. A calf
thus vaccinated will yield from 18 to 24 grams of vaccine material.
This is now thoroughly triturated and mixed with six times its weight
of a sterilised solution of 50 per cent. chemically pure glycerine
in distilled water. The resulting emulsion is aseptically stored in
sealed tubes in a cool place. For four weeks it is carefully examined
bacteriologically until the glycerine has absolutely killed any
possible germ that may have obtained entrance. When by agar plates it
is demonstrably sterile it is ready for distribution.

_Pasteur's Treatment of Rabies._ Rabies is a disease affecting dogs (in
Western Europe) and wolves (in Russia), and can be transmitted to other
animals and man, infection being carried by the bite of a rabid animal.
It takes two chief forms: (1) furious rabies and (2) paralytic rabies.
The former is more common in dogs. The animal becomes restless, has a
high-toned bark, and snaps at various objects. Sometimes it exhibits
depraved appetite; spasms of the throat follow, and these soon develop
into convulsions, which are followed by coma and death. In man the
incubation period is fortunately a very long one, averaging about forty
days. Nervous irritability is the first sign; spasms occur in the
respiratory and masticatory muscles, and the termination is similar to
rabies in the dog. The symptom of fear of water is a herald of coming
fatality.

Although a number of the workers at the Pasteur Institute and elsewhere
have addressed themselves to the detection of a specific microbe, none
has as yet been found, although, in the opinion of Pasteur, such an
agent may be suspected as the cause.

Pathologically rabies and tetanus (see page 168) are closely allied
diseases, and the recent remarkable additions to our knowledge of the
latter disease only make the similarity more evident. There are in
rabies three chief sets of _post-mortem_ signs. First, and by far the
most important, are the changes in the nervous system. Here we find
patches of congestion in the brain, and breaking down of the axis
cylinders of the nerves. The stomach, in the second place, exhibits
hæmorrhagic changes, not unlike acute for arsenical poisoning. Thirdly,
the salivary glands show a degenerative change in a breaking down of
their secreting cells. Roux has pointed out that in life the saliva
of a mad dog becomes virulent three days before the appearance of the
symptoms of disease.

We may now turn to the method of treatment which was introduced by
Pasteur. Before his time cauterisation of the wound was the only
means adopted. If more than half an hour has elapsed since the bite,
cauterisation is of little or no avail. The basis for Pasteur's
treatment was the difference in virulence obtainable in spinal cords
infected with rabies. Pasteur found that drying the cord led to a
lessening of its virulence, just as certain other conditions increased
its virulence. Next he established the fact that subcutaneous injection
of a weak virus, followed up with doses of ever-increasingly virulent
cords, immunised dogs against infection or inoculation of fully
virulent material. From this he reasoned that if he could establish
a standard of weakened virulence he would have at hand the necessary
"vaccine" for the treatment of the disease.

[Illustration: SUSPENDED SPINAL CORD

In drying jar containing Calcium Chloride]

Subsequent research and skilled technique resulted in a method of
securing this standard, which he found to be a spinal cord dried for
fourteen days. The exact details are as follows: The spinal cords
of two rabbits dead of rabies are removed from the spinal canal in
their entirety by means of snipping the transverse processes of
the vertebrae. Each cord is divided into three more or less equal
pieces, and each piece, being snared by a thread of sterilised silk,
is carefully suspended in a sterilised glass jar. At the bottom of
the jar is a layer, about half an inch deep, of sterilised calcium
chloride. The jars are then removed to a dark chamber, where they are
placed at a temperature of 20-22° C. in wooden cases. Here they are
left to dry. Above each case is a tube of broth, to which has been
added a small piece of the corresponding cord, in order to test for
any organismal element that may by chance be included. In case of the
slightest turbidity in the broth, the cord is rejected. Fourteen series
of cords are thus suspended on fourteen consecutive days. The first,
second, and third are found to be of practically equal virulence,
but from the third to the fourteenth the virulence proportionately
decreases, and on the fifteenth day the cord would be practically
innocuous and non-virulent. When treatment is to be commenced,
obviously the weakest--that is, the fourteenth day--cord is used to
make the "vaccine," and so on in steadily increasing doses (as regards
virulence) up to and including a third-day cord. The fourteenth-day
cord is therefore taken, and a small piece cut off and macerated in 10
cc. of sterile broth, which are placed in a conical glass and covered
with two layers of thick filter-paper, the glass with its covering
having been previously sterilised by dry heat. When the patient bitten
by the rabid animal is prepared, 3 cc. of this broth emulsion of spinal
cord are inoculated by means of a hypodermic needle into the flanks
or abdominal wall. On the following day the patient returns for an
inoculation of a cord of the thirteenth day, and so on until a rabid
cord emulsion of the first three days has been inoculated. As a matter
of practice, the dosage depends upon the three recognised classes of
bites, viz. (1) bites through clothing (least severe); (2) bites on the
bare skin of the hand; (3) bites upon the face or head, most severe
owing to the vascularity of these parts. An example of each, which the
writer was permitted to take in the Pasteur Institute, may be here
added to make quite clear the entire practice. (See page 258.)

It may be well to add the returns of inoculation made at the Pasteur
Institute, Rue Dutot, Paris, as above described. They are as follows:

        No. of Persons  No. of  Rate of
  Year.  inoculated.   Deaths. Mortality.

  1886      2,671        25      0.94
  1887      1,770        14      0.79
  1888      1,622         9      0.55
  1889      1,830         7      0.38
  1890      1,540         5      0.32
  1891      1,559         4      0.25
  1892      1,790         4      0.22
  1893      1,648         6      0.36
  1894      1,387         7      0.50
  1895      1,520         2      0.13
  1896      1,308         4      0.30
  1897      1,521         6      0.39

Pasteur's treatment of rabies by inoculation of emulsions of dried
spinal cord is, therefore, a "vaccination" of attenuated virus,
resulting in antitoxin formation, to the further protection of the
individual against rabies.

One further example of the modern application of the principles of
active acquired immunity may be shortly mentioned. We refer to the
cholera and plague vaccinations. The vaccination in small-pox is an
inoculation of the _virus of the disease_; the rabies inoculation is
a transmission of the _vital products of the disease attenuated_; the
plague and cholera vaccinations are inoculations of _pure cultures of
living virus_ from outside the body. Inoculating cholera virus against
cholera has been made illegal, as variolation was in 1840. But Haffkine
has prepared two vaccines. The weak one is made from pure cultures of
Koch's spirillum of Asiatic cholera, attenuated by growth to several
generations on agar or broth at 39°C. The strong one is from similar
culture the virulence of which has been increased. One cubic centimetre
of the first vaccine is injected hypodermically into the flank, and
the second vaccine three or four days afterwards. The immunisation is
prophylactic, not remedial, and its action takes effect five or six
days after the second vaccine has been injected.

INOCULATION TREATMENT FOR PERSONS AFFECTED WITH RABIES

  ------------------------------
  1. FOR THOSE BITTEN THROUGH
       CLOTHES.
  -------------+--------+-------
  Days of      |Doses of|Dates
  Treatment.   |Emulsion|of Cord
               |per cc. |Drying.
  -------------+--------+-------
   1 at 11 A.M.|   3    |  14
   1    "    " |   3    |  13
   2    "    " |   3    |  12
   2    "    " |   3    |  11
   3    "    " |   3    |  10
   3    "    " |   3    |   9
   4    "    " |   3    |   8
   4    "    " |   3    |   7
   5    "    " |   3    |   6
   5    "    " |   3    |   6
   6    "    " |   3    |   5
   7    "    " |   3    |   5
   8    "    " |   3    |   4
   9    "    " |   2    |   3
  10    "    " |   3    |   5
  11    "    " |   3    |   5
  12    "    " |   3    |   4
  13    "    " |   3    |   4
  14    "    " |   3    |   3
  15    "    " |   3    |   3
       ...     |  ...   |  ...
       ...     |  ...   |  ...
       ...     |  ...   |  ...
       ...     |  ...   |  ...
       ...     |  ...   |  ...
       ...     |  ...   |  ...
       ...     |  ...   |  ...
       ...     |  ...   |  ...
  -------------+--------+-------
  2. FOR THOSE BITTEN ON
       UNCOVERED SKIN OF HANDS,
       ETC.
  -------------+--------+-------
  Days of      |Doses of|Dates
  Treatment.   |Emulsion|of Cord
               |per cc. |Drying.
  -------------+--------+-------
   1 at 11 A.M.|   3    |  14
   1    "    " |   3    |  13
   2    "    " |   3    |  12
   2    "    " |   3    |  11
   3    "    " |   3    |  10
   3    "    " |   3    |   9
   4    "    " |   3    |   8
   4    "    " |   3    |   7
   5    "    " |   3    |   6
   5    "    " |   3    |   6
   6    "    " |   3    |   5
   7    "    " |   3    |   5
   8    "    " |   3    |   4
   9    "    " |   2    |   3
  10    "    " |   3    |   5
  11    "    " |   3    |   5
  12    "    " |   3    |   4
  13    "    " |   3    |   4
  14    "    " |   3    |   3
  15    "    " |   3    |   3
  16    "    " |   3    |   5
  17    "    " |   3    |   4
  18    "    " |   3    |   3
       ...     |  ...   |  ...
       ...     |  ...   |  ...
       ...     |  ...   |  ...
       ...     |  ...   |  ...
       ...     |  ...   |  ...
  -------------+--------+-------
  3. FOR THOSE BITTEN ON FACE
       OR HEAD.
  -------------+--------+-------
  Days of      |Doses of|Dates
  Treatment.   |Emulsion|of Cord
               |per cc. |Drying.
  -------------+--------+-------
   1 at 11 A.M.|   3    |  14
   1    "    " |   3    |  13
   1 at  3 P.M.|   3    |  12
   1    "    " |   3    |  11
   2 at 11 A.M.|   3    |  10
   2    "    " |   3    |   9
   2 at  3 P.M.|   3    |   8
   2    "    " |   3    |   7
   3 at 11 A.M.|   3    |   6
   3    "    " |   3    |   6
   4    "    " |   3    |   5
   5    "    " |   3    |   5
   6    "    " |   3    |   4
   7    "    " |   2    |   3
   8    "    " |   3    |   4
   9    "    " |   3    |   3
  10    "    " |   3    |   5
  11    "    " |   3    |   5
  12    "    " |   3    |   4
  13    "    " |   3    |   4
  14    "    " |   3    |   3
  15    "    " |   3    |   3
  16    "    " |   3    |   5
  17    "    " |   3    |   4
  18    "    " |   3    |   3
  19    "    " |   3    |   5
  20    "    " |   3    |   4
  21    "    " |   3    |   3
  -------------+--------+-------

In plague the same plan has been followed. Luxurious crops of
Kitasato's plague bacillus are grown on ordinary nutritive media plus
large quantities of fat. The fat of milk, as clarified butter, is that
generally used. Under the globules of fat flakes of culture grow like
stalactites, hanging down into the clear broth. These are in time
shaken to the bottom, and a second crop grows on the under-surface
of the fat. In the course of a month perhaps half a dozen such crops
are obtained and shaken down into the fluid, until the latter assumes
an opaque milky appearance. This is now, unlike the cholera vaccine,
exposed to a temperature of 70° C., by which the microbes are killed.
The culture contains all the toxins, and the dose is 3 cc. This
preparation has the advantage of being easily prepared, obtainable
in large quantities, and requires no animals in its preparation.
When inoculated it produces local pain and swelling at the site of
inoculation, and general reactive symptoms such as fever. From a
careful analysis of the results of this inoculation, it is shown that
the efficacy of the prophylactic depends upon the virulence of the
bacillus culture from which the vaccine is prepared, and upon its dose
and ability to produce a well-marked febrile reaction. It appears to be
more effective in the prevention of deaths than of attacks.

The anti-typhoid vaccination is another example of inoculation to
secure active immunity. It is needless, perhaps, to point out that all
these vaccinations, except rabies, are prophylactic, and not curative.

_Passive Immunity; Preparation of Antitoxins._ We must now consider
the question of passive immunity. This, it will be remembered, may
be defined as a protection (against a bacterial disease) produced by
inoculation, not of the disease itself, as in small-pox inoculation,
nor yet of its weakened toxins, as in rabies, but of the _antitoxins_
produced in the body of an animal suffering from that particular
disease. Examples of this treatment are increasing every year, and the
term "antitoxin" has now become almost a household word. The chief
examples are to be found in diphtheria, tetanus, streptococcus, and
pneumococcus.

To be of value, antitoxins must be used as early as possible, before
tissue change has occurred and before the toxins have, so to speak,
got the upper hand. When the toxins are in the ascendency the patient
suffers more and more acutely, and may succumb before there has been
time for the formation in his own body of the antitoxins. If he can
be tided over the "crisis," theoretically all will be well, because
then his own antitoxin will eventually gain the upper hand. But in the
meantime, before that condition of affairs, the only way is to inject
antitoxins prepared in some animal's tissues whose disease began at an
earlier date, and thus _add_ antitoxins to the blood of our patient,
early in the disease, and the earlier the better, for, however soon
this is done, it is obvious that the toxins begin their work earlier
still. It should not be necessary to add that general treatment must
also be continued, and indeed local germicidal treatment, _e. g._, of
the throat in diphtheria and the poisoned wound in tetanus. Further,
in a mixed infection, as in glandular abscesses with diphtheria, it
must be borne in mind that the antitoxin is specific and may therefore
probably fail in such mixed cases.

After these preliminary remarks we will now consider shortly some of
the methods employed for the production of antitoxins. An animal is
required from whose body a considerable quantity of blood can be drawn
without injurious effect. Moreover, it must be an animal that can stand
an attack of such diseases as diphtheria and tetanus. Such an animal is
the horse. Now, by injecting into the horse (_a_) living organisms of
the specific disease, but in non-fatal doses, or (_b_) dead cultures,
or (_c_) filtered cultures containing no bacteria and only the toxins,
we are able to produce in the blood of the horse first the toxins and
then the antitoxins of the disease in question. The non-poisonous
doses of living organisms can be weakened, or, as we say, attenuated,
by various means. Dead cultures have not been much used to produce
immunity except by Pfeiffer. In actual practice the third method is
much the most general, viz., filtering a fluid culture free from the
bacteria, and then inoculating this in ever-increasing doses. The
preparation of diphtheria antitoxin may be taken as an example, but
what follows would be equally applicable to other diseases, such as
tetanus.

1. _To Obtain the Toxin._ First grow a pure culture of the
Klebs-Löffler bacillus of diphtheria in large flasks containing
"Löffler's medium," or a solution made by mixing three parts of blood
serum with one of beef broth and adding one per cent. of common salt
(Na Cl) and one per cent. of peptone. An alkaline medium is preferable.
The flask was thoroughly sterilised before use, and is now plugged with
sterile cotton-wool and incubated at 77° C. for three or four weeks.
Pure air may be passed over the culture periodically, thereby aiding
the growth. After the lapse of about a month a scum of diphtheria
growth will have appeared over the surface of the fluid. This is now
filtered into sterilised flasks, and some favourable antiseptic added
to ensure that nothing foreign to the toxin shall flourish, and the
flasks are kept in the dark. Here, then, we have the product, the
toxin, ready for injection into the horse.

[Illustration: Flask used for the Preparation of the Toxin of
Diphtheria]

2. _Immunisation of the Horse._ It is evident that only healthy horses
are of service in providing healthy antitoxin, even as healthy children
are necessary in arm-to-arm vaccination. To provide against any
serious taint the horse is tested for glanders (with mallein) and for
tuberculosis (with tuberculin). The dose of the injection of toxin is
at the commencement about 1/10 cc., or a little more. The site of the
inoculation is the apex of the shoulder, which has been antiseptically
cleaned. A mere prick is the whole operation. After the first injection
there is generally a definite febrile reaction and a slight local
swelling. From 1/10 or 1/2 cc. the dose is steadily increased, until
at the end of two or three months[86] perhaps as much as 300 cc. (or
even half a litre) may be injected without causing the reaction which
the initial injection of 1/10 cc. caused at the outset. This shows an
acquired tolerance of the tissues of the horse to the toxic material.
After injecting 500 cc. into the horse without bad effect, the animal
has a rest of four or five days.

3. _To Obtain the Antitoxin._ During this period of rest the
interaction between the living body cells of the animal and the toxins
results in the production in the blood of an antitoxin. By means of a
small sterilised cannula, five, or eight, or even ten litres of blood
are drawn from the jugular vein of the horse into sterilised flasks or
jars. The top of the jar is closed by two paper coverings before it is
sterilised. Then it is again covered with a further loose one. Before
use the loose one is removed and replaced by a metal (zinc) lid, which
has been separately sterilised. This metal lid contains an aperture
large enough for the tube which conveys the blood from the cannula to
pass through. The tube, therefore, passes through the metal lid and two
paper covers, which it was made to pierce. When enough blood has passed
into the vessel the tube is withdrawn, and the metal lid slightly
turned. Thus the contained blood is protected from the air.[87]

The jar containing the blood (which contains the antitoxin) is next
placed in a dark, cool cellar, where it stands for two or three days.
During this time the blood naturally coagulates, the corpuscles falling
as a dense clot to the bottom, and the faintly yellow serum rising
to the top. The serum, or _liquor sanguinis_, averages about 50 per
cent. of the total blood taken. Sometimes antiseptics are added with
a view to preservation. It is generally filtered before bottling for
therapeutic use, and sometimes examined bacteriologically as a test of
purity.

4. _The Use of Antitoxins._ The antitoxins are now ready for injection
into the patient who has contracted diphtheria, and in whose blood
toxins are in the ascendency and under which the individual may
succumb. They are injected in varying doses, as we have already
pointed out.[88] The general result is that mortality has been greatly
lessened, and that in fatal cases there has been a considerable
lengthening of the period of life. Moreover, the whole clinical course
of the disease has been greatly modified, and suffering lessened.[89]




CHAPTER VIII

_BACTERIA AND DISEASE_


Probably the most universally known fact respecting bacteria is that
they are related in some way to the production of disease. Yet we have
seen that it was not as disease-producing agents that they were first
studied. Indeed, it is only within comparatively the latest period
of the two centuries during which they have been more or less under
observation that our knowledge of them as causes of disease has assumed
any exactitude or general recognition. Nor is this surprising, for
although an intimate relationship between fermentation and disease had
been hinted at in the middle of the seventeenth century, it was not
till the time of Pasteur that the bacterial cause of fermentation was
experimentally and finally established.

In the middle of the seventeenth century men learned, through the eyes
of Leeuwenhoek, that drops of water contained "moving animalcules." A
hundred years later Spallanzani demonstrated the fact that putrefaction
and fermentation were set up in boiled vegetable infusions when
outside air was admitted, but when it was withheld from these boiled
infusions no such change occurred. Almost a hundred years more passed
before the epoch-making work of Tyndall and Pasteur, who separated
these putrefactive germs from the air. Quickly following in their
footsteps came Davaine and Pollender, who found in the blood of animals
suffering from anthrax the now well-known specific and causal bacillus
of that disease. Improvements in the microscope and in methods of
cultivation (Koch's plate method in particular) soon brought an army
of zealous investigators into the field, and during the last twenty
years first this disease and then that have been traced to a bacterial
origin. We may summarise the vast mass of historical, physiological,
and pathological research extending from 1650 to 1898 in three great
periods: the period of detection of living, moving cells (Leeuwenhoek
and others in the seventeenth century); the period of the discovery of
their close relationship to fermentation and putrefaction (Spallanzani,
Schulze, Schwann, in the eighteenth century); and, thirdly, the period
of appreciation of the rôle of bacteria in the economy of nature and
in the production of disease (Tyndall, Pasteur, Lister, Koch, in the
nineteenth).

But we must look less cursorily at the growth of the idea of bacteria
causing disease. More than two hundred years ago Robert Boyle
(1627-91), the philosopher, who did so much towards the foundation
of the present Royal Society, wrote a learned treatise on _The
Pathological Part of Physic_. He was one of the earliest scientists to
declare that a relationship existed between fermentation and disease.
When more accurate knowledge was attained respecting fermentation,
great advance was consequently made in the etiology of disease. The
preliminary discoveries of Fuchs and others between 1840 and 1850
had relation to the existence in diseased tissues of a large number
of bacteria. But this was no proof that such germs _caused_ such
diseases. It was not till Davaine had inoculated healthy animals with
bacilli from the blood of an anthrax carcass, and had thus produced
the disease, that reliance could be placed upon that bacillus as the
_vera causa_ of anthrax. Too much emphasis cannot be laid upon this
idea, that unless a certain organism _produces_ in healthy tissues
the disease in question, it cannot be considered as proven that the
particular organism is related to the disease as cause to effect. In
order to secure a standard by which all investigators should test their
results, Koch introduced four postulates. Until each of the four has
been fulfilled, the final conclusion respecting the causal agent must
be considered _sub judice_. The postulates are as follows:

(_a_) The organism must be demonstrated in the circulation or tissues
of the diseased animal.

(_b_) The organism thus demonstrated must be cultivated in artificial
media outside the body, and successive generations of a _pure culture_
of that organism must be obtained.

(_c_) Such pure cultures must, when introduced into a healthy and
susceptible animal, produce the specific disease.

(_d_) The organism must be found and isolated from the circulation or
tissues of the inoculated animal.

It is evident that there are some diseases--for example, cholera,
leprosy, and typhoid--which are not communicable to lower animals, and
therefore their virus cannot be made to fulfil postulate (_c_). In
such cases there is no choice. They cannot be classified along with
tubercle and anthrax. Bacteriologists have little doubt that Hansen's
bacillus of leprosy is the cause of that disease, yet it has not
fulfilled postulates (_b_) and (_c_). Nor has the generally accepted
bacillus of typhoid fulfilled postulate (_c_), yet by the majority it
is _provisionally_ accepted as the agent in producing typhoid. Hence
it will be seen that, though there is an academical classification
of causal pathogenic bacteria according as they respond to Koch's
postulates, yet nevertheless, there are a number of pathogenic bacteria
which are looked upon as causes of disease provisionally. Anthrax and
tubercle, with perhaps the organisms of suppuration, tetanus, plague,
and actinomycosis, stand in the first order of pathogenic germs. Then
comes a group awaiting further confirmation. It includes the organisms
related to typhoid, cholera, malaria, leprosy, diarrhœa, and
pneumonia. Then comes in a third category, a long list of diseases,
such as scarlet fever, small-pox, rabies, and others too numerous to
mention, in which the nature of the causal agent is still unknown.
Hence it must not be supposed that every disease has its germ, and
without a germ there is no disease. Such universal assertions, though
not uncommonly heard, are devoid of accuracy.

In the production of bacterial disease there are two factors. First,
there is the body tissue of the individual; secondly, there is the
specific organism.

Whatever may be said hereinafter with regard to the power of
micro-organisms to cause disease, we must understand one cardinal
point, namely, that bacteria are never more than causes, _for the
nature of disease depends upon the behaviour of the organs or tissues
with which the bacteria or their products meet_ (Virchow). Fortunately
for a clear conception of what "organs and tissues" mean, these have
been reduced to a common denominator, the cell. Every living organism,
of whatever size or kind, and every organ and tissue in that living
organism, contains and consists of cells. Further, these cells are
composed of organic chemical substances which are not themselves alive,
but the mechanical arrangement of which determines the direction and
power of their organic activity and of their resistance to the specific
agents of disease. With these facts clearly before us, we may hope to
gain some insight into the reasons for departure from health.

The normal living tissues have an inimical effect upon bacteria.
Saprophytic bacteria of various kinds are normally present on exposed
surfaces of skin or mucous membrane. Tissues also which are dead or
depressed in vitality from injury or previous disease, but which are
still in contact with the tissues, afford an excellent nidus for the
growth of bacteria. Still these have not the power, unless specific, to
thrive in the normal living tissue. It has been definitely shown that
the blood fluids of the body have in their fresh state the germicidal
power (_alexines_) which prevents bacteria from flourishing in them.
Such action does undoubtedly depend in measure upon the number of germs
as well as their quality, for the killing power of blood and lymph must
be limited. Buchner has pointed out that the antagonistic action of
these fluids depends in part possibly upon phagocytosis, but largely
upon a chemical condition of the serum. The blood, then, is no friend
to intruding bacteria. Its efforts are to a certain extent seconded
by the lymphoid tissue throughout the body. Rings of lymphoid tissue
surround the oral openings of the trachea (windpipe) and œsophagus
(gullet); the tonsils are masses of lymphoid tissue. Composed as it is
of cells having a germicidal influence when in health, the lymphoid
tissue may afford formidable obstruction to intruding germs.

All the foregoing points in one direction, namely, that if the tissues
are maintained in sound health, they form a very resistant barrier
against bacteria. But we know from experience that a full measure of
health is not often the happy condition of human tissues; we have, in
short, a variety of circumstances which, as we say, _predispose_ the
individual to disease. One of the commonest forms of predisposition
is that due to _heredity_. Probably it is true that what are known as
hereditary diseases are due far more to a hereditary predisposition
than to any transmission of the virus itself in any form. _Antecedent
disease_ predisposes the tissues to form a nidus for bacteria;
_conditions of environment or personal habits_ frequently act in the
same way. Damp soils must be held responsible for many disasters to
health, not directly, but indirectly, by predisposition; dusty trades
and injurious occupations have a similar effect. Any one of these three
different influences may in a variety of ways affect the tissues and
increase their susceptibility to disease. Not infrequently we may get
them combined. For example, the following is not an unlikely series of
events terminating in consumption (tuberculosis of the lungs):--(_a_)
The individual is predisposed by inheritance to tuberculosis; (_b_)
an ordinary chronic catarrh, which lowers the resisting power of the
lungs, may be contracted; (_c_) the epithelial collections in the air
vesicles of the lung--_i. e._, dead matter attached to the body--afford
an excellent nidus for bacteria; (_d_) owing to occupation, or
personal habits, or surroundings, the patient comes within a range of
tubercular infection, and the specific bacilli of tubercle gain access
to the lungs. The result, it is needless to state, will be a case of
consumption more or less acute according to environment and treatment.

The _channels of infection_ by which organisms gain the vantage-ground
afforded by the depressed tissues are various, and next to the
maintenance of resistant tissues they call for most attention from the
physician and surgeon. It is in this field of preventive medicine--that
is to say, preventing infective matter from ever entering the tissues
at all--that science has triumphed in recent years. It is, in short,
applied bacteriology, and therefore claims consideration in this place.

1. _Pure Heredity._ By this term may be understood the actual
transmission from the mother to the unborn child of the specific virus
of the disease. That such a conveyance may occur is generally admitted
by pathologists, but it is impossible to enter fully into the matter
in such a book as the present. Summarily we may say that, though
this sort of transmission is possible, it is not frequent, nor is
disease appreciably spread through such a channel. Sixty per cent. of
consumptives, it has been estimated, have tuberculous progenitors, and
this is the highest figure. Many would be justified from experience in
placing it at half that number.

2. _Inoculation_, or inserting virus through a broken surface of
skin, is itself a sufficiently obvious mode of infection to call for
little comment. Yet it is under this heading that a word must be
said of that remarkable application of preventive medicine known as
the antiseptic treatment of wounds. When Lord Lister was Professor of
Surgery in Glasgow, he was impressed with the greatness of the evil
of putrefaction in wounds, which was caused, not by the oxygen of the
air, as Liebig had declared, but by the entrance into the wound of
fermentative organisms from the air. This was demonstrated by Pasteur,
who pointed out that they could not arise _de novo_ in the wound. Hence
it appeared to Lister that these fermentative bacteria which produce
putrefaction in wounds must either be kept out of the wound altogether,
or killed, or their action prevented, in the wound. To keep air away
from wounds is an almost impossible task, and thus it came about that
wounds were dressed with a solution of carbolic acid.

From time to time examples occur of bacterial disease being directly
inoculated in wounds made with polluted instruments, or in cuts made by
contaminated broken glass, or in gunshot wounds. Tetanus is, of course,
one of the most marked examples.

3. _Contagion_ is a term which has suffered from the many ways in which
it has been used. Defined shortly and most simply, we should say a
disease is _contagious_ when it can be "caught" by contact, through the
unbroken surfaces, between diseased and healthy persons. Ringworm is an
example, and there are many others.

4. _The Alimentary Canal: Food._ The recent Royal Commission on
Tuberculosis has collected a large mass of evidence in support of the
view that tubercle may be spread by articles of food. Milk and meat
from tuberculous animals naturally come in for the largest amount of
condemnation. To these matters we refer elsewhere.

5. _The Respiratory Tract: Air._ The air may become infected with germs
of disease from dusty trades, dried sputum, etc. If such infected air
be inhaled, pathogenic results will follow, especially if the bacteria
are present in sufficient numbers, or meet with devitalised, and
therefore non-resisting, tissues.

These, then, are the five possible ways in which germs gain access to
the body tissues. The question now arises, How do bacteria, having
obtained entrance, set up the process of disease? For a long time
pathologists looked upon the action of these microscopic parasites in
the body as similar to, if not identical with, the larger parasites
sometimes infesting the human body. Their work was viewed as a
devouring of the tissues of the body. Now, it is well known that,
however much or little of this may be done, the specific action
of pathogenic bacteria is of a different nature. It is twofold.
We have the action of the bacteria themselves, and also of their
products or toxins. In particular diseases, now one and now the other
property comes to the front. In bacterial diseases affecting or being
transmitted mostly by the blood, it is the toxins which act chiefly.
The convenient term _infection_ is applied to those conditions in
which there has been a multiplication of living organisms after they
have entered the body, the word _intoxication_ indicating a condition
of poisoning brought about by their products. It will be apparent
at once that we may have both these conditions present, the former
before the latter, and the latter following as a direct effect of the
former. Until intoxication occurs there may be few or no symptoms, but
directly enough bacteria are present to produce in the body certain
poisons in sufficient amount to result in more or less marked tissue
change, then the symptoms of that tissue change appear. This period of
latency between infection and the appearance of the disease is known
as the _incubation period_. Take typhoid, for example. A man drinks a
typhoid-polluted water. For about fourteen days the bacilli are making
headway in his body without his being aware of it. But at the end of
that incubation period the signs of the disease assert themselves.
Professor Watson Cheyne and others have maintained that there is some
exact proportion between the number of bacteria gaining entrance and
the length of the incubation period.

Speaking generally, we may note that pathogenic bacteria divide
themselves into two groups: those which, on entering the body, pass
at once, by the lymph or blood stream, to all parts of the body,
and become more and more diffused throughout the blood and tissues,
although in some cases they settle down in some spot remote from the
point of entrance, and produce their chief lesions there. Tubercle
and anthrax would be types of this group. On the other hand, there is
a second group, which remain almost absolutely local, producing only
little reaction around them, never passing through the body generally,
and yet influencing the whole body eventually by means of their
ferments or toxins. Of such the best representatives are tetanus and
diphtheria. The local site of the bacteria is, in this case, the local
manufactory of the disease.

Whilst the mere bodily presence of bacteria may have mechanical
influence injurious to the tissues (as in the small peripheral
capillaries in anthrax), or may in some way act as a foreign body and
be a focus of inflammation (as in tubercle), the real disease-producing
action of pathogenic bacteria depends upon the _chemical poisons_
(toxins) formed directly or indirectly by them. Though within
recent years a great deal of knowledge has been acquired about the
formation of these bodies, their exact nature is not known. They
are allied to albuminous bodies and proteoses, and are frequently
described as tox-albumens. It may be found, after all, that they are
not of a proteid nature. Sidney Martin has pointed out that there
is much that is analogous between the production of toxins and the
production of the bodies of digestion. Just as ferments are necessary
in the intestine to bring about a change in the food by which the
non-soluble albumens shall be made into soluble peptones and thus
become absorbed through the intestinal wall, so also a ferment may be
necessary to the production of toxins. Such ferments have not as yet
been isolated, but their existence in diphtheria and tetanus is, as we
have seen, extremely likely. However that may be, it is now more or
less established that there are two kinds of toxic bodies, differing
from each other in their resistance to heat. It may be that the one
most easily destroyed by heat is a ferment and possibly an originator
of the other. A second division which has been suggested for toxic
bodies, and to which reference has been made, is _intracellular_ and
_extracellular_, according to whether or not the poison exists within
or without the body of the bacillus.

Lastly, we may turn to consider the action of the toxins on the
individual in whose body-fluids they are formed. It is hardly necessary
to say that any action which bacteria or toxins may have will depend
upon their virulence, in some measure upon their number, and not
a little upon the channel of infection by which they have gained
entrance. It could not be otherwise. If the virulence is attenuated,
or if the invasion is very limited in numbers, it stands to reason
that the pathogenic effects will be correspondingly small or absent.
The influence of the toxins is twofold. In the first place (i.) they
_act locally upon the tissues_ at the site of their formation, or
at distant points by absorption. There is inflammation with marked
cell-proliferation, and this is, more or less rapidly, followed by
a specific cell-poisoning. The former change may be accompanied
by exudation, and simulate the early stages of abscess formation;
the latter is the specific effect, and results, as in leprosy and
tubercle, in infective nodules. The site in some diseases, like typhoid
(intestinal ulceration, splenic and mesenteric change) or diphtheria
(membrane in the throat), may be definite and always the same. But, on
the other hand, the site may depend upon the point of entrance, as in
tetanus. The distant effects of the toxin are due to absorption, but
what controls its action it is impossible to say. We only know that we
do find pathological conditions in certain organs at a distance and
without the presence of bacteria. We have a parallel in the action of
drugs; for example, a drug may be given by the mouth and yet produce
a rash in some distant part of the body. In the second place (ii.)
toxins produce _toxic symptoms_. Fever and many of the _nervous
conditions resulting_ from bacterial action must thus be classified.
We have, it is true, the chemical symptoms of the pathological tissue
change, for example, the large spleen of anthrax or the obstruction
from diphtheritic membrane. But, in addition to these, we have general
symptoms, as fever, in which after death no tissue change can be formed.

We may now consider briefly some of the more important types of disease
produced by bacteria:

+1. Tuberculosis.+[90] As far back as 1794 Baillie drew attention to
the grey miliary nodules occurring in tuberculous tissue which gave
rise to the term "tubercles." This preliminary matter was confirmed by
Bayle in 1810.

In 1834 Laennec described all caseous deposits as "tubercles,"
insisting upon four varieties:

(1) Miliary, which were about the size of millet seeds, and in groups;

(2) Crude, miliary tubercles in yellow masses;

(3) Granular, similar to the last, but scattered;

(4) Encysted, a hard mass of crude tubercle with a fibrous or
semi-cartilaginous capsule.

The tubercle possesses in many cases a special structure, and certain
cell-forms frequently occur in it and give it a characteristic
appearance. The central part of the tubercle usually contains _giant
cells_ with numerous nuclei. The uninuclear cells are partly lymphoid,
partly large epithelial or endothelial cells; these are called
_epithelioid_ cells.

It was not till 1865 that the specific nature of tuberculosis was
asserted by Villemin. Burdon Sanderson (1868-69) in England confirmed
his work, and it was extended by Connheim, who a few years later laid
down the principle that all is tubercular which by transference to
properly constituted animals is capable of inducing tuberculosis, and
nothing is tubercular unless it has this capability.

Klebs (1877) and Max Schiller (1880) described masses of living
cells or micrococci in many tuberculous nodules in the diseased
synovial membrane and in lupus skin. In 1881 Toussaint declared
he had cultivated from the blood of tubercular animals and from
tubercles an organism which was evidently a micrococcus, and in the
same year Aufrecht stated that the centre of a tubercle contained
small micrococci, diplococci, and some rods. But it was not till the
following year, 1882, that Koch discovered and demonstrated beyond
question the specific _Bacillus tuberculosis_.

It is now held to be absolutely proved that the introduction of the
bacillus, or its spores or products, is the one and only essential
agent in the production of tuberculosis. Its recognised manifestations
are as follows:

  Tuberculosis in the lungs = _acute or chronic phthisis_;
        "      in the skin = _lupus_[1];
        "      in the mesenteric glands = _Tabes mesenterica_;
        "      in the brain = _hydrocephalus_;
        "      in lymphatic glands = _Scrofula_.[91]

The disease may occur generally throughout the body or locally in the
suprarenal capsules, prostate, intestine, larynx, membranes of the
heart, bones, ovaries, pleura, kidneys, spleen, testicles, Fallopian
tubes, uterus, etc.

We may summarise the history of the pathology of tubercle thus:

  1794. Baillie drew attention to grey miliary nodules occurring
  in tuberculosis, and called them "tubercles."

  1834. Laennec described four varieties: _miliary_; _crude_;
  _granular_; _encysted_.

  1843. Klencke produced tuberculosis by intravenous injection
  of tubercular giant cells.

  1865. Villemin demonstrated infectivity of tubercular matter
  by inoculation of discharges; Connheim, Armanni, Burdon
  Sanderson, Wilson Fox, and others showed that nothing but
  tubercular matter could produce tuberculosis.

  1877. Living cells were found in tubercles, "micrococci"
  (Klebs, Toussaint, Schiller).

  1882. Koch isolated and described the specific bacillus, and
  obtained pure cultivations (1884).

_The Bacillus of Koch_, 1882. Delicate cylindrical rods, measuring
1.5-4 micromillimetres in length and about .2 µ in breadth; non-motile.
Many are straight with rounded ends; others are slightly curved. They
are usually solitary, but may occur in pairs, lying side by side or in
small masses. They are chiefly found in fresh tubercles, more sparingly
in older ones. Some lie within the giant cells; others lie outside;
shorter in tissue sections of bovine tuberculosis, but longer in the
milk (Crookshank).

When stained they appear to be composed of irregular cubical or
spherical granules within a faintly stained sheath. In recent lesions
the protoplasm appears more homogeneous, and takes on the segmented or
beaded character only in old lesions, pus, or sputum.

Morphological differences are found under different circumstances, and
within limits variation occurs according to the environment.

_Cultivation on Various Media._ Koch inoculated _solid blood serum_
with tubercular matter from an infected lymphatic gland of a
guinea-pig, and noticed the first signs of growth in ten or twelve days
in the form of whitish, scaly patches. These enlarged and coalesced
with neighbouring patches, forming white, roughened, irregular masses.
Nocard and Roux showed that by adding 5/8 per cent. of glycerine to the
media commonly used in the laboratory, such as nutrient agar or broth,
the best growth is obtained.

On _glycerine broth_ or _glycerine agar_ abundant growth appears at the
end of seven or eight days. By continuous sub-culture on glycerine agar
the virulence of the bacillus is diminished. But in fifteen days after
inoculation of the medium the culture equals in extent a culture of
several weeks' age on blood serum.

Sub-cultures from glycerined media will grow in ordinary broth without
glycerine (Nocard, Roux, Crookshank).

In _alkaline broth_ to which a piece of boiled white of egg was added
Klein obtained copious growths, and found that continued sub-culturing
upon this medium also lessens the virulence.

_Description of Cultivations:_--On glycerine agar minute white colonies
appear in about six days, raised and isolated, and coalescing as time
advances, forming a white lichenous growth, fully developed in about
two months.

On glycerine broth a copious film appears on the surface of the liquid,
which if disturbed falls to the bottom of the flask as a deposit.

_Spore Formation._ In very old cultivations spore-like bodies can be
observed both in stained and unstained preparations, but neither the
irregular granules within the capsule nor the unstained spaces between
the granules are spores (Babes and Crookshank). That the bacilli
possess spores is believed on account of the following facts:

1. That tubercular sputum, when thoroughly dried, maintains its
virulent character (Koch, Schill, Fischer, etc.). No sporeless bacillus
is known which can survive through drying.

2. That tubercular matter and cultures survive temperature up to 100°
C. Non-spore-bearing bacilli and micrococci are killed by being exposed
for five minutes to a temperature of 65-70° C., whereas spores of other
bacilli withstand much higher temperatures.

3. Tubercular sputum distributed in salt solution does not lose its
virulence by being kept at 100° C. for one or two minutes; sporeless
bacilli certainly would (Klein).

4. A solution of per-chloride of mercury does not kill the tubercle
bacilli, as it does sporeless bacilli (Lingard and Klein).

Koch and many bacteriologists have declared the bacillus to be a "true
parasite." Koch based this view upon the belief which he entertained
that the bacillus can grow only between 30° C. and 41° C., and
therefore in temperate zones is limited to the animal body and can
originate only in an animal organism. "They are," he said, "true
parasites, which cannot live without their hosts. They pass through the
whole cycle of their existence in the body." But at length Koch and
others overcame the difficulties and grew the bacillus as a saprophyte.

Schottelius[92] has observed that tubercle bacilli taken from the lung
of phthisical persons buried for years still retains its virulence and
capability of producing tuberculosis upon inoculation. He further shows
that tubercular lung kept in soil (enclosed in a box) shows a marked
rise in temperature. Klein quotes these experiments as indications that
"_tubercle bacilli are not true parasites, but belong to the ectogenic
microbes which can live and thrive independent of a living host_."

It has now been abundantly proved that the bacillus of tuberculosis is
capable of accommodating itself to circumstances much less favourable
than had been supposed, especially as regards temperature.

_Temperature of Growth of Bacillus._ 30-41° C. have been laid down by
Koch as the limits of temperature at which the bacillus will grow in
culture medium outside the body. The generally accepted temperatures as
most favourable to the growth of the bacillus are between 36° C. and
38° C.

Sir Hugh Beevor, however, was able to grow the bacillus upon glycerine
agar at 28° C. (82° F.), obtaining an ample culture which developed
somewhat more slowly than on blood serum, and to a less extent than
at 37° C. In both Beevor succeeded in growing the bacillus at a lower
temperature even than on agar, viz., at a temperature rarely above 60°
F. Sheridan Delepine and others have also been successful in obtaining
growths at room temperature both in summer and winter.

Although, speaking generally, there is an actual cessation of growth at
low temperature, the bacillus may be exposed to very low temperatures
for a considerable time without losing its power of again becoming
active when returned to a favourable environment (Woodhead).

_The Relation of the Bacillus to the Disease._ All four of Koch's
postulates have been fulfilled in the case of _Bacillus tuberculosis_.
Hence we are dealing with the specific cause of the disease. Yet,
whilst this is so, we may usefully ask ourselves: _How_ does the
bacillus set up the changes in normal tissues which result in
tubercular nodules? In arriving at a solution of this problem we are
materially aided if we bear in mind the fact that such an organism
in healthy tissues has a double effect. First, there is an ordinary
inflammatory irritation, and secondly, there is a specific change
set up by the toxins of the bacillus. Directly the invading bacilli
find themselves in a favourable nidus they commence multiplication.
In three or four days this acts as an irritant upon the surrounding
connective-tissue cells, which proliferate, and become changed into
large cells known as _epithelioid cells_. At the periphery of this
collection of epithelioid cells we have a congested area. This change
has been accomplished by the presence of the bacilli themselves. The
production of their specific poisons changes the epithelioid cells
in the centre of the nodule, some of which become fused together,
whilst others expand and undergo division of nucleus. By this means we
obtain a series of large multi-nucleated cells named _giant cells_.
If the disease is very active, these soon caseate and break down
in the centre. In a limb we get a discharge; in a lung we get an
expectoration. Both discharge and expectoration arise from a breaking
down of the new cell formation. Previously to breaking down we have in
a fully developed nodule healthy tissue, inflammatory zone, epithelioid
cells, giant cells, containing nuclei and bacilli. The sputum or the
discharge will, during the acute stage of the disease at all events,
contain countless numbers of the bacilli, which may thus be readily
detected, and their presence used as evidence of the disease. It is
obvious that if the centre of the nodule degenerates and comes away as
discharge a cavity will be left behind. By degrees this small cavity
may become a very large one, as is frequently the case in the lung,
which particularly lends itself to such a condition. Hence, though at
the outset a tubercular lung is solid, at the end it is hollow.

[Illustration: BACILLUS TUBERCULOSIS

(In sputum from a case of phthisis, "consumption" of the lungs)

× 1000

_By permission of the Scientific Press, Limited_]

[Illustration: BACILLUS TUBERCULOSIS

(The bacilli are arranged within the giant cell)

× 1000]

[Illustration: STREPTOCOCCUS PYOGENES

(From broth culture)

× 1000

_By permission of the Scientific Press, Limited_]

[Illustration: BACILLUS ANTHRACIS

(From splenic blood of cow)

× 1000

_By permission of the Scientific Press, Limited_]

The exact period of giant-cell formation depends on the rapidity
of the formative processes. Thus different conditions occur. Inside
the giant cells the bacilli are arranged in relation to the nuclei
in one of three ways: (_a_) polar, (_b_) zonal, or (_c_) mixed. The
breaking down of the nodule is partly due to the cell-poisons, and
partly because the nodule is non-vascular, owing to the fact that new
capillaries cannot grow into the dense nodule, and the old ones are all
occluded by the growth of the nodule.

From the local foci of disease the tubercle process spreads chiefly by
three channels:

(_a_) _By the lymphatics_, affecting particularly the glands. Thus
we get tuberculosis set up in the bronchial, tracheal, mediastinal,
and mesenteric glands, and it is so frequently present as to be
a characteristic of the disease. This is the common method of
dissemination in the body.

(_b_) _By the blood-vessels_, by means of which bacilli may be carried
to distant organs.

(_c_) _By continuity of tissues_, infective giant-cell systems
encroaching upon neighbouring tissues, or discharge from lungs or
bronchial glands obtaining entrance to the gullet and thus setting up
intestinal disease also.

It has been abundantly proved that the respiratory and digestive
systems are principally affected by Koch's bacillus. Wherever the
bacilli are arrested, they excite formation of granulations or miliary
tubercular nodules, which increase and eventually coalesce. The
lymphatic glands which collect the lymph from the affected region are
the earliest affected, always the nearest first, and then the disease
appears to be appreciably stopped on its invading march. Each lymphatic
gland acts as a temporary barrier to progress until the disease has
broken its structure down. It remains local, in spite of increase in
number and importance of the foci of disease, as long as the bacilli
have not gained access to the blood stream.

_Toxins and Tuberculin._ Koch, Crookshank, and Herroun, Hunter, and
others have isolated products from pure cultures of the tubercle
bacillus. These have comprised chiefly _albumoses_, _alkaloids_, and
various _extractives_. Koch's observations led him to suppose that
in pure cultures of tubercle a substance appeared having healing
action on tuberculosis, and an extract of this in glycerine he termed
"tuberculin." It was made as follows: A veal broth containing peptone
and glycerine was inoculated with a pure culture of the bacillus and
incubated at 38° C. for six or eight weeks. An abundant growth with
copious film formation appeared. The culture was then concentrated by
evaporation over a water-bath until reduced to about one-tenth of its
volume.

[Illustration: FLASK USED IN THE PREPARATION OF TUBERCULIN]

The announcements in 1890 and 1891 to the effect that a "cure" had
been discovered for consumption will be remembered. The hopes thus
raised were unfortunately not to be realised. Koch advocated injections
of this tuberculin in cases of skin tubercle (lupus) and consumptive
cases. In many of these benefit was apparently derived, but its general
application was not founded upon any substantial basis. Dead tissue,
full of bacilli, could not thus be got rid of; nor could the career of
the isolated bacilli distributed through the body be thus checked.

Tuberculin has, however, found a remarkable sphere of usefulness in
causing reaction in animals suffering from tuberculosis. Indeed,
tuberculin is the most valuable means of diagnosis that we possess
(MacFadyen). When injected (dose, 30-40 centigrammes) it causes a
rise of one and a half to three degrees. The fever begins between the
twelfth and fifteenth hour after injection, and lasts several hours.
The duration and intensity of the reaction have no relation to the
number and gravity of the lesions, but the same dose injected into
healthy cattle causes no appreciable febrile reaction. The tuberculous
calf reacts just as well as the adult, but the dose is generally 10-20
centigrammes. Injections of tuberculin have no troublesome effect on
the quantity or quality of the milk of cows or on the progress of
gestation.

_Tuberculosis of Animals._ Cattle come first amongst animals liable
to tubercle. Horses may be infected, but it is comparatively rare,
and among small ruminants the disease is rarer still. Dogs, cats, and
kittens may be easily infected. Amongst birds, fowls, pigeons, turkeys,
and pheasants, the disease assumes almost an epidemic character.
Especially do animals in confinement die of tubercle, as is illustrated
in zoölogical gardens. Respecting the lesions of bovine tuberculosis,
it will be sufficient to say that nothing is more variable than the
localisation or form of its attacks. The lungs and lymphatic glands
come first in order of frequency, next the serous membranes, then the
liver and intestines, and lastly the spleen, joints, and udder (Nocard).

The anatomical changes in bovine tubercle are mostly found in the
lungs and their membranes, the pleuræ. It also affects the internal
membrane lining, the abdomen and its chief organs, the peritoneum,
and the lymphatic glands. In both these localities a characteristic
condition is set up by small grey nodules appearing, which increase
in size, giving an appearance of "grapes." Hence the condition is
called grape disease, or _Perlsucht_. The organs, as we have said, are
equally affected, and when we add the lymphatic glands we have a fairly
complete summary of the form of the disease as it occurs in cattle. As
has been clearly pointed out by Martin, Woodhead, and others in their
evidence before the Royal Commission, the organs, glands, and membranes
are the sites for tubercle, not the muscles (or "meat"). This latter
is most liable to convey infection when the butcher smears it with the
knife which he has used to remove tubercular organs.

As regards the udder in its relation to milk infection, it may be
desirable to state that the initial lesion, according to Nocard and
Bang, takes the form of a progressive sclerosis. The interlobular
connective tissue, normally scanty, becomes thickened, fibrous, and
infiltrated by minute miliary granulations. The granular tissue is
thus "smothered by the hypertrophy and fibrous transformation of the
interstitial connective tissue" (Nocard). The walls of the ducts are
thickened and infiltrated, the lumen frequently dilated by masses of
yellow caseous material. On the whole it may be said that tubercle
of the udder is rare. Usually only one quarter is attacked, and by
preference the posterior. For some time the milk remains normal,
but gradually it becomes serous and yellow, and contains coagula
holding numbers of bacilli. Lastly, it becomes purulent and dries up
altogether. While the milk is undergoing these changes the lesion of
the udder is becoming more marked, the tissue becomes less supple, and
the toughness increases almost to a wooden hardness.

The general anatomical characteristics of the disease are similar to
those occurring in man.

The percentage of cattle suffering from tubercle varies. In Germany it
appears to vary from 2 to 8 per cent. of all cattle, in Saxony 17 to
30 per cent., in England 22 per cent. approximately (in London 40 per
cent.), in France 25 per cent. Lowland breeds are much more infected
than mountain breeds, which possess stronger constitutions.

Tuberculosis of the _pig_ is less common than that of cattle, but not
so rare as that of the calf (Nocard). In nine out of ten cases the pig
is infected by ingestion, particularly when fed on the refuse from
dairies and cheese factories. The disease follows the same course as
in cattle. The finding of the bacillus is difficult, and the only safe
test is inoculation (Woodhead).

_Sheep_ are very rarely tuberculous by nature, though there is evidence
to believe that very long cohabitation with tuberculous cattle would
succeed in transmitting tuberculosis to some sheep.

Tuberculosis in the _horse_ is relatively very rare. It attacks the
organs of the abdominal cavity, especially the glands; it affects the
lung secondarily as a rule. The cases are generally isolated ones, even
though the animal belongs to a stud. Nocard holds that the bacillus
obtained from the pulmonary variety is like the human type, whilst the
abdominal variety is more like the avian bacillus.

Nocard says[93]:

  "If the dog can become tuberculous from contact with man, the
  converse is equally true. Infection is at any rate possible
  when a house-dog scatters on the floor, carpet, or bed, during
  its fit of coughing, virulent material, which is rendered
  extremely dangerous by drying, especially for children,
  its habitual playmates. The most elementary prudence would
  recommend the banishment from a room of every dog which coughs
  frequently, even though it only seems to be suffering from
  some common affection of the bronchi or lung."

Tuberculosis is a common disease among the _birds_ of the poultry-yard:
poultry, pigeons, turkeys, pea-fowl, guinea-fowl, etc. They are
infected almost exclusively through the digestive tract, generally by
devouring infected secretions of previous tubercular fowls. Whatever
the position or form of avian tuberculosis, the bacilli are present in
enormous numbers, and are often much shorter and sometimes much longer
than those met with in tuberculous mammalia, and grow outside the
body at a higher temperature (43° C.). They are also said to be more
resistant and of quicker growth. The species is probably identical with
Koch's bacillus, though there are differences. In the nodule, which is
larger than in human tuberculosis, there are few or no giant cells, and
it does not so readily break down.

Nocard and others have demonstrated the fact that the _Bacillus
tuberculosis_ of Koch is the common denominator in all tubercular
disease, whatever and wherever its manifestations, in all animals. The
bacillus, they hold, may, however, experience profound modifications
by means of successive passages through the bodies of divers species
of animals. But if the modifications which it undergoes as a result of
transmissions through birds, for example, are profound enough to make
the bacillus of avian tubercle a peculiar variety of Koch's bacillus,
they are not enough, it is generally believed, to make these bacilli
two distinct species.

We may, therefore, take it for granted that tuberculosis is one and the
same disease, with various manifestations, common to man and animals,
intercommunicable, and having but one _vera causa_: the _Bacillus
tuberculosis_ of Koch.

_The Prevention of Tuberculosis._ At the present time much attention is
being directed to the administrative personal control of tuberculosis.
How greatly this is needed in so preventable a disease is evident from
a perusal of the following quotation from the Registrar-General's
reports. (See opposite page.)

These figures show a marked decline in the three worst forms of the
disease. But this decline is apparently less marked in tabes than
in phthisis or tubercular meningitis, _i. e._, less in the kind of
tubercle due to the ingestion of infected milk. Fortunately the State
is beginning to realise its duty in regard to preventive measures. The
abolition of private slaughter-houses, the protection of meat and milk
supplies, the seizure of tuberculous milch cows, and such like measures
fall obviously within the jurisdiction of the State rather than the
individual, and claim the earnest and urgent attention of the public
health departments of states.[94]

ENGLISH DEATH-RATES FROM ALL TUBERCULAR DISEASES

THE FOLLOWING IS A TABLE OF DEATH-RATES TO A MILLION LIVING

(ENGLAND), 1877-1897 (_Reg.-Gen. Annual Reports_):--

  ------------------+-----+-----+-----+-----+-----+-----+-----+-----+-----+-----+-----
                    |1877 |1878 |1879 |1880 |1881 |1882 |1883 |1884 |1885 |1886 |1887
  ------------------+-----+-----+-----+-----+-----+-----+-----+-----+-----+-----+-----
  Tabes Mesenterica | 316 | 348 | 300 | 370 | 284 | 313 | 289 | 310 | 251 | 300 | 253
  Tubercular        |     |     |     |     |     |     |     |     |     |     |
    Meningitis      | 319 | 338 | 322 | 330 | 276 | 264 | 262 | 264 | 253 | 257 | 236
  Phthisis          |2079 |2111 |2021 |1869 |1825 |1850 |1880 |1827 |1770 |1739 |1615
  Other Forms       | 126 | 124 | 116 | 129 | 145 | 153 | 160 | 170 | 157 | 177 | 179
  ------------------+-----+-----+-----+-----+-----+-----+-----+-----+-----+-----+-----
      Total         |2840 |2921 |2759 |2698 |2530 |2580 |2591 |2571 |2431 |2473 |2283
  ------------------+-----+-----+-----+-----+-----+-----+-----+-----+-----+-----+-----

  ------------------+-----+-----+-----+-----+-----+-----+-----+-----+-----+-----
                    |1888 |1889 |1890 |1891 |1892 |1893 |1894 |1895 |1896 |1897
  ------------------+-----+-----+-----+-----+-----+-----+-----+-----+-----+-----
  Tabes Mesenterica | 240 | 269 | 265 | 251 | 242 | 265 | 192 | 243 | 196 | 201
  Tubercular        |     |     |     |     |     |     |     |     |     |
    Meningitis      | 239 | 234 | 240 | 247 | 227 | 226 | 211 | 222 | 210 | 213
  Phthisis          |1568 |1573 |1682 |1599 |1468 |1468 |1385 |1398 |1307 |1341
  Other Forms       | 174 | 183 | 189 | 203 | 199 | 186 | 185 | 200 | 179 | 175
  ------------------+-----+-----+-----+-----+-----+-----+-----+-----+-----+-----
      Total         |2221 |2259 |2376 |2300 |2136 |2145 |1973 |2063 |1892 |1930
  ------------------+-----+-----+-----+-----+-----+-----+-----+-----+-----+-----

_Tabes mesenterica_ is tuberculosis of the alimentary canal and
mesenteric lymph glands.

_Tubercular meningitis_ is the name of the same disease as it affects
the membranes of the brain (acute hydrocephalus).

_Phthisis_ is the term applied to "consumption," or tubercle in the
lungs.

But personal hygiene and the prevention of the transmission of the
disease depend very largely indeed upon the mass of the population.
Hence we hail with satisfaction the recent endeavours to educate public
opinion. In order to make this matter very simple indeed, we have
placed in a footnote a series of statements embodying some of the chief
facts which every individual in our crowded communities should know.[95]

[Illustration: BACILLUS OF DIPHTHERIA]

+Diphtheria+ (Klebs-Löffler Bacillus, 1882-1884). Diphtheria is an
infective disease characterised by a variety of clinical symptoms, but
commonly by a severe inflammation followed by a fibrous infiltration
(constituting a _membrane_) of certain parts. The membrane ultimately
breaks down. The parts affected are the mucous membrane of the fauces,
larynx, pharynx, trachea, and sometimes wounds and the inner wall of
the stomach. The common sign of the disease is the membrane in the
throat; but muscle weakness, syncope, albuminuria, post-diphtheritic
paralysis, convulsions, and many other symptoms guide the physician in
diagnosis and the course of the disease.

The _Bacillus diphtheriæ_ was isolated from the many bacteria found
in the membrane by Löffler. Klebs had previously identified the
bacillus as the cause of the disease. It is a slender rod, straight
or slightly curved, and remarkable for its beaded appearance; there
are also irregular and club-shaped forms. It differs in size according
to its culture medium, but is generally 3 or 4 µ in length. In the
membrane which is its strictly local habitat in the body--indeed, the
bacillus is found nowhere else in the body--it almost invariably shows
parallel grouping, lying between the fibrin of the membrane, and most
largely in its deeper parts. Here it is mixed with other bacilli,
micrococci, staphylococci, and streptococci, all of which are present
and performing their part in complicating the disease. The bacillus
possesses five negative characters; namely, it has no spores, threads,
or power of mobility, and does not produce liquefaction or gas. It
stains with Löffler's methylene blue, and shows metachromatic granules
and polar staining. Its favourable temperature is blood-heat, though
it will grow at room temperature. It is aërobic, and, indeed, prefers
a current of air. Löffler contrived a medium for cultivation which has
proved most successful. It is made by mixing three parts of ox-blood
serum with one part of broth containing 1 per cent. of glucose, 1
per cent. of peptone, and 1/2 per cent. of common salt; the whole is
coagulated. Upon this medium the Klebs-Löffler bacillus grows rapidly
in eighteen or twenty hours, producing scattered "nucleated" round
white colonies, becoming yellowish. It grows well in broth, but without
producing either a pellicle or turbidity; it can grow on the ordinary
media, though its growth on potato is not visible; on the white of egg
it flourishes extremely well.

It retains its vitality in cultures and sometimes in the throat for
months. Three or four weeks is the average length of time for its
existence in the membrane, but, owing to the difficulty of killing it
_in situ_, it may live on for as long as a year. All the conditions in
the throat--mucous membrane, blood-heat, moisture, air--are extremely
favourable to the bacillus; but it is very materially modified in
virulence. It is secured for diagnostic purposes by one of two methods:
(_a_) Either a piece of the membrane is detached, and after washing
carefully examined by culture as well as the microscope; or (_b_)
a "swab" is made from the infected throat and cultured on serum,
and incubated at 37° C. for eighteen hours and then microscopically
examined. Both methods--and there is no further choice--present some
difficulties owing to the large number of bacteria found in the throat.
Hence a negative result must be accepted with reserve.

We have already referred at some length to the question of _toxins_ in
diphtheria, and need not dwell further upon that matter. Still a word
or two may be said here summarising the general action of the bacillus.
Locally it produces inflammatory change with fibrinous exudation and
some cellular necrosis. In the membrane a ferment is probably produced
which, unlike the localised bacilli, passes throughout the body and
by digestion of the proteids produces albumoses and an organic acid
which have the toxic influence. The toxins act on the blood-vessels,
and nerves, and muscle fibres of the heart, and many of the more highly
specialised cells of the body. Thus we get degenerative changes in
the kidney, in cells of the central nervous system, in the peripheral
nerves (post-diphtheritic paralysis), and elsewhere, these pathological
conditions setting up, in addition to the membrane, the signs of
the disease. The bacillus is pathogenic for the horse, ox, rabbit,
guinea-pig, cat, and some birds. Cases are on record of supposed
infection of children by cats suffering from the disease. The horse, it
will be remembered, yields the antitoxin which has saved so many lives
(_Metropolitan Asylums Board Report_, 1896).

The influence of drainage, milk, and schools must not be forgotten
by sanitary authorities any more than the essential importance of
adequate isolation hospital accommodation. Mr. Shattock's experiments
on the effect of sewer air upon attenuated Klebs-Löffler bacilli have
been mentioned (see p. 105). Nevertheless there can be no doubt that
emanations from defective drains have a materially predisposing effect,
not, it is true, upon the bacilli, but upon the tissues. Sore throats
thus acquired are _par excellence_ the site for the development of
diphtheria.

The influence of school attendance has claimed the recent attention of
the Medical Officer of the London School Board and the Medical Officer
of the administrative County of London. In London since 1881 there has
been a marked increase of diphtheria, which has occurred, though in a
much less degree, throughout England and Wales.

The Registrar-General has only classified diphtheria as a separate
disease since 1855, when the death-rate per 1,000,000 in England and
Wales was stated as 20. The following are the figures for four decades
up to 1895:

  AVERAGE DEATH-RATE PER MILLION OF THE POPULATION FROM
  DIPHTHERIA IN ENGLAND AND WALES AND IN LONDON (IN DECADES
  1856-95)

                England and
                   Wales.          London.

  1856-65          246.9           225.4
  1865-75          124.8           123.5
  1875-85          129.0           176.7
  1885-95          210.6           421.4

From these figures the extraordinary increase during the last few years
is clearly demonstrated.

Sir Richard Thorne Thorne, in 1891, drew attention to the influence
of damp soils and schools upon diphtheria. In 1894 Mr. Shirley
Murphy, Medical Officer to the London County Council, reported that
there had been an increase in diphtheria mortality in London at
school ages (three to ten) as compared with other ages since the
Elementary Education Act became operative in 1871; that the increased
mortality from diphtheria in populous districts, as compared with
rural districts, since 1871, might be due to the greater effect of
the Education Act in the former; and that there was a diminution of
diphtheria in London during the summer holidays at the schools in 1893,
but that 1892 did not show any marked changes for August.

In 1896 Professor W. R. Smith, the Medical Officer to the London School
Board, furnished a report[96] on this same subject of school influence,
in which he produces evidence to show that the recrudescence of the
disease in 1881-90 was greatest in England and Wales at the age of
two to three years, and in London at the age of one to two years, in
both cases _before school age_; that age as an absolute factor in the
incidence of the disease is enormously more active than any school
influence, and that personal contact is another important source of
infection.

Although it is said that "statistics can be made to prove anything,"
there can be little doubt that both of these reports contain a great
deal of truth; nor are these truths incompatible with each other. They
both emphasise _age_ as a great factor in the incidence of the disease,
and whatever affects the health of the child population, like schools,
must play, directly or indirectly, a not unimportant part in the
transmission of the disease.

_The Pseudo-diphtheria Bacillus._[97] Löffler and Hoffman described a
bacillus having the same morphological characters as the true _Bacillus
diphtheriæ_, except that it had no virulence. Roux believes this is
merely an attenuated diphtheria bacillus. It is frequently found
in healthy throats. The chief differences between the real and the
pseudo-bacillus are:

1. The pseudo-bacillus is thicker in the middle than at the poles, and
not so variable as the _Bacillus diphtheriæ_. Polar staining is absent.

2. Its growth on potato reveals cream-coloured colonies visible in a
couple of days; the real bacillus is invisible.

3. The pseudo-bacillus will not grow at all anaërobically in hydrogen,
but the _Bacillus diphtheriæ_ is able to do so.

4. There is the great difference in virulence.

+Suppuration.+ This term is used to designate that general breaking
down of cells which follows acute inflammation. An "abscess" or
"gathering" is a collection, greater or smaller, of the products of
suppuration. The word _pus_ is generally used to describe this matter.
We may have such an advanced inflammatory condition in any locality
of the body, and it will assume different characters according to its
site. Hence there are connected with suppuration, as causal agents, a
variety of bacteria. Pus is not matter containing a pure culture of
any specific species, but, on the contrary, is generally filled with a
large number of different species. The most important are as follows:

1. _Staphylococcus pyogenes aureus._ These are micrococci arranged in
groups, which have been likened to bunches of grapes. They are the
common organisms found in pus, and were with other auxiliary bacteria
first distinguished as such by Professor Ogston, of Aberdeen. There are
several forms of the same species, differing from each other in colour.


Thus we have the _S. pyogenes aureus_ (golden yellow), _albus_ (white),
_citreus_ (lemon), and others. They occur commonly in nature, in
air, soil, water, on the surface of the skin, and in all suppurative
conditions. The _aureus_ is the only one credited with much virulence.
It occurs in the blood in blood-poisoning (septicæmia, pyæmia), and is
present in all ulcerative conditions, including ulcerative disease of
the valves of the heart.

The _Staphylococcus cereus albus_ and _S. cereus flavus_ are slightly
modified forms of the _S. pyogenes aureus_, and are differentiated from
it by being non-liquefying. They produce a wax-like growth on gelatine.

_Staphylococcus pyogenes aureus_, the type of the family, is grown in
all ordinary media at room temperature, though more rapidly at 37° C.
Liquefaction sets in at a comparatively early date, and subsequently
we have in the gelatine test-tube cultures a flocculent deposit of a
bright yellow amorphous mass, and in gelatine plates small depressions
of liquefaction with a yellow deposit. It renders all media acid, and
coagulates milk. Its thermal death-point in gelatine is 58° C. for ten
minutes, but when dry considerably higher. It is a non-motile and a
facultative anaërobe; but the presence of oxygen is necessary for a
bright colour. Its virulence readily declines.

2. _Streptococcus pyogenes._ In this species of micrococcus the
elements are arranged in chains. Most of the streptococci in pus,
from different sources, are one species, having approximately the
same morphological and biological characters. Their different effects
are due to different degrees of toxic virulence; they are always more
virulent when associated with other bacteria, for example, the Proteus
family.

The chains vary in length, consisting of more elements when cultured
in fluid media. They multiply by direct division of the individual
elements, and in old cultures it has been observed that the cocci vary
in form and size. This latter fact gave support to the theory that
streptococcus reproduced itself by _arthrospores_, or "mother-cells."

[Illustration: TYPES OF STREPTOCOCCUS]

In culture upon the ordinary media streptococcus is comparatively
slow-growing, producing minute white colonies on or about the sixth
day. It does not liquefy gelatine, and remains strictly localised
to the track of the inoculating needle. Like the staphylococcus, it
readily loses virulence. The thermal death-point is, however, lower:
54° C. for ten minutes. Marmorek has devised a method by which the
virulence may be greatly increased, and he holds that it depends upon
the degree of virulence possessed by any particular streptococcus as to
what effects it will produce. By the adoption of Marmorek's methods
attempts have been made to prepare an antitoxin.

_Streptococcus pyogenes_ has been isolated from the membrane of
diphtheria, and from small-pox, scarlet fever, vaccinia, and other
diseases. In such cases it is not the causal agent, but merely
associated with the complications of these diseases. Suppuration and
erysipelas are the only pathological conditions in which the causal
agency of streptococcus has been sufficiently established.

3. The _Bacillus pyocyaneus_ occurs in green pus, and is the cause of
that colouration. Gessard was the first to prove its significance, and
he describes two varieties.

[Illustration: MICROCOCCUS TETRAGONUS]

It is a minute, actively motile, non-sporulating bacillus, which
occasionally complicates suppuration and produces green pus. Oxygen
is necessary for pigmentation, which is due to two substances:
_pyocyanin_, a greenish-blue product extracted with chloroform, and
_pyoxanthose_, a brown substance derived from the oxidation of the
former pigment. Both these colours are produced in cultivation outside
the body. On gelatine the colour is green, passing on to olive. There
is liquefaction. On potato we generally obtain a brown growth (compare
_Bacillus coli_, _B. mallei_, and others). The organism grows rapidly
on all the ordinary media, which it has a tendency to colour throughout.

It will be remembered that when speaking of the antagonism of
organisms, we referred to the inimical action of _Bacillus pyocyaneus_
upon anthrax.

4. _Micrococcus Tetragonus._ This species occurs in phthisical
cavities and in certain suppurations in the region of the face. It is a
micrococcus usually in the form of small tetrads. A capsule is always
present and sometimes discernible.

5. _Bacillus coli communis_ and many putrefactive germs commonly
occur in suppurative conditions, but they are not restricted to such
disorders (see p. 64).

[Illustration: DIPLOCOCCUS OF NEISSER]

6. _Micrococcus gonorrhœæ_ (Neisser, 1879). This organism is more
frequently spoken of as a diplococcus. It occurs at the acute stage
of the disease, but is not readily differentiated from other similar
diplococci except by technical laboratory methods. Each element
presents a straight or concave surface to its fellow. A very marked
concavity indicates commencing fission. The position which these
diplococci take up in pus is _intracellular_, and arranged more or
less definitely around the nucleus. Difficulty has often been found
in cultivating this organism in artificial media outside the body.
Wertheim and others have suggested special formulæ for the preparation
of suitable media, but it is a very simple matter to secure cultures
on agar plates smeared with human blood from a pricked finger. The
plate is incubated at 37° C. At the end of twenty-four hours small
raised grey colonies appear, which at the end of about four days
show adult growth. The margin is uneven, and the centre more opaque
than the rest of the colony. This diplococcus is readily killed, and
sub-cultures must be frequently made to retain vitality and virulence.
Light, desiccation, and a temperature of 55° C. all act germicidally.
The organism stains readily in Löffler's blue, but is decolourised
by Gram's method. It is more or less strictly parasitic to man. Its
shape, size, character of growth, and staining properties assist in
differentiating it from various similar diplococci.[98]

+Anthrax.+ This disease was one of the first in which the causal agency
of bacteria was proved. In 1849 Pollender found an innumerable number
of small rods in the blood of animals suffering from anthrax. In 1863
Davaine described these, and attributed the disease to them. But it was
not till 1876 that Koch finally settled the matter by isolating the
bacilli in pure culture and describing their biological characters.

It is owing in part to its interesting bacterial history, which opened
up so much new ground in this comparatively new science, that anthrax
has assumed such an important place in pathology. But for other
reasons, too, it claims attention. It appears to have been known in the
time of Moses, and was perhaps the disease described by Homer in the
First Book of the _Iliad_. Rome was visited by it in 740 B.C.

Anthrax is an acute disease, affecting sheep, cattle, horses, goats,
deer, and man. Cats, white rats, and Algerian sheep are immune. Swine
become infected by feeding on the offal of diseased cattle (Crookshank).

The _post-mortem_ signs are mainly three: The _spleen_ is greatly
enlarged and congested, is friable to the touch, and contains enormous
numbers of bacilli; the _skin_ may show exudations forming dark
gelatinous tumours; and the _blood_ remains fluid for some time
after death, is black, tar-like, contains bubbles of air, and shows
other degenerative changes in the red corpuscles, whilst the _small
blood-vessels_ contain such vast quantities of bacilli that they may be
ruptured by them. Particularly is this true in the peripheral arteries.
Many of the organs of the body show marked congestion.

[Illustration: BACILLUS OF ANTHRAX AND BLOOD CORPUSCLES]

[Illustration: THREADS OF BACILLUS ANTHRACIS, SHOWING SPORES]

Clinically there is rise of temperature and rapid loss of muscular
power. The _bacilli_ of anthrax are square-ended rods 1 µ broad and
4-5 µ long. In the tissues of the body they follow the lines of the
capillaries, and are irregularly situated. In places they are so
densely packed as to form obstructions to the onward flow of blood.
In cultures they are in chains end to end, having as a rule equal
inter-bacillary spaces. In cultures long filaments and threads occur.
The exact shape of the bacillus depends, however, upon two things: the
staining and spore formation. Both these factors may very materially
modify the normal shape. The spores of anthrax are oval endospores,
produced only in the presence of free oxygen, and at any temperature
between 18 and 41° C. On account of requiring free oxygen, they are
formed only outside the body. The homogeneous protoplasm of the
bacillus becomes granular; the granules coalesce, and we have spores.
Each spore possesses a thick capsule, which enables it to resist many
physical conditions which kill the bacillus. When the spore is ripe or
has exhausted the parent bacillus, it may take on a resting stage, or
under favourable circumstances commence germination, very much after
the manner of a seed. The spores may infect a farm for many months;
indeed, cases are on record which appear to prove that the disease on
a farm in the autumn may by means of the spores be carried on by the
hay of the following summer into a second winter. Thus, by means of the
spores, the infection of anthrax may cling to the land for very long
periods, even for years. Spores of anthrax can withstand 5 per cent.
carbolic acid or 1-1000 corrosive sublimate for more than an hour; even
boiling does not kill them at once, whilst the bacilli without their
spores are killed at 54° C. in ten minutes. When the spores are dry
they are much more resistant than when moist. Hence the persistence of
the anthrax bacillus is due to its spores.

The bacillus is aërobic, non-motile, and liquefying. Broth cultures
become turbid in thirty-six hours, with nebulous masses of threads
matted together. The pellicle which forms on the surface affords an
ideal place for spore formation.

Cultures in the depth of gelatine show a most characteristic growth.
From the line of inoculation delicate threads and fibrillæ extend
outwards horizontally into the medium. Liquefaction commences at the
top, and eventually extends throughout the tube. On gelatine plates
small colonies appear in thirty-six hours, and on the second or third
day they look, under a low power of the microscope, like matted hair.
The colonies after a time sink in the gelatine, owing to liquefaction.
On potato, agar, and blood serum anthrax grows well.

_Channels of Infection._ 1. _The Alimentary Canal._ This is the usual
mode of infection in animals grazing on infected pasture land. A soil
suitable for the propagation of anthrax is one containing abundance
of air and proteid material. Feeding on bacilli alone would probably
not produce the disease, owing to the germicidal effect of the gastric
juice. But spores can readily pass uninjured through the stomach and
produce anthrax in the blood. Infected water as well as fodder may
convey the disease. Water becomes infected by bodies of animals dead
of anthrax, or, as was the case once at least in the south-west of
England, by a stream passing through the washing-yard of an infected
tannery. Manure on fields, litter in stalls, and infected earth may
all contribute to the transmission of the disease. Darwin pointed out
the services which are performed in superficial soils by earthworms
bringing up casts; Pasteur was of opinion that in this way earthworms
were responsible for continually bringing up the spores of anthrax
from buried corpses to the surface, where they would reinfect cattle.
Koch disputed this, but more recently Bollinger has demonstrated the
correctness of Pasteur's views by isolating anthrax contagium from five
per cent. of the worms sent him from an anthrax pasture. Bollinger also
maintains that flies and other insects may convey the disease from
discharges or carcasses round which they congregate.

Alimentary infection in man is a rare form, and it reveals itself in
a primary diseased state known as _mycosis intestinalis_, an inflamed
condition of the intestine and mesenteric lymph glands.

2. _Through the Skin._ Cutaneous anthrax goes by the name of _malignant
pustule_, and is caused by infective anthrax matter gaining entrance
through abrasions or ulcers in the skin. This local form is obviously
most contracted by those whose occupation leads them to handle hides
or other anthrax material (butchers and cleaners of hides). Two or
three days after inoculation a red pimple appears, which rapidly passes
through a vesicular stage until it is a pustule. Concomitantly we have
glandular enlargement, general malaise, and a high temperature. Thus
from a local sore a general infection may result. Unless this does
occur, the issue will not be fatal, and the bacilli will never gain
entrance into the blood or be anything but local.

3. _Respiratory Tract._ In man this is the commonest form of all, and
is well known under the term "wool-sorters' disease," or pulmonary
anthrax. This mode of infection occurs when dried spores are inhaled in
processes of skin-cleaning. It frequently commences as a local lesion
affecting the mucous membrane of the trachea or bronchi, but it rapidly
spreads, affecting the neighbouring glands, which become greatly
enlarged, and extending to the pleura and lung itself. Such cases, as a
rule, rapidly end fatally.

From what has been said, it will be clear that anthrax carcasses are
better not opened and exposed to free oxygen. An extended _post-mortem_
examination is not necessary. Burning the entire carcass in a
crematorium would be the ideal treatment. As such is not generally
feasible the next best thing is to bury the carcass deeply with lime
below and above it, and rail in the area to prevent other animals
grazing off it.

A very small prick will extract enough blood to examine for the anthrax
bacilli which are driven by the force of the blood-current to the small
surface capillaries. This occurs, of course, only when the disease has
become quite general, for in the early stage the healthy blood limits
the bacilli to the internal organs. In such cases examination of the
blood of the spleen is necessary.

Anthrax covers a wide geographical area all over the world, and no
country seems altogether exempt. In Germany as many as 3700 animals
have been lost in a single year. About 900 animals were attacked in
1897 in Great Britain.

+Plague.+ This disease, like anthrax and leprosy, has a long historical
record behind it. As the Black Death it decimated the population of
England in the fourteenth century, and visited the country again in
epidemic form in the middle of the seventeenth century, when it was
called the Great Plague. Now, it is highly probable that these two
scourges and the recent epidemic in the East are all forms of one and
the same disease. As a matter of fact, it is difficult to be sure what
was the exact pathology of a number of the grievous ailments which
troubled our country in the Middle Ages, but from all accounts bubonic
plague and true leprosy were amongst them. The former came and went
spasmodically, as is its habit; the latter dragged through the length
of several centuries.

[Illustration: BACILLUS OF PLAGUE]

The distribution of plague at the present time is fortunately a
somewhat limited one, namely, a definite area in Asia known as the
"Plague Belt." From Mesopotamia, as a sort of focus, the disease
spreads northwards to the Caspian Sea, westwards to the Red Sea,
southwards as far as Central India, and eastwards as far as the China
Sea. This constitutes the "belt," but the disease may take an epidemic
form, and is readily, though very slowly, conveyed by infection or
contagion. It appears to be infectious by means of infective dust, and
contagious by prolonged and intimate contact with the plague-stricken.
Rats have been accused of conveying the disease from port to port,
and even infecting man. It is clear that rats are not the only agency
acting in this way. Nevertheless it is true that rats contract the
disease more readily than any other animals, and that when suffering
from it they may spread the infection. How it is thus spread it is not
known. Drs. Cantlie and Yersin have pointed out that previously to an
epidemic of plague rats die in enormous numbers.

The bacteriology of plague is almost the latest addition to the
science. Kitasato, of Tokio, demonstrated the cause of plague to be a
bacillus during the Hong Kong epidemic in 1894. This was immediately
confirmed by Yersin, and further proved by the isolation in artificial
media of a pure culture of a bacillus able to cause the specific
disease of bubonic plague.

The bacillus was first detected in the blood of patients suffering from
the disease. It takes the form of a small, round-ended, oval cell, with
marked polar staining, and hence having an appearance not unlike a
diplococcus. In the middle there is a clear interspace, and the whole
is surrounded with a thick capsule, stained only with difficulty.
The organisms are often linked together in pairs or even chains, and
exhibit involution forms. In culture the bacillus is even more coccal
in form than in the body.

The plague bacillus grows readily on the ordinary media at blood-heat,
producing circular cream-coloured colonies with a wavy outline, which
eventually coalesce to form a greyish film. The following negative
characters help to distinguish it: No growth occurs on potato, milk is
not coagulated, and gelatine is not liquefied; Gram's method does not
stain the bacillus, and there are no spores; the bacillus is readily
killed by heat and by desiccation over sulphuric acid at 30° C. Both
in cultures and outside the body the bacillus loses virulence. To this
may be attributed possibly the variety of forms of the plague bacillus
which differ in virulence. On gaining entrance to the human body the
bacillus affects in particular two organs, the spleen and the lymph
glands. The latter become inflamed in groups, commencing frequently
with those in the armpit (axillary) or groin (inguinal). The spleen
suffers from inflammatory swelling, which may affect other organs
also. In both places the bacilli occur in enormous numbers. Kitasato
considers that the bacillus may enter the body by the three channels
adopted by anthrax, namely, the skin, alimentary canal, and respiratory
tract.

Haffkine has prepared a vaccine to be used as a prophylactic. He grows
a pure culture of Kitasato's bacillus in broth upon the surface of
which some globules of fat ("ghee") have been placed. The bacillus
grows upon this fat in copious stalactite form. From time to time this
growth is shaken down, until after five or six weeks the shaken broth
appears milky. The contained bacilli are killed by heating the fluid to
70° C. for one hour. The resultant is the vaccine, of which the dose is
3 cc. Haffkine believes that inoculated persons in India have suffered
twenty times less than non-inoculated living under the same conditions.

Plague is essentially a "filth disease," and it is frequently preceded
by famine. Temperature and overcrowding exert an influence upon it. The
areas affected by the disease in the Middle Ages, in the seventeenth
century, and in 1894-96 are alike in being characterised by filth and
overcrowding. There is little fear, speaking generally, of the plague
ever flourishing under Western civilisation, where the conditions are
such that even when it appears there is little to encourage or favour
its development.

+Leprosy.+ This ancient disease is said to have existed in Egypt 3500
B.C., and was comparatively common in India, China, and even in parts
of Europe 500 B.C. We know it has existed in many parts of the world
in the past, in which regions it is now extinct. Some of the earliest
notices we have of it in this country come from Ireland, and date
back to the fifth and sixth centuries. Even at that period of time
also various classical descriptions of the disease had been written
and various decrees made by the Church councils to protect lepers and
prevent the spread of the disease, which was often looked upon as
a divine visitation. In the tenth century leprosy was prevalent in
England; it reached its zenith in the thirteenth century, or possibly
a little earlier, and declined from that date to its extinction in the
sixteenth. But even two hundred years later leprosy was endemic in the
Shetlands, and it is recorded that in 1742 there was held a public
thanksgiving in Shetland on account of the disappearance of leprosy.

At one time or another there were as many as two hundred institutions
in the British Isles for the more or less exclusive use of lepers.
Many of these establishments were of an ecclesiastical or municipal
character, and probably the exact diagnosis of diseases was a somewhat
lax matter. Bury St. Edmunds, Bristol, Canterbury, London, Lynn,
Norwich, Thetford, and York were centres for lepers. Burton Lazars and
Sherburn, in Durham, were two of the more famous leper institutions.

At the present time the distribution of the disease is mostly Asiatic.
Norway contains about 1200 lepers, Spain approximately the same number.
Scattered through Europe are perhaps another 600 or 700, in India
100,000, and a large number in Japan. The Cape possesses a famous leper
hospital on Robben Island, with a number of patients. The disease is
also endemic in the Sandwich Islands.

Descriptions of the pathological varieties of leprosy have been very
diverse. The classification now generally adopted includes three forms:
the _tuberculated_, the _anæsthetic_ (or maculo-anæsthetic), and the
_mixed_. _Lepra tuberculosa_ is that form of the disease affecting
chiefly the skin, and resulting in nodular tuberculated growth or a
diffuse infiltration. It causes great disfigurement. The anæsthetic
form causes a destruction of the nerve fibres, and so produces
anæsthesia, paralysis, and what are called "trophic" changes. Not
infrequently patches occur on the skin, which appear like parchment,
owing to this trophic change. Bullæ may arise. When the tissue change
is radical or far advanced, considerable distortion may result. The
mixed variety of leprosy, as its name implies, is a mixture of the two
other forms.

The _Bacillus lepræ_ was discovered by Hansen in 1874. He found it in
the lepra cells in the skin, lymph glands, liver, spleen, and thickened
parts of the nerves. It is common in the discharges from the wounds of
lepers. It is conveyed in the body by the lymph stream, and has rarely
been isolated from the blood (Köbner).

[Illustration: BACILLUS OF PLAGUE (B. PESTIS BUBONICÆ)

(From liver of rat)

× 1000

_By permission of the Scientific Press, Limited_]

[Illustration: BACILLUS OF LEPROSY (HANSEN)

(From the tissues of a leper)

× 1000]

[Illustration: STREPTOTHRIX ACTINOMYCES (RAY FUNGUS)

× 700]

[Illustration: BACILLUS MALLEI (GLANDERS)

× 1000]

The bacillus is present in enormous numbers in the skin and tissues,
and has a form very similar indeed to _Bacillus tuberculosis_. It
is a straight rod, and showing with some staining methods marked
beading, but with others no beading at all. It measures 4 µ long and
1 µ broad. Young leprosy bacilli are said to be motile, but old ones
are not. Neisser has maintained that the bacillus possesses a capsule
and spores. The latter have not been seen, but Neisser holds that
this is the form in which the bacillus gains entrance to the body.
There is a characteristic which fortunately aids us in the diagnosis
of this disease in the tissues, and that is the arrangement of the
bacilli, which are rarely scattered or isolated, but gathered together
in clumps and colonies. Bordoni-Uffreduzzi and Campania claim to have
isolated the bacillus and grown it on artificial media, the former
aërobically on peptone-glycerine-blood-serum, at 37° C., the latter
anaërobically. But no other worker has been able to do this. Hence we
are not able to study the bacteriology of leprosy at all completely,
nor have inoculation experiments proved successful. Nevertheless there
is little doubt that leprosy is a bacterial disease produced by the
bacillus of Hansen. Bordoni-Uffreduzzi maintains that the parasitic
existence of the _Bacillus lepræ_ may alternate with a saprophytic
stage. This may be of importance in the spread of the disease. There
is evidence in support of the non-communicability of the disease by
heredity or contagion. Segregation does not appear always to result
in a decline of the disease, as we should expect if it were purely
contagious. Ehlers, of Copenhagen, has, however, as recently as 1897,
reaffirmed his belief in the contagiousness of leprosy; Virchow, on
the other hand, has declared that it is not highly contagious. There
is evidence to show that persons far advanced in the disease may live
in a healthy community and yet not infect their immediate neighbours.
Indeed, the transmission of the disease is still an unsolved problem.
Mr. Hutchinson suggests diet, particularly uncooked or putrid fish, as
a likely channel; on the other hand, leprosy appears in districts where
no fish is eaten. Deficiency of salt, telluric and climatic conditions,
racial tendencies, social status, poverty, insanitation, drinking
water, even vaccination, have all secured support from various seekers
after the true channel by which the bacillus gains entrance to the
human body. The real mode of transmission is, however, still unknown.
The decline and final extinction of leprosy in the British Islands was
probably due in part to the natural tendency of the disease, under
favourable hygienic circumstances, to die out, and in part to a general
and extensive social improvement in the life of the people, to a
complete change in the poor and insufficient diet, and to agricultural
advancement, improved sanitation, and land drainage.

At the Leprosy Congress held in Berlin in 1897, Hansen again emphasised
his belief that segregation was the cause of the decline of leprosy
wherever it had occurred. But there appears to be some evidence to
show that leprosy has declined where there has been no segregation
whatever, and therefore, however favourable to decline such isolation
may be, it would seem not to be actually necessary to the decline. At
the same Congress Besnier declared in favour of the infective virus
being widely propagated by means of the nasal secretion. Sticker states
that the nasal secretion contains millions of lepra bacilli, especially
in the acute stage of the disease, and Besnier and Sticker have
pointed out how frequently and severely the septum nasi and skin over
the nose are affected in leprosy. Several leprologists in India have
recorded similar observations. These facts appear to support Besnier's
contention that the disease is spread by nasal secretion.

We may fitly add here the conclusions arrived at by the English Leprosy
Commission[99] in India:

  "1. Leprosy is a disease _sui generis_; it is not a form
  of syphilis or tuberculosis, but has striking etiological
  analogies with the latter.

  "2. Leprosy is not diffused by hereditary transmission, and,
  for this reason and the established amount of sterility among
  lepers, the disease has a natural tendency to die out.

  "3. Though in a scientific classification of diseases leprosy
  must be regarded as contagious, and also inoculable, yet the
  extent to which it is propagated by these means is exceedingly
  small.

  "4. Leprosy is not directly originated by the use of any
  particular article of food, nor by any climatic or telluric
  conditions, nor by insanitary surroundings, neither does it
  peculiarly affect any race or caste.

  "5. Leprosy is indirectly influenced by insanitary
  surroundings, such as poverty, bad food, or deficient drainage
  or ventilation, for these by causing a predisposition increase
  the susceptibility of the individual to the disease.

  "6. Leprosy, in the great majority of cases, originates _de
  novo_, that is, from a sequence or concurrence of causes and
  conditions dealt with in the Report, and which are related to
  each other in ways at present imperfectly known."

The practical suggestions of the Commission for preventive treatment
included voluntary isolation, prohibition of the sale of articles of
food by lepers, leper farms, orphanages, and "improved sanitation and
good dietetic conditions" generally. Serum-therapy has been attempted
on behalf of the French Academy of Medicine, but without success. Many
forms of treatment ameliorate the miserable condition of the leper, but
up to the present no curative agent has been found.

[Illustration: DIPLOCOCCUS OF PNEUMONIA]

+Pneumonia.+ Some of the difficulty which has surrounded the
bacteriology of inflammation of the lungs is due to the confusion
arising from supposing that attacks of the disease differed only
in degree. Pneumonia, however, has various forms, arising now from
one cause, now from another. The specific or croupous pneumonia is
associated with two organisms: Fraenkel's diplococcus and Friedländer's
pneumo-bacillus. Several other bacteria have from time to time been
held responsible for pneumonia, a streptococcus receiving, at one
time, some support. But whilst opinion is divided on the rôle of
various extraneous and concomitant bacteria in lung disease, importance
is attached to Fraenkel's and Friedländer's organisms.

The _diplococcus_ of Fraenkel is a small, oval diplococcus found in the
"rusty" sputum of croupous pneumonia. It is non-motile, non-liquefying,
and aërobic. When examined from cultures the diplococci are frequently
seen in chains, not unlike a streptococcus, and there is some reason
to suppose that this form gave rise to the belief that it was another
species; when examined from the tissues it possesses a capsule, but
in culture this is lost. It is difficult to cultivate, but grows on
glycerine agar and blood serum at blood-heat. On ordinary gelatine at
room temperature it does not grow, or, if so, very slightly. The ideal
fluid is a slightly alkaline liquid medium, and in twenty-four hours a
powdery growth will occur in such broth. On potato there is apparently
no growth. It rapidly loses its virulence on solid media, and is said
to be non-virulent after three or four sub-culturings. A temperature
of 54-58° C. for a few minutes kills the bacteria, but not the toxin.
This, however, is removed by filtration, and is therefore probably
intracellular. It is attenuated by heating to 70° C.

Fraenkel's diplococcus occurs, then, in the acute stage of pneumonia,
in company with streptococci and staphylococci. It also occurs in the
blood in certain suppurative conditions, in pleurisy and inflammation
of the pericardium, and sometimes in diphtheria, and therefore it is
not peculiar to pneumonia.

There is one other point to which attention should be drawn. Fraenkel's
organism is said to be frequently present in the saliva of healthy
persons. Pneumonia depresses the resistant vitality of the tissues,
and thus affords to the diplococcus present in the saliva an excellent
nidus for its growth.

_Friedländer's Pneumo-bacillus_ is a capsulated oval coccus, assuming
the form of a small bacillus. It is inconstant in pneumonia, unequally
distributed, and scarce; it is aërobic, and facultatively anaërobic;
it occasionally occurs in long forms and filaments; it is non-motile,
non-liquefying, and has no spores; it does not stain by Gram's
method, which stain is therefore used for differential diagnosis; it
will grow fairly well in ordinary gelatine at 20° C.; and it is a
denitrifying organism, and also an actively fermentative one, even
fermenting glycerine. It is not unlike _Bacillus coli communis_, and to
distinguish it from that organism we may remember that the _B. coli_
is motile, never has a capsule, produces indol, and does not ferment
glycerine.

[Illustration: BACILLUS OF INFLUENZA]

+Influenza.+ In 1892, during the pandemic of influenza, Pfeiffer
discovered a bacillus in the bronchial mucus of patients suffering from
the disease. It is one of the smallest bacilli known, and frequently
occurs in chains not unlike a streptococcus. Carron obtained the same
organism from the blood. In the bronchial expectoration it can retain
its virulence for as long as a fortnight, but it is quickly destroyed
by drying. The bacillus is aërobic, non-motile, and up to the present
spores have not been found. It grows somewhat feebly in artificial
media, and readily dies out. Blood serum, glycerine agar, broth, and
gelatine have all been used at blood-heat. It does not grow at room
temperature. Pfeiffer's bacillus appears most abundantly at the height
of the disease, and disappears with convalescence. It is said not to
appear in any other disease.

+Yellow Fever.+ Sternberg and Havelburg have both isolated bacilli
from cases of yellow fever; but the organism discovered by Sanarelli,
the _Bacillus icteroides_, is now accepted as the causal agent of the
disease. It is a small, short rod, with round ends, and generally
united in pairs; it has various pleomorphic forms; it grows well on all
the ordinary media; it is killed in sea-water at 60° C., and also by
direct sunlight in a few hours.

+Diarrhœa of Infants.+ From time to time different organisms have
been isolated in this diseased condition. _Bacillus coli_ and _B.
enteriditis sporogenes_ (Klein) have been held responsible for it. W.
D. Booker, of Johns Hopkins University, sums up an extended research
into the question as follows:

  "No single micro-organism is found to be the specific exciter
  of the summer diarrhœa of infants, but the affection is
  generally to be attributed to the result of the activity of
  a number of varieties of bacteria, some of which belong to
  well-known species, and are of ordinary occurrence and wide
  distribution, the most important being the streptococcus and
  _Proteus vulgaris_.

  "The first step in the pathological process is probably
  an injury to the epithelium from abnormal or excessive
  fermentation, from toxic products of bacteria, or from other
  factors.

  "Bacteria exert a direct injury upon the tissues in some
  instances, whereas in others the damage is brought about
  indirectly through the production of soluble poisons."

+Actinomycosis.+ This disease affects both animals and man. As
Professor Crookshank points out, it has long been known in this
country,[100] but its various manifestations have been mistaken for
other diseases or have received popular names.

Here we can only mention the most outstanding facts concerning
the disease. It is caused by the "ray fungus," or _Streptothrix
actinomyces_, which, growing on certain cereals, often gains entrance
to the tissues of man and beast by lacerations of the mucous membrane
of the mouth, by wounds, or by decayed teeth. Barley has been the
cereal in question in some cases. The result of the introduction of the
parasite is what is termed an "infective granuloma." This is, generally
speaking, of the nature of an inflammatory tumour composed of round
cells, epithelioid cells, giant cells, and fibrous tissue, forming
nodules of varying sizes. In some cases they develop to large tumours,
in others they soon break down. Actinomycosis in some ways closely
resembles tuberculosis in its tissue characters.

In the discharge or pus from human cases of the disease small
sulphur-yellow bodies may be detected, and these are tufts of "_clubs_"
which are the broken-down rays of the parasite; for in the tissues
which are affected the parasite arranges itself in a radiate manner,
growing and extending at its outer margin and degenerating behind. In
cattle the centre of the old ray becomes caseated, like cheese, or even
calcified, like a stone. In the human disease abundant "_threads_"
are formed as a tangled mass in the middle of the colony. As clubs
characterise the bovine actinomycosis, so threads are a feature of
the human form of the disease. But in both there is a third element,
namely, _small round cells_, called by some spores, by others simply
cocci. Authorities are not yet agreed as to the precise significance
and rôle of these round cells. The life-history of the micro-organism
may be summed up thus:

  "The spores sprout into excessively fine, straight or sinuous,
  and sometimes distinctly spirilliform threads, which branch
  irregularly and sometimes dichotomously. The extremities of
  the branches develop the club-shaped bodies. The clubs are
  closely packed together, so that a more or less globular body
  is formed, with a central core composed of a dense mass of
  threads" (Crookshank).

Possibly these clubs represent organs of fructification, and produce
the spores. These latter are, it is believed, set free in the vicinity
of the ray, and create fresh centres of disease.

In _man_ the disease manifests itself in various parts according to the
locality of entrance. When occurring in the mouth it attacks the lower
jaw most frequently. In one recorded case the disease was localised to
the bronchi, and did not even extend into the lungs. It was probably
contracted by inhalation of the parasite. The disease may spread
to distant parts by means of the blood stream, and frequently the
abscesses are apt to burrow in various directions.

In the _ox_ the disease remains much more localised, and frequently
occurs in the lower jaw, palate, or tongue. In the last site it is
known as "wooden tongue," owing to the hardness resulting. The skin
and subcutaneous tissues are also a favourite seat of the disease,
producing the so-called wens or clyers so commonly seen in the fen
country (Crookshank). Actinomycosis in cattle is specially prevalent
in river valleys, marshes, and on land reclaimed from the sea. The
disease occurs at all seasons, but perhaps more commonly in autumn and
winter. It is more frequently met with in young animals. The disease is
probably not hereditary nor readily communicated from animal to animal.

Actinomyces may be cultivated, like other parasitic diseases, outside
the body. Gelatine, blood serum, agar, glycerine agar, and potato have
been used for this purpose. After a few days on glycerine agar at the
temperature of the blood little white shining colonies appear, which
increase and coalesce. In about ten days' time the culture often turns
a bright yellow, though it may remain white or even take on a brown
or olive tint. The entire mass of growth is raised dry and crinkled,
and composed almost exclusively of threads. In its early stage small
bacillary forms occur, and in its later stage coccal forms. True clubs
never occur in pure cultures, although the threads may occasionally
show bulbous endings.

+Glanders+ in the horse and ass, and sometimes by communication in
man also, is caused by a short, non-motile, aërobic bacillus, named,
after the old Roman nomenclature (_malleus_), _Bacillus mallei_. It
was discovered in 1882 by Löffler and Schütz. It is found in the
nasal discharge of glandered animals. In appearance the bacillus is
not unlike _B. tuberculosis_, except that it is shorter and thicker.
The beading of the bacillus of glanders, like that in tubercle, does
not denote spores. _B. mallei_ can be cultivated on the usual media,
especially on glycerine agar and potato. On the latter medium it
forms a very characteristic honey-like growth, which later becomes
reddish-brown.

In the horse glanders particularly affects the nasal mucous membrane,
forming nodules which degenerate and emit an offensive discharge.
From the nose, or nasal septum, as a centre, the disease spreads to
surrounding parts. It may also occur as nodules in and under the skin,
when it is known as "farcy." Persons attending a glandered animal may
contract the disease, often by direct inoculation.

_Mallein_ is a substance analogous to tuberculin, and is made by
growing a pure culture of _Bacillus mallei_ in glycerine-veal broth
in flat flasks, with free access of calcined air. After a month's
growth the culture is sterilised, filtered, concentrated, and mixed
with an equal volume of a .5 per cent. solution of carbolic. The dose
is 1 cc., and it is used, like tuberculin, for diagnostic purposes.
If the suspected animal reacts to the injection, it is suffering
from glanders. Reaction is judged by three signs, namely, a rise of
temperature 2-3° C., a large "soup-plate" swelling at the site of
inoculation, and an enlargement of the lymphatic glands.

_Swine fever_, _foot-and-mouth disease_, _chicken cholera_,
_dysentery_, _rinderpest_, and other diseases of animals have
microorganisms intimately related to them.

There is a group of diseases due to the presence in the blood or
tissues of _hæmatozoa_, that is, protozoa which can live and perform
their function in the blood. Amongst these are malaria, sleeping
sickness, and other tropical diseases in man, and surra and various
hæmatozoa in horses, fish, frogs, or rats.

_Malaria._ Although a _Bacillus malariæ_ has been described as the
cause of this disease, it is now almost universally supposed that the
true cause is a protozoan parasite. In 1880 Laveran first described
this organism, and the discovery was confirmed by Marchiafava, Celli,
and others. Laveran claimed that it occurred in four different forms
during the progress of its life-history:

(_a_) _Spherical or Irregular Bodies_ attached to the blood corpuscle,
or free in the blood plasma. They are a little smaller than the
blood-cells, and may or may not contain pigment. They eventually
invade the corpuscles, possess more pigment, and lose their amœboid
movement. Within the red blood corpuscles they increase in size until
they reach the adult stage.

(_b_) _Segmentation Forms_, often assuming a rosette shape, follow
next. They are pigmented, are possibly a sporing stage, and are finally
set free in the blood.

(_c_) _The Crescents_, or _Semilunar Bodies_, are free in the blood,
but motionless. They are colourless, have a distinct membrane, and
generally show a little pigment about the middle; they taper towards
the poles. They appear in the blood after the fever has existed for
some time, occurring chiefly, sometimes only, in the quotidian and
malignant types of malaria.

(_d_) _The Flagellated Bodies_ apparently occur only in the blood
outside the body. They are extracorpuscular bodies, and possess several
long flagella, and are therefore actively motile. They are derived from
the crescents or irregular intracorpuscular bodies.

What is the precise significance of these various forms and
modifications of them is not at present known. Possibly the semilune
is a resting stage inside the body, and the flagellated body another
similar stage outside. Attempts to cultivate the parasite outside the
body have failed. There is a good deal of evidence to show that the
mosquito is the host outside the human body. There may be different
forms and varieties of parasite, if not actually different species,
causing the diverse forms of clinical malaria.

The above account of diseases caused by bacteria does not profess to
be in any sense exhaustive. It is merely illustrative. It reveals
some of the disease-producing powers of micro-organisms. There are a
large number of other diseases in which bacteria have been found. They
are not the causes, but only accidentally present or associated with
"secondary infection." Variola (small-pox), scarlet fever, and measles
are excellent examples. It is possible that the danger at the present
time is rather in the direction of supposing that every disease will
readily yield its secret to the bacteriologist. Such, of course, is
not the case. Nevertheless, as in the past, so in the future, constant
research and patient investigation is the only hope we have for the
elucidation of truth in respect to the causes of disease.




CHAPTER IX

_DISINFECTION_


The object of modern bacteriology is not merely to accumulate tested
facts of knowledge, nor only to learn the truth respecting the biology
and life-history of bacteria. These are most important things from a
scientific point of view. But they are also a means to an end; that
end is the prevention of preventable diseases and the treatment of any
departure from health. In a science not a quarter of a century old much
has already been accomplished in this direction. The knowledge acquired
of, and the secrets learned from, these tiny vegetable cells which have
such potentiality for good or evil have been, in some degree, turned
against them. When we know what favours their growth and vitality and
virulence, we know something of the physical conditions which are
inimical to their life; when we know how to grow them, we also know how
to kill them.

We have previously made a cursory examination of the methods which are
adopted for opposing bacteria and their products in the tissues and
body fluids. We must now turn to consider shortly the modes which may
be adopted in preventive medicine for opposing bacteria outside the
body.

It will be clear at once that we may have varying degrees of
opposition to bacteria. Some substances kill bacteria, and they are
known as _germicides_; other substances prevent their development and
resulting septic action, and these are termed _antiseptics_. The
word _disinfectant_ is used more or less indiscriminately to cover
both these terms. A _deodorant_ is, of course, a substance removing
the odour of evil-smelling putrefactive processes. Here, then, we
have the common designations of substances able to act injuriously on
bacteria and their products outside, or upon the surface of, the body.
But a moment's reflection will bring to our minds two facts not to be
forgotten. In the first place, an antiseptic applied in very strong
dose, or for an extended period, may act as a germicide; and, _vice
versâ_, a germicide in too weak solution to act as such may perform
only the function of an antiseptic. Moreover, the action of these
disinfecting substances not only varies according to their own strength
and mode of application, but it varies also according to the specific
resistance of the protoplasm of the bacteria in question. Examples
of the latter are abundant, and readers who have only assimilated
the simple facts set forth in these pages are aware that between the
bacillus of diphtheria and the spores of anthrax there is an enormous
difference in power of resistance. In the second place, reflection
will enable us to recall what has already been said, when discussing
the requirements necessary for bacterial growth, respecting the
physical conditions injurious to development. In a _cold temperature_,
as a general rule, bacteria do not multiply with the same rapidity
as at blood-heat. Within the limits of a _moist perimeter_ the air
is, to all intents and purposes, germ-free. Direct _sunlight_ has a
definitely germicidal effect in the course of time upon some of the
most virulent bacteria we know. Here, then, are three examples of
physical agents--low temperature, moist perimeter, sunlight--which, if
strong enough in degree, or acting for a long enough period of time,
become first antiseptics and then germicides. Yet for a limited period
they have no injurious effect upon bacteria. These are simple points,
and call for little comment, yet the pages of medical and sanitary
journals reveal not a few keen controversies upon the injurious action
of certain substances upon certain bacteria owing to the discrepancies,
of necessity arising, between results of different skilled observers
who have been carrying out different experiments with different
solutions of the same substance upon different protoplasms of the
same species of bacteria. We feel no doubt that in these pioneering
researches much labour has been to some extent misspent, owing to the
neglect of a common denominator. Only a more accurate knowledge of
bacteria or a recognised standard for disinfecting experiments can ever
supply such common denominator.

Species of bacteria for comparative observation-experiments upon
the action of chemical or physical agents must be not only the same
species, but cultured under the same conditions, and treated by the
agent in the same manner, otherwise the results cannot be compared upon
a common platform, or with any hope of arriving at exactly the same
conclusions.

Sir George Buchanan laid down, in 1884, a very simple and suitable
standard of what true disinfection meant, viz., the _destruction of the
most stable known infective matter_. Such a test is high and difficult
to attain unto; nevertheless, it is the only satisfactory one.
Obviously many substances which are useful antiseptics in practical
life would fall far short of such a standard, yet for true and complete
disinfection such an ideal is the only adequate one.

Quite recently three or four workers at Leipzig[101] have drawn up
simple directions, the adoption of which would considerably assist
in securing a common standard for disinfectant research. They are as
follows:

1. In all comparative observations it is imperative that _molecularly
equivalent_ quantities of the reagents should be employed.

2. The bacteria serving as test objects should have equal power of
resistance.

3. The numbers of bacteria used in comparative observation should be
approximately equal.

4. The disinfecting solution must be always used at the same
temperature in comparative experiments.

5. The bacteria must be brought into contact with the disinfectant with
as little as possible of the nutrient material carried over. (This
obviously will depend upon the object of the research.)

6. After having been exposed to the disinfectant for a fixed time, they
should be freed from it as far as possible.

7. They should then be returned in equal numbers to the respective
culture medium most favourable to the development of each, and kept at
the same, preferably the optimum, temperature for their growth.

8. The number of surviving bacteria capable of giving rise to colonies
in solid media must be estimated after the lapse of equal periods of
time.

We may now turn from general principles to mention shortly some of
the commoner methods and substances adopted to secure efficient
disinfection. They are all divisible, according to Sir George
Buchanan's standard, into two groups:

1. Heat in various forms;

2. Chemical bodies in various forms.

It should at the outset be understood that we desire in practical
disinfection to inhibit or kill micro-organisms without injury to, or
destruction of, the substance harbouring the germs for the time being.
If this latter is of no moment, as in rags or carcasses, burning is the
simplest and most thorough treatment. But with mattresses and beddings,
bedclothes and garments, as well as with the human body, it is obvious
that something short of burning is required.

1. From the earliest days of bacteriology heat has held a prominent
place as a disinfector. But it is only in comparatively recent times
that it has been fully established that _moist heat_ is the only really
efficient form of heat disinfection. Boiling at atmospheric pressure
(100° C.) is the oldest form of moist heat disinfection, and because
of the simplicity of its application it has gained a large degree of
popularity. But it must not be forgotten that mere boiling (100° C.)
may not effectually remove the spores of all bacilli. Besides, boiling
is not applicable to furniture, mattresses, and such-like frequently
infected objects. For many of these _hot-air ovens_ were used in the
early days. But it was found that such disinfection was no disinfection
at all, for not only did it leave many organisms and spores untouched,
but the degree of temperature was rarely, if ever, uniform throughout
the substance being treated.

The failures following in the track of these methods were an indication
of the need of some form of moist heat, viz., steam.

Here it will be necessary to digress for a moment into some of the
characters of steam. When water is heated certain molecular changes
take place, and at a certain temperature (100° C., 212° F.) the water
becomes steam, or _vapour_, and on very little cooling will condense.
But if the vapour is heated, it will become practically a gas, and
will not condense until it has lost the whole of the heat, _i. e._,
the heat of making water into vapour plus the heat of making vapour
into gas. A gas proper is, then, the vapour of a liquid of which the
boiling-point is substantially below its actual temperature. But we
know that the temperature at which it boils depends upon the pressure
to which it is subjected (Regnault's law). Hence in reality "steam at
any temperature whatever may be a vapour proper, provided the pressure
is such as prevents the liquid from boiling below that temperature."
In such a condition of vapour it is termed _saturated steam_. But if
it is at that same pressure further heated, it becomes practically a
gas, and is called _superheated steam_. The former can condense without
cooling; the latter cannot so condense at the same pressure. Saturated
steam condenses immediately it meets the object to be disinfected,
and gives out its latent heat; superheated steam acts by conduction,
and not uniformly throughout the object. Its advantage is that it
dries moistened objects. As a disinfecting power, superheated steam
is much less than saturated steam. There is one further term which
must be defined, namely, _current steam_. This is steam escaping from
a disinfector as fast as it is admitted, and may be at atmospheric or
higher temperatures. The disinfecting temperature which is now used as
a standard is _an exposure to saturated steam of 115° C. for fifteen
minutes_.

A number of different kinds of apparatus have been invented to
facilitate disinfection to this standard on a large scale. Most
sanitary authorities of importance are now supplied with some form
of steam disinfector, though many are unable to go to the expense of
high-pressure disinfectors. Professor Delépine has pointed out[102]
that a current of steam at low pressure may completely disinfect.
Whilst such simple current-steam machines have thus been demonstrated
as efficient bactericides, for all practical purposes it is important
to have disinfectors capable of giving temperatures considerably
above 100° C., of simple construction, having steam power of uniform
temperature and rapid penetration, and containing, when in action, a
minimum of superheated steam. In addition to these characters of a
first-rate steam disinfector, two other important points should be
borne in mind, namely, the air must be completely ejected from the
disinfection chamber before the results due to steam are obtained, and
some sort of automatic index giving a record of each disinfection is
indispensable.

We may turn from these general principles to mention shortly some of
the types of steam disinfectors most commonly in use. They are four,
namely, the Washington Lyon, the Equifex (Defries), the Thresh, and the
Reck.

_Washington Lyon's_ apparatus consists of an elongated boiler having
double walls, with a door at each end. The body of the apparatus is
jacketed. The whole is large enough to admit of bedding and mattresses,
and generally is so arranged that one end opens into one room, and
the other end opens into another room. This convenient position
admits of inserting infected articles from one room and receiving
them disinfected into the other room. Possible reinfection is thereby
prevented. Steam is admitted into the jacket at a pressure of between
twenty and twenty-five pounds, and is generally twenty pounds in the
interior of the cylinder. At the end of the operation a partial vacuum
is created, by which means much of the moisture on the articles may
be removed. In some cases a current of warm air is admitted before
disinfection in order to diminish the extent of condensation.

The _Equifex_ (Defries) contains no steam jacket, but coils of pipes
are placed at the top and bottom of the apparatus, with the object
of imparting to the steam as much heat as is lost by radiation
through the walls of the disinfecting chamber, and at the same time
of preventing undue condensation. The air is first removed by a
preliminary current of steam, after which steam at a pressure of ten
pounds is intermittently introduced and allowed to escape. The object
of this proceeding is to remove air from the pores of the articles to
be disinfected by the sudden expansion of the film of water previously
condensed on their surface. The apparatus introduced by _Dr. Thresh_
was constructed with a view of overcoming the objection to some of
the other machines that bulky articles retained a large percentage
of moisture, thus necessitating the use of some additional drying
apparatus. A central chamber receives the articles to be disinfected,
and is surrounded by a boiler containing a solution of calcium chloride
at a temperature of 225° F. This is heated by a small furnace, and
the steam given off (218-300° F.) is conducted into the central
chamber. The steam is not confined under any pressure except that of
the atmosphere. When the steam has passed for a sufficient length
of time, it is readily diverted into the open air. Hot air is now
introduced, and at the expiration of an hour the articles may be taken
out disinfected and as dry as they were when inserted. The apparatus
is comparatively inexpensive, and not of a complicated nature. The
current steam is saturated, and at a temperature a few degrees above
the boiling-point. Many experiments have been performed with this
apparatus, and there is now a large amount of evidence in favour of it
and current steam disinfection.

_Reck's_ apparatus is another kind of saturated steam disinfector,
which resembles the Equifex, but differs from it in employing steam as
a current.

It is probable that many other forms of steam disinfector will be
invented, and each will have its enthusiastic supporters. Even at the
time of writing some excellent results are announced from America.

2. The effects of _chemical substances_ as solutions, or in spray
form, upon bacteria have been observed from the earliest days of
bacteriology. To some decomposing matter or solution a disinfectant
was added and sub-cultures made. If bacteria continued to develop,
the disinfection had not been efficient; if, on the other hand, the
sub-culture remained sterile, disinfection had been complete. From such
rough-and-ready methods large deductions were drawn, and it is hardly
too much to say that no branch of bacteriology contains such a vast
mass of unassimilated and unassimilable statements as that relating to
research into disinfectants. Most of the tabulated and recorded results
are conspicuous in having no standard as regards bacterial growth. Yet
without such a standard results are not comparable.

Silk threads, impregnated with anthrax spores, were placed in bottles
containing carbolic acid of various strengths, and at stated periods
threads were removed and placed in nutrient media, and development or
otherwise observed. But, as Professor Crookshank[103] has pointed out,
this method is fallacious, the thread being still wet with the solution
when transferred to the medium, and thus modified in culture, possibly
even inhibited altogether. It is unnecessary for us here to discuss
every mode adopted by investigators in similar researches. We may just
mention that the most approved methods at the present time are based
upon two simple plans of exposure. In one we use a known volume of
recent broth culture of an organism grown under specified conditions.
To this is added a measured quantity of the antiseptic. At stated
periods loopfuls of the broth and antiseptic mixture are sub-cultured
in fresh-sterilised broth, and resulting development or otherwise
closely observed. The other method is practicable when we are dealing
with volatile bodies. In such cases a standard culture is made of the
organism in broth at a standard temperature. Into this are dipped
small strips of sterilised linen. When thoroughly impregnated these
are removed from the broth and subsequently dried over sulphuric acid
in a vacuum at 38° C. These may now be exposed for a longer or shorter
period to the fumes of the antiseptic in question, and broth cultures
made at the end of the exposure. It is obvious that a very large number
of modifications are possible of these two simple devices for testing
the bactericidal power of chemical substances. It should be remembered
that here, perhaps, more than anywhere else in bacteriological
research, careful control experiments are absolutely necessary.

_Mineral acids_ (nitric, hydrochloric, sulphuric), especially
concentrated, are all germicides.

The _halogens_--chlorine, bromine, iodine, and fluorine--are, all four,
disinfectants, but not used in practice. They are named in their order
of power as such.

A number of separate bodies, such as _chloroform_ and _iodoform_, have
been much advocated as antiseptics. The cost of the former and odour
of the latter have, however, greatly militated against their general
adoption.

_Chloride of lime_ is a powerful disinfectant. Professor Sheridan
Delépine and Dr. Arthur Ransome have demonstrated its germicidal
effect as a solution applied directly to the walls of rooms inhabited
by tuberculous patients.[104] It may also be used in solid form for
dusting decomposing matter.

_Mercuric chloride_ (corrosive sublimate) has been an accepted
germicide for some time. But the experiments of Behring, Crookshank,
and others have proved that the weaker solutions cannot be relied
upon. This is, in part, due to the fact that it forms in albuminous
liquids an albuminate of mercury which is inactive. Dilute solutions
have the further disadvantage of being unstable. Various authorities
recommend a solution of 1-500 as a germicide, and much weaker solutions
are, of course, antiseptic. An ounce each of corrosive sublimate
and hydrochloric acid in three gallons of water makes an efficient
disinfectant.

_Potassium permanganate_ is, of course, the chief substance in Condy's
fluid, as _zinc chloride_ is in Burnett's disinfecting fluid. A 5 per
cent. of the former and a 2-1/2 per cent. of the latter are germicidal.

_Boracic acid_ is one of the most useful antiseptics with which to
wash sore eyes, or preserve tinned foods or milk. It is not a strong
germicide, but an unirritating and effective wash. Many cases of its
addition to milk have found their way into the law courts, owing to
cumulative poisoning, and it should only be used with the very greatest
care as a food preservative.

_Carbolic acid_ has come into prominence as an antiseptic since its
adoption by Lister in antiseptic surgery. It is cheap, volatile, and
effective. One part in 400 is antiseptic, and 1 in 20 germicidal. As
a wash for the hands the former is used, and a weaker solution for
the body generally. Carbolic soap and similar toilet combinations are
now very common. At one time it appeared as if corrosive sublimate
would oust carbolic from the first place as an antiseptic solution,
but a large number of experiments have confirmed opinion in favour
of carbolic. Professor Crookshank found that carbolic acid, 1 in 40,
acting for only one minute is sufficient to destroy _Streptococcus
pyogenes_, _S. erysipelatis_, and _Staphylococcus pyogenes aureus_,
and in the strength of 1 in 20 carbolic acid completely sterilised
tubercular sputum when shaken up with it for one minute.

_Creosol_, a member of the phenol series, is a good disinfectant, and
the active element in lysol, Jeye's fluid, creoline, izal, and creosote.

_Sulphurous acid_ is one of the commonest disinfectants employed for
fumigation--the old orthodox method of disinfecting a room in which a
case of infective disease has been nursed. It is evolved, of course, by
burning sulphur. For each thousand cubic feet from one to five pounds
of sulphur is used, and the walls may be washed with carbolic acid.
Dr. Kenwood carried out some experiments in 1896[105] which appear to
support the disinfecting power of sulphur fumes. He found that the
_Bacillus diphtheriæ_ was not killed, though markedly inhibited, when
the sulphurous gas (SO_{2}) did not much exceed .25 per cent. But the
bacillus was killed where the sulphur fumes exceeded .5 per cent. Both
these results had reference to the SO_{2} in the air in the centre of
the room at a height of four feet, and after the lapse of four hours.
There can be little doubt that fuming a sealed-up room with sulphur
fumes in a moist atmosphere, and leaving it thus for twenty-four hours,
is generally, if not always, efficient disinfection. It will kill
the bacillus of diphtheria, though not always more resistant germs.
Moreover, its simplicity of adoption is greatly in its favour. Anyone
can readily apply it by purchasing a few pounds weight of ordinary roll
sulphur and burning this in a saucer in the middle of a room which has
had all its crevices and cracks in windows and walls blocked up with
pasted paper. _Nitrous fumes_ may also be used in this way.

Recently _formalin_ has come much in favour as a room disinfectant.
Formalin is a 40 per cent. solution of formaldehyde in water, a gas
discovered by Hofmann in 1869. This gas is a product of imperfect
oxidation of methyl alcohol, and may be obtained by passing vapour
of methyl alcohol, mixed with air, over a glowing platinum wire or
other heated metals, such as copper and silver. It is the simplest
of a series of aldehydes, the highest of which is palmitic aldehyde.
Its formula is CH_{2}O, and it is a colourless gas with a pungent
odour, and having penetrating and irritating properties, particularly
affecting the nasal mucous membrane and the eyes of those working with
it. It is readily soluble in water, and in the air oxidises into formic
acid (CH_{2}O_{2}). This latter substance occurs in the stings of bees,
wasps, nettles, and various poisonous animal secretions. Formalin
is a strong bactericide even in dilute solutions, and, of course,
volatile. A solution of 1 to 10,000 is said to be able to destroy
the bacilli of typhoid, cholera, and anthrax. A teaspoonful to ten
gallons of milk is said to retard souring. When formalin is evaporated
down, a white residue is left known as paraform. In lozenge form this
latter body is used by combustion of methylated spirit to produce the
gas. Hence we have three common forms of the same thing--_formalin_,
_formic aldehyde_, _paraform_--each of which yields _formic acid_, and
thus disinfects. The vapour cannot in practice be generated from the
formalin as readily as from the paraform.

By a variety of ingenious arrangements formic aldehyde has been tested
by a large number of observers during the last two or three years. We
may refer to three modes of application. 1. _The sprayer_ (Equifex
apparatus) produces a mixture of air and solution for spraying walls,
ceilings, floors, and sometimes garments. 2. _The autoclave_ (Trillat's
apparatus). In this a mixture of a 30-40 per cent. watery solution
of formaldehyde and calcium chloride (4-5 per cent.) is heated under
a pressure of three or four atmospheres, and the almost pure, dry
gas is conducted through a tube passing through the keyhole of the
door into the sealed-up room. 3. _The paraform lamp_ (the Alformant).
The principle of this lamp is that the hot, moist products from
the combustion of methylated spirit act upon the paraform tablets,
converting them into gas. Most of the conclusions derived from
experiments with these three different forms of apparatus are the same.
It is agreed that the gas is harmless to colours and metal and polished
wood. The vapour acts best in a warm atmosphere. As for its action on
bacteria, it compares favourably with any other disinfectant. In 1 per
cent. solution formalin destroys non-spore-bearing bacteria in thirty
to sixty minutes.

Many observers have decried formaldehyde on account of its professed
lack of penetrating power. Professor Delépine, however, states[106]
that it possesses "penetration powers probably greater than those
of most other active gaseous disinfectants. _Bacillus coli_, _B.
tuberculosis_, _B. pyocyaneus_, and _Staph. pyogenes aureus_ were
killed in dry or moist state, even when protected by three layers of
filter paper." In Professor Delépine's opinion, the vapours of
phenol, izal, dry chlorine, and sulphurous acid have, under the same
conditions, given inferior results.

We may now shortly summarise the foregoing facts respecting antiseptics
and disinfection in the simplest terms possible to afford facility to
the uninitiated in practical application:

_To disinfect a room_, seal up cracks and crevices, and burn at least
one pound of roll sulphur for every 1,000 cubic feet of space.[107]
Many authorities recommend four or five pounds of sulphur to the same
space. Let the room remain sealed up for twenty-four hours.

_To disinfect walls_, wash with chloride of lime solution (1-100) or
carbolic acid (1-40). This latter solution may be used to wipe down
furniture. Either or both may be used _after_ sulphur fuming. Formic
aldehyde may also be used by lamp or autoclave.

_To disinfect bedding_, _etc._, the steam sterilisation secured in
a Thresh, Equifex, or Lyon apparatus is the best. Rags and infected
clothing, unless valuable, should be burnt.

_To disinfect garments and wearing apparel_, they should be washed in a
disinfectant solution, or fumed with formic aldehyde.

_To disinfect excreta or putrefying solutions_, enough disinfectant
should be added to produce _in the solution or matter being
disinfected_ the percentage of disinfectant necessary to act as such.
Adding a small quantity of antiseptic to a large volume of fluid or
solid is as useless as pouring a small quantity of antiseptic down a
sewer with the idea that such treatment will disinfect the sewage. The
mixture of the disinfectant with the matter to be disinfected must
contain the standard percentage for disinfection. Chloride of lime is a
common substance for use in this way. Potassium permanganate (1-100)
and carbolic (1-100), and many manufactured bodies containing them, are
also widely used. Drs. Hill and Abram recommend[108] that the excreta
and disinfectant be thoroughly mixed and stand for at least half an
hour. For various reasons they particularly advise _chinosol_ as the
most convenient disinfectant for this specific purpose.

_Antiseptics for wounds._ Carbolic acid (1-40) or corrosive sublimate
(1-1,000) are commonly used in surgical practice. Boracic acid is one
of the most unirritating antiseptics which are known. It may be used in
saturated watery solution (1-30) or dusted on copiously as fine powder.
It is especially applicable in open wounds, and as an eye-wash.

_To disinfect hands and arms._ Operating surgeons are those to whom it
is a most urgent necessity to cleanse hands and arms antiseptically.
Carbolic acid (1-20, or 1-40) is used for this purpose.

It is hardly necessary to add that in a case of infectious disease
occurring in a household many of these modes of application, perhaps
all of them, must be adopted. Formalin is probably the best gaseous
disinfectant which we have, but its use does not, and should not,
preclude the simultaneous adoption of other methods.




APPENDIX


It is proposed to add one or two notes on certain technical points in
bacteriological work, with a view to assisting those medical men not
able to obtain the advantages of a well-equipped laboratory, and yet
desirous of occasionally attempting some practical bacteriology.


1. _General Examination._ All fluids may be examined for bacteria in
two chief ways:

(_a_) A small quantity may be placed on a cover-glass or slide, dried
over a lamp or bunsen flame, and stained with aniline dyes for a few
minutes. It is then ready for microscopic examination. It is obvious
that the result will generally be a _mixture_ of bacteria, for which
differentiating stains may be used (Gram, Ziehl-Neelsen, etc.).

(_b_) A minute drop of the suspected fluid may be added to various
fluid media (broth, liquefied gelatine, etc.) and then plated out
upon small sterilised sheets of glass. In the course of two or three
days the contained bacteria will reveal themselves in characteristic
colonies, which may be examined, and if possible sub-cultured, and
carefully studied.


_Double-Staining Methods._ These are various, and are used when it is
desired to stain the bacteria themselves one colour, and the matrix or
ground substance in which they are situated another colour. Three of
the commoner methods are those of Ehrlich, Neelsen, and Gram. They are
as follows:


_Ehrlich's Method._ "Five parts of aniline oil are shaken up with
100 parts distilled water, and the emulsion filtered through
moistened filter paper. A saturated alcoholic solution of fuchsine,
methyl-violet, or gentian-violet is added to the filtrate in a
watch-glass, drop by drop, until precipitation commences. Cover-glass
preparations are floated in this mixture for fifteen to thirty minutes,
then washed for a few seconds in dilute nitric acid (one part nitric
acid to two of water), and then rinsed in distilled water. The stain is
removed from everything except the bacilli; but the ground substance
can be after-stained brown if the bacilli are violet, or blue if
they have been stained red" (Crookshank, _Bacteriology and Infective
Diseases_, p. 89).


_Gram's Method._ The primary stain in this method is a solution of
aniline gentian-violet (saturated alcoholic solution of gentian-violet
30 cc., aniline water 100 cc.), which stains both ground substance and
bacteria in purple. The preparation is next immersed in the following
solution for half a minute or a little more:

  Iodine                  1  part
  Potassium iodide        2  parts
  Distilled water       300  parts

In this short space of time the iodine solution acts as a mordant of
the purple colour in the bacteria, but not in the ground substance.
Hence, if the preparation be now (when it has assumed a _brown_
colour) washed in alcohol (methylated spirit), the ground substance
slowly loses its colour and becomes clear. But the bacteria retain
their colour, and thus stand out in a well-defined manner. Cover-glass
preparations decolourise more quickly than sections of hardened tissue,
and they should only be left in the methylated spirit until no more
colour comes away. The preparation may now be washed in water, dried,
and mounted for microscopic examination, or it may be double-stained,
that is, immersed in some contrast colour which will lightly stain the
ground substance. Eosin or Bismarck brown are commonly used for this
purpose. The former is applied for a minute or two, the latter for
five minutes, after which the specimen is passed through methylated
spirit (and preferably xylol also) and mounted. The result is that the
bacteria appear in a dark purple colour on a background of faint pink
or brown. Carbol-thionine blue, picro-carmine, and other stains are
occasionally used in place of the aniline gentian-violet, and there
are other slight modifications of the method.


_Ziehl-Neelsen Method._ Here the primary stain is a solution of
carbol-fuchsin:

  Fuchsin                                              1 part
  Absolute alcohol                                    10 parts
  5 per cent. aqueous solution of carbolic acid      100 parts

It is best to heat the dye in a sand-bath, in order to distribute the
heat evenly. The various stages in the staining process are as follows:
(_a_) The cover-glass with the dried film upon it is immersed in the
hot stain for one to three minutes. (_b_) Remove the cover-glass from
the carbol-fuchsin, and place it in a capsule containing a 25 per cent.
solution of sulphuric acid to decolourise it. Here its redness is
changed into a slate-grey colour. (_c_) Wash in water, and alternately
in the acid and water, until it is of a faint pink colour. (_d_) Now
place the cover-glass for a minute or two in a saturated aqueous
solution of methylene-blue, which will counter-stain the decolourised
ground substance blue. (_e_) Wash in water. (_f_) Dehydrate by rinsing
in methylated spirit, dry, and mount. A pure culture of bacteria will
not necessarily require the counter-stain (methylene-blue). Sections
of tissue may require twenty to thirty minutes in the primary stain
(carbol-fuchsin). This stain is used for tubercle and leprosy. With
a little practice the staining of the bacillus of tubercle when
present in pus or sputum becomes a very simple and accurate method of
diagnosis. A small particle of sputum or pus is placed between two
clean cover-glasses and thus pressed between the thumb and finger into
a thin film. This is readily dried and stained as above, the bacillus
of tubercle appearing as a delicately-beaded red rod with a background
of blue.


_Bacteriological Diagnosis._ The following points must be ascertained
in order to identify any particular micro-organism:

(1) Its morphology, bacillus, coccus, spirillum, etc.; the presence or
absence of involution forms.

(2) Motility by the unstained cover-glass preparation ("hanging drop");
note presence of flagella.

(3) Presence of spores, their appearance and position.

(4) Whether or not the organism stains with Gram's method.

(5) The character of the growth upon various media (gelatine, agar,
milk, potato, broth); the presence or absence of liquefaction in the
gelatine culture; its power of producing acid, gas, or indol.

(6) Whether it is aërobic or anaërobic.

(7) Its colour in cultivation.

(8) If it is a disease-producing organism under examination, its
effect upon the animal tissues and the course of the disease should be
observed.

There are other points of importance, but the above are essential to a
right conclusion.


_Diagnosis in Special Diseases:_

(1) _Diphtheria._ This disease may be bacteriologically diagnosed with
a minimum of apparatus and equipment. By means of a swab a rubbing
from a suspected throat is readily obtained. This may be examined by
the microscope, or sub-cultured on favourable medium. Blood serum is
perhaps the best, but, as Hewlett remarks, "If no serum tubes can be
had, an egg may be used. It is boiled hard, the shell chipped away from
one end with a knife sterilised by heating, and the inoculation made
on the exposed white surface; the egg is then placed, inoculated end
down, in a wine-glass of such a size that it rests on the rim and does
not touch the bottom. A few drops of water may with advantage be put at
the bottom of the glass to keep the egg-white moist. The preparation
is kept in a warm place for twenty-four to forty-eight hours and
then examined." The examination, of course, consists in staining and
preparing for the microscope and observing the form, arrangement, and
characters of the organism or organisms present. A small piece of the
membrane may be detached, washed in water, and stained for the bacilli.

(2) _Tubercle_ (Ziehl-Neelsen's stain, _vide supra_).

(3) _Typhoid_ (_Enteric Fever_).

_Widal's Reaction._ This diagnostic test depends upon the effect
which the blood of a person suffering from typhoid fever has upon the
_Bacillus typhosus_. The effect is twofold. In the first place, the
actively motile _B. typhosus_ becomes immotile; and secondly, there
is an agglutination, or grouping together in colonies, of the _B.
typhosus_. Neither of these features occurs if healthy human blood is
brought into contact with a culture of the typhoid bacillus. There
are various ways in which this "serum diagnosis" can be carried out.
The simplest and quickest method is as follows: To ten drops of a
twenty-four or forty-eight-hours-old neutral broth culture of the
typhoid bacillus one drop of the blood serum to be tested is added.
The serum and culture are rapidly mixed in the trough of a hollow
ground slide (such as is used for the "hanging drop"), and a single
drop is taken, placed upon an ordinary clean slide, and a cover-glass
superimposed. The positive reaction of agglutination and immotility,
if the blood comes from a case of typhoid fever, will probably appear
within fifteen or twenty minutes. The fluid culture of typhoid may be
taken from an agar culture as well as from broth. In both cases it may
be desirable to filter through ordinary filter paper to remove any
normally agglutinated masses of bacilli before commencing the test.

In his first experiments Widal used a test-tube in the following
manner: The blood to be tested is diluted by one part of it being added
to fifteen parts of broth in a test-tube. The mixture is inoculated
with a drop of a typical _Bacillus typhosus_ culture. The tube is then
incubated at 37° C. for twenty-four hours, after which it is examined.
If the reaction be positive, the broth appears comparatively clear, but
at the bottom of the test-tube a more or less abundant sediment will
be found. This is due to the clumps of bacilli having fallen owing to
gravity. If, on the other hand, the reaction is negative, the broth
will appear more or less uniformly turbid.

For the _apparatus_ required to carry out the simpler methods of
bacteriological work reference should be made to the standard
laboratory text-books, which furnish all necessary details. A good
microscope, with a 1/12 oil immersion lens, is, of course, essential.
This can now be obtained for about £16 (Beck, Swift, Baker, Watson,
etc.), and the other necessary apparatus is readily obtainable of Baird
and Tatlock, Hatton Garden, E. C., and other makers.




FOOTNOTES:

  [1] _The Contemporary Review_, November, 1897, p. 719.

  [2] Some notable exceptions are found in the work of the Bath
  and West of England Society, Lord Vernon's model dairy, and
  the Essex County Council Bacteriological Teaching Laboratory.

  [3] We propose throughout to use the term _bacterium_ (pl.
  _bacteria_) in its generic meaning, unless especially stated
  to the contrary. It will also be synonymous with the terms
  _microbe_, _germ_, and _micro-organism_. The term _bacillus_
  will, of course, be restricted to a rod-shaped bacterium.

  [4] Migula has recently (1896) suggested that the
  Schizomycetes should be subdivided into _Coccaceæ_,
  _Bacteriaceæ_, _Spirillaceæ_ (spirilla, spirochæta),
  _Chlamydobacteriaceæ_ (Streptothrix, Crenothrix, Cladothrix),
  and _Beggiatoa_.

  [5] A one-twelfth oil immersion lens is requisite for the
  study of the lower bacteria.

  [6] A _flagellum_ is a hair-like process arising from the
  poles or sides of the bacillus. It must not be confused with
  a _filament_, which is a thread-like growth of the bacillus
  itself.

  [7] A "pure culture" is a growth in an artificial medium
  outside the body of one species of micro-organism only.

  [8] Some pathogenic germs (suppuration and typhoid) can
  withstand freezing for weeks.

  [9] G. J. Romanes, _Darwin and After Darwin_, vol. ii., 231.

  [10] It will be observed that there is a marked difference
  between the effects of dry heat and moist heat. Moist heat is
  able to kill organisms much more readily than dry, owing to
  its penetrating effect on the capsule of the bacillus. Dry
  heat at 140° C. (284° F.), maintained for three hours, is
  necessary to kill the resistant spores of _Bacillus anthracis_
  and _B. subtilis_, but moist heat at fifty degrees less will
  have the same effect. It is from data such as these that in
  laboratories and in disinfecting apparatus moist heat is
  invariably preferred to dry heat. For with the latter such
  high temperatures would be required that they would damage the
  articles being disinfected. Koch states the following figures
  for general guidance: Dry heat at a temperature of 120° C.
  (248° F.) will destroy spores of mould fungi, micrococci,
  and bacilli in the absence of their spores; for the spores
  of bacilli 140° C. (284° F.), maintained for three hours, is
  necessary; moist heat at 100° C. (212° F.) for fifteen minutes
  will kill bacilli and their spores.

  [11] Water from a house cistern is rarely a fair sample. It
  should be taken from the main. If taken from a stream or still
  water, the collecting bottle should be held about a foot below
  the surface before the stopper is removed.

  [12] The _cubic centimetre_ (cc.) is a convenient standard of
  fluid measurement constantly recurring in bacteriology. It is
  equal to 16-20 drops, and 28 cc. equal one fluid ounce.

  [13] The gelatine is reduced to liquid form by heating in
  a water-bath. Before inserting the suspected water it is
  essential that the gelatine be under 40° C, or thereabouts, in
  order not to approach the thermal death-point of any bacteria.

  [14] _Micro-organisms in Water_ (1894).

  [15] Report on the Micro-organisms of Sewage, Reports to L. C.
  C., 1894, No. 216.

  [16] Harben Lectures, 1896.

  [17] Report on the Metropolitan Water Supply.

  [18] The methods adopted for making a quantitative and
  qualitative examination of sewage are precisely analogous
  to those used in milk research. Dilution with sterilised
  water previous to plating out on gelatine in Petri dishes
  is essential (1 cc. to 10,000 cc. of sterile water, or some
  equally considerable dilution), otherwise the large numbers
  of germs would rapidly liquefy and destroy the film. Special
  methods must be used for the isolation of special organisms;
  phenol-gelatine, Elsner medium, indol reaction, "shake"
  cultures, Parietti broth, etc., must often be resorted to for
  special bacteria. Spores of bacteria may always be numerically
  estimated by adding the suspected water or sewage to gelatine,
  and then heating to 80° C. for ten minutes before plating out.
  This temperature removes the bacilli, but leaves the spores
  untouched.

  [19] The bacilli of typhoid can live in crude sewage (Klein),
  but only for a very short period. When sewage is diluted
  with large quantities of water the case is very different.
  _Bacillus coli_ flourishes in sewage.

  [20] Annual Report of the Medical Officer of the Local
  Government Board, 1897-98, p. 210.

  [21] John Tyndall, F.R.S., _Floating Matter of the Air_.

  [22] Flügge has lately attempted to demonstrate that an air
  current having a velocity of four metres per second can remove
  bacteria from surfaces of liquids by detaching drops of the
  liquid itself.

  [23] Hewlett and Thomson graphically demonstrated the
  bactericidal power of the nasal mucous membrane by noting
  the early removal of _Bacillus prodigiosus_, which had been
  purposely placed on the healthy Schneiderian membrane of the
  nose.

  [24] _Pathological Society of London, Transactions_, 1897.

  [25] _Annali d'Igiene Sperimentale_, vol. v. (1895), fasc. 4.

  [26] _Public Health_, vol. x., No. 4, p. 130 (1898).

  [27] Flügge, _Grundriss der Hygiene_, 1897.

  [28] _Zeitschrift für Hygiene_, vols. xxiv.-xxvi.

  [29] _Annales de Micrographie._

  [30] E. A. Schäfer, F.R.S., _Text-book on Physiology_, vol.
  i., p. 312.

  [31] The unorganised ferments are frequently otherwise
  classified than as above, not according to the locality, but
  according to the function. The chief are these:--_amylolytic_,
  those which change starch and glycogen (amyloses) into sugars,
  _e. g._, ptyalin, diastase, amylopsin; _proteolytic_, those
  which change proteids into proteosis and peptones, _e. g._,
  trypsin, pepsin; _inversive_, those which change maltose,
  sucrose, and lactose into glucose, _e. g._, invertin;
  _coagulative_, those which change soluble proteids into
  insoluble, _e. g._, rennet; _steatolytic_, those which split
  up fats into fatty acids and glycerine, _e. g._, steapsin.

  [32] A chemical change obtained by the action of sulphuric or
  some other acid, or by the influence of _diastase_.

  [33] _Bacteriology and Infective Diseases_, Appendix.

  [34] E. C. Hansen, _Studies in Fermentation_ (Copenhagen), p.
  98.

  [35] _Proc. Royal Soc. of Edin._, xxxvii., pt. iv., p. 759.

  [36] E. A. Schäfer, _Text-book of Physiology_, vol. i., p. 25
  (W. D. Halliburton).

  [37] "Denitrifying" means reducing _nitrates_.

  [38] R. Warington, M.A., F.R.S., _Journ. Roy. Agricultural
  Soc. Eng._, series iii., vol. viii., pt. iv., pp. 577 _et seq._

  [39] The saltpetre beds of Chili and Peru are an excellent
  example of the industrial application of these facts. Nitrates
  are there produced from the fæcal evacuations of sea-fowl in
  such quantities as to form an article of commerce. A like
  form of utilisation of the action of these bacteria was once
  practiced on the continent of Europe. Economic application is
  also seen in the treatment of sewage referred to elsewhere.

  [40] The course of nitrification may be followed by means
  of chemical tests. 1. The disappearance of ammonia. 2. The
  appearance of nitrite. 3. Its disappearance. 4. Appearance of
  nitrate.

  [41] Professor Warington, in Report IV. (p. 526) of his
  admirable series of papers on the subject, draws attention to
  Müntz's criticism that the nitrifying organisms only oxidise
  from nitrogenous matter to nitrites, and not from nitrites
  to nitrates. Müntz held that the conversion of nitrite into
  nitrate is brought about by the joint action of carbonic
  acid and oxygen. Professor Warington's experiments, however,
  clearly illustrate that the production of nitrates from
  nitrites in an ammoniacal solution can be determined by the
  character of the bacterial culture with which the solution is
  seeded, and that in a solution of potassium nitrite conversion
  into nitrate can be determined by the introduction of the
  nitric organism. Professor Warington still adheres to the
  opinion, in favour of which he has produced so much evidence,
  that the formation of nitrates in the soil is due to the
  nitric organism which soil always contains.

  [42] British Association for the Advancement of Science,
  Bristol, 1898, Presidential Address.

  [43] British Association for the Advancement of Science,
  Bristol, 1898, Presidential Address.

  [44] Sir John Lawes and Sir Henry Gilbert (_Times_, December
  2, 1898), have pointed out that the addition of nitrates only
  would be of no permanent use to the wheat crop. They rely upon
  thorough tillage and proper rotation of crops as the means of
  improving the nitrogen value of the soil.

  [45] Geddes, _Nature_, xxv., 1882.

  [46] Sir Henry Gilbert, F.R.S., _The Lawes Agricultural Trust
  Lectures_, 1893, p. 129.

  [47] _Ibid._, p. 140.

  [48] This has been denied recently in the official report
  by the chemist of the Experimental Farm to the Minister of
  Agriculture at Ottawa (_Report_, 1896, p. 200).

  [49] It has already been pointed out that the nitrifying
  bacteria, though able to live on organic matter, do not
  require such either for existence or for the performance of
  their function.

  [50] Lehmann and Neumann, p. 305.

  [51] The conditions requisite for an outbreak of enteric fever
  were, according to Pettenkofer, (_a_) a rapid fall (after a
  rise) in the ground water, (_b_) pollution of the soil with
  animal impurities, (_c_) a certain earth temperature, and
  lastly (_d_) a specific micro-organism in the soil. These
  four conditions have not, particularly in England, always
  been fulfilled preparatory to an epidemic of typhoid. Yet the
  observations necessary for these deductions were a definite
  step in advance of mere dampness of soil.

  [52] _Supplement to the Report of the Medical Officer of the
  Local Government Board_, 1887, p. 7.

  [53] _Report of Medical Officer to Local Government Board_,
  1895-1896, Appendix.

  [54] H. L. Russell, _Dairy Bacteriology_, p. 46.

  [55] _Bureau of Animal Industry Reports_, 1895-1896.

  [56] _British Medical Journal_, 1895, vol. ii., p. 322.

  [57] _British Medical Journal_, 1895, vol. ii., p. 322.

  [58] _Journal of Comparative Pathology_, vol. x. (1897), pp.
  150-189.

  [59] E. W. Hope, M.D., D.Sc., _Report of the Health of
  Liverpool during 1897_, p. 40.

  [60] S. Rideal and A. G. R. Foulerton conclude, from a series
  of experiments, that boric acid (1-2,000) and formaldehyde
  (1-50,000) are effective preservatives for milk for a period
  of twenty-four hours, and that these quantities have no
  appreciable effect upon digestion or the digestibility of
  foods preserved by them (_Public Health_, May, 1899, pp.
  554-568).

  [61] _Report from Wisconsin Agricultural Experiment Station_,
  1896.

  [62] Jenner Institute of Preventive Medicine (First Series
  _Transactions_).

  [63] _Centralblatt für Bakteriologie_, etc., II. Abteilung.

  [64] _A Manual of Bacteriology, Clinical and Applied_, p. 397.

  [65] Hewlett asserts that butter may contain from two to
  forty-seven millions of bacteria per gram.

  [66] Such pure cultures for such purposes are in the United
  States termed "starters," because they start the process of
  special ripening. For the sake of convenience the term will be
  used here.

  [67] The Essex County Council is one of the few public
  bodies in England which have undertaken pioneer work in
  this department of industry. Under the leadership of Mr.
  David Houston, a course of elementary instruction in dairy
  bacteriology as applied to modern dairy practice is given in
  the County Biological Laboratory at Chelmsford.

  [68] _Report of Storr's Agricultural Experiments Station,
  State of Connecticut_, 1895.

  [69] "Observations on Cheddar Cheese Making," _Reports of Bath
  and West and Southern Counties Society_, 1898, pp. 163-171.
  Mr. Lloyd's Reports to the West of England Society since
  1892 contain various points respecting the application of
  bacteriology to cheese-making.

  [70] _Journal of Bath and West of England Society_, 1893,
  1895, and 1897.

  [71] _New York Medical Record_, 1894.

  [72] _British Medical Journal_, 1896, ii., p. 760 _et seq._

  [73] _Special Report of the Medical Officer to the Local
  Government Board on Oyster Culture, etc._, 1896.

  [74] Royal Commission on Tuberculosis, _Report_, 1895, pt. i.,
  p. 13.

  [75] _Ibid._, p. 18.

  [76] _British Medical Journal_, 1895, vol. ii., p. 513.

  [77] It should be distinctly understood that this table
  is merely schematic and provisional. The details of toxin
  production and its effect are still open to revision and
  amendment.

  [78] Sidney Martin, M.D., F.R.S., F.R.C.P., _Croonian Lectures
  delivered before the Royal College of Physicians_, June, 1898.

  [79] It is impossible here to enter into a detailed
  consideration of the various views held with regard to the
  formation of antitoxins. It is needless to remark that the
  whole matter is one of abstruse technicality and intricacy.
  These antitoxic bodies gradually increase in the blood and
  tissues, and their action falls into two groups: (_a_)
  _antitoxic_, which counteract the effects of the poison
  itself; and (_b_) _antimicrobic_, which counteract the effects
  of the bacillus itself. "In one and the same animal the blood
  may contain a substance or substances which are both antitoxic
  and antimicrobic, such, for example, as occurs in the process
  of the formation of the diphtheria and tetanus antitoxic
  serums" (Sidney Martin).

  [80] Types of bodies possessing positive chemiotaxis for
  bacteria are the salts of potassium, peptone, glycerine.

  [81] Negative chemiotaxis is illustrated in alcohol, and free
  acids, and alkalies.

  [82] The friend of Addison and Pope, who married Mr.
  Edward Wortley Montagu in 1712, and on his appointment to
  the ambassadorship of the Porte in 1716 went with him to
  Constantinople. They remained abroad for two years, during
  which time Lady Wortley Montagu wrote her well-known Letters
  to her sister the Countess of Mar, Pope, and others.

  [83] Crookshank, _History and Pathology of Vaccination_.

  [84] An exhaustive account of vaccine may be found in the
  Milroy lectures delivered in 1898 at the Royal College of
  Physicians by S. Monckton Copeman, M.D.

  [85] Crookshank, _Bacteriology and Infective Diseases_;
  Virchow, _The Huxley Lecture_, 1898.

  [86] To shorten this period Dr. Cartwright Wood has adopted
  a plan by which time may be saved, and 200 cc. injected say
  within the first two or three weeks. This is accomplished by
  using a "serum toxin" (containing albumoses, but not ferments)
  previously to the broth toxin, an ingenious method which we
  cannot enter into here.

  [87] At the conclusion of the operation the cannula is removed
  from the jugular vein, and the wound is closed by the valvular
  character of the slit in the skin and vein and the elasticity
  of the wall of the vein. No stitching or dressing is required.
  Indeed, it is striking to observe in the horse an entire
  absence of pain throughout the proceedings.

  [88] The term _unit_ is used as a standard measurement. This
  means the amount of antitoxin which will just neutralise ten
  times the minimum fatal dose of the toxin in a guinea-pig
  (250 grams toxin to kill on the fourth day). If 1 cc. of the
  antitoxic serum is required for this, one unit is contained in
  1 cc.; if 0.01 cc. is sufficient, then 100 units are contained
  in the cc. Not less than 1500 units should be administered for
  a dose, and repeated every twelve hours. In severe cases two
  or three times this amount may be given.

  [89] The value of antitoxin treatment in diphtheria is
  discussed in the _Brit. Med. Jour._, 1899, pp. 197 and 268, by
  E. W. Goodall, M.D.

  [90] A detailed study of tuberculosis from its pathological
  and bacteriological aspect will be found in _La Tuberculose
  et son Bacille_, pt. i., Straus, Professeur à la Faculté de
  Médecine de Paris.

  [91] For differences of virulence between these conditions
  of pulmonary tubercle see Lingard, _Local Government Board
  Report_, 1888, p. 462.

  [92] _Centralblatt. f. Bact. und Parasit._, vol. vii., p. 9.

  [93] _Animal Tuberculosis_, p. 129.

  [94] See the _Harben Lectures_, November, 1898, by Sir Richard
  Thorne Thorne, Medical Officer to the Local Government Board;
  also the _Report of the Royal Commission on Tuberculosis_,
  1896-98.

  [95] 1. Tuberculosis is a disease mainly affecting the lungs
  (_consumption_, _decline_, _phthisis_) of young adults and the
  bowels of infants (_tabes mesenterica_). It may affect any
  part of the body, and its manifestations are very various. It
  also affects animals, particularly cattle, by whom it may be
  transmitted to man.

  2. _Its direct cause_ is a microscopic vegetable cell, known
  as the _Bacillus tuberculosis_, discovered by Koch in 1882.
  This fungus requires to be magnified some hundreds of times
  before it can even be seen. When it gains entrance to the
  weakened body it sets up the disease, which is an _infectious_
  disease, though different in degree to the infectiousness of,
  say, measles.

  3. _Trade influence and occupation_, in some cases,
  undoubtedly predispose the individual to tubercle. Cramped
  attitudes, exposure to dampness or cold, ill ventilation, and
  exposure to inhalation of dust of various kinds, all act in
  this way. In support of the evil effect of each of these four
  conditions much evidence could he produced.

  4. _Overcrowding_ has a definite influence in propagating
  tubercular diseases. The agricultural counties without big
  towns, like Worcestershire, Herefordshire, Buckinghamshire,
  and Rutland, are the counties having the lowest mortality
  from tuberculosis; whilst the crowded populations in
  Northumberland, South Wales, Lancashire, London, and the
  West Riding suffer most. Speaking more particularly, the
  overcrowded areas of London, such as St. Giles', Strand,
  Holborn, and Central London generally, show very high
  tubercular death-rates.

  5. _Tuberculosis is not increasing._ During the last thirty
  years it has shown, with few exceptions, a steady decline
  in all parts of England. "Consumption" is most fatal in
  comparatively young people (fifteen to forty-five years),
  whilst "tabes" and other forms of tubercle are fatal chiefly
  to young children. These forms have not declined so much as
  the lung form. The mortality in consumption of males has since
  1866 been in excess of that of females. The age of maximum
  fatality from consumption is _later_ than in the past, which
  is probably due to improved hygiene and treatment.

  6. _This decline has been due_, not to any special repressive
  measures--for few or none have been carried out--but to a
  general and extensive social improvement in the life of the
  people, to an increase of knowledge respecting tuberculosis
  and hygiene, to an enormous advance in sanitation, and to more
  efficient land drainage.

  7. _Not all persons are equally liable to consumption_, some
  being much more susceptible than others. We have mentioned
  the predisposing influence of certain trades. There is also
  heredity, which acts, as we have said, in transmitting a
  tubercular _tendency_, not commonly the actual virus of
  the disease; there is, thirdly, the debilitating effect of
  previous illness or chronic alcoholism; there is, fourthly,
  the habitual breathing of rebreathed air; and, fifthly, there
  are the conditions of the environment, like dampness and
  darkness of the dwelling. Such influences as these weaken the
  resisting power of the tissues, and thus afford a suitable
  nidus for the bacillus conveyed in milk or by the inspiration
  of infected dust.

  8. _Consumption is curable_ if taken in time. In cases where
  the lungs are half gone, and consist of large cavities, it is
  obvious that curability is out of the question. But if the
  disease can be properly treated in its earliest stages, there
  is considerable likelihood of recovery.

  9. _The breath is not dangerous_, as far as we know, but there
  is danger from discharges of any kind from any infected part,
  whether lungs or bowels; for such discharges, when dry, may
  readily pollute the air, and either the bacilli or spores be
  inhaled into the lungs.

  10. _The chief channels of personal infection or the spread
  of the disease amongst a community_ are two: (_a_) dried
  tubercular sputum (or other tubercular discharges); (_b_)
  infected milk or meat. So long as the former remains wet or
  moist, infection cannot take place. It is, of course, better
  to destroy it completely. As for milk and meat, boiling the
  former and thoroughly cooking the latter will remove all
  danger.

  11. _The expectoration is infective._ This is one of the
  commonest modes of infection, and to it is held to be due
  the large amount of respiratory tuberculosis (consumption,
  phthisis). The expectoration from the lungs must contain, from
  the nature of the case, a very large number of bacilli. As
  a matter of fact, a single consumptive individual can cough
  up in a day millions of tubercle bacilli. When expectoration
  becomes dry, the least current of air will disseminate the
  infective dust, which can by that means be readily reinspired.
  Expectoration on pavements and floors, as well as on
  handkerchiefs, may thus become, on drying, a source of great
  danger to others. The discharges from the bowels of infants
  suffering from the disease also contain the infective material.

  12. _Milk_, though a much more likely channel for conveyance
  of tubercle than meat, is only or chiefly virulent when the
  udder is the seat of tuberculous lesions. The consumption of
  such milk is only dangerous when it contains a great number of
  bacilli and is ingested in considerable quantity. Practically
  the danger from using raw milk exists only for those persons
  who use it as their sole or principal food, _e. g._, young
  children. All danger is avoided by boiling or pasteurising the
  milk.

  At the same time there is an increasing amount of evidence
  forthcoming at the present time which goes to prove that milk
  is not infrequently tainted with tubercle (see p. 195). The
  tuberculin test should be applied to all milch cows, and the
  infected ones isolated from the herd. Milk supplies should be
  more strictly inspected even than cowsheds.

  13. There are several methods by which _meat infection can
  be prevented_. In the first place, herds should be kept
  healthy, and tubercular animals isolated. Cowsheds and byres
  should be under sanitary supervision, especially as regards
  overcrowding, dampness, lack of light, and uncleanliness.
  Public slaughter-houses under a sanitary authority would
  undoubtedly be most advantageous. Meat inspection should also
  be more strictly attended to; efficient cooking, and avoidance
  of "roll" meat which has not been thoroughly cooked in the
  middle.

  14. _Consumptive patients may diminish their disease._ Dr.
  Arthur Ransome[95a] has laid down five axioms of hygiene for
  phthisical patients which, if followed, would materially
  improve the condition of such persons. At Davos, St. Moritz,
  Nordrach and other places where they have been practised, the
  beneficial change has been in many cases extraordinary:

  (1) Abundance of light, nutritious, easily digested food,
  which must comprise a large allowance of fat; small meals, but
  frequent;

  (2) An almost entirely open-air life, with as much sunshine as
  can be obtained;

  (3) Suitable clothing, mostly wool;

  (4) Cleanliness and bracing cold-water treatment;

  (5) Mild but regular exercise.

  15. _Consumptive patients may also assist in preventing the
  spread of the disease._ In the first place, they should
  follow the hygienic directions just mentioned, because such
  conditions fulfilled will materially lessen the contagiousness
  of such patients; next, the expectoration must never be
  allowed to get dry. A spitting-cup containing a little
  disinfectant solution (one teaspoonful of strong carbolic acid
  to two tablespoonfuls of water) should always be used, or the
  expectoration received into paper handkerchiefs which can be
  burnt. Spoons, forks, cups, and all such articles should be
  thoroughly cleaned before being used by other persons. The
  patient should not sleep in company with another, but occupy,
  if possible, a separate bedroom.

  Isolation hospitals for consumptives, as for patients
  suffering from diphtheria, are now being established.

  16. _House influence_ has some effect, both directly and
  indirectly, upon tubercular diseases. Damp soils, darkness,
  and small cubic space in the dwelling-house exert a very
  prejudicial effect upon tubercular patients. Sir Richard
  Thorne Thorne[95b] has described the favourable house for such
  persons as one built upon a soil which is dry naturally or
  freed by artificial means from the injurious influences of
  dampness and of the oscillations of the underlying subsoil.
  The house itself should be so constructed as to be protected
  against dampness of site, foundations, and walls. Upon at
  least two opposite sides of the dwelling-house there should be
  enough open space to secure ample movement of air about it,
  and free exposure to sunlight. Lastly, it should be possible
  to have free movement of air by day and night through all
  habitable rooms of the house. It is clear that many inhabited
  houses could not stand to these tests; but effort should be
  made to approach as near to such a standard as possible.

  17. _Sunlight and fresh air_ are the greatest enemies to
  infection.

  18. _Disinfection is necessary after death from phthisis_,
  and should be as complete as after any other infective
  disease. Compulsory notification of fatal cases and compulsory
  disinfection have been officially ordered by the Prussian
  Government. In this country also absolute disinfection should
  always be insisted upon after phthisis. Walls, floors,
  carpets, curtains, etc., should be strictly sterilised.
  Professor Delepine recommends spraying with 1-100 solution of
  chloride of lime.

  [95a] Arthur Ransome, M.D., F.R.S., _Treatment of Phthisis_.

  [95b] _Practitioner_, vol. xlvi.

  [96] _Journal of State Medicine_, vol. iv. (1896), p. 169.

  [97] For a fuller statement see _Trans. Jenner Institute_
  (First Series), pp. 7-32.

  [98] See _Trans. Jenner Institute_ (First Series), A. G. R.
  Foulerton, pp. 40-81.

  [99] Dated 1890-91. The Commissioners were the late Beaven
  Rake, M.D., G. A. Buckmaster, M.D., the late Professor
  Kanthack, of Cambridge, the late Surgeon-Major Arthur Barclay,
  and Surgeon-Major S. J. Thomson.

  [100] _Bacteriology and Infective Diseases_ (1896), p. 144.
  Professor Crookshank's Reports to the Agricultural Department
  of the Privy Council constitute the most complete account of
  this disease hitherto published.

  [101] _Zeitschr. f. Hyg. und Inf. Krank._, xxv.

  [102] _Journal of State Medicine_, December, 1897, p. 561.

  [103] _Bacteriology and Infective Diseases_, p. 35.

  [104] _British Medical Journal_, 1895 (February), p. 353.

  [105] _British Medical Journal_, 1896 (August), p. 439.

  [106] _Journal of State Medicine_, 1898 (November), p. 541.

  [107] The measurement of cubic space is of course made by
  multiplying together in feet the length, breadth, and height
  of a room.

  [108] _British Medical Journal_, 1898 (April), p. 1013.


  INDEX


  Abscess formation, 296-301

  Acetous fermentation, 115, 127

  Actinomycosis, 316

  Aërobic organisms, 26

  Agar, 21

  Air, bacteriology of, 96-110

  -- examination of, 96-99

  -- of sewers, 105

  -- expired, 102

  -- bacteria and gravity, 106

  -- standard of bacteria in, 108

  -- pathogenic bacteria in, 109

  -- passages, bacteria in, 103

  Alcohol, formation of, 115

  Alcoholic fermentation, 115, 117

  Alexines, 249, 268

  Alformant lamp for disinfection, 334

  Algæ in water, 53

  Ammoniacal fermentation, 115

  Amylolytic ferments, 115

  Anaërobic organisms, 132

  -- methods of culture, 139-142

  -- in hydrogen, 139, 140

  -- in glucose-agar, 142

  -- in Fränkel's tube, 140

  -- in Buchner's tube, 141

  Aniline dyes, 44

  Antagonism of organisms, 33

  Anthrax, 19, 26, 30, 34, 245

  -- pathology of, 301

  -- spores of, 302

  -- bacillus of, 302

  Antiseptics, 323, 332

  -- definition of, 322

  -- some of the chief, 332

  Antitoxins, 245-250

  -- preparation of, 259

  -- use of, 263

  -- unit of, 263

  Appendix, 337

  Arthrospores, 17

  Artificial purification of water, 73

  Ascospores, 120

  Asiatic cholera, 65

  Association of organisms, 31

  Attenuation of virulence, 36


  Bacillus, definition of, 11

  -- aceti, 34, 129

  -- acidi lactici, 131, 185, 190

  -- amylobacter, 132

  -- anthracis, 26, 31, 34, 110, 301-305

  -- aquatilis, 53

  -- butyricus, 133

  -- coli communis, 32, 56, 58-62, 64, 67, 86, 88, 108, 151, 194, 237, 299,
       315

  -- cyanogenus, 193

  -- diphtheriæ, 201, 212, 244, 289-296

  -- enteriditis sporogenes, 60, 86, 87, 316

  -- erythrosporus, 53

  -- fluorescens liquefaciens, 43, 53, 64, 86

  -- fluorescens non-liquefaciens, 53, 15

  -- of cholera, 65-69

  -- of diarrhœa, 203, 204

  -- of influenza, 315

  -- lactis erythrogenes, 193

  -- lactis pituitosi, 193

  -- lactis viscosus, 193

  -- liquefaciens, 53, 151

  -- of leprosy, 308-313

  -- of glanders (mallei), 299, 319

  -- mesentericus, 86, 151

  -- mycoides, 151

  -- of malignant œdema, 19, 172

  -- No. 41, 219

  -- pasteurianum, 130

  -- of scarlet fever, 202

  -- of symptomatic anthrax, 19, 171, 172

  -- of plague, 306-308

  Bacillus prodigiosus, 34, 151, 193, 238

  -- pyocyaneus, 7, 34, 64, 110, 299

  -- pyogenes fœtidus, 34

  -- radicicola, 164

  -- saponacei, 193

  -- subtilis, 31, 86, 108

  -- synxanthus, 194

  -- of tetanus, 19, 168-171

  -- termo, 53

  -- of tubercle, 110, 212, 225, 274-291

  -- typhosus, 41, 50, 55-62, 212

  -- ubiquitous, 53

  -- of yellow fever, 316

  Bacteria, action of, 26

  -- in sewage, 84

  -- and wheat supply, 161

  -- and fixation of nitrogen, 160-166

  -- in cheese-making, 220-227

  -- in the dairy, 215-227

  -- products of, 240, 241

  -- and disease, 264-321

  -- the higher, 11, 33

  -- in soil, 137-177

  Bacterial action, 26

  -- treatment of sewage, 90

  Bacteroids, 166

  Beer diseases, 134

  Berkefeld filter, 52

  Biogenesis, 3

  Biology of bacteria, 1-36

  Bitter fermentation, 191

  Blood serum, 22

  Blue milk, 193

  Boracic acid, 205, 331

  Bread, bacteria in, 238

  Broth, 21

  Brownian movement, 14

  Bubonic plague, 306-308

  Buchner's tube, 141

  Butter, bacteria in, 213, 214

  -- examination of, 214

  -- bacterial flavouring of, 215

  Butyric fermentation, 115, 132, 191


  Carbol-fuchsin, 44

  Carbol-gelatine, 62

  Carbolic acid as a germicide, 332

  Caries, dental, 104

  Chamber, moist, 41

  Channels of infection in disease, 269

  -- in tubercle, 289

  Cheese, bacteria in, 220

  Chemical products of bacteria, 241

  Chemical substances as disinfectants, 329

  -- and bacteriological examination of water compared, 51

  -- tests for nitrification, 158

  Chemiotaxis, 15, 248

  Chicken cholera, 320

  Chinosol as a disinfectant, 336

  Chloride of lime as a germicide, 331

  Cholera, 65-68

  -- diagnosis of, 68

  -- and filtration, 75

  -- and milk, 200

  Chromogenic bacteria, 193, 241

  Cladothrix, 8, 33

  Clark's process, 73

  Classification, 7

  Coccus, definition of, 8

  Colon bacillus, _see_ B. coli communis

  Comma bacillus, 66

  Commensalism, 162

  Composition of bacteria, 12-14

  Conditions affecting bacteria in water, 70

  -- in milk, 186, 187

  Contagion, 270

  Corrosive sublimate as disinfectant, 331

  Counter (Wolfhügel), 49

  Cover-glass preparations, 44

  Cream, bacteria in, 213

  Crenothrix polyspora, 53

  Creosol as a germicide, 332

  Cultivation beds, 92

  Culture media, 20

  -- anaërobic, 139

  -- hanging drop, 44

  -- plate, 40-43

  -- pure, 20, 46

  -- shake, 62


  Decomposition bacteria, 149

  Denitrifying bacteria, 143, 149

  Dental caries, 104

  Deodorants, 323

  Desiccation, 26

  Diagnosis, 339

  Diarrhœa of infants, 175, 316

  Diphtheria, 243-245, 289-296

  -- bacillus of, 289

  -- toxins of, 244, 293

  -- and milk supply, 201

  -- and school influence, 294, 295

  -- pseudo-bacillus of, 296

  Diplococcus, definition of, 8

  Diplococcus of gonorrhœa, 300

  -- in pneumonia, 313

  Directions for estimating disinfectants, etc., 324

  Disease, production of, 264

  Diseases of beer, 134

  -- of plants, 35

  -- of animals, 316-320

  -- conveyed by water, 81

  -- and soil, 173-177

  Disinfectants, 322, 331

  Disinfection, 322-336

  -- of a room, 335

  -- of walls, 335

  -- of bedding, 335

  -- of garments, 335

  -- of excreta, 335

  -- of wounds, 336

  -- of hands, 336

  Domestic purification of water, 79

  Dunham's solution, 69

  Dysentery, 319


  Earth temperatures and disease, 175

  Economic bacteria, 145

  Egg cultures, 340

  Elsner's medium, 62

  Endospores, 17

  Enteric fever, _see_ Typhoid

  Enzymes, 114

  Equifex disinfector, 328

  -- sprayer, 334

  Erysipelas, 299

  Examination, bacteriological--

  -- air, 96-99

  -- cholera, 68

  -- diphtheria, 290, 340

  -- leprosy, 309

  -- meat, 234

  -- milk, 227

  -- sewage, 80

  -- soil, 138

  -- tetanus, 170

  -- tubercle, 276, 339

  -- water, 43-48

  -- yeasts, 119, _et seq._

  Extracellular poisons, 244, 273


  Fermentation, 111-136

  -- acetous, 115, 127

  -- alcoholic, 115, 117

  -- ammoniacal, 115, _see_ under Soil

  -- butyric, 115, 132, 191

  -- lactic acid, 115, 130, 190

  Ferments, organised, 114, 115

  Ferments, unorganised, 114, 115

  -- chromogenic, 193

  -- curdling, 191

  -- bitter, 191

  -- slimy, 192

  -- soapy, 193

  Films, 123

  Filter, domestic, 79

  Filter-beds, 74

  Filtration, milk, 206

  -- method of air examination, 99

  -- sand, 77-79

  Fission, 16

  Fixing specimens, 45

  Flagella, 15

  -- staining, 63

  Food, bacteria in, 179, 180

  Foot-and-mouth disease, 320

  Formaldehyde and formalin, 205, 206, 333

  Forms of bacteria, 8

  Fränkel's tube, 140

  -- pneumococcus, 314

  Friedländer's pneumo-bacillus, 315


  Gas, production of, 62, 241

  Gathering-ground, 38

  Gelatine, 21

  -- carbol, 62

  -- liquefaction of, 44

  Gemmation, 119

  Gentian-violet, aniline, 44

  Germicidal temperatures, 30, 207

  Germicides, 322, 331

  Glanders, 319

  Gonorrhœa, 300

  Gram's method, 44, 338

  Gravity, influence on bacteria, 106

  Gypsum block, 121


  Hæmatozoa, 320

  Hanging drop cultivations, 44

  Hansen's method of dilution, 123

  Heat as steriliser, 30, 326

  Heredity, 268, 269

  Hesse's method of air examination, 98

  Higher bacteria, 11

  High yeasts, 125

  Hot air steriliser, 31

  Hydrogen cultivation, 139

  Hydrophobia, treatment of, 253


  Ice-cream, bacteria in, 236-238

  -- examination of, 236

  Immunity, 240-263

  -- acquired, 247, 250

  -- active, 250

  -- artificial, 250

  -- natural, 250

  -- passive, 250

  Incubation period, 271

  Incubators, 22

  Indol, formation of, 61

  -- testing for, 61

  Industries and bacteria, 135

  Influenza, 315

  Intracellular poisons, 34

  Inversive ferments, 115

  Involution forms, 12, 66


  Kipp's apparatus for producing hydrogen, 27, 139

  Klebs-Löffler bacillus, 289

  Koch's plate method, 40

  -- postulates, 266

  -- comma bacillus, 66

  -- bacillus of tubercle, 276


  Lactic acid fermentation, 115, 130, 185, 190, 221, 226

  Lactose, 190

  Leguminosæ, fixation of nitrogen by, 163

  Leprosy, 308-313

  Leptothrix, 8, 33

  Leuconostoc, 17

  Light, influence upon bacteria, 24-26, 70

  Liquefaction of gelatine, 44, 241

  Low yeasts, 125

  Lymph, glycerinated calf, 252

  Lyon's, Washington, disinfector, 328


  Maceration industries, 135

  Malaria, 177, 320

  Malignant œdema, 19, 172

  Mallein, 319

  Mastitis, 184

  Measles, 321

  Meat, 234

  Media, culture, 20

  Merismopedia, 11

  Method of examination, 43-47

  Metropolitan water supply, 72

  Miasmatic diseases, 176

  Micrococcus, definition of, 8

  -- agilis, 16

  -- aquatilis, 53

  -- casei amari, 226

  Micrococcus Freudenreichii, 192

  -- gonorrhœæ, 299

  -- tetragonus, 299

  -- viscosus, 192

  Milk, bacteriology of, 178-213

  -- absorptivity of, 180

  -- sources of pollution, 181-184

  -- number of bacteria in, 185

  -- influence of temperature upon, 186

  -- influence of time of standing, 187

  -- fermentation bacteria in, 189

  -- constitution of, 189-195

  -- disease-producing power of, 195

  -- and tuberculosis, 197-199, 228, 290

  -- and typhoid, 199

  -- and cholera, 200

  -- and diphtheria, 201

  -- and scarlet fever, 202

  -- and thrush, 203

  -- methods of preservation of, 205

  -- and added antiseptics, 205, 206

  -- filtration of, 206

  -- sterilisation of, 207

  -- pasteurisation of, 208-213

  -- products, bacteria in, 213-227

  -- examination of, 227

  -- and economic bacteria, 215-220

  -- sterile, 181

  -- kinds of bacteria in, 188

  -- chromogenic, 193

  -- cooling processes, 207, 209

  Moist chamber, 41

  Moisture necessary for bacteria, 23

  Motility, 14

  Moulds, 116

  Mycoderma aceti, 127


  Nasal passages, bacteria in, 103

  Natural purification of water, 69

  Needles, platinum, 22, 24

  Nitrates, 147, 154

  Nitric organism, 157, 158

  Nitrification, 76, 143-159

  -- chemistry of, 144-148

  -- stages in, 145

  -- bacteria of, 152

  Nitrifying organisms, cultivation of, 156

  Nitrogen, fixation of, 144, 160-168

  Nitrous organism, 154, 158

  Nodules on roots, bacteria in, 163


  Oidium albicans, 203

  Oxygen necessary for bacteria, 26

  Oysters and bacteria, 229-234


  Paraform for disinfection, 334

  Parasitism, 27, 162

  Parietti's method, 62

  Pasteurisation of milk, 208-213

  Pasteur's treatment of rabies, 253-258

  Perlsucht, 283

  Petri dishes, 50

  Phagocytosis, 247

  Phosphorescence, 26, 241

  Pigment, formation of, 241

  Place of bacteria in nature, 5

  Plague, 306-308

  Plant diseases, 35

  Plate cultures, 40-46

  Platinum needles, 22, 24

  Pleomorphism, 12

  Pneumo-bacillus, 315

  Pneumococcus, 313

  Pneumonia, 313

  Polymorphism, 12

  Postulates, Koch's, 266

  Potato medium, 22

  Pouchet's aëroscope, 96

  Proteolytic ferments, 115

  Proteus family, 86, 180, 297

  -- vulgaris, 60, 86, 151, 194

  -- zenkeri, 60

  Pseudo-diphtheria bacillus, 296

  Ptomaines, 179

  Pure culture, 20, 46

  Purification of water--

  -- natural, 69

  -- artificial, 73

  Pus, 296

  Putrefaction, 143-149

  Pyocyanin, 299

  Pyoxanthose, 299


  Quantitative standard for water bacteria, 48, 49

  -- air bacteria, 107

  -- milk bacteria, 188

  -- soil bacteria, 137

  Quarter evil, 19, 171


  Rabies, treatment of, 253

  -- forms of, 253

  -- pathology of, 254

  -- results of treatment, 256

  Reck's disinfector, 328

  Reproduction of bacteria, methods of, 16

  Retting, 135

  Rinderpest, 320


  Saccharomycetes, biology of, 119-121

  -- methods of examination, 122

  -- anomalous, 122

  -- apiculatus, 127

  -- aquifolii, 127

  -- cerevisiæ, 117, 124, 126

  -- conglomeratus, 126

  -- ellipsoideus I., 126

  -- ellipsoideus II., 126, 134

  -- exiguus, 127

  -- Hansenii, 127

  -- illicis, 127

  -- Ludwigii, 122

  -- mycoderma, 127

  -- pastorianus I., 127, 134

  -- pastorianus II., 127

  -- pastorianus III., 127, 134

  -- pyriformis, 127

  Salicylic acid as antiseptic, 205, 206

  Saprophytes, 27, 166

  Sarcina, 10

  Scarlet fever, 202, 321

  Sedgwick's method of air analysis, 99

  Sedimentation, 71, 73

  Septic processes, 296

  -- tank, 90

  Sewage, organisms in, 84

  -- bacterial treatment of, 89

  Sewer air, 87

  -- and toxicity of bacteria, 105

  Shake culture, 62

  Shell-fish and bacteria, 229, 234

  Small-pox, 251, 321

  Soil, bacteriology of, 137

  -- examination of, 138

  -- kinds of bacteria in, 142-145

  -- and its relation to disease, 173, 176, 177

  Species of bacteria, 29

  Spirillum, definition of, 11

  -- of cholera, 66

  Spontaneous generation, 2

  Spores, kinds of, 17-19

  -- resistance of, 19, 278

  -- staining of, 19

  -- of yeasts, 122

  Staining methods, 45

  Staphylococcus, 10, 296

  -- cereus albus, 297

  -- pyogenes aureus, 88, 108, 297

  Steam as a disinfector, 326

  -- disinfectors, 327-329

  -- steriliser, 326

  -- saturated, 327

  -- superheated, 327

  Steam current, 327

  Sterilisation, 29-31

  -- methods of, 30, 31

  Streptococcus, 9, 297

  -- pyogenes, 299

  -- Hollandicus, 192

  Streptothrix, 317

  Structure of bacteria, 8

  Sulphurous acid as a germicide, 332

  Suppuration, 296-301

  Swine fever, 320

  Symbiosis, 162

  Symptomatic anthrax, 171


  Table of economic bacteria in soil, 145

  Temperature, influence of, on bacteria, 23

  Tetanus, 19, 168, 245

  -- toxin of, 168

  Thresh's disinfector, 328

  Thrush, 203

  Tissues, effect of, on bacteria, 267

  Tobacco-curing, 136

  Toxins, 28, 241-247, 272

  Tuberculin, 281

  Tuberculosis, 274-292

  -- pathology of, 274

  -- varieties of, 275

  -- history of, 276

  -- conveyed by the air, 104

  -- and the milk supply, 196-198, 290

  -- giant cells in, 275

  -- bacillus of, 276

  -- cultivation of bacillus of, 277

  -- spores of, 277

  -- relation of bacillus to disease, 279

  -- toxins of, 281

  -- of animals, 283

  -- prevention of, 286-292

  -- disinfection in cases of, 292

  -- decline of, 288

  -- and overcrowding, 288

  -- channels of infection in, 289

  -- expectoration in, 290

  Tuberculosis and house influence, 291

  -- and glanders, 319

  Typhoid fever, 56

  -- bacillus of, 55-58

  -- micro-pathology, 57

  -- bacillus compared with B. coli, 58

  -- bacillus in sewage, 59

  -- bacillus in drinking water, 60

  -- tests for bacillus of, 61-64

  -- and soil, 173, 176

  -- conveyed by the air, 105

  -- and milk supply, 199

  Tyrotoxicon, 205, 237


  Unit of antitoxin, 263

  Urea, 148


  Vaccination, 251-253

  Vaccines, plague, 257, 259

  -- cholera, 257

  -- small-pox, 251

  Vaccinia, 251

  Variolation, 250, 251

  Virulence increased, 32

  -- diminished, 36


  Water, bacteria in, 37-84

  -- number of bacteria in, 38

  -- examination of, 39-52

  -- disease organisms found in, 55

  -- natural purification of, 69

  -- artificial purification of, 73

  -- filtration of, 74

  -- domestic purification of, 79

  -- pollution of, 82

  Wheat supply and bacteria, 161

  Widal reaction, 63, 340

  Wooden tongue, 318

  Wool-sorters' disease, 305


  Yeasts, 116

  Yellow fever, 316


  Ziehl-Neelsen stain, 44, 340


  [Illustration]

  The Science Series


  Edited by Professor J. MCKEEN CATTELL, Columbia University,
  with the cooperation of FRANK EVERS BEDDARD,
  F.R.S., in Great Britain.

  Each volume of the series will treat some department of
  science with reference to the most recent advances, and will
  be contributed by an author of acknowledged authority.
  Every effort will be made to maintain the standard set by the
  first volumes, until the series shall represent the more important
  aspects of contemporary science. The advance of
  science has been so rapid, and its place in modern life has
  become so dominant, that it is needful to revise continually
  the statement of its results, and to put these in a form that is
  intelligible and attractive. The man of science can himself
  be a specialist in one department only, yet it is necessary for
  him to keep abreast of scientific progress in many directions.
  The results of modern science are of use in nearly every profession
  and calling, and are an essential part of modern
  education and culture. A series of scientific books, such as
  has been planned, should be assured of a wide circulation,
  and should contribute greatly to the advance and diffusion of
  scientific knowledge.

  The volumes will be in octavo form, and will be fully illustrated
  in so far as the subject-matter calls for illustrations.


  G. P. PUTNAM'S SONS, NEW YORK & LONDON

  THE SCIENCE SERIES

  (Volumes ready, in press, and in preparation.)

  +The Study of Man.+ By Professor A. C. HADDON, M.A., D.Sc., Royal
  College of Science, Dublin. Illustrated. 8^o, $2.00

  +The Groundwork of Science.+ A Study of Epistemology. By ST.
  GEORGE MIVART, F.R.S. 8^o, $1.75

  +Rivers of North America.+ A Reading Lesson for Students of Geography
  and Geology. By ISRAEL C. RUSSELL, LL.D., Professor of Geology
  in the University of Michigan. Illustrated. 8^o, $2.00

  +Earth Sculpture.+ By Professor JAMES GEIKIE, F.R.S., University of
  Edinburgh. Illustrated. 8^o, $2.00

  +Volcanoes.+ By T. G. BONNEY, F.R.S., University College, London.
  Illustrated. 8^o, $2.00

  +Bacteria.+ By G. NEWMAN, M.D., F.R.S., Demonstrator of Bacteriology
  in King's College, London. Illustrated. 8^o.

  +The Stars.+ By Professor SIMON NEWCOMB, U.S.N., Nautical Almanac
  Office, and Johns Hopkins University.

  +Meteors and Comets.+ By Professor C. A. YOUNG, Princeton University.

  +The Measurement of the Earth.+ By Professor T. C. MENDENHALL,
  Worcester Polytechnic Institute, formerly Superintendent of the U. S.
  Coast and Geodetic Survey.

  +Earthquakes.+ By Major C. E. DUTTON, U.S.A.

  +Physiography; The Forms of the Land.+ By Professor W. M. DAVIS,
  Harvard University.

  +The History of Science.+ By C. S. PEIRCE.

  +General Ethnography.+ By Professor DANIEL G. BRINTON, University
  of Pennsylvania.

  +Recent Theories of Evolution.+ By J. MARK BALDWIN, Princeton
  University.

  +Whales.+ By F. E. BEDDARD, F.R.S., Zoölogical Society, London.

  +The Reproduction of Living Beings.+ By Professor MARCUS HARTOG,
  Queen's College, Cork.

  +Man and the Higher Apes.+ By Dr. A. KEITH, F.R.C.S.

  +Heredity.+ By J. ARTHUR THOMPSON, School of Medicine, Edinburgh.

  +Life Areas of North America: A Study in the Distribution of
  Animals and Plants.+ By Dr. C. HART MERRIAM, Chief of the Biological
  Survey, U. S. Department of Agriculture.

  +Age, Growth, Sex, and Death.+ By Professor CHARLES S. MINOT,
  Harvard Medical School.

  +Bacteria.+ Dr. J. H. GLADSTONE.

  +History of Botany.+ Professor A. H. GREEN.

  +Planetary Motion.+ G. W. HILL.

  +Infection and Immunity.+ GEO. M. STERNBERG, Surgeon-General U.S.A.