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  pepsine → pepsin
  bibliographic xref (9) → (19)

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  Casenin – Caseuin
  Kentniss – Kenntnis – Kenntniss
  resistant – resistent
  c.c. – c.cm.
  ureaæ – ureæ
  ae – æ




                                  THE
                      PUERING, BATING & DRENCHING
                               OF SKINS




    _Scientia et potentia humana in idem coincidunt, quia ignoratio
    causæ destituit effectum. Natura enim non nisi parendo vincitur: et
    quod in contemplatione instar causæ est, id in operatione instar
    regulæ est._


    Knowledge and human power are synonymous, since the ignorance of
    the cause frustrates the effect; for nature is only subdued by
    submission, and that, which in contemplative philosophy corresponds
    with the cause, in practical science becomes the rule.--BACON,
    _Aphorism III_.




                                  THE
                      PUERING, BATING & DRENCHING
                               OF SKINS


                                  BY

                          JOSEPH TURNEY WOOD

           DIRECTOR OF TURNEY BROTHERS, LIMITED, NOTTINGHAM;
  EXAMINER IN LEATHER TANNING, DRESSING OF SKINS, AND LEATHER DYEING
              TO THE CITY AND GUILDS OF LONDON INSTITUTE;
          MEMBER OF THE INTERNATIONAL ASSOCIATION OF LEATHER
                         TRADES CHEMISTS, ETC.


                           33 ILLUSTRATIONS


                            [Illustration]


                                London
                  E. & F. N. SPON, Ltd., 57 HAYMARKET

                               New York
                SPON & CHAMBERLAIN, 123 LIBERTY STREET

                                 1912




                               DEDICATED
                                  TO
                            SIR JOHN TURNEY

                              THE VETERAN
                                OF THE
                        LIGHT LEATHER INDUSTRY
                                  IN
                        GRATEFUL ACKNOWLEDGMENT




CONTENTS


                                                                    PAGE

        LIST OF ILLUSTRATIONS                                         ix

        PREFACE                                                       xi

     I. DESCRIPTION OF THE PUERING AND BATING PROCESS                  1

    II. THE CHEMISTRY OF BATING                                       23

   III. THE PHYSICS OF BATING                                         55

    IV. THE BACTERIOLOGY OF THE BATE                                  87

     V. ACTION OF ENZYMES                                            127

    VI. ORIGINAL PAPERS ON BATING                                    147

   VII. ARTIFICIAL BATES                                             178

  VIII. PATENTS                                                      193

    IX. DRENCHING                                                    233

     X. ORIGINAL PAPERS ON DRENCHING                                 246

    XI. BIBLIOGRAPHY                                                 282

        CONCLUSION                                                   292

        INDEX                                                        295




ILLUSTRATIONS


  FIG.                                                              PAGE

   1. Curve showing removal of lime by washing                         6

   2. Cubical “truck” for measuring skin                     _facing_  8

   3. Puer wheel                                                "     13

   4. Front elevation of puer wheel                                   13

   5. End elevation and section of puer wheel                         14

   6. Sir John Turney’s scudding machine                     _facing_ 17

   7. Curves of ash contents during puering                           38

   8. Diagram of volumenometer                                        62

   9.    "       connexions of electrometric apparatus                76

  10. Electrometric apparatus                                         78

  11. Apparatus for measuring degree of falling                       83

  12. Curves obtained by measuring apparatus                      85, 86

  13. Improved apparatus for measuring degree of falling              86

  14. Various forms of bacteria in puer liquors              _facing_ 88

  15. _B. coli commune_                                         "     88

  16. _B. erodiens_ (Becker)                                    "     97

  17. Plate culture from fresh puer                             "     97

  18.   "     "     puer wheel                                  "    103

  19. _B. putrificus_                                           "    103

  20. Organisms in pigeon dung, × 1000                               104

  21. Pure culture of bacillus (_d_) from sweated skin      _facing_ 106

  22.  "      "          "     (_e_)  "          "              "    106

  23. _B. butyricus_ (Hueppe)                                   "    112

  24. _Spirillum volutans_ (Kutscher), stained to show flagellæ "    112

  25.      "          "          "      unstained preparation   "    112

  26. Curves showing rate of hydrolysis                              129

  27. Organisms in bran drench, × 1000                               240

  28. Chains of bran bacteria, × 1000                                240

  29. Bran fermentation, advanced stage                              241

  30. _B. furfuris_, α                                      _facing_ 269

  31. _B. furfuris_, β                                          "    269

  32. Cultures of α in glucose gelatin                               268

  33. Gas curve (_B. furfuris_)                                      277




PREFACE


The present volume is the outcome of a desire to preserve the numerous
notes which I have made during over twenty years’ work at the practical
and scientific study of bating. It has been my wish to complete the
investigation of this important process in leather manufacture, for,
as Lord Allerton has paradoxically remarked:[1] “Good leather is
made before the skins go into the tan liquor at all,” but owing to
circumstances having drawn me more and more to the commercial side of
the business, I have been compelled to abandon this project.

  [1] Report of First Conference of Leather Trade Chemists, 1897,
  p. 146.

When learning the trade as an apprentice every fault in the leather was
attributed to this part of the work, and the troubles and miseries of
the “puer shop” first caused me to take up the study of puering. I was
determined to know the causes underlying the process. Puering is not
only a filthy and disgusting operation, but is prejudicial to health,
and in the nature of it is attended by more worry and trouble than all
the rest of the processes in leather making put together.

By giving a résumé of the work done up to the present time, I hope to
be of assistance to the younger generation of Tanning Chemists, to some
of whom is reserved the privilege of carrying on the work.

I think it may now be said, at any rate, that the solution of the
problem of constructing an artificial bate on scientific principles,
which will replace the present crude methods, is well within sight.
The principal obstacles are, on the one hand, the inertia of English
manufacturers; on the other hand, the class of labour employed in
puering is not of the highest order of intelligence. Innovations in
most things are resisted, partly because they necessitate changes in
the method of working, and partly because of the innate conservatism
of human nature. It is certainly a significant fact that although
most of the pioneer work on this subject was done in England, the
practical side has been taken up in Germany, and by freely spending
money on large trials in the works they have enabled the manufacture of
artificial bates to be developed on a commercial scale.

In 1886, while studying Chemistry under Professor Frank Clowes, I
began to examine microscopically the various liquors of a light
leather factory, and more especially the bran drenches. At that time
I knew nothing of bacteriology, for the simple reason that little but
pathological work in this line was being done in England. Through
the kindness of Professor Clowes, I obtained an introduction to Mr.
Adrian Brown (now professor at the University of Birmingham), and in
his laboratory at Burton-on-Trent I saw the first pure cultivations
of _Bacterium Aceti_ which he had isolated, and of which he had
completely studied the chemical action.[2] I had there an opportunity
of seeing the methods and apparatus employed. I also had the benefit
of Mr. Brown’s advice in commencing a systematic study of the process
of drenching. Professor Percy Frankland, then at Dundee, advised me
further as to the microscope and other matters. My warmest thanks are
due to these friends for directing me in the right way.

  [2] Jour. Chem. Soc., Mar. 1886.

As a result of my first investigations, on December 11, 1889, I read a
short paper entitled “Methods of Bacteriological Research--with some
account of Bran Fermentation,” before the Society of Chemical Industry.

The way in which this paper was received led to a further research into
the nature of bran fermentation in conjunction with Mr. W. H. Willcox,
B.Sc. (now Senior Analyst to the Home Office), by which the action of
the bran drench was thoroughly investigated, and the results published
in the Journal of the Society of Chemical Industry, May 31, 1893.

This was followed, on June 30, 1897, by a paper “On a Pure Cultivation
of a Bacillus Fermenting Bran Infusions,” also in conjunction with Dr.
Willcox.

In 1898, in reply to a publication of Director Eitner, of Vienna, I
published in the Leather Trades’ Review (November 15), a résumé of the
whole subject, entitled “The Rationale of Drenching.”

Already, in the first paper above named (“Methods of Bacteriological
Research”), I had called attention to the bacteria of the bate prepared
from dogs’ dung, and in a paper entitled “Fermentation in the Leather
Industry,”[3] developed this aspect of the subject, and first pointed
out the influence of enzymes in bating. I therefore decided to study
the phenomena occurring in the bate in the same way as I had studied
drenching.

  [3] Jour. Soc. Chem. Ind., 1894. p. 218.

The work was begun in 1895, and, as it was likely to occupy an
indefinite time, the first instalment, entitled “Notes on the
Constitution and Mode of Action of the Dung Bate in Leather
Manufacture,” was published November 30, 1898; while “Further Notes on
the Action of the Dung Bate” was published on November 30, 1899.

In these papers I indicated the lines on which a culture of bacteria
might be practically applied to the bating of skins, and gave the
composition of a liquid which, while acting as a nutrient medium for
the bacteria, contained at the same time most of the active chemical
compounds of dog dung.

Meanwhile, Dr. Popp and Dr. Becker, in Frankfort a/M, were
investigating independently the bacteria of dog dung, and conceived the
idea of employing them commercially. My dear friend Franz Kathreiner,
of Worms,[4] put me in communication with these gentlemen, and we
were thus enabled to work in conjunction. As a result of our combined
labours, an artificial bate, called “Erodin,” was put upon the market.
This will be fully treated of in the chapter on Artificial Bates.

  [4] Died, April 1905.

I shall give first a short account of bating, and then sum up as
briefly as possible the present state of our knowledge of the process,
afterwards giving an account of the more important of the various
patents which have been taken out for artificial bates.

Although the book is divided into separate sections for convenience,
it is obvious that we cannot separate chemistry from physics, nor
bacteriology from chemistry, nor enzyme action from all three.

My own papers are printed as read. The Bibliography does not profess to
be complete, but includes most of the works consulted.

No one realizes more than I how incomplete the work is, and how much
research still remains to be done in order to complete it.

Thanks to the efforts of the Leather Industries Department of the
University of Leeds, and the Technical College of the Leather Sellers’
Company, Bermondsey, the era of Rule of Thumb is passing, and there is
little doubt that the work that is being done in these institutions
will be translated into practical use in the factories by the coming
generation.

I wish to express my special thanks to Mr. DOUGLAS J. LAW, and to Dr.
H. J. S. SAND for assistance in preparing the notes for publication,
to Dr. J. GORDON PARKER, Director of the Leather Sellers’ Technical
College, London, for the description of the bating of hides, and to
PROFESSOR KRÀL, of Prague, for some of the photographs of bacteria.

    JOSEPH T. WOOD.

    Nottingham: _January 1912_.




PUERING, BATING, AND DRENCHING OF SKINS




CHAPTER I.

THE PUERING AND BATING PROCESS.


    “Beizen sind Stoffe die mit dem Kalk nicht nur ein chemische
    Verbindung einzugehen im Stande sind, wodurch derselbe löslich und
    somit vollständig unschädlich wird, sondern auch den gegenseitigen
    festen Schluss der einzelnen Wandungen der Zellenelemente
    mechanisch lockern, den ganzen Bau der Haut in sich nachgiebiger
    machen und so die Verschiebbarkeit der einzelnen Hautgebilde
    erhöhen.”--J. C. H. LIETZMANN, 1862.

    “Bates are materials which are not only able to enter into chemical
    combinations with the lime, whereby it becomes soluble, and is
    thereby rendered completely harmless, but they also mechanically
    loosen the cementing substance of the separate cell elements, and
    render the whole structure of the hide more pliable, and thus
    increase the mobility of the various parts.”

The object of bating or puering is to render the skins, and the
resulting leather, soft and supple. Skins which have undergone the
liming process, must be thoroughly freed from lime before going into
the tan liquors, and, for light and soft leathers, they must be reduced
or “brought down,” so that the elasticity or resilience of the skin
fibres is got rid of, and the skin, when tanned, can be stretched
without springing back. This is usually done in the case of light
leathers, by passing the skins through a bate or puer, composed of an
infusion of dogs’ dung in water at a temperature of 35° to 40° C.,
until the required result is obtained. This condition is known to the
workman by the feel of the skin. A good indication is, that the skin
when “down” retains the impression of the thumb and finger if squeezed.
A properly puered skin, when dropped on the floor, will also be
perfectly flaccid, the folds lying closely together. It may, however,
be said that it is only by experience and a kind of instinct that the
exactly correct condition of the skin can be judged.

I know of no very early works on leather manufacture giving an account
of bating. It was a “secret process,” and the results obtained depended
almost entirely upon the judgment of the operator; and this judgment
was frequently in error, owing to the fact that he did not understand
what took place in the bate.

The earliest account I have been able to find is in a book[5] in the
possession of Mr. Seymour-Jones, of Wrexham, entitled “The Art of
Tanning and Currying Leather, with an Account of all the Different
Processes made use of in Europe and Asia for Dying Leather Red and
Yellow, Collected and Published at the Expense of the Dublin Society,
to which are added Mr. Philippo’s Method of Dying the Turkey Leather as
approved of by the Society for the Encouragement of Arts, etc., and for
which he had a Reward of £100, and their Gold Medal, for the Secret.
Also the New Method of Tanning invented by the late David Macbride,
M.D., London. Reprinted for J. Nourse, on the Strand, Bookseller to His
Majesty, 1780.”

  [5] There is a copy in the British Museum and also in the Patent
  Office Library.

In the chapter entitled “Alumed Calf Skins for Bookbinding” (p. 138),
after the limed skins have been fleshed, the writer continues--

“To alum them, put into a large vat, three or four pails of dogs’ turd
(this dogs’ turd is called alum); on this they fling a large pail of
water to dilute it; this done, the workman goes into the vat, and, with
his wooden shoes, tramples it, filling the vat half full of water. The
_alumer_, on his part, pours water out of his boiler into this vat,
mixing it with the cold water, after which he flings in the skins,
stirring them and turning them for some moments with great sticks.”

The work is described pretty much as now practised, the puer tub being
kept at a uniform heat by constantly taking out liquor, heating it, and
returning it. But neither these goods, nor morocco, are put through a
“drench” _after_ the puer, as we do now; they are scudded on the flesh,
and well washed several times in clear water before being tanned.

*Morocco Leather* (p. 204).--The dry skins are soaked three or four
days, “pared” on the beam, and unhaired in weak lime pits one month.
At Nicosia they put the skins into lime, reduced to powder, for twenty
days in summer, or twenty-five to thirty in winter; out of the lime,
the skins are well washed and drained. The author continues--

“The river work finished, the skins are put into the _dogs’ confit, or
mastering_; for every four dozens of skins they add one bucket of dogs’
excrement, containing fourteen or fifteen quarts, which is worked up
with their hands into a kind of pap and well diluted. The skins are
flung in, stirred and worked in the _mastering_ for some minutes, then
turned and left to rest.

“They remain about twelve hours in the mastering, which opens them,
and takes off the rawness, disposes them to relax, fill and ferment.
This excrement, by its alkaline parts, also cleanses them and takes out
the grease, which would hinder them from taking the colour. I shall
hereafter speak of _bran mastering_.

“At Diarbekir, they make use of these masterings in a different way.
Whilst the skins are drying, they fill great hollows made in the
earth, like our lime pits, with dogs’ dung, which is diluted to the
consistence of honey, or of thin pap, in which they soak the skins for
eight days in winter, and three in summer, treading them each day with
the feet. They are taken out of this fecal matter, and well washed with
fresh water, after which another mastering is made with bran diluted
with water, in which the skins are soaked six days in winter, and three
in summer, observing to tread them each day with the feet, the same as
in the dogs’ mastering; they are then taken out, washed in fresh water,
and prepared for dyeing.”

From these primitive methods the majority of tanners, even of the
present day, have departed but little, except that, instead of stirring
the goods and liquor with a stick, a paddle is used, and the process
thus shortened considerably. Before describing the operation of
puering, it will be best to describe the preliminary washing of the
skins, because, although limed skins may be entered direct into the
puer and brought down quite satisfactorily, in this case more puer and
more time are required, hence it is usual to get rid of the bulk of the
lime by washing them in water before putting them into the puer. In
some cases very dilute hydrochloric or other acid is used, in order to
shorten the time of watering.

It is now well known that lime cannot be entirely removed from skins by
washing in water, no matter how long the washing be continued. A limed
skin containing 4·6 per cent. CaO, calculated on the dry weight, was
found to contain after washing--

    -----------+------------------+---------------
      Time of  | Per cent. CaO on | Per cent. lime
      washing  |     dry skin     |    removed
    -----------+------------------+---------------
      1  hour  |       3·05       |      34
      2  hours |       2·20       |      52
      3   "    |       1·75       |      62
      6   "    |       1·55       |      66
      7   "    |       1·55       |      66
     24   "    |       1·50       |      67
    -----------+------------------+---------------

[Illustration: Fig. 1.]

If the washing be continued, a less amount of lime is removed in each
successive period of time, so that it is evident a point is soon
reached at which it becomes a waste of time to continue the washing.
In practice this point is reached in about two hours. The progress
of the washing is best shown by the curve in the diagram (Fig. 1),
in which the ordinates represent the percentage of lime (CaO) in the
dry skin, and the abscissæ time in hours. It will be seen that the
character of the curve is a hyperbola; such a curve only approaches
a line (representing in the example chosen about 1-1/2 per cent. of
lime) asymptotically--in other words, it is impossible to wash out all
the lime except by an infinite number of changes of water, since each
washing removes a less amount than the previous one. The above is a
typical case of the washing of limed grains from the splitting machine
in the manufacture of skivers. These contain from 4 to 5 per cent. of
lime on the dry skin, and, after washing in water in a paddle for six
hours, analysis shows them to contain still about from 1·5 per cent.
to 1·9 per cent. of lime (CaO).[6]

  [6] Some further analyses of grains at Trent Bridge gave the
  following results:--

                                    Water   CaO    CaO on
                                                  dry skin
    Grain from splitting machines    76·3   1·14    4·8
    The same after washing 24 hrs.   86·0   0·22    1·57
    The same after puering            --     --     0·80
    Another grain                    79·7   1·06    5·20
    The same after deliming as   }
      completely as possible with}    --     --     0·45
      HCl                        }
    Skin in natural condition        64·0    --     0·125

  M. C. Lamb has found in sheep grains split for skivers the following
  amounts:--

                                     Ash    CaO  CaO in Ash
    Grain from splitting machine     9·3    3·2     34·8
    The same after washing           4·1    1·5     35·1
    The same after puering           3·1    0·9     29·1
    The same after bran drench       1·7    0·55    32·4

  The figures are all percentages calculated on the dry weight of the
  skin.

  Munro Payne (Tanners’ Year Book, 1905, p. 75) gives the following
  amounts of lime as Ca(OH)_{2} in limed hides, calculated on dry
  weight at 212° F.:--

    Direct from limes                max.  3·859 per cent.
      "      "                       min.  2·836    "
    Limed for buff                         4·621    "
    Limed for tanning                      3·7659   "
    Ditto after bating                     0·689    "
    Calf limed                             2·601    "
    Calf bated                             0·1215   "
    Goat limed                             5·613    "
    Goat bated                             1·268    "

The effect of washing depends on the character of the water (hard or
soft), and also on its temperature. Hard waters should have a small
quantity of clean lime added to them before entering the goods, in
order to remove dissolved CO_{2}, which, by carbonating the lime on
the surface of the skins, renders the grain harsh and the subsequent
puering difficult.

With regard to temperature, it is preferable to employ cold water
until the bulk of the lime is out, since this dissolves more lime than
warm water. 100 c.c. of saturated lime water, at 10° C., contains
0·134 grm. CaO. 100 c.c. at 40° C. contains 0·1119 grm. CaO. Moreover,
a comparatively small rise of temperature causes a considerable
decomposition in a fully limed skin, by which the skin substance is
rendered more soluble, and consequently lost for the purposes of the
tanner. For a fully limed skin the limit of temperature is about 82° F.
(28° C.), whereas a skin free from lime or alkali may be submitted to
a temperature of 120° F. (49° C.) without damage to the fibre. Lamb
prefers a short washing of half an hour in water at 35° to 38° C., for
the reason that the increased temperature causes the goods to become
more flaccid, but this condition is attained at the expense of the loss
of skin substance we have mentioned.

[Illustration: Fig. 2.--Cubical Truck.]

In order to save time and water, the following method is adopted. The
goods are measured by means of a cubical truck on wheels (Fig. 2); it
holds 250 kilos of wet skins (550 lb.). Four of such trucks are placed
in a wash wheel, and a stream of water from a 1-in. pipe turned on. The
goods are run from three-quarters of an hour to one hour; the water is
then stopped, and 4000 c.c. of commercial hydrochloric acid (18° Bé.)
is run in slowly, in a very diluted state, through a perforated lead
pipe. After all the acid has run in, the wheel is run for half an hour,
then water turned on again for half an hour in order to wash away
the calcium chloride produced. During this last washing, hot water is
admitted at the back of the wheel through a perforated pipe, in order
to raise the temperature of the goods, so that they do not enter the
puer wheel in a cold condition, and thus lower the temperature of the
puer liquor. The goods are now ready for puering.

The chemical action of the acid is a very simple one, and is expressed
by the formula--

    Ca(OH)_{2} + 2HCl = CaCl_{2} + 2H_{2}O
        74     + 73·1 = 110·9    +  36[7]

  [7] From the molecular weights it will be seen that 74 grm. of
  calcium hydrate, equivalent to 56 grm. of lime (CaO), require 73 grm.
  of HCl gas for neutralization. This quantity of gas is contained
  in 265 grm. of commercial hydrochloric acid of 18° Bé., or about
  230 c.c.--that is, for 100 grm. CaO, 410 c.c. of 18° Bé. acid are
  required; hence for the 100 kilos wet grain containing 400 grm. CaO,
  1640 c.c. of acid are required for _complete neutralization_. It is
  impracticable to use this amount of acid, for the reasons stated
  above.

It will be noted that the quantity of acid used is only about one-tenth
of that required to neutralize completely the lime in the skin. If a
greater amount of acid be used the skins begin to absorb acid before
the lime in the interior is neutralized; but, provided the lime on the
surface of the skins is removed, that remaining in the interior is
forced out during the course of the bating process (see Chapter III.).
If more acid were used, then it would require to be added so slowly
that time would be wasted unnecessarily in doing the work.

With regard to the use of other acids for deliming, Lamb considers
formic acid preferable to hydrochloric, and states that the removal
of lime is more effectively accomplished by its use. He attributes
a pulling down action to the calcium formate produced, so that less
formic acid is required than would be equivalent to hydrochloric,
merely regarded as a solvent for lime. We shall refer to this point
again in Chapter II. It may be stated here that the author has obtained
better results by the use of a mixture of formic and acetic acid, in
equal proportions, than from either acid alone, but that the cost of
working is considerably greater than with hydrochloric acid.

Lactic acid is in fairly common use as a deliming agent. In practice
1 per cent. lactic acid (50 per cent. strength) calculated on the wet
weight of pelt, is used, added gradually in small quantities. This does
not remove the whole of the lime, but sufficient for the skins to puer
quickly.

Some tanners measure their acid per dozen skins. In a case which came
under my notice, 1200 c.c. 90 per cent. formic acid were being used for
ten dozens medium goat skins at a temperature of 35° C., time 1-1/4
hours.

The following table, due to Professor H. R. Procter,[8] gives the
cost of dissolving 1 lb. of lime at present approximate prices of
the various acids. The dissociation constant K shows their relative
“strength;”[9] the equivalent, Eq., the weight in lb. of 100 per cent.
acid required to dissolve 28 lb. of lime. Ordinary wet limed hides,
unhaired and fleshed, contain only about 4 lb. lime per 1000 lb. wet
weight.

  [8] Tanners’ Year Book, 1911, p. 59.

  [9] See Chapter III.

COST OF ACIDS TO REMOVE 1 LB. LIME.

    ------------+------+-----------+----------+----------+----------
                |      |           |   Cost   | Strength | Cost for
        Acid    |  Eq. |     K     | per cwt. | per cent.| 1 lb. CaO
    ------------+------+-----------+----------+----------+----------
                |      |           |  _s. d._ |          |   _d_.
    Hydrochloric| 36·5 | say 200   |    3 3   |   31·5   |    1·4
    Sulphuric   | 49·0 |    "      |    4 0   |   95·0   |    0·8
    Oxalic      | 63·0 | 0·1       |   30 4   |   99·0   |    8·1
    Formic      | 46·0 | 0·0214    |   35 0   |   87·4   |    7·0
    Lactic      | 90·0 | 0·0138    |   26 0   |   49·7   |   18·0
    Acetic      | 60·0 | 0·0018    |   18 0   |   40·0   |   10·0
    Butyric     | 88·0 | 0·00115   |   21 0   |   82·8   |    8·0
    Boracic     | 62·0 | 0·00000001|   27 0   |   99·0   |    6·5
    ------------+------+-----------+----------+----------+----------

It will be seen from the above table that sulphuric is the cheapest
acid to use; but, owing to the insoluble nature of the calcium
sulphate, it is best to use hydrochloric acid. Procter has suggested
using a mixture of sulphuric acid and common salt in molecular
proportions, so as to avoid the iron which is generally present as an
impurity in commercial hydrochloric acid. The author has tried this,
and found it perfectly satisfactory. For a complete discussion of
chemical deliming, see Procter’s “Principles of Leather Manufacture,”
chapter xiii.

Quite recently butyric acid, which is now being manufactured on a
commercial scale by the process of Dr. Effront (Brussels), has come
into use for deliming.[10] and is likely to become a useful acid, as it
can be manufactured cheaply. According to Parker, it dissolves less
skin substance than formic, acetic or lactic acids, which observation
is confirmed by Thuau. The quantity required per 100 lb. of ordinary
washed sheep skin is about 1/2 lb. of the 80 per cent. acid. It is used
in exactly the same way as other acids, i.e. it should be added in
small quantities, suitably diluted, at successive intervals of time.

  [10] See also L’Acide Butyrique dans la Tannerie, Urbain J. Thuau, Le
  Cuir, Aug. 1910. Also Collegium, 1910, pp. 347, 363.

Acid salts are also used for deliming, and Procter suggested sodium
bisulphate NaHSO_{4} (“Principles,” p. 155). More recently, sodium
bisulphite (NaHSO_{3}) has been recommended;[11] the skins are tumbled
for half an hour with a dilute solution, which combines with the lime
according to the following equation--

    (1) Ca(OH)_{2} + NaHSO_{3} = CaSO_{3} + NaOH + H_{2}O

if sulphides are present the reaction is--

    (2) Na_{2}S + 2NaHSO_{3} = H_{2}S + 2 Na_{2}SO_{3}

  [11] Le déchaulage des Peaux en tripe, Ettore Guisiana (Turin), Coll.
  1910, p. 14.

After this treatment the theoretical quantity of hydrochloric acid,
diluted with plenty of water, is run in through the axle of the
tumbler, and the skins run another half hour. The reactions are--

    (1) CaSO_{3}     + 2HCl = CaCl_{2} + SO_{2} + H_{2}O

    (2) Na_{2}SO_{3} + 2HCl = 2NaCl    + SO_{2} + H_{2}O

The sulphurous acid evolved in this way slightly swells the skins, and
also removes stains due to the alkaline polysulphides; it is also said
to remove salt stains.

[Illustration: Fig. 3.--Puer Wheel, with Cover Removed.]

[Illustration: Fig. 4.--Elevation of Puer Wheel (back).]

A more interesting process, introduced by Dr. Gr. Eberle[12] as a
bating process, but which is really a deliming process, consists in
using organic acids in the form of their anhydrides, lactones, or
lactides. During the process these anhydrides, lactones, or lactides,
are gradually decomposed with formation of the free acids, which in
a nascent condition unite directly with the lime in the skins. The
anhydrides of acetic, propionic, butyric and lactic acid have been
tried, the lactone of γ-oxybutyric acid

    CH_{2} CH_{2}
                 \
                  O, and the lactide of lactic acid.
                 /
        CH_{2} CO

  [12] Verfahren zum Beizen von Haüten, Ledertechnische Rundschau, No.
  24, 1910. Coll. 1910, p. 372.

After washing and deliming, the goods, warmed to a temperature of 90°
to 100° F., are now measured in the truck above described, and put as
quickly as possible into the bating paddle. This is constructed to
carry two trucks. The figures (Figs. 3, 4 and 5) show a modern paddle
adapted for puering light skins, such as goat, sheep, splits (grains),
either with dung or with an artificial bate.

[Illustration: Fig. 5.--End Elevation of Puer Wheel.]

The inside dimensions are: length, 4 ft. 6 in.; width, 4 ft.; depth,
2 ft. 6 in. A copper pipe of two coils is provided at the back,
through which steam is passed for maintaining the temperature of the
wheel--the goods are prevented from coming in contact with this pipe
by a perforated board. Another steam pipe, jointed so that it may be
turned in and out of the paddle, is provided for heating up the wheel
to the required temperature before beginning the bating. This pipe is
not shown in the drawing. The thermometer can be read while the paddle
is in motion. A suitable speed to keep the goods in motion is twenty
revolutions per minute. The whole wheel is covered in, for the purpose
of preserving the heat, and also to exclude light and air, which are
prejudicial to the action of the bacteria. Such a paddle, or wheel,
has a capacity of 200 gallons liquor, or 900 litres, and will bate 25
dozens of average grains, weighing in the wet state 1100 lb. or 500
kilos.

The dog dung, which is usually obtained from hunting kennels, and of
which the composition is given in Chapter II., is simply diluted with
water in the paddle;[13] five buckets, weighing about 165 lb., are
required for the above quantity of goods.[14]

  [13] When the puer is dirty it should be diluted with water and
  strained through a bag, or the diluted puer may be put into a tub and
  the heavy grit and dirt allowed to fall to the bottom of the vessel.
  Puer from hunting kennels is usually clean enough to use without this
  process.

  [14] The quantities of puer used vary considerably in different
  works. At Trent Bridge, 10 to 15 lb. of wet puer from hunting kennels
  are used per 100 lb. of wet pelt. In a German works, 15 kilos of
  fairly dry dog dung are used per 100 kilos of pelt (Blösse). In an
  American works, two buckets (one bushel) wet dog dung were used to
  puer 10 dozens of 8 lb. calf skins. This is equal to about 62 lb. of
  puer to 100 lb. of skin, which seems an excessive quantity. In an
  Italian works, two kilos dry dung were used to 20 skins of sheep,
  equivalent to about four kilos per 100. In an English tannery,
  100 hides for harness leather required four bushels by measure of
  pigeon dung, costing 1_s._ 6_d._ per bushel (one bushel equals eight
  gallons).

The amount used depends on the state of the goods, the prevailing
weather, and, further, on the quality of the puer.[15] When the
temperature is maintained at 40° C., ordinary limed goods, which have
been washed, come down in from one to three hours.[16]

  [15] A German firm, who supply dry dog and pigeon dung for bating
  purposes, recommend that the material should be spread in a warm room
  at 30° C., and moistened up to 80 per cent. of its weight with an
  infusion of oat straw, and continuously turned over until completely
  softened. Other materials may be used for the fermenting fluid in
  place of the oat straw infusion, such as broth made from waste
  fleshings, whey, or even a simple inorganic solution, such as is used
  for cultivating bacteria, consisting of--

    2 parts potassium phosphate.
    1 part magnesium sulphate.
    1 part calcium nitrate.
    1·5 part calcium chloride.
    100 parts water.

  This procedure is neither more nor less than an application of the
  principles of bacterial cultivation and propagation, shown to be
  practically useful in the bating process by Professor H. Becker, of
  Frankfort, and by the Author in various publications (see Chapter
  VII.).

  [16] There is considerable variation in the length of time required
  to bring down the skins. Lamb states that half an hour is frequently
  sufficient, but at Trent Bridge 2–3 hours are generally required. The
  causes of the variation are not altogether clear, but depend partly
  on the previous history of the goods (fellmongering, liming, etc.),
  and partly on the water used.

During puering, the lime soaps in the skin are decomposed, and the
fat set free--in an estimation of the fat, 0·151 per cent. on the wet
skin was found before puering, and 2·48 per cent. after puering. The
fat thus set free can be removed to a great extent by scudding; but in
modern practice this is not pushed to extremes, as the grain is liable
to be damaged by excessive scudding. The fat is afterwards removed from
the dry leather by means of benzine or other solvent, employed in a
suitable apparatus.

[Illustration: Fig 6.--Sir John Turney’s Scudding Machine for Sheep
Grains.]

*Scudding.*--When the goods are “down,” they are ready for scudding.
This operation in the case of grains is performed either by hand
over the beam, or by means of a special scudding machine.

The scudding machine figured (Fig. 6) was invented by Sir John Turney
in 1880,[17] and is the only type of machine which will safely scud
split skins. It will readily be understood that the usual type of
scudding machine, in which a spiral knife is used, would tear the
delicate structure of the grain. The Turney scudding machine works
with hard, circular brushes, which revolve against a wooden roller
carrying the skin. At the same time a continuous stream of water, at
a temperature of 110° F., is sprayed upon the skins from a perforated
pipe.

  [17] The original Turney scudding machine, was invented in 1871,
  and patented in that year. The specifications are numbered 1351,
  and 3310, 1871. It was also patented in America. Specification No.
  131,480, dated September 17, 1872. Improvements in apparatus for
  cleaning and scouring hides and skins.

After scudding, the skins are washed in cold water for about ten
minutes. This checks the action of the puer, and also washes away any
loose dirt. They are then ready to pass on to the drench (Chapter IX.),
or to the pickling process, or direct to the tan liquor, according to
the kind of leather required.

*The Bating of Hides for Harness or Dressing Purposes.*--The hides
intended for harness leather, or for dressing purposes, are usually
bated with hen or pigeon manure. The exact process varies in different
tanyards, being dependent upon the condition of the pelt prior to
bating, as to whether it is bated directly after fleshing or receives
a preliminary deliming. The process is also dependent upon the amount
of bating action required for the specific purpose prior to tanning,
but the following three methods are in actual use, and may be taken as
typical methods:--

_Harness Backs._--About a hundredweight of hen manure is put into
a suitable tub or vat capable of holding about fifty gallons; some
thirty gallons of water, at a temperature of about 100° F., is poured
on to the manure, and the whole is thoroughly stirred up by means of
a wooden stick or plunger, working the manure about in order to mix
it to a consistence of thin soup. The vat is now stored in a warm
place, so that the temperature is kept about 70° F.--it is customary to
store this in the boiler-house, or some similar warm room. It should
be stirred two or three times a day, until it begins to show signs of
working; two days is generally sufficient for this.

A pit is now prepared, with the necessary amount of water; and, by the
aid of a steam pipe, or other similar means, the temperature of the
water is raised to 70°, and the contents of the bate tub are now added.
This should be poured into the pit through a sack, or a canvas filter,
so as to keep back stones, solids, feathers, and other extraneous
matters. The pit is now thoroughly plunged, and the goods entered;
these should be handled up three to four times a day for two days. At
the end of two days the goods are usually scudded, by working the back
on an ordinary unhairing beam with an unhairing knife. They then go
into a fresh pit containing a new solution of bate made up in the same
manner, and, when sufficiently bated, which generally takes about three
days, the goods are removed from the pit, scudded again by working them
over with a slate scudding tool, when they are ready for tanning. Some
tanners give them a bath of boracic acid prior to their going into
the tanning liquor; this has the effect of materially brightening the
colour. The second pack of harness backs go into the same liquor--which
must be re-warmed to a temperature of 70°--in which they are handled as
before for about two days, and are finished off in a new liquor made as
above described.

It will be understood, therefore, that in a tanyard, working regularly,
the pack of backs go first into an old bate for two days, and are then
finished off in a freshly made one; so that each pack gets two days in
an old pit, and one or more days in the new. The bate in the tub may
also have a second water, or be strengthened.

_Dressing Hides._--For these goods a somewhat stronger bating effect
is required, and pigeon manure is therefore frequently used. The
preparation of the bating liquor is as described above, both as regards
quantity and temperature of the water and time of maceration.

The whole process may be carried out exactly as described for harness
backs, but the following is an illustration of another method in
practical use. Imagine four ordinary bating pits, worked in a round.
The first pit is very old, having had three packs of hides through it;
the second having had two packs, and the third one pack; and the fourth
is made new. The goods, after unhairing and fleshing, and washing, are
put into the first pit quite cold, are handled in this three times on
the first day, and afterwards put into the second pit, the temperature
of the second pit having been previously raised to 65°--the cold pelt
going into this pit reduces the temperature to about 60°. They are
handled in this pit twice, and remain overnight; and the third day they
go into the third pit, the temperature of which is raised to 70° before
the goods go in. They are raised, scudded thoroughly on the grain,
and new liquor is made from new bating liquor prepared as described
above; the goods are now entered, handled at least twice a day, and,
if insufficiently bated, are left overnight. By the next day they will
probably be sufficiently “down” to permit of them going on to the
scudding beam and thence on to tanning.

In the event of any hide being insufficiently bated--in other words,
if the “bone” has not completely disappeared in the inner part of
the hide--they are returned once more for three or four hours, the
temperature of the pit being raised to 70° with a steam coil.

By this system, which is known as a system of “sets,” it will be
noticed that the pits are worked in a round of four; the first pit,
or oldest, being used more or less as a wash pit, the actual bating
taking place in the second, third and fourth pits. No pit has more
than four packs passing through it, and the temperature never rises
above 70°, or, at the outside, 75°. The reason of the comparatively low
temperature is due to the fact that in pit bating, if the temperature
is raised above 75°, the grain of the hide becomes affected before the
internal “bone” is sufficiently soft, which results in a tender grain,
which considerably affects the resulting staining or dyeing which the
leather usually receives before it is put on the market.

_The Third Process_.--This process is suitable for either harness or
dressing hides, and differs from the above-described process only in
the fact that instead of bating the hides in pits, a latticed drum is
used in order to keep the hides in constant motion. This consists of
a large pit, in which a latticed drum revolves two-thirds immersed in
the liquor. The bate liquor is made up as usual, and the temperature
of the liquor raised to 75° or 80° F.; the hides are then placed in
the latticed drum, which revolves at from four to six revolutions per
minute, and, if the goods are put in the drum in the morning, and
if the temperature is kept at about 70° F., the hides are generally
sufficiently bated by evening. They may then be taken out of the drum,
scudded, and left suspended in boracic acid overnight, and can then be
taken to the liquor next morning.

Where there is considerable motion, even the heaviest hides will stand
a temperature of 75°, and even 80°. Some tanners who use the latticed
drum method, do not place their hides in the drum until the afternoon;
they are drummed for about an hour in the cold bate, and left
overnight; next morning the temperature of the bate is raised to 75°,
and the drum started; they are then removed from the drum towards the
afternoon, when sufficiently “down” to admit of them passing forward
into the next process.

The above are three typical methods in common use in England, but many
modifications of these processes exist. Everything depends upon the
amount of lime in the hide, on the number of haulings or handlings
given to the goods, and the temperature or the conditions under which
the hides are bated, so that no hard-and-fast lines can be laid down.
Much also depends upon the method of liming used, prior to the bating.
It must be borne in mind that the bating of hides, and even of kips,
differs materially from the bating of goat and sheep skins, as one has
to deal with double or even treble the substance. If the bate is worked
at a temperature of even 70° or 75°, there is the danger of the grain
being seriously affected before the bate has penetrated to the interior
of the pelt, unless the goods are kept in constant motion.

Instead of a latticed drum, large paddles are used in some factories.
If the pit is large enough the paddle causes both hides and liquor to
revolve, and thus gives the required movement necessary for a regular
and even “bating effect” to be obtained.




CHAPTER II.

THE CHEMISTRY OF BATING.


    “And now, as we cannot understand the _frame_ of a _Watch_, without
    taking it into pieces; so neither can Nature be well _known_,
    without a _resolution_ of it into its _beginnings_, which certainly
    may be best of all done by _Chymical Methods_”--JOS. GLANVILL, 1668.

Professor H. R. PROCTER, in his “Principles of Leather Manufacture,”
1903, p. 153, has given a very complete account of chemical deliming,
and also of the bating and puering processes. Meunier and Vaney, “La
Tannerie,” 1903, give a general review of our knowledge of these
processes up to that date. These accounts are extremely useful, but
treat the subject in a general manner.

I propose to treat of the particular case of puering by means of an
infusion of dogs’ dung in water, as I believe this will give an insight
into all the other bating processes.

So far as the purely chemical action of the bate goes, it consists in
the solution of the lime contained in the skins,[18] thus setting free
a certain portion of skin substance which was combined with the lime,
and the subsequent solution of more or less of this skin substance.

  [18] Limed sheep grains, or Pelts, which have been limed for
  splitting, contain from 3 per cent. to 6 per cent. CaO on the dry
  skin (see footnote, p. 7), and about 80 per cent. of water. In
  a typical case of a fully limed grain, the lime was found to be
  distributed as follows:--

    Free uncombined lime (CaO)       1·7 per cent. on the dry skin
    CaO combined with skin           2·5 "
    CaO as carbonate, or other salts 1·4 "
                                     -----
                                     5·6

  Thus, 75 per cent. of the total lime in the skin was in the caustic
  state. If the skin be cut during the progress of the bating
  operation, and a solution of phenolphthalein applied to the cut
  surface, the course of bating may be followed, and it will be found
  that the caustic lime disappears from the skin in a very short
  period of time, accompanied by the sudden collapse of the fibres. We
  shall treat of this more fully in Chapter III. After puering, the
  lime (CaO) contained in the skin, amounts to 0·5 to 0·9 per cent.,
  calculated on the dry skin. This lime is in a perfectly neutral
  state, and is more or less in combination with the fibre of the skin.
  The exact state in which the lime exists in the skin _after_ puering
  is not known, and would form a very interesting subject for research.

If a fresh puer be made and boiled for half an hour, then allowed to
cool to 95° F., it will be found to remove the lime from the skin
in a very similar way to an ordinary bate, but it has not so rapid
a reducing action as an unboiled bate. In this case both bacteria
and enzymes are destroyed, so that the action may be put down to the
chemical constituents of the bate.

*Chemical Composition of the Puer.*--The mineral constituents of the
fæces, both in dogs and human beings, are well known, owing to the
study of the processes of digestion and nutrition in physiological
laboratories; but the organic constituents are yet little known, and
the sum of the weight of those at present estimated is far from the
total of these matters present.

It is a mistake to suppose that the fæces represent the residue only
of the food taken. Strassburger[19] estimates that bacteria alone
account for nearly one-third of the dry matter. In addition, the
intestinal mucous membrane is an important excretory channel for lime,
magnesia, iron and phosphoric acid, as has been shown by analyses of
fæces from men and dogs, during prolonged fasting.[20] Even when no
nitrogenous food is eaten, the dry fæces always contain from 4 to 8 per
cent. of nitrogen; in a dog fed on meat it amounts to 6·5 per cent.

  [19] Schmidt, and Strassburger, Die Fæces des Menchen (Hirschwald,
  Berlin), 1901.

  [20] Lambling, Précis de Biochimie, 1911, p. 221.

In making a chemical analysis of the fæces, the mineral matter is
estimated in the ash in the usual way for ash of organic substances,
with special precaution, on account of the phosphates present. The
material is first charred, the salts removed by acetic acid; the acetic
acid solution is then decanted, the residue washed with distilled
water, and the combustion then completed. The acetic solution and
washings are added to the final ash, the whole evaporated to dryness,
and gently ignited to decompose the acetates. (Cf. Bull. 46, U.S. Dept.
of Agriculture, Washington, 1899.)

In estimating the fats, the dry puer is ground up with sand, then
extracted with ether in a Soxhlet. The fats are present in four
states:--1, neutral fats; 2, free fatty acids; 3, alkali soaps, soluble
in ether; 4, small quantities of lime and magnesia soaps remaining
in the residue in the Soxhlet. For details as to the separation and
estimation of these, Lewkowitsch’s “Oils, Fats and Waxes” (Macmillan)
should be consulted.

The separation and estimation of the organic constituents in the puer,
is one of the most difficult problems of physiological chemistry, and
to include the methods used would demand a special treatise beyond the
scope of the present volume. Hoppe-Seyler’s “Physiological Chemistry,”
and Allen’s “Commercial Organic Analysis,” vol. iv., may be consulted;
and there is a mass of useful information in Dr. René Gaultiers’
“Précis de Coprologie Clinique” (Paris, Baillière et Fils, 1907).

It is proposed to give here the results of a number of analyses of dog
dung made by the author and others, and to discuss the action of the
various bodies upon the skins. Many more experiments and much research
requires to be done, before the _complete_ action of the bate is clear,
but only by the method of experiment with the various bodies present
can the problem eventually be solved.

Analysis of raw puer from hunting kennels (Quorn) showed the
approximate composition of 1000 grm. of raw puer, containing 150
grm.[21] of dry matter to be as follows:--

  [21] Another sample contained 136 grm. dry matter.

                                                      Grammes

    Sodium chloride and sodium sulphate                 2·1
    Sodium ammonium phosphate                          14·0
    Earthy phosphates, principally Ca_{3}(PO_{4})_{2}  33·6
    Ferric phosphate                                    0·87
    Calcium sulphate                                    1·94
    Silicic acid                                        3·40
    Calcium in solution as CaO                          1·42
    Total P_{2}O_{5} in solution                        4·00
    Non-volatile acids (as lactic)                      3·00
    Volatile acids (as acetic)                          2·20
    Amines (as ammonia)                                 6·20
    Enzymes                                             3·66
    Nitrogenous bodies not included in the above,
        consisting of complex amido-acids, leucin,
        tyrosin, xanthin, and other purine bases,
        indol and skatol                               17·00
    Organic matter, cellulose, etc. mostly insoluble,
        including fats and soaps                       70·00

On the dry matter the amounts extracted by various solvents were:--

                                           Per cent.
    Water                                    24·03
    Alcohol                                  22·27
    CS_{2}                                   15·57
    C_{2}HCl_{3} (Tri-chlorethylene)         14·45
    Petrol. ether                            13·23

No carbohydrates (sugars) capable of reducing Fehling’s solution were
found in the puers examined.

The ash[22] of the dry fæces from dogs fed on a flesh diet varies from
20 to 34·27 per cent., with the following percentage composition:--

  [22] Gaultier. See also analysis given in Chapter VI.

    Silica                 7·04
    CO_{2}                 4·62
    SO_{3}                 7·37
    Fe_{2}O_{3}            4·22
    CaO                   25·29
    P_{2}O_{5}            26·41
    MgO                   15·52
    Cl                     1·50
    Alkali                 5·53

In a dog weighing 30 kilos., fed on a mixed diet of 500 grm. meat and
200 grm. starch, 78·6 grm. dry fæces were obtained containing 23·76 per
cent. ash of the following percentage composition:--

    CaO                    22·3
    P_{2}O_{5}             25·4
    Fe_{2}O_{3}            10·6
    MgO                     9·8
    SO_{3}                  5·0
    Cl                      0·2
    Alkali                  1·1
    Insol. in HCl          21·8

It is obvious that the composition of the fæces will vary with the food
given, and in this connexion it may be stated that the puer used was
obtained from dogs fed on a mixture of boiled horseflesh and oatmeal
porridge, about equal quantities. Sometimes charcoal and cabbage are
mixed with the food, in this case the puer is very dark coloured. The
dark colour so produced is to be carefully distinguished from the dark
colour caused by decomposition of the puer.

We cannot leave out of account the urinary products, which, in the case
of dung from hunting kennels, are always present, though in varying
quantities.

The total nitrogen in 1520 c.c. of urine was found[23] to be 15·9 grm.

  [23] Abderhalden, Physiological Chemistry.

Each 100 grm. total nitrogen was distributed as follows:--

    Urea                     85·9
    NH_{3}                    4·1
    Creatinine                3·3
    Uric acid                 0·5
    N in other compounds      5·7

The inorganic compounds were:--

                            Per cent.
    Sulphur (as sulphate)     3·31
    P_{2}O_{5}                3·98
    Cl                        6·30

The urine of carnivora is acid, that of herbivora is neutral or
alkaline.

Urea, which is the chief nitrogenous constituent, is very quickly
fermented by several species of bacteria, and entirely decomposed into
ammonium carbonate, so that it is this latter body which acts in the
bate.

Uric acid exists in the urine of the carnivora, but the quantity is
small compared with the urea. Herbivora excrete, mostly, hippuric acid

                              NHC, H_{5}O
                             /
  (benzoyl-glycocoll,  CH_{2}             ),
                             \
                              COOH

and this difference may account to some extent for the difference in
bating property, that of the excreta of herbivora being very small as
compared with the carnivora, such as the dog. In this respect there
are differences which are difficult to account for; for instance, the
excreta of the lion, fed entirely on a meat diet, was not found to have
such a powerful bating effect as ordinary dog puer. Even after keeping
for several weeks, to allow the bacterial flora to develop, the result
was the same.

The analysis of the lion’s dung gave the following results:--

                              Per cent.
    Water                       59·2
    Ash                         21·1
    Lime (CaO)                  10·3
    Phosphates (P_{2}O_{5})     10·67
    Organic matter              19·7

Dog dung contains some non-volatile bases of the purine group, such as
xanthin (C_{5}H_{4}N_{4}O_{2}) and guanin (C_{5}H_{5}N_{5}O), but the
action of these bodies in the bate, has not been studied. It is also
extremely probable that these and similar bodies are formed _during_
the bating by a process of auto-digestion of various proteids.[24] The
quantity of _fat_ in dry puer, amounts to 10–11 per cent. Cholesterol
was present.[25] Part of the fat is in the form of lime and magnesia
soaps, but part in the form of an emulsion, which probably plays an
important part in the puering process. The function of the fatty
constituents of puer is one of the problems on which further research
is necessary.

  [24] Cf. Mann. Chemistry of the Proteids, 1906, p. 432.

  [25] The cholesterol was obtained by extracting the dry puer
  with ether, saponifying with alcoholic potash. Shaking out the
  unsaponifiables with ether, evaporating to dryness. The residue was
  taken up with absolute alcohol, decolorized with animal charcoal.
  On cooling, characteristic crystals of cholesterol separated.
  Hoppe-Seyler finds from 1 per cent. to 4 per cent. cholesterol in
  fæces. See also Gaultier, Coprologie Clinique, p. 160.

The colouring matters of the puer are nearly all derived from bile
products. Most of them are found in the petroleum extract, which
contains far more of the colouring matters than the chloroform extract.

The following bile colouring matters have been prepared by Merck,
of Darmstadt: bilihumin, biliprasin, bilirubin, bilifuscin,
biliverdin.[26] So far as has been ascertained by the author, their
effect in the bating is nil, but, in so far as they impart colour to
the pelt, they are detrimental.

  [26] For qualitative tests for the colouring matters, see Gaultier,
  Précis de Coprologie Clinique, p. 159.

Experiments on the action of bile are given in the paper reprinted in
Chapter VI.

Recently Eberle[27] has proposed to add a proportion of gall juice
(bile) to a bate containing pancreatic enzymes, with a view of
energizing the latter. This part of the subject will be dealt with in
Chapter V.

  [27] English Patent, 21202, 1909. See also Chapter VIII.

*The Reactions of the Puer.*--The organic acids are present principally
in the form of the sodium or calcium salts. The intestinal juices
contain sodium carbonate, but this is neutralized by lactic acid found
in the intestines, so that the excreta usually have an acid reaction.
The reaction of a fresh puer liquor is faintly acid to litmus, but
this acid is immediately neutralized by the introduction of the skins,
so that the rest of the bating process takes place in a neutral or
alkaline medium, and the quantity of lime removed by the free acids is
probably small.

The acidity of a filtered puer liquor made up freshly and titrated with
N/5 soda, and phenolphthalein as indicator, was found to equal 10 c.c.
N/1 acid per 1000 c.c. of bate. In the volume of liquor used, namely,
900 litres, this would neutralize only 250 grm. of lime (CaO).

The determination of the exact acidity or alkalinity of puer liquors
by ordinary methods with indicators is not easy, as the end reactions
are not sharp. The liquor above described was alkaline to methylorange
and litmus, although acid to phenolphthalein, and the results also vary
somewhat, according to the quantity of bate used for titration, and
according to the strength of the acid or alkali used. The method used,
both at the Trent Bridge laboratory and Messrs. Doerr and Reinhart’s
laboratory at Worms, is as follows:--

The puer liquor is filtered through a Schleicher and Schulls hard
filter, No. 602, 18-1/2 cm. diam.; 50 c.c. are taken, 4 drops of
phenolphthalein solution (10 gr. dissolved in 300 c.c. alcohol) added,
and titrated with N/5 acid or alkali as the case may be.

If the acidity be determined by adding excess of alkali and titrating
back, a larger amount of acid is found than by direct titration. In the
above-mentioned case, an indirect titration showed an acidity equal to
18 c.c. N/10 acid per 100 c.c. of liquor.

On adding the alkali to a fresh filtered puer liquor, a flocculent
precipitate is produced, which appears to be due to the decomposition
of proteid compounds of weak organic acids,[28] the alkali uniting with
them and setting the proteids free. As these compounds are undoubtedly
decomposed by the lime in the skins, it seems probable that the acidity
available for neutralizing lime may be greater than that shown by
direct titration.

  [28] Probably syntonin, or bodies of a similar nature (see Allen.
  Comm. Org. Anal. iv. p. 4). The supposition is supported by the
  fact that solutions of puer have a slight laevorotatory action on
  polarized light.

  See also note on Van Liers paper, Coll. 376, 1909, p. 823.

A method which was suggested by me for the estimation of the acidity
of tan liquors may be applied also to the examination of the bate
liquors, namely, the method by which the electric potential between a
hydrogen electrode dipping into the bate liquor, and a standard calomel
electrode, is used to determine the neutral point (for details see
Chapter III.), using the potentiometer devised by Dr. H. J. S. Sand.[29]

  [29] See paper entitled, “The Employment of the Electrometric Method
  for the Estimation of the Acidity of Tan Liquors,” by H. J. S. Sand,
  Ph.D. D.Sc., and D. J. Law, B.Sc. F.I.C., J.S.C.I. 1911, p. 3; also
  Part II., by Wood, Sand and Law, J.S.C.I. 1911, p. 872.

Puer liquors, titrated in this manner with N/10 soda or HCl until a
potential of 0·69 volts is reached (at which point phenolphthalein
turns from colourless to red), gave the following results per 100 c.c.
filtered liquor.

  ---+-----------------+--------+---------------------------------
     |                 |        |          {Alkali}
  No.|  Description of |Reaction|c.c. N/10 {  or  } Required for
     |      Liquor     |        |          { Acid } Neutralization
  ---+-----------------+--------+---------------------------------
   1 |{New puer       }|  Acid  |           7·4  alkali
     |{before goods   }|        |
     |                 |        |
   2 |{The same after }|Alkaline|           0·57 acid
     |{goods          }|        |
     |                 |        |
   3 |{New puer       }|  Acid  |           8·1  alkali
     |{before goods   }|        |
     |                 |        |
   4 |{The same after }|Alkaline|           3·25 acid
     |{goods          }|        |
     |                 |        |
   5 |{Used puer after}|Alkaline|           5·00 acid
     |{goods          }|        |
     |                 |        |
   6 |Spent puer       |Alkaline|           6·6  acid
  ---+-----------------+--------+---------------------------------

Determinations made in this way are more accurate than the colorimetric
method above described. Puer liquors after use are found by the
electrometric method generally on the alkaline side, whereas such
liquors show neutral to phenolphthalein owing to some action of the
bate constituents on the indicator.

PUER LIQUORS IN ELECTROMETRIC APPARATUS.

    ---+--------+---------+-------+---------+-------------------------
       |        | c.c. of |       | c.c. of |
       |  P.D.  |N/10 KOH |  P.D. |N/10 HCl |
       | Before |required   After |required |
    No.| Skins  |   for   | Skins |   for   | Description of Liquor
       |        | 50 c.c. |       | 50 c.c. |
       |  volt  |liquor to|  volt |liquor to|
       |        |0·69 volt|       |0·69 volt|
    ---+--------+---------+-------+---------+-------------------------
    A1 | 0·622  |   1·95  |   ..  |    ..   | New puer
    A2 |   ..   |   ..    | 0·747 |   1·9   | A1 after 2 packs skins
    B1 |  ·607  |   3·7   |   ..  |    ..   | New puer
    B2 |   ..   |   ..    |  ·770 |   3·1   | B1 after 3 packs skins
    C1 |  ·580  |   4·3   |   ..  |    ..   | New puer
    C2 |   ..   |   ..    |  ·762 |   2·8   | C1 after 2 packs skins
    D1 |  ·580  |   5·2   |   ..  |    ..   | New puer
    D2 |   ..   |   0·8   |  ·680 |    ..   |{D1 after 1 pack skins
       |        |         |       |         |{(still on the acid side
       |        |         |       |         |{ of 0·69)
    E1 |  ·570  |   6·2   |   ..  |    ..   | D2 + 4 buckets more puer
    E2 |   ..   |   ..    |  ·720 |   0·8   | E1 after 1 pack skins
    F1 |  ·610  |   3·0   |   ..  |    ..   | New puer
    F2 |   ..   |   0·5   |  ·680 |    ..   |{F1 after 1 pack skins
       |        |         |       |         |{(still on the acid side
       |        |         |       |         |{of 0·69)
    ---+--------+---------+-------+---------+-------------------------

The potential at the neutral point, using an auxiliary electrode
filled with N/1 potassium chloride, was 0·69 volts. Potentials below
this point, therefore, indicate an acid reaction, and, conversely,
potentials above 0·69 volts indicate an alkaline reaction. The
apparatus is thus very useful in following the course of the bating
process. The initial potential also enables the hydrion concentration
of the bate liquor to be determined directly (see Chapter III.).
It was found that during the bating of a pack of skins the hydrion
concentration of the liquor diminished from 10^{-5·2} to 10^{-7·4}
normal.

Such acidities as are found by this method, e.g. 7–8 c.c. N/10 acid per
100 c.c., are too great if made up by taking a solution of the free
acids. The ionization must be almost entirely repressed by the presence
of sufficient neutral salts of the same acids in the bating liquid, in
order to obtain a similar result to the puer.

The table opposite gives the values found for a series of puer liquors,
made with dog manure from hunting-kennels.

The reaction of puer liquors (expressed in c.c. of N/1 alkali or acid
per 1000 c.c. of bate), in a series in which the washed goods were run
for one hour in old puer, out of which goods had been taken, and then
transferred to a freshly-made puer, was as follows:--

    ----------+----------------+--------------
       Bate   |  Before Goods  |  After Goods
    ----------+----------------+--------------
     Old puer | Neutral        | 28 c.c. acid
     Fresh    | 10 c.c. alkali | Neutral
     Old puer | Neutral        | 40 c.c. acid
     Fresh    | 11 c.c. alkali | Neutral
    ----------+----------------+--------------

These figures were obtained by the ordinary colorimetric method
described on p. 32.

Adding together the alkalinity of the bate run away, and the acidity of
the new bate which is neutralized, the total for 1 and 2 = 38 c.c. N/1
acid, and for 3 and 4, 51 c.c. N/1 acid.

Multiplying by 810 (volume of bate less volume of dry skin), we find
that the skins have neutralized 30·78 and 41·31 litres N/1 acid
respectively. Since one litre of N/1 acid will neutralize 28 grm. of
lime, it follows that the lime removed by the bate was in one case
862 grm., in the other case 1157 grm.

The 500 kilos, of wet washed grains introduced into the paddle contain
approximately 0·5 per cent. of lime CaO, equivalent to 2500 grm.
altogether. As will be seen from the figures given in table, p. 35, the
free acids present in the fresh bate are only capable of neutralizing
10 × 810 c.c. of normal alkali, and this is equivalent to 225 grm. of
CaO, or 9 per cent. of the total lime.[30]

  [30] Eitner, Der Gerber, 566, p. 77, states that little or no
  neutralization of lime by acids or acid salts takes place in the bate.

In addition to this, a considerable amount of lime is removed from
the skins which is not found afterwards in solution, but which is
precipitated in an insoluble form during the course of the bating
process. In an experiment to determine the amount of this insoluble
lime, the following figures were obtained:--

LIME IN PUER LIQUOR, GRAMS PER LITRE CaO.

                    Before Skins  After Skins
    Soluble lime        0·19         0·485
    Insoluble lime      0·13         0·485
                        ----         -----
                        0·32         0·970

It will be noted that the amount of insoluble lime has increased to a
greater extent than the soluble lime, showing that part of the lime
from the skins has been precipitated in an insoluble form. The increase
of lime in the solution is 0·295 grm. per litre of soluble lime, and
0·355 grm. per litre of insoluble lime, a total increase of 0·650 grm.;
that is, the ratio of soluble lime to insoluble lime in the original
puer is 1·46 : 1. In the used puer it is 1 : 1, so that in the above
case more than half the lime removed from the skins (54·5 per cent.)
has been precipitated in an insoluble form, some of it in the form of
calcium phosphate, and the remainder probably in the form of calcium
oxalate. Crystals of calcium oxalate may be seen under the microscope
in the puer residues.

The oxalic acid is produced by bacterial action, as has been shown by
Zopf[31] and Banning[32]; but it is difficult to estimate it in the
bate, and a reliable analytical method of doing so requires devising.

  [31] Ber. Deutsch. Botan. Ges. 1900, xviii. 32, J.S.C.I. 1900, 386.

  [32] Centr. Bakt. Parasitenk, ii. Abt. viii., 393, etc., J.S.C.I.
  1902, 1151.

A further 30 per cent. to 40 per cent. of the lime in the skins is
removed by the chemical action of the complex amines of the organic
acids, and by the mechanical effect of the paddle or drum, so that the
final result is more or less as follows:--

                                      Per cent.
    Lime neutralized by free acids        9
    Lime dissolved by complex amines     25
    Lime precipitated                    30
    Lime remaining in the skins          36
                                        ---
                                        100

The lime remaining in the skins is not in the caustic state, but
principally in the form of neutral salts (see footnote to p. 24).

[Illustration: Fig. 7.]

Some of these neutral salts appear to be absorbed by the skin during
the course of the bating, for, on examining pieces of the same skin at
frequent intervals during the operation, and determining the ash, it
was found that a minimum point for ash content was reached in about ten
minutes, after which the ash actually increased. The results are shown
in the following table, and also graphically by the curves, Fig. 7--in
which A is a “grain” previously washed as free from lime as possible;
B, an unwashed “roan.” The effect of the absorption of inorganic matter
is very noticeable in both cases. A part of the effect only is apparent
since a certain amount of skin substance passed into solution.

VARIATION OF ASH PERCENTAGE DURING PUERING.

    -----------------+--------------------------------
            --       |  Ash, per cent. on Dried Skin
    -----------------+----------------+---------------
     Time of Puering | Washed Delimed | Limed Roan (B)
       in Minutes    |   Grain (A)    |
    -----------------+----------------+---------------
            0        |      2·88      |     7·03
            5        |      2·91      |      --
           10        |      3·20      |     3·54
           15        |      4·80      |      --
           20        |      4·08      |     4·24
           25        |      4·29      |      --
           30        |      4·85      |     5·19
           35        |      4·59      |      --
           40        |      4·70      |     5·45
           45        |      4·60      |      --
           50        |      4·45      |     5·67
           55        |      4·42      |      --
           60        |       --       |     5·00
    -----------------+----------------+---------------

The following analyses give the amount of lime in solution before and
after the bating of the skins, in grams per litre of filtered puer
liquor:--

    ---+-----------------+------------+-----------+---------------
    No.|      Bate       |Before Goods|After Goods|Increase of CaO
    ---+-----------------+------------+-----------+---------------
     1 |Fresh puer       |   0·108    |    0·62   |     0·512
     2 |  "    "         |   0·34     |    0·72   |     0·38
     3 |  "    "         |   0·20     |    0·52   |     0·32
     4 |  "    "         |   0·19     |    0·49   |     0·30
     5 |Old puer         |   0·54     |    0·84   |     0·30
     6 |{Old puer, goods}|   0·98     |    1·38   |     0·40
       |{not washed.    }|            |           |
     7 |{French puer    }|   0·308    |    0·548  |     0·24
       |{shop.          }|            |           |
    ---+-----------------+------------+-----------+---------------

The limit of the quantity of lime in solution in a normal puer, in
the form of calcium salts, appears to be about 1 grm. per litre. If
more lime be presented to the liquor, the goods stop coming down. If
now fresh dung be added, they will continue to come down, but the
quantity of lime in solution does not increase; the excess of lime is
precipitated, partly as phosphate and partly as oxalate, in the way
stated above.

The type of reaction by which the lime is dissolved is similar to that
occurring between ammonium chloride and lime, and is expressed by the
formula--

    2NH_{4}Cl + Ca(OH)_{2} = CaCl_{2} + 2NH_{3} + 2H_{2}O

and in the case of the calcium carbonate in the skins--

    2NH_{4}Cl + CaCO_{3} = CaCl_{2} + (NH_{4})_{2}CO_{3},

so that every molecule of lime neutralized, liberates two molecules of
ammonia.

Jean states that the production of ammonia progresses regularly during
the puering, and that when the free ammonia reaches 0·2 grm. per litre
the bate is unfit for further use. But if the excess of ammonia be
neutralized by the addition of phosphoric acid, which also precipitates
the lime brought in by the skins, the bate may be kept in constant
use for some time. Jean found 0·06 grm. NH_{3} per litre in the puer
as made up for use, and, after one lot of skins had been put through,
0·086 grm. per litre; after two lots of skins, 0·135 grm. per litre. In
ordinary puer wheels, as used at Trent Bridge, we find--

                    (_a_)   (_b_)
    Before goods    0·0816  0·0850 grm. NH_{3} per litre
    After    "      0·0833  0·0799

so that there is little or no difference before and after puering in
this case.

Part of the ammonia formed escapes into the air; a further portion
unites with the acids which are being formed by the action of bacteria
in the bate (see Chapter IV.), and it is also probable that part
combines in some way with the calcium salts in the bate.

The actual reactions taking place are of course much more complicated
than this simple case, because, instead of ammonia and ammonium
chloride, we have a number of complex salts formed by combination
of organic acids with substituted ammonia derivatives, such as
methylamine, ethylamine, etc. The chief of these are ethylamine and
methylamine butyrates, and lactates, and probably propionates,
although I have not been able to separate the latter.[33]

  [33] Fitz (Berichte, 1876–1884, see Herfeldt, J.S.C.I. May 31, 1895),
  has shown that lactates in dung are decomposed by bacteria, the chief
  product being propionic acid.

The reaction with the butyrate is represented by the equation--

      2C_{2}H_{5}NH_{2}C_{3}H_{7}COOH + CaO

    = 2C_{2}H_{5}NH_{2} + C_{3}H_{7}COO
                                       \
                                        Ca + H_{2}O
                                       /
                          C_{3}H_{7}COO

by which it will be seen that the amine is set free just in the same
way as ammonia in the equation on p. 40.

In order to ascertain if the action of the amines was the same as that
of salts of ammonia, I prepared the following compounds and tested
their action on skins at 37° C., a control piece of the same skin being
kept in water at the same temperature. The time in each case was one
hour. All the solutions were neutral.

    1. Ethylamine lactate: skin swells slightly, not “down” equal to
    puer.

    2. Ethylamine propionate: skin moderately fallen, not equal to puer.

    3. Ethylamine butyrate: about the same as Exp. 1.

    4. Trimethylamine butyrate: very similar action to Exp. 3 and to
    ammonium butyrate.

The results correspond very closely with those obtained in previous
experiments with various salts of ammonia (see Chapter VI.), and
justify us in assuming that in all essential respects the action is
similar. The free amines enter into fresh combinations with acids
which are produced by bacterial action, and this process goes on until
all the nutrient material is exhausted.

*The Role of Phosphates in the Bate.*--There is no doubt that the
phosphates in the bate play an important part, but exactly in what
manner they act is not yet known. One of the principal effects is the
part they play as “buffers,” in preventing brusque changes of the
hydrion concentration during the bating process. This has been pointed
out by Soerensen in the case of enzyme reactions. The phosphates in
dung are mixtures, which are capable of fixing both acids and bases;
and so the small quantities of these bodies, which are produced by the
splitting up of the organic matter, are taken up or released as the
case may be.

The chemistry of the phosphates is one of the most complicated branches
of inorganic chemistry, and, as a consequence, the determination of
the constitution of the various phosphates in dung is an extremely
difficult matter, and demands a lengthy research. For instance,
besides the salts directly derived from the three phosphoric acids,
HPO_{3}, H_{3}PO_{4} and H_{4}P_{2}O_{7}, phosphates exist which are
probably derived from hypothetical di-, tri-, or meta-phosphoric
acid, _n_HPO_{3}, and a few salts have been isolated, which are
perhaps derived from the hypothetical acids P_{4}O_{7}(OH)_{6} and
P_{10}O_{19}(OH)_{12} (Watt’s Dict., art. “Phosphates”). Including
the double salts, there are more than 16 different calcium salts of
phosphoric acids. The normal lime salt Ca_{3}(PO_{4})_{2} is very
slightly soluble in water, but its solubility is increased by the
presence of various organic substances such as exist in dung, and part
of the soluble phosphates found in the bate are undoubtedly nothing
more than this salt in solution. This fact has been utilized in the
manufacture of the artificial bate Erodin (see Chapter VII.).

The phosphoric acid in the puer is partly precipitated by the lime in
the skins, and hence diminishes during the bating process. In some
cases practically the whole of the phosphoric acid disappears from the
solution.

The following experiment will give an idea of the amount of lime
precipitated as phosphate. A filtered puer liquor was analysed for lime
and phosphoric acid, before and after the skins were passed through.
The results were, in grm. per litre--

                            Before   After
    Soluble lime as CaO     0·364    0·540
    P_{2}O_{5}              0·424    0·328

Increase of lime, 0·176 grm.; diminution of P_{2}O_{5}, 0·096 grm.
Calculated to calcium phosphate Ca_{3}(PO_{4})_{2}, this amount of
phosphoric acid has combined with 0·114 grm. CaO. Assuming that the
ratio of soluble to insoluble lime is the same as given, p. 36, then
the lime is distributed as follows:--

                                     Grm.
    Increase of soluble CaO          0·176
       "      "  insol. CaO          0·210
    Lime as Ca_{3}(PO_{4})_{2}       0·114
    Hence " " oxalate (?)            0·096

That is, of the lime precipitated, 54 per cent. is phosphate, 46 per
cent. oxalate.

In another puer containing before use 0·383 grm. P_{2}O_{5} per litre,
only traces of phosphates were found in solution _after_ goods had been
puered in the liquor, and in some analyses by Jean (39) the following
figures were obtained:--

GRAMS OF PHOSPHORIC ACID PER LITRE.

    Fresh dung after four days’ maceration      0·082
    Bate after one pack of skins                0·036
    Bate after two packs of skins               0·018

Although these figures are less than those found at Trent Bridge, they
confirm the fact that the soluble phosphates diminish during the bating
process.

The phosphates in solution thus diminish during the bating, and are
found in the insoluble matter which separates out. A small portion of
the lime remaining in the skins is also converted into phosphate by
the action of the bate. In an experiment to determine this, a portion
of the same skin was taken before and after puering. The pieces
were dried, ashed, the ash dissolved in dilute nitric acid, and the
phosphates precipitated by ammonium molybdate. In the skin before
puering no phosphates were present, but in the skin after puering there
was a small amount, though not sufficient to weigh.

The action of ammonium phosphate on the lime in the skin is very small.
A skin was treated with a 0·1 per cent. solution of ammonium phosphate
at 100° F. for one hour. The CaO in the dry skin was estimated, and
found to be--

                            Per cent.
    Before the experiment     1·93
    After   "      "          1·45

A considerable amount of calcium phosphate was found in the skin after
the experiment.

Other chemical compounds existing in the puer, or formed by the
action of bacteria (principally _B. coli commune_, see Chapter IV.),
are indol, skatol and a number of aromatic oxyacids, principally
para-oxyphenyl-propionic acid, a little para-oxyphenylacetic acid and
skatol carbonic acid. In addition, tyrosin, leucin, tryptophan and
mercaptans have been separated.[34]

  [34] Rettger, Amer. Jour. of Physiol., viii. p. 284; Koch’s
  Jahresbericht, 1903, p. 112.

With these bodies no experiments on skin have been made, so far as I am
aware, except with indol and skatol. Kathreiner found that these had a
slight reducing action on skin, so that one may say they play some part
in the puering.

The action of the bile salts, glycocholate and taurocholate of soda,
also needs investigation. These have an indirect effect in the puer,
as they favour the development of some species of bacteria (chiefly
_coli_) and hinder the growth of others.

Some action has also been ascribed to sulphuretted hydrogen, but in the
puer liquors which I have examined no H_{2}S was found, either before
or after the skins were entered.

It will be seen that the ammonia compounds in the bate are not of
themselves particularly fitted for the purpose of removing lime[35]
from the skins, but owing to bacterial action (which we shall treat of
in Chapter IV.), acids are produced which combine with the ammonia,
and in this way the small quantity of these compounds originally
present is continually being regenerated while the bating is in
progress. Ammonia is set free by the lime in the skins. It is then
neutralized by acids produced by bacteria, and thus acts as a carrier
for the acids, and the bate remains in a nearly neutral condition. As
the lime increases in the liquid the action of the bacteria diminishes,
and finally the alkalinity becomes too great to allow the bacterial or
chemical action to proceed further.

  [35] Deliming with acids when carefully done takes out more lime than
  the puer, without in any way injuring the skin, but when tanned with
  sumac the bellies and axilla are harsh, and of a browner colour than
  in a puered skin. Analysis showed no more lime in the brown portions
  than in the centre of the skin, where the colour was quite bright and
  satisfactory.

It will be noted that the _concentration_ of the active salts in the
bate is extremely small. If the amine compounds be assumed to consist
of ethylamine butyrate or lactate, the concentration of the solution is
approximately 1 grm. per litre; it is important that the concentration
of salts should not greatly exceed this amount. I have found by
experiment with ammonium chloride solutions, that the best reducing
action is provided by a concentration of 0·7 to 1 grm. NH_{4}Cl per
litre; if the concentration be raised to 2 or 3 grm. per litre, the
skins become “leathery” and do not fall properly. The alkalinity must
not be greater than 3–5 c.c. N/10 per 100 c.c. bate, for the bate to
work at its best.

*Solution of Skin Substance during the Puering.*--The determination
of the total skin substance dissolved by the puer is best done by
Kjeldahl’s method before and after the goods.[36]

  [36] Vide Procter, L.I.L.B., 1908, p. 64.

The difference in the total nitrogen found multiplied by 5·6 gives the
amount of skin substance dissolved by the bate, assuming the amount of
nitrogen in the dry ash-free skin to be 17·8 per cent. If very great
accuracy be required, a small correction for nitrogen, brought into
solution from the puer itself, is necessary.[37] This correction must
be ascertained for the particular puer used by actual experiment.

  [37] In a blank experiment to determine this, 0·0476 grm. N per litre
  was found to be brought into solution from the puer.

The following figures give the results in grams per litre obtained in
the puering of sheep grains. 50 c.c. of the filtered puer liquor are
slightly acidified with sulphuric acid, evaporated nearly to dryness
and Kjeldahled in the usual way.

                                            Mean
    Total nitrogen before skins   {0·2632}  0·2604
                                  {0·2576}
     "       "     after    "     {0·4928}  0·4844
                                  {0·4760}
    Difference                       --     0·2240

Equivalent to 1·254 grm. skin substance per litre. This was somewhat
over a kilogram of skin substance for the paddle in question, and equal
to 1 per cent. of the dry ash-free skin.

As to the differentiation of the dissolved skin substance into
albumoses, peptones, monamino acids, diamino acids, ammonia, etc., a
modification of Stiasny’s method[38] for the examination of soaks and
old limes may be used.

  [38] Stiasny, On Old Limes. Collegium 1910, p. 181.

The method is based on the fact, discovered by Schiff, that
formaldehyde reacts with amino acids, forming methylen-amino
acids, which are distinctly acid and allow a sharp titration with
phenolphthalein as indicator, while the amino acids themselves react
almost neutral. Soerensen has worked out a method on this basis for the
determination of different amino acids, and for tracing the course of
hydrolysis of albuminous matters.

Instead of using phenolphthalein as an indicator, the electrometric
apparatus of Sand (see p. 76) is employed. 50 c.c. of the filtered puer
liquor are put into a beaker, the hydrogen electrode is immersed in
the liquor, and the potential difference (P.D.) observed; this gives
the hydrogen ion concentration of the solution. 10 c.c. of neutral
formaldehyde solution (40 per cent.) are added, and the P.D. again
observed; it will be found to diminish rapidly, but soon becomes
constant, indicating that the reaction is a quick one.

The increase of acidity, as shown by the lowering of the potential
difference, is due to the acidity developed by the combination of
the formaldehyde with the amino acids forming methylen-amino acids
of appreciable hydrion concentration. The amount of such acids is
estimated by titrating with N/10 caustic soda solution until the P.D.
rises to the same voltage as that originally found. The following
figures were found in an experiment:--

                                  Original Puer    The same
                                     Liquor       After Goods
    π (volts)                         0·61           0·69
    π after 10 c.c. formalin          0·53           0·54
    N (Kjeldahl), grm. per litre      0·3136         0·5936
    Increase of N                      --            0·2800
    N/10 soda for 50 c.c. to
      original voltage                7·0           11·6
    c.c. increase N/10 soda            --            4·6
    ∴ 1 c.c. N/10 soda = mg N          --            3·05

A preferable method is to add decinormal acid or alkali to the original
liquor until the P.D. of 0·69 is reached, at which point the liquor
will be neutral to phenolphthalein, and, after adding formaldehyde, to
titrate with N/10 soda until the P.D. of 0·69 is again reached.

The factor which connects the amount of decinormal soda required for
the titration, after the addition of formaldehyde, with the total
nitrogen as determined by Kjeldahl’s method, will afford information as
to the extent of the hydrolysis undergone by the proteid matter, in the
same manner as Stiasny (loc. cit.), has proposed to differentiate the
dissolved proteid matter in lime liquors. As hydrolysis proceeds the
percentage of nitrogen in the molecule increases, being at its maximum
in the ultimate nitrogenous product ammonia; the factor, therefore,
becomes less as hydrolysis becomes more advanced.

    For ammonia     1 c.c. N/10 soda             = 1·4 mg. N
     "  hydrolized gelatin[39]                   = 2·9  "
     "  Witte peptone completely hydrolized[39]  = 3·6  "
     "  lysin                                    = 2·8  "
     "  arginin                                  = 5·6  "
     "  histidin                                 = 4·2  "

  [39] Stiasny. Collegium 1910, p. 184.

We may conclude that the skin substance dissolved in the puer liquor is
hydrolized almost as completely as gelatin is by boiling with sulphuric
acid.

I have previously pointed out that dilute acids dissolve a certain
amount of skin substance (see p. 157), and in this connexion, Dr.
Georges Abt has given me the results of some experiments, on the
solubility of skin in various organic acids, which he made in Vienna.
Pieces of skin, weighing 40 grm. in the wet state, were allowed to
remain for one month in N/10 solutions of the acids. The N was then
determined, by Kjeldahl’s method, with the following results, expressed
as per cent. of the wet skin dissolved:--

                            Per cent.
    Acetic acid dissolved     0·645
    Lactic  "       "         2·27
    Butyric "       "         0·577
    Formic  "       "         1·47

It will be seen that butyric acid dissolved the least amount of skin,
lactic acid dissolving close upon four times as much.

*Scud.*--A certain amount of skin substance comes away in the “scud.”
This is the liquid squeezed out of the skin by the pressure of the
scudding knife after puering.

The liquid has the same composition as the puer liquor out of which the
goods have been taken, and in addition contains large quantities of
pigment granules, wool roots, and some skin substance, which together
constitute the so-called “filth” of the skin. Analysis of a scud from
English sheep grains showed only 0·164 per cent. N, equivalent to about
1 per cent. skin substance (9·15 grm. per litre). Fat, 7·9 grm. per
litre.

Eberle and Krall have recently[40] analysed the fatty matter which
adheres to the men’s knives in scudding lamb skins for gloving work.
They obtained the following results:--

  [40] Ueber die Zusammensetzung des beim Beizen von Lammfellen
  mit Hundekot abfällenden “festen Schmutzes”: ein Beitrag zur
  Beizenfrage.--Coll., 1911, p. 445.

                                          Per cent.
    Water                                   29·7
    Fat                                     42·0
    Fatty acids combined with lime           6·6
    Albuminous matter soluble in water       3·8
    Hair and insoluble albuminous bodies    14·4
    Ash (containing 57 per cent. CaO)        3·5

The fat had a--

    Melting-point                          40–44°
    Saponification number            about   121
    Iodine number                           31·6
    Hehner value                            91·9
    Acid value                               9·3

Dr. Fahrion found, in a sample of the fat extracted with ether:--

                                       Per cent.    Iodine number.
    Unsaponifiable                       47·6           27·3
    Fatty acids (sol. in petrol ether)   39·3           30·2
    Oxy acids (sol. in ether)            13·5           13·4

The figures obtained for the fat therefore agree closely with those for
wool fat.

*Action of the Bird-Dung Bate.*--The depleting action of the pigeon-
and hen-dung bate is very similar to that of the puer, or dog-dung
bate; but the bating process with these materials, as we have seen
(p. 18), is carried out at a lower temperature, and is consequently
more prolonged. The principal difference between the two bates appears
to be a chemical one, due to the fact that bird dung contains all the
urinary products which are present only to a small extent in the
dung of mammals. In birds uric acid is the chief stage in nitrogenous
katabolism, the mechanism of its formation being a process of synthesis
in the liver (Halliburton). Urea is also present in considerable
amount, and does not appear to be so easily decomposed as the urea
in animal urine.[41] As we shall show in the next chapter, urea, and
probably also urates, greatly facilitate the permeability of gelatine,
and to this fact may be ascribed the more gradual action of bird-dung
bates. If we attempt to bate hides with dog-dung, the grain of the hide
is found to be attacked and destroyed before the bate has penetrated
to the interior of the hide. On the other hand, a bird-dung bate may
be used at a temperature of 38° to 40° C. for the puering of skins
destined for light leather, but its action is not so favourable as that
of the puers.

  [41] Urea is present to the extent of 5 per cent. in samples of
  Peruvian guano.

COMPOSITION OF BIRD EXCRETA.

    ------------------------+---------------+-------+-------+------
                            |     Pigeon    |  Hen  | Duck  | Goose
    ------------------------+---------------+-------+-------+------
    Moisture                | 58·32 | 56·08 | 60·88 | 46·65 | 77·08
    Organic matter†         | 28·25 | 19·56 | 19·22 | 36·12 | 13·44
    Phosphates              |  2·69 |  2·54 |  4·47 |  3·15 |  0·89
    Carbonate and sulphate} |  1·75 |  3·08 |  7·85 |  3·01 |}
      of calcium          } |       |       |       |       |} 2·94
    Alkaline salts          |  1·99 |  0·82 |  1·09 |  0·32 |}
    Silica and sand         |  7·00 | 17·92 |  6·69 | 10·75 |  5·65
    ------------------------+-------+-------+-------+-------+------
                            |100·00 |100·00 |100·00 |100·00 |100·00
    ------------------------+-------+-------+-------+-------+------
    † Containing nitrogen } |       |       |       |       |
      equal to ammonia    } |  1·75 |  1·21 |  0·74 |  0·85 |  0·67
    ------------------------+-------+-------+-------+-------+------

Macadam[42] states that pigeon dung is the most concentrated. Hen
manure contains the largest proportion of phosphates, and is followed
by duck droppings. That of the goose is the least valuable. The
preceding table is taken from his paper.

  [42] Manures, Natural and Artificial, W. Ivison Macadam. Jour. Soc.
  Chem. Ind. 1888, p. 79.

Procter[43] quotes the following, as a mean of 40 analyses of pigeon
dung by Schulze:--

  [43] Principles of Leather Manufacture, p. 179.

                         Per cent.
    Water                  21·00
    Nitrogen                2·53
    Phosphoric acid         1·79
    Potash                  1·46

He remarks that the action of bird dung is more penetrating, but less
softening and loosening than that of dog dung, and this effect may be
explained by what has been said above.

Unfortunately, far less work has been done on the bird-dung bate than
on the puer, and there is a wide field open for research in this
direction.




CHAPTER III.

THE PHYSICS OF BATING.


    “The most important aspect of any phenomenon, from a mathematical
    point of view, is that of a measurable quantity.” --MAXWELL.

It is impossible in the space of a short chapter to give an adequate
explanation of the physical changes taking place during the puering
process. An outline only can be given, and perhaps a few signposts to
indicate for those interested, in what direction further work can be
usefully done. It is obvious that if more be attempted, a treatise on
physics and physical chemistry would be required; such a work is beyond
the scope of the present volume. It is to be hoped that the whole of
the questions dealt with here, including the physical chemistry of skin
and of the whole tanning process, will shortly be fully treated by the
master hand of our greatest tanning chemist, Professor H. R. Procter.

Since tanning, in the earlier stages, is for the greater part a
physical absorption, or colloidal co-precipitating action,[44] the
physical state of the skin fibre, or condition of the skin before it
enters the tanning liquor, is of the greatest importance. In fact,
the whole of the operations to which the skin is subjected previous
to tanning are directed towards changing its physical state, and the
chemical changes undergone are small and principally hydrolytic.

  [44] Stiasny, Kritische und Experimentelle Beiträge zur Aufklärung
  der Gerbvorgänge. Collegium 1908, p. 117.

  Wood, Compounds of Gelatin and Tannin. Collegium 1908, p. 494.

The two most important physical influences over which we have control
are pressure and temperature.

The pressure during puering is practically constant, viz. the
atmospheric pressure, but, as we shall see later, a diminution of
pressure is favourable to falling or depletion. It would, therefore,
be interesting to conduct experiments on puering in vacuo, or under
reduced pressure.

Before considering the effect of temperature and the changes in the
volume of the skin during puering, it will be well to consider the
properties of the skin and the puer liquor.

In the skin prepared for tanning, practically all the keratin and
epidermis and soluble matters are got rid of, and we have a mass of
fibres which are composed of collagen, a proteoïd, which by prolonged
boiling with water is converted into gelatin.

Neither the chemical formula nor the molecular weight of collagen is
known with certainty, but, from a series of ultimate analyses, the
change of collagen into gelatin is represented by Hofmeister[45] by the
following equations:--

  [45] Allen, Comm. Org. Analysis, iv. p. 459.

    C_{102}H_{149}N_{31}O_{38} + H_{2}O = C_{102}H_{151}N_{31}O_{39}
             collagen                             gelatin

from which it will be seen that he assigns a molecular weight of 2416
to collagen, and 2434 to gelatin. Other considerations, however, make
it probable that the molecular weight is double the above figures.

Gelatin is a typical colloid, and we may consider that skin is
practically a colloid with a structure (a very important point, as
we shall see later), but behaving in many ways as gelatin. The puer
solution is to a large extent colloidal. Therefore, both the skin
and the puer are in the colloidal state. In the typical crystalloid
solution of an electrolyte, the dissolved body is separated into its
molecules, and to a large extent into individual ions, while, in the
colloid state, the units of distribution are either large and often
conjugated molecules, or more frequently minute particles composed
of many molecules united by cohesive attraction.[46] In the case of
the skin the molecules are not free to move, but are held in place by
the structure of the skin, and the fibres thus act as semi-permeable
membranes, with capillary spaces between them, in which water and other
fluids are merely held by capillary attraction.

  [46] Procter, Colloidal Chemistry. Brit. Assoc. Rep. 1908.

If filtered puer liquor is put into a vessel closed by a membrane of
skin, and the whole immersed in clear lime water, the puer solution
becomes turbid in a short time, but the outer solution remains clear,
showing that the skin is permeable to the lime solution (crystalloid),
but not to the puer solution (colloid). It was observed, however,
that the acids contained in the puer diffused through the membrane;
this was shown by the addition of a few drops of phenolphthalein to
the lime solution, when the pink colour disappeared after a short
time in the neighbourhood of the skin. These phenomena are due to
osmosis. It is the fundamental property of all animal membranes, to
allow some substances to pass through them more easily than others.
In many cases, such membranes, while freely permeable to water, are
practically impermeable to certain substances in solution, and play
the part of sieves in directing and controlling the diffusion. In the
case of skin the phenomena are complicated by the fact that the skin
combines chemically with many substances in solution, and thus we do
not always know what part to assign to chemical combination and what to
the osmotic phenomena.

Procter has shown (Colloid. chem. Beihefte 1911, ii., pp. 243–284)
that, while gelatin is very permeable as such to solutions of acids
and salts, there may be formed in the presence of excess of acid a
hydrolysable chemical complex of the nature of a salt, in which the
gelatin functions as base, and which is probably less permeable to
acids and their salts than the neutral gelatin. The conditions would
then be similar to those which obtain when solutions of an acid and
its salt are separated by a movable membrane, which is permeable for
the acid and water but not for the salt solution. From the organized
structure of the skin surface, it is unlikely that osmosis takes
place between the skin itself and the outer solution, with the two
surfaces of the skin as semi-permeable membrane. Osmotic action is most
likely to occur in the interior of the skin, between the skin fibres
themselves and the interfibrillar spaces. The colloids in the puer
solution, which constitute a large proportion of its material, cannot,
from their nature, penetrate the skin. This may be shown by the
above-mentioned experiment. From this it is reasonable to assume that
the lime is not actually dissolved from the interior of the skin by the
puer acids, but that solution takes place for the most part after it
has diffused out into the puer solution. It is probable, however, that
part of the bodies of acidic character present in the puer are capable
of penetrating the skin fibre, as has been explained above.

The intensity of the osmotic action of puer upon skin must depend upon
the quantity of substances contained in it, to which skin substance
acts as an impermeable membrane, and which on that account induces an
osmotic pressure between the outer puer solution and the solutions
held in the skin fibre. The effect of puering does not necessarily
imply the actual expulsion of water from the skin--in fact, well puered
skin may quite possibly contain as much water as it did in the swelled
condition. The difference consists in the manner in which the water
is held in the skin, and its freedom to move from parts which are
submitted to pressure. In the swollen skin, the fibres may be conceived
as swollen by the water and holding it in the same manner as a gelatin
jelly; after puering, the fibres are “fallen,” and the water, hitherto
held by them, surrounds them in the liquid form.

The osmotic pressure[47] of a solution of concentration _c_,
temperature T, and pressure _p_, is the difference of pressure
exerted on both sides of a semi-permeable membrane in thermodynamic
equilibrium, having on the one side the solution under the above
condition, and on the other side the pure solvent under the pressure
_p__{0} of its own saturated vapour. On this definition the osmotic
pressure of a normal solution is over 22 atmospheres, or 330 lb. per
sq. in.; and since a saturated lime solution is about 1/20 normal,
its osmotic pressure is about 1·1 atmospheres, or 16 lb. per sq.
in.--this represents the force causing the lime to diffuse into water
in which the skin is placed. The puer solution being of a colloidal
nature, exerts practically no osmotic pressure, and since it contains
substances capable of entering into combination with lime, the latter
is removed from the surface very quickly. The curve representing the
removal of lime by water has been given in Chapter I., p. 6. That for
puer is not of such a simple character (see Fig. 7, p. 38), but it
will be seen that the greater part of the lime is removed during the
first 10 minutes. The curve is plotted for percentage of ash, since
the lime is no longer in a caustic condition but in the form of salts.
It is remarkable that the percentage of ash, after reaching a minimum,
_increases_ considerably. This phenomenon still requires investigation.

  [47] M. Planck, Zeit. f. Phys. Chem. xlii. p. 584 (1903).

*Density of Skin.*--Coming to the consideration of the volume of skin
and its changes during puering, we know that the volume _v_ is the
reciprocal of the density, i.e.--

    _v_ = 1/δ,

and therefore

    δ = 1/_v_.

Carini[48] has carried out exhaustive experiments on the density of
skin during tanning, but, so far as I am aware, little or no work has
been done as to the effect of puering on the density of raw skin.

  [48] Sull’ appliccazione della bilancia idrostatica per il controllo
  della concia delle pelli, Milan, 1903.

The usual methods of determining density are well known,[49] and
consist in weighing the body first in air, then in water or other
liquid. If _m_ be the weight of the body, and it loses the weight _w_
when weighed in water--

    δ = _m_/_w_.

  [49] _See_ Kohlrausch, An Introduction to Physical Measurements,
  1894, p. 43. _See also_, Weighing Hides in Water, C. E. Parker and
  G. H. Russell, Tanners’ Year-Book 1905, p. 45.

For experiments on skin, instead of weighing in water it has been found
more convenient to use a simple volumenometer, which was devised by Mr.
Douglas J. Law (see Coll. 1911, p. 230.)

[Illustration: Fig. 8.--Volumenometer for Raw Skin.]

The apparatus (Fig. 8) consists of the two vessels A and B, connected
by means of thick rubber tubing to the burette C, of which the top is
enlarged to a bulb. The bottle A, which was specially made for us by
Messrs. Townson and Mercer, London, is of about 1 litre capacity, and
the wide mouth is closed by a stopper, accurately ground to fit the
neck, and extending down to the bottom of the neck. The upper part
of the stopper is elongated to a tube, which is closed by the tap G.
The vessel A is also fitted with a tap E. The vessel B serves as a
reservoir, and is used to adjust the level of the liquid in the burette
C by means of the tap at F. To find the volume of a piece of skin, the
method of procedure is as follows. The bottle A is filled with water
up to the neck, and the stopper D, carefully greased, is inserted.
The tap E is then closed, and the burette is filled with water. Then,
with the taps G and E open, the bottle A is filled with water up to D
by raising the burette. G and E are then closed, and by opening the
tap F the level of the water in the burette is adjusted to zero when F
is again closed. G and E are then opened again, and, by lowering the
burette, the water in A is allowed to fall below the level of the neck
of the bottle. E is then shut, the stopper D removed, and the piece
of skin is carefully introduced, avoiding air bubbles. The stopper
D is then replaced with the tap open, and, by opening E and raising
the burette, the water is allowed to come up to the stopper D again.
The taps G and E are then closed, and the volume of the piece of skin
read off directly from the burette scale. Volumes up to 50 c.c. are
measurable as described above, but larger volumes may be measured if a
known volume of water is run from the burette into the reservoir before
introducing the skin.

By using various solutions in the apparatus instead of pure water,
the real swelling or contracting effect of these upon the skin may be
observed. The skin is introduced into the bottle A, and the solution
adjusted to G, which is then closed; then, by leaving the tap E open,
the real swelling or contraction of the skin is measured by the rise or
fall of the liquid in the burette.

Petroleum or other liquid may be used instead of water; in some cases,
the use of petroleum is more advantageous.

The density of dry gelatin as determined by Lüdeking is 1·412, which
is not greatly different from that of skin. Carini gives the following
figures for ox-hide:--

    Skin with hair                          1·450
     "   depilated with lime                1·425
     "   depilated with sodium sulphide     1·441

but does not give details how these figures were obtained, and if
corrected for ash and fat.

The density of limed and puered sheepskin, determined by drying
the skin over sulphuric acid until the weight was constant, then
determining the volume in petroleum, gave the following results:--

    Limed skin         1·2335
    Puered  "          1·2590     I.

By determining the volume of the wet skin in the volumenometer, and the
per cent. of water on drying the skin, the calculated densities were--

    Limed skin         1·438
    Puered  "          1·300     II.

Correcting for ash and fat, the dry ash-free skins had the densities--

    Limed skin         1·397
    Puered  "          1·335    III.

The density of the wet limed skin was 1·063, but the density calculated
from the results (I.) above is 1·0475; from this, it is evident the
fibres of the swollen limed skin undergo compression on swelling, or
that the water contained in them is in a state of compression, in the
same way as gelatin swollen with water occupies a less volume than the
sum of the volumes of gelatin and water. Lüdeking[50] found for 10 per
cent. gelatin jelly δ = 1·069; δ calculated was 1·041. He attributed
the whole of the compression to the water, so that 1 c.c. of water in a
10 per cent. gelatin jelly occupied a volume of 0·96069 c.c. See also
par. 4 p. 68.

  [50] Wiedemann’s Annal d. Physik, xxxv. p. 352 (1888).

It may be of interest to give the figures used in the preceding lines
in tabular form.

    ---------------------------------------+----------+------------
                                           |Limed skin|Puered skin
    ---------------------------------------+----------+------------
    Density of wet skin                    |   1·063  |   1·053
    Per cent. of water found               |  80·63   |  78·2
        "        "     calculated          |  79·50   |  79·9
        "     of ash on wet skin           |   1·71   |   1·17
        "     of fat  "    "               |   0·151  |   2·6
    Density of dry ash-free skin substance |   1·3970 |   1·3356
    ---------------------------------------+----------+------------

In addition to the diminution of the density of the skin by puering,
the increase of fat is very marked.

The percentage of water referred to the wet skin may be calculated from
the formula

    ((_v__{2} - _v__{1})/(1 - _v__{1})) x 100

    where _v__{1} = specific volume of the dry skin.
          _v__{2} = specific volume of the wet skin.

The specific volume of the dry skin (_v__{1}) may be calculated from
the specific volume of the wet skin (_v__{2}), the percentage of water
(_w_) being known by the following formula:--

    _v__{1} = (_v__{2} - (_w_/100))/(1 - (_w_/100)),

and by comparing the volume thus obtained with the volume determined by
direct experiment, the amount of contraction is ascertained. In the
case we have been considering (viz. limed skin with 80 per cent. water)

    _v__{2} calculated = 0·955
    _v__{2} found      = 0·941

The specific volume of the dry skin

    _v__{1} calculated = 0·6955
    _v__{1} found      = 0·8112

The difference for the dry skin is surprising, and considerably
greater than anticipated, but has been verified by a large number of
experiments. It may be remarked that the exact determination of the
density of skin by drying out pieces is liable to error on account of
the non-homogeneity of the pieces. There appears to be some chemical
combination between the water and the skin very similar to that
between water and alcohol, and although the figures obtained in the
above experiments by this method are accurate, it is not certain that
they represent the true density. It is possible that the interior of
the skin may still contain some moisture, and, in order to arrive at
absolute certainty, it would be necessary to powder the skin, and
ascertain its density in a finely divided condition.

When this is done, the results obtained are very much more concordant
than when pieces of skin are used. The following results were obtained
for the density of dry hide powder (as used for analysis by the
I.A.L.T.C.):--

    In petroleum ether         1·2568
    In carbon tetrachloride    1·2570
    In alcohol                 1·2580
    Mean                       1·2572

This is equivalent to a specific volume _v__{1} = 0·7954.

The density of the powdered skin may also be determined in the air
volumenometer (Say; Kopp. Kohlrausch Physical Measurements, p. 53).

*Swelling and Falling.*--The skin, in its living condition on the
healthy animal, is the most supple and perfect of coverings, and in
producing soft leathers it is the object of the tanner to retain this
supple condition. To do this, the swollen fibres must, as we have
seen, be got back to their natural state. The phenomena of swelling
and depletion of skin have been discussed by Körner,[51] and recently
Prof. H. R. Procter[52] has published a paper which goes into the whole
theory of colloidal swelling. In order to understand depletion, which
is the opposite of swelling, it is necessary to consider what takes
place when a body like skin is swollen. I am indebted to Dr. Th. Körner
for the following summary of the phenomena of swelling. There are three
types of swelling: 1. Capillary attraction; 2. Endosmose; 3. Molecular
imbibition.

  [51] Th. Körner, 10 Jahres. Bericht, d. Deutsch. Gerberschule zu
  Freiberg, 1899, p. 32.

  [52] Procter, Ueber die Einwirkung Verdünnter Sauren und Salzlösungen
  auf Gelatine, Kolloid Chem. Beihefte, 1911, ii. p. 243.

The last named is of the greatest importance in tanning. Körner (loc.
cit.) enumerates certain principles governing molecular swelling.

1. A body capable of swelling, when put into water, absorbs a
_definite_ quantity of the water up to a maximum, which cannot be
exceeded. (C. Ludwig.)

2. The maximum of swelling depends upon the chemical composition of
the body, on its cohesion and elasticity, and on the temperature and
interior pressure of the liquid. (C. Ludwig.)

3. Power of resistance to swelling increases from the exterior to the
interior, according to a parabolic law; i.e. the external layers of the
body attain the maximum swelling sooner than the internal portions. (L.
Mathiessen and A. Schwarz.)

4. The volume of the swollen body is smaller than its original volume,
plus that of the liquid absorbed. (Quincke.)

5. Swelling is accompanied by development of heat.[53]

  [53] Duvernoy, E. Wiedemann, and Lüdeking, Wied. Ann. xxv. 1885, p.
  145.

The production of heat is simply due to the contraction, and not to any
chemical phenomenon, such as hydration.[54] This explains a fact well
known to tanners, viz. that skins swell in cold water and “fall” in
warm water. Riecke[55] concludes that the degree of swelling, _m__{2}/M
(where _m__{2} = mass of water absorbed, M = mass of the body swollen),
in a space filled with aqueous vapour, unsaturated, is a function of
the pressure and temperature.

  [54] Rodewald. Thermodynamik der Quellung, Zeit. f. Phys. Chem. xxiv.
  1897, p. 193.

  [55] Zur Lehre von d. Quellung, Wied. Ann. liii. 1894, p. 564.

The velocity of swelling (Pascheles) may be expressed by the formula--

    _d_Q/_dt_ = (M - Q)K

where M = maximum of swelling, Q = amount of swelling in the time _t_,
and K = a constant.

The differential quotient _d_Q/_dt_ gives the velocity for each moment,
and it will be seen that the swelling becomes slower and slower as the
maximum is approached. Thus, the law of the velocity of swelling is
identical with that of the velocity of inversion of cane sugar, itself
an application of the law of masses.[56]

  [56] Nernst and Schönfliess. Einleitung in die mathematische
  Behandlung der Naturwissenschaft, München 2 Aufl. 67.

For every process of swelling, the constant K must be determined
experimentally from observation of M, and it may be shown that--

    K = 1/_t_ log(M/(M - Q))

whence the value of K may be calculated for each series of
determinations.

On the manner in which water is held in swollen colloid bodies, three
hypotheses have been put forward.[57]

  [57] See also Colloidal Chemistry, H. R. Procter, M.Sc., Brit. Assoc.
  Reports, Dublin, 1908.

1. The hypothesis that colloids have a structure in the form of a
honeycomb. (Bütschli.)

2. The water is absorbed at the surface of colloids in a specially
condensed form.[58]

  [58] Wilhemy. Pogg. Ann. cxix. pp. 121, 122.

The water forms, with the swollen body, a “solid solution.” (Nägeli.)

Swelling, and its opposite, contraction, are connected with the surface
tension between the swelling or contracting bodies and the surrounding
solution. With diminished surface tension, the surfaces of contact
between the two become greater, i.e. swelling takes place, with
simultaneous diminution of the volume of the whole system, and vice
versa.

For the absorption phenomena which occur, the following relation holds
good: When a substance in solution diminishes the surface tension at
the dividing surface, its concentration is increased; it is absorbed.
When a substance in solution increases the surface tension at the
dividing surface, its concentration is diminished.

In the system water-hide substance the researches of Wiedemann and
Lüdkeing have shown that swelling is accompanied by evolution of
heat. Since a rise of temperature is favourable to a system formed by
absorption of heat, it therefore hinders swelling and vice versa. This
is confirmed by practical experience, and most bating operations are
conducted at temperatures between 35° and 40° C.

With regard to the influence of pressure on swelling, a similar law
holds good as for temperature, which may be expressed as follows.
When a chemical system is compressed at constant temperature, its
equilibrium is shifted in that direction by which the reaction is
accompanied by diminution of volume. Quincke, however, has shown
what appears to be a paradox, namely, that swelling is accompanied
by diminution of volume, i.e. the swelling substance, plus the water
taken up, occupies a smaller space than the sum of the two constituents
taken together; therefore, an increase of pressure must be favourable
to swelling, and conversely a diminution of pressure is favourable to
“falling.”

We must clearly bear in mind that in all cases of swelling, it is the
entry of water into the system which is the cause of the swelling, and
since puering is a process which acts in a contrary direction, i.e. the
skin falls, this means that water is expelled from the system, skin
plus water. By direct determination, from 3 to 8 per cent. of water is
expelled during puering. Calculations made from density determinations
are masked by the fact, above referred to, that the density of the skin
changes during puering.

A fallen skin contains less water than when in the swollen condition,
but the difference between the percentages of water in the skin in the
two states is only relatively small, and obviously insufficient in
itself to account for the great difference in the physical properties
of the skin in the two conditions. It is evident, therefore, that
the state in which the water is held in the two stages must be
different. It is reasonable to suppose that, in the case of swollen
skin substance, the whole of the capillary cavities of the organized
structure of the skin are completely filled with water, which, owing to
its incompressible nature, confers upon the skin elastic and unyielding
properties which it always possesses in this condition. The water,
entering the organized cell structure in the first place by osmotic
pressure, is fixed there and confers upon the skin, in part, its own
property of incompressibility. It is a well-recognized fact that,
during the puering process, large quantities of the skin substance are
dissolved by the action of the enzymes in the bate, and it is probable
that the finer organized structure is first attacked. The walls of the
cells which hold the water are partly destroyed, leaving the skin with,
quite possibly, the same amount of water contained in it as a whole,
but with the water dispersed at large throughout its interior structure
instead of being held in the fibres. The result of this is, that when a
small area of the puered skin is subjected to local pressure the water
in the portion undergoing compression is free to move into the adjacent
portions of the skin, and there is no force acting upon it, except that
of capillarity, to cause it to return to that area when the pressure
is removed from it. When a portion of a puered skin is compressed,
the surrounding part is always swollen to an equivalent amount. That
this view is, in the main, correct is supported by an observation of
Abt (Paris), that, while cell nuclei may be demonstrated in the layers
near the epidermis of an unpuered skin, they are entirely absent from a
puered skin, showing that the puer has completely dissolved the nuclear
structure of the cells.

The swelling caused by alkalis is of a different nature to the swelling
caused by acids. Procter has pointed out[59] that alkaline swelling is
not repressed by sodium chloride, even when caused by sodium hydrate,
but is repressed by sufficient concentration of the hydroxyl ion in the
outer solution.

  [59] Colloidal Chemistry, p. 19.

Müller[60] points out that jellies containing certain non-electrolytes
(glucose, glycerine, alcohol) render the diffusion of soluble matter
much slower than pure jellies of the same concentration, but the
presence of urea favours the permeability of gelatin and agar jellies.
The differences in the velocity of diffusion of various salt solutions
through jellies of the same strength, are thus not due altogether to
the greater diffusibility of the particular salt solution, but are to
be attributed to the influence of the salt on the permeability of the
colloidal medium. Limed skin treated with a 1 per cent. solution of
urea at 43° C. is depleted or “falls,” but both the solution and the
skin remain alkaline.

  [60] Allgemeine Chem. d. Kolloide, p. 13.

Přibram[61] has recently shown that the swelling and contraction of
muscle are to be ascribed to absorption and expulsion of water from
the cells of the muscle; the rapid changes taking place, are brought
about by changes in the concentration and composition of the liquid
components of the muscle. Lactic and phosphoric acids are produced with
enormous rapidity, and of relatively high concentration; during a short
period these cause instantaneous changes in the swelling of the muscle
cells, followed by a return to their original state. For the evidence
of this the original paper must be consulted, but it goes to show that
the equilibrium of the system--colloids, electrolytes, water--depends
on the proportions and quantities of each of these constituents. The
phenomena we are considering thus conform to the general law of mass
action (Guldberg and Waage).

  [61] Přibram, Die Bedeutung der Quellung und Entquellung für
  physiologische und pathologische Erscheinungen. Kolloid Chem.
  Beihefte, ii. p. 1.

*Influence of Solid Matter in the Bate.*--It was first pointed out by
Wood (J.S.C.I. 1899, p. 991) that a filtered puer liquor had less
action on the skin than an unfiltered liquor containing much finely
suspended matter.

On adding an inert solid (kaolin) to the filtered bate, the action was
hastened. No doubt much of the colloidal matter, and with it some of
the enzymes, are removed by filtration, but nevertheless, the suspended
particles in the bate have some effect on the process.

In a most interesting paper, Perrin[62] has recently shown, that
the granules in suspension in a colloidal liquid function like the
invisible molecules of a perfect gas with a molecular weight of 3·3
x 10^{-9}. Such granules are endowed with the molecular (Brownian)
movement, and therefore may exert a mechanical effect on the fibres
of the skin. Although the mass of the particles is large[63] compared
with molecular dimensions, they are small enough to penetrate the
pores of the skin, and where puering is carried too far, may become
deposited beneath the fine hyaline layer, and thus render it cloudy and
unsuitable for fine colours.

  [62] Perrin, Kolloid Chem. Beihefte, i. p. 221.

  [63] A particle of gamboge in a colloidal solution has a mass 10^9
  times that of a molecule of hydrogen.

*Hydrogen ion Concentration of Puer Liquors.*--It has already been
shown, in Chapter II., that fresh puer liquors have a certain acidity
(7 c.c. N/10 per 100 c.c.) at the commencement of the operation, but
that at the end they are alkaline (3 c.c. N/10 per 100 c.c.). If an
artificially acid liquor be made by diluting hydrochloric acid until it
shows the same number of c.c. by titration, it will be found far too
“strong,” and will swell the skins. This brings us to the consideration
of what is meant by the strength of acids.

Procter and Jones[64] have drawn attention to the point in their paper
on “Acids in Tan Liquors.” As is well known, the ionic theory affirms
that degree of acidity depends on the concentration of hydrogen ions, a
strongly acid solution being one in which the hydrion concentration is
great, an alkaline solution one in which it is extraordinarily minute,
and if we adopt pure water as our standard of neutrality, a neutral
solution is one in which the hydrion concentration is approximately
10^{-7} normal. A normal solution of hydrogen ions would contain 1 gram
of hydrogen ions per litre; in the case of hydrochloric acid this would
equal 1·35 N/1 HCl.

  [64] Acids in Tan Liquors, Journ. Soc. of Chem. Ind., 1910, p. 1354.

Sand and Law,[65] and Wood, Sand and Law,[66] have described the mode
of estimating the hydrogen ion concentration in tan liquors directly
by means of the electrometric method, and this method is especially
applicable to the estimation of the hydrogen ion concentration in
puer liquors. It can also be used to titrate the liquors, and we have
already given some of the results in Chapter II.

  [65] Journ. Soc. Chem. Ind., 1911, p. 3.

  [66] Ibid., 1911, p. 872.

The method is based on the theory of Nernst, that the difference of
potential between a metal plate and the solution of one of its salts
into which the metal is dipping, depends on the osmotic pressure of
the free ions of that metal in the solution (in other words, on the
concentration of the solution). In the case of hydrogen ions, we use
a plate of platinum coated with platinum black and saturated with
hydrogen, and the difference of potential depends, therefore, on the
concentration of the hydrogen ions in solution.

[Illustration: Fig. 9.--Diagram of Connexions of Electrometric
Apparatus.]

The accompanying figure (Fig. 9) shows the hydrogen electrode I, and
the auxiliary electrode II, drawn to scale,[67] on the right, whereas
the electrical apparatus is explained diagrammatically on the left.

  [67] An improved form of hydrogen electrode devised by Dr. H. Sand
  may now be obtained from Universitäts-mecanikes Fritz Köhler,
  Leipzig. It is shown at I in Fig. 10.

The principle of the method of measurement consists in connecting the
two ends P and Q, of a sliding rheostat to the terminals of a dry cell,
D, and balancing the potential-difference to be measured against the
potential-difference between one end, P, and the slider, S, by means
of a special form of enclosed capillary electrometer, E. The value of
this potential-difference is read directly on a delicate voltmeter, V.
The connexions, which are found ready-made in the box, have been drawn
out, whereas, those to be made by the operator are shown by dotted
lines. The steps to be taken by the latter, consist first in taking off
the capillary electrometer and manipulating it in such a manner, that
on returning it into position the capillary may be partly filled with
a thread of mercury and partly with the acid. The terminals, X and Y,
marked battery + and −, are connected to a dry cell, and the terminals,
Z and U, marked cathode and auxiliary respectively, to the hydrogen and
calomel electrode. Very careful insulation of the connexion between the
terminal marked auxiliary and the calomel electrode is necessary. The
hydrogen is passed through the hydrogen electrode until a constant P.D.
between it and the calomel electrode is obtained. This P.D. is measured
by moving the slider up and down until no movement of the mercury in
the capillary electrometer is observed on depressing the key K marked
electrometer.

Fig. 10 is a view of the apparatus as set up for the titration of
a puer liquor. H is a cylinder of compressed hydrogen;[68] I, the
hydrogen electrode, dipping into the beaker C containing the liquor for
titration; _b_, the burette, containing N/10 acid or alkali; II, the
auxiliary electrode, the capillary of which is also seen dipping into
the beaker C; P, the potentiometer box containing the sliding rheostat
S and electrometer E; D, a dry battery. The acid or alkali is run in
from the burette until the voltmeter shows 0·69 volts, indicating that
the liquid is neutral or has a hydrogen ion concentration of 10^{-7}.

  [68] It is necessary to use pure hydrogen. This may now be obtained
  commercially from the Knowles’ Oxygen Co., Wolverhampton.

[Illustration: Fig. 10.--Electrometric Apparatus.]

The following table shows some results obtained on puer liquors before
and after skins have been put through. _p__{H+} has been called by
Sorensen the exponent of the hydrogen ion concentration C, and is
defined by the equation

    _p__{H+} = log(1/C)

i.e. it is equivalent to the logarithm of the reciprocal of the factor
of normality of the solution with respect to the hydrogen ions.[69]
_p__{H+} for pure water or a neutral solution is 7, corresponding
to 0·69 volts. The measurements were made on filtered puer liquors,
using a N/1 KCl calomel electrode as auxiliary, the capillary of the
electrode being filled with 3·5N potassium chloride:--

  [69] The formula for calculating _p__{H+} for the N/1 auxiliary
  electrode used is as follows:--

    Since π = (0·283 + 0·0575 log(1/C) volt)

    log(1/C) = _p__{H+} = (π - 0·283)/0·0575

    π = potential difference found

    C = concentration of hydrogen-ions.


    ---+------------------+--------+---------
       |   Before goods.  |   After goods.
       +--------+---------+--------+---------
    No.| Volts. | _p__{H+}| Volts. | _p__{H+}
    ---+--------+---------+--------+---------
     1 |  0·560 |   4·7   |  0·755 |   8·16
     2 |  0·585 |   5·16  |   --   |    --
     3 |  0·600 |   5·4   |  0·658 |   6·44
     4 |  0·595 |   5·35  |  0·710 |   7·35
     5 |  0·600 |   5·4   |  0·725 |   7·6
     6 |   --   |    --   |  0·770 |   8·35
    ---+--------+---------+--------+---------

No. 6 was a very old “spent” liquor. The mean hydrogen ion
concentration before goods was 0·588 volt, i.e. the concentration
was 10^{-5·32} normal equivalent to 0·00000479 grm. per litre of
hydrogen ions. Therefore the value of _p__{H+} was 5·32. A solution of
hydrochloric acid of the same strength by titration consumed 0·7 c.c.
N/1 alkali per 100 c.c. Measured by the electrometric apparatus, it
showed 0·410 volt, corresponding to _p__{H+} = 2·1, or a hydrion
concentration of ·0079N. In other words, the HCl solution has an
acidity or strength 1600 times that of the puer liquor.

The mean hydrogen ion concentration of the liquors after goods was
0·000000076 grm. per litre, corresponding to 0·715 volt and _p__{H+} =
7·12, i.e. the liquor was alkaline to a slight extent. For comparison
saturated lime-water gave a reading of 1·01 volt, corresponding to
_p__{H+} = 12·5.

The hydrogen ion concentration is of the greatest importance for the
proper action of the enzymes in the bate;[70] we shall, however, treat
of this in Chapter VII.

  [70] _Vide_ Sœrensen, Sur la mesure et l’importance de la
  concentration des ions hydrogène dans les réactions enzymatiques.
  Comp. rend. Lab. de Carlsberg, 8me. vol. 1^{ere} livraison,
  Copenhagen, 1909.

*Conductivity of Puer Liquors.*--It was thought of interest to examine
the electrical conductivity of puer liquors in actual use, in the hope
that the numbers obtained might give some useful indications. It was
found that the conductivity increased, as might be expected from the
lime going into solution, but the difficulties of the method render it
of less use than ordinary chemical analysis. The results of a typical
liquor are given here as a record--

Conductivity (K) of liquor before goods

    0·00316(1/(ohm × cm.))

Conductivity (K) of liquor after goods

    0·00423(1/(ohm × cm.))

The difficulty of expressing the complex reactions of puering
numerically is, we have seen, very great, for, as Minot[71] says, “with
human minds constituted as they actually are, we cannot anticipate that
there will ever be a mathematical expression for any organ or even a
simple cell, although formulæ will continue to be useful for dealing
now and then with isolated details. Nevertheless, the value of graphic
methods to every student of science has been immense.”

  [71] Minot, Address to Amer. Assoc. for Advancement of Science,
  Minneapolis, Dec. 29, 1910. Nature, 1911, p. 96.

It has long been my endeavour to express quantitatively the degree to
which a skin has fallen. My friend Dr. Sand has suggested that this may
be done by subjecting a piece of the skin successively to increasing
and then decreasing pressures, and measuring the thickness under each
load. Experiments carried out with the apparatus described below show
that a limed skin treated in this way is first compressed, and then on
releasing the pressure recovers more or less of its former thickness,
according to the amount of plumping it has received, i.e. it shows a
certain amount of resilience. A well-puered sheep-skin, on the other
hand, shows no resilience at all, i.e. on releasing the pressure the
whole of the compression persists. In the case of an ox-hide subjected
to a bate of hen-dung, a slight recovery takes place on releasing the
pressure. This accords with the fact that it will never be possible
to puer a thick ox-hide so effectively as a thin sheep-skin. A piece
of india-rubber, on the other hand, is completely resilient, i.e. it
wholly recovers its thickness on releasing the pressure. The relative
thickness of the same skin in the limed and puered conditions under
varying loads is also of interest. The process of puering may, as
a rule, be taken to reduce a limed skin to between two-thirds and
one-half of its thickness in the swollen condition. If both limed and
puered skin be then subjected to the same load, the puered skin will at
first be compressed very much more than the limed one. This is probably
due to the expulsion from it of water, held simply by capillary
attraction. On further increasing the load, however, the compression
decreases greatly in the case of the puered skin; with both limed and
puered skin increase of compression ultimately becomes practically
proportional to increase of pressure, and is slightly greater with the
former than with the latter.

The table gives representative results obtained on the same sheep-skin
(roan) in the limed and in the puered condition. These results are
expressed graphically in Fig. 12.

    ------------+--------------------------+-------
        Load    |      Thickness in mm     |   Δ
     per sq.cm. +------------+-------------+-------
       in grm.  | Limed skin | Puered skin |
    ------------+------------+-------------+-------
           0    |    3·45    |    1·78     |  1·67
          20    |    3·43    |    1·58     |  1·85
          40    |    3·33    |    1·43     |  1·90
          60    |    3·28    |    1·35     |  1·93
          80    |    3·22    |    1·23     |  1·99
         100    |    3·15    |    1·13     |  2·02
         120    |    3·08    |    1·08     |  2·00
         140    |    3·03    |    1·05     |  1·98
         160    |    2·95    |    1·01     |  1·94
         180    |    2·90    |     ·98     |  1·92
         200    |    2·83    |     ·95     |  1·88
         300    |    2·73    |     ·88     |  1·85
       All off  |    2·98    |     --      |   --
         380    |    2·61    |     ·82     |  1·79
         600    |    2·43    |     ·76     |  1·67
       All off  |     --     |     ·76     |   --
    ------------+------------+-------------+-------

    Δ = difference in thickness of the skin--i.e. compression under the
    same load.

[Illustration: Fig. 11.--Apparatus for Measuring Degree of Falling

    _m_ = micrometer dial.

     JJ = jaws between which the skin is placed.

    _b_ = balance-weight attached to cord passing over pulleys _p p_,
    to counterbalance weight of frame _f f_.

    _k_ = knife edge supporting pan and weights _w_.]

Fig. 11 shows the apparatus[72] that was employed to obtain these
results. It consists essentially of a commercial form of micrometer
for measuring the thickness of leather. To one of its jaws a pan for
weights is attached, by means of the frame _f f_, in such a manner as
to secure a perfectly straight pull. The weights of the frame and pan
are counterbalanced in the manner shown by a counterpoise _b_. The
delicacy of measurement may be increased by inserting larger jaws in
the form of suitably fashioned disks, but even when this is done the
results are to a certain extent vitiated by the rather considerable
friction of the micrometer.

  [72] The apparatus was constructed by Mr. W. Linney, of the
  University College, Nottingham.

  Since this apparatus was constructed Professor H. R. Procter has
  informed me that an appliance devised by Mr. Anderson, of the Leeds
  University, for measuring the thickness of leather under varying
  pressures, has been in use in the leather industries department
  for some months for the purpose of measuring the proportion of the
  permanent to elastic compression.

[Illustration: Fig. 12.--Curve obtained by Measuring Apparatus.

(The dotted lines show the pressure on taking off the load.)]

An apparatus free from this fault is shown in Fig. 13. It consists
essentially of a counterbalanced lever A, to which the upper jaw J is
rigidly attached. By means of a sliding weight W, any desired load,
from zero upwards, may be put on this jaw. The lever carries a very
delicate spirit level, which allows it to be set accurately horizontal
in every experiment. The lower jaw is movable vertically between
parallel guides, and its position is controlled by the screw-wheel S
which bears a divided circle on its circumference. The position of this
wheel, and therefore of the lower jaw, may be accurately read on the
vernier _v_. In every experiment it is adjusted so as to make the upper
lever accurately horizontal.

[Illustration: Fig. 13.--Improved Apparatus for Measuring the Degree of
Falling. (Sand.)]




CHAPTER IV.

THE BACTERIOLOGY OF THE BATE.


    “Omne vivum ex ovo.”--HARVEY (1578–1675).

When a drop of liquid from a puer wheel in use is examined under the
microscope[73] with 1/12 o.i. objective, it is seen to be swarming with
bacteria.

  [73] A good microscope, with 1/12 oil-immersion objective and Abbe
  condenser, is necessary for bacteriological work. The new dark-ground
  condenser made by James Swift and Son, London, is extremely useful
  for examining living bacteria under high magnifications.

The majority are short rods (bacilli), but other forms, cocci and
spirilli, are seen in lesser numbers. Most of these bacteria move
briskly in the liquid; as the temperature of the slide sinks, their
movements become slower, and finally cease. The illustration, Fig. 14,
shows the various forms of bacteria observed by the author in puer
liquors × 1000 diam.

[Illustration: Fig. 14.--Various Forms of Bacteria in Puer Liquors.]

The living bacteria are best examined in a drop culture in the
following manner. A clean cover-glass, of the proper thickness for the
objective to be used, is laid upon a black glass plate. With a platinum
loop, previously heated to redness in the flame, a drop of sterile
physiological salt solution (0·6 to 0·75 per cent.) or sterile broth
is placed in the centre of the cover-glass. With a platinum needle a
minute quantity of the puer liquor is stirred into the drop. A slide
with a depression in the centre is taken, the edge of the depression
painted round with vaseline, and pressed over the cover-glass, so that
the drop is exactly central. If the whole be now turned smartly over,
the drop will hang central in the hollow space.

If the ring of vaseline is continuous, and the cover well pressed
down, the drop is preserved from evaporation, and the bacteria may be
examined in their natural condition--best on the edge of the drop.

For illuminating the drop culture, the concave mirror is used, and a
small diaphragm without condenser; whereas, for stained preparations,
the flat mirror is used in conjunction with the Abbe condenser.

If the cover-glass be carefully removed, and dried under a bell glass,
the culture may be preserved in a dry condition, or may be stained and
mounted.[74]

  [74] The most recent method is to preserve the slides without
  cover-glasses: a drop of cedar oil is placed directly on the
  preparation, and, after examination, carefully washed off with xylol.
  Care should be taken not to touch the preparation with the fingers.

If the dried preparation is on a cover-glass, it should be held in the
fingers (prepared side upwards), and passed slowly three times through
the flame of a Bunsen burner. By holding the preparation in this way,
the exact temperature for proper fixation is obtained.

A drop of fuchsin stain, or gentian violet, is allowed to remain on the
preparation for five minutes; wash off the superfluous dye with water,
and examine, either in the wet state or after drying and mounting in
balsam.

[Illustration: Fig. 15.--B. Coli Commune. Stained to show Flagellae.]

For a detailed account of the technique of staining and mounting, the
following works may be consulted:--

  Methods and Formulæ. P. W. Squire. (Churchill.)

  Taschenbuch für den bakteriologischen Praktikanten. Dr. Rudolf
  Abel. (Stubers-Verlag, Würzburg.)

  Technique Microbiologique. Nicolle and Remlinger. (Octave Doin,
  Paris.)

  Practical Bacteriology. Kanthack and Drysdale. (Macmillan, London.)

The recent researches of Tissier and Metchnikoff have shown that
the flora of the intestines, both of men and animals, consist very
largely of anaerobic bacteria. These have been overlooked in previous
researches, owing to imperfect means of studying this class of
organisms. Indeed, in one work on the microbes of the alimentary canal
of the dog, no mention was made of them, whereas they are all very
active.

Most of these organisms, and the new methods by which they have been
isolated, are fully described in a new work entitled “Les Anærobies,”
by M. Jungano and A. Distaso, of the Pasteur Institute, Paris.[75]

  [75] Masson et Cie, Publishers.

The following bacteria have up to the present been isolated from dung
(mostly dog dung), and studied in pure cultures:--

   1. _Micrococcus ureae_ (Cohn). (Pasteur.)
   2.      "       _fulvus_ (Cohn).
   3.      "       _prodigiosus._
   4.      "       _ureae liquefaciens._
   5. _Bacterium sulphureum._
   6.      "     _coli commune._ (Fig. 15.)
   7.      "     _coli anindolicum._
   8. _Bacterium coli anaerogenes._
   9.      "     _furfuris_ α (Wood). (Fig. 30.)
  10.      "     _furfuris_ β (Wood).  (Fig. 31.)
  11. _Bacillus fluorescens putridus._
  12.      "         "          "      _liquefaciens._
  13.      "    _subtilis._
  14.      "    _saprogenes_ (Herfeld), three varieties.
  15.      "    _butyricus_ (Hueppe). (Fig. 23.)
  16.      "    _putrificus._ (Fig. 19.)
  17.      "    _pyocyaneus._
  18.      "    _janthinus._
  19.      "    _coprogenes fœtidus._
  20.      "    _pyogenes fœtidus_ (a variety of _B. coli_).
  21.      "    _zenkeri._
  22.      "    _magnus._
  23.      "    _spinosus._
  24.      "    _liquefaciens_ (Eisenberg, Frankland).
  25.      "    _amylobacter_ (Van Tieghem).
  26.      "    _acidi paralactici._
  27.      "    {I.  } Isolated from horse manure by Severin,
  28.      "    {II. } Centr. Bl. f. Bakt. (2), i., 97.
  29.      "    {III.}
  30.      "    from horse dung (anaerobic) Severin, Centr. Bl. f.
                    Bakt. (2) iii., 708.
  31.      "    from horse dung (anaerobic), No. 3, ditto.
  32.      "    _oedematis maligni_ (Vibrion Septique, Pasteur).
  33.      "    _mesentericus vulgatus._
  34.      "    _lactis aerogenes._
  35.      "    _cavicida_ (Brieger).
  36.      "    _albuminis_ (Bienstock).
  37.      "    _Bienstockii._
  38.      "    _tenuis._
  39.      "    _enteritidis sporogenes_ (Klein).
  40.      "    _lactis acidi_ (Ankerschmid, 1905).
  41.      "    _megatherium._
  42.      "    _cadaveris sporogenes_ (Klein) said to be identical
                    with No. 16.
  43.      "    _thermophilus._ (Houston).
  44.      "    _a._ from puer. See p. 162.
  45.      "    _b._  "    "        "
  47. _Bacillus mycoides._
  48–61. 14 species isolated from dog and pigeon dung by Prof.
             H.  Becker. Zeit. f. Offentlich. Chemie. Heft xxiii.
             Jahrgang X. p. 447, includes _B. erodiens_ (Fig. 16).
  62. _Sarcina fimentaria_ (Lehmann and Neumann).
  63. _Streptococcus_ from sewage. (Houston.)
  64.      "          _brevis._
  65.      "          _longus._
  66.      "          _pyogenes._
  67.      "          _liquefaciens coli._ (Gamgee Phys. Chem. 2.)
  68. _Streptothrix_ from stable manure. (Severin, 6.)
  69. _Spirillum serpens_ (Kutscher).
  70.      "     _tenue_       "
  71.      "     _undula_      "
  72.      "     _volutans_    " (Figs. 24 and 25).
  73.      "     from pig dung. Smith, Centr. Bl. f. Bakt. 16, (1),
                    124
  74–76. _Vibrio_, three species isolated by Kutscher.
  77. _Clostridium butyricum_ (Prazmowski), said to be identical with
                    No. 25.
  78. _Streptococcus faecalis._ Sidney Martin, 37 and 38; Ann. Rep.
               Loc. Gov. Board, 1907–9; Nature, March 3, 1910, p. 22.
  79. _Bacillus bifidus._
  80.      "    _perfringens._
  81.      "    _bifermentans._
  82.      "    _funduliformis_ (Veillon).
  83.      "    _capillosus._
  84.      "    _sporogenes._
  85.      "    _ventriosus._
  86.      "    _rodella III._
  87. _Staphylococcus parvulus._
  88. _Diplococcus orbiculus._
  89. _Coccobacillus preacutus._
  90. _Coccobacillus oviformis._
  91. _Bacillus faecalis alkaligenes_ (Petruschky).

     (79–90 are anaerobic bacteria, described and figured by
                   Jungano and Distaso.)

It will be surmised from the above list, to which additions are still
being made, that the flora of the intestines is pretty extensive, and,
consequently, the study of the part played by the various species of
bacteria is a long and difficult one.

The methods of isolating these bacteria, and the compositions of the
media employed, would demand a treatise on bacteriology; but, for
general purposes, a good liquid medium for the cultivation of puer
bacteria is a gelatin peptone broth, made by digesting 10 grm. gelatin
with 6-1/2 grm. 80 per cent. lactic acid in 100 c.c. water under
pressure for three hours, neutralizing with ammonia, adding 1 grm.
potassium phosphate, making up to 1000 c.c., and filtering. A sterile
infusion of fresh dung may be used, but it is troublesome to prepare
and not easy to get uniform in strength or composition. The culture
liquids are left slightly alkaline, an alkalinity equal to 0·0636 per
cent. Na_{2}CO_{3} or 12 c.c. N/1 soda per litre. The amount of alkali
may be increased to 0·15 per cent. Na_{2}CO_{3} without affecting the
growth of the bacteria. Of solid media, 10 per cent. nutrient gelatin,
or in summer 15 per cent., is good if used at temperatures below 25° C.
For higher temperatures, up to 39° and 40°, nutrient agar is required.
The best nutrient gelatin for general work is made according to Klein’s
formula.[76] For media in general, a most useful compendium is Abel’s
Taschenbuch.

  [76] Klein, E., Micro-organisms and Disease (Macmillan), p. 22. See
  also, Kanthack and Drysdale, Practical Bacteriology (Macmillan 1896),
  p. 86.

The number of bacteria in fresh fæces varies greatly, but is of the
order of 10,000,000 per grm. of dry matter, capable of developing in
nutrient gelatin. Of this number, about 100,000 are spore-bearing
organisms. This estimate applies to healthy animals; in a diseased
condition, the numbers vary enormously.

Dr. A. C. Houston found in raw London sewage from 3,000,000 to
9,000,000 microbes per c.c., of which more than one-tenth were
gelatin-liquefying organisms. There were only about 300 spores of
aerobic bacteria, about 100,000 B. coli, 100 B. enteritidis sporogenes,
and streptococci, in one gram of fæces.

With the object of ascertaining the effect of the various species of
bacteria contained in the dung upon skins, a large number have been
isolated, and the effect of pure cultures in different media has been
tried upon skin.[77] A number of the results have been published in
the Journal of the Society of Chemical Industry. Professor H. Becker,
who has done a great deal of this part of the work, is of opinion that
the principal organisms concerned in the bating exist in the dog’s
intestines, and belong to the group of coli bacteria. These are very
widely distributed bacteria, and are found in the large intestines of
mammals, and, as a consequence, in almost all soils, and in the mud of
rivers and lakes. The principal variety is B. coli commune.

  [77] The subject is not so far removed from direct human interest as
  one might suppose. For many years Metchnikoff, in Paris, has been
  studying the bacterial flora of the human intestines. His theory is,
  that old age is caused by the poisonous products of these intestinal
  bacteria, and he proposes to counteract the effects of these
  poisonous organisms, by introducing into the system large quantities
  of lactic acid bacteria, either in the form of tabloids, or in the
  form of sour milk cultures.

Lortet found it, along with other organisms, in the mud of the Lake of
Geneva, at a spot where the water was chemically very pure. Dr. A. C.
Houston, the bacteriologist of the Metropolitan Water Board, enumerates
sixteen varieties of this organism, 80 per cent. of which produced acid
and gas in lactose-peptone cultures, indol in peptone-water cultures,
and when grown in milk produced acid and clot. The bacterium (Fig. 15)
resembles that of typhoid fever, and has frequently been mistaken for
it. It is, however, much more resistent to destructive influences. It
is a short bacillus, possessing flagellae, by which it moves more or
less rapidly.

B. coli forms short rods 0·8 µ wide, 1 to 3 µ long. It moves somewhat
slowly by means of flagellæ, which may be demonstrated by staining with
Loffler’s method.[78] It grows equally well in absence or presence of
air, that is, it is a facultative anaerobe. Although it will grow at
room temperature, the optimum growth is at 37° C. In plate cultures the
appearance of the colonies below the surface of the gelatin is quite
different from that of the surface colonies. The former are small
round colonies, about the size of a pin head; the latter spread into a
whitish iridescent film, with irregular edges.

  [78] For the demonstration of flagellæ, the material is taken from
  a culture on solid media--a young agar culture is best. Make a
  dilution by Soyka’s method (p. 103), dry and fix on a cover-glass as
  described, p. 88, then proceed as follows:--

  1. Mordant with a mixture of 10 c.c. 20 per cent. tannin solution,
  5 c.c. cold saturated ferrous sulphate, 1 c.c. fuchsin or wool-black
  solution. This solution is put on the preparation, which is then
  heated for one minute until steam is given off.

  2. Wash under the tap, then in alcohol.

  3. Stain with anilin-water-fuchsin solution, prepared by dissolving
  the dye in anilin-water and adding 1 per cent. of a 1 per cent.
  solution of caustic soda until the liquid begins to go cloudy.

  4. Wash off the stain with distilled water.

B. coli does not liquefy the gelatin. When grown in nutrient solutions
containing sugars, it produces much acid, and at the same time gases
are given off, consisting of CO_{2} and hydrogen. If the growth in this
solution be allowed to continue a secondary fermentation ensues, and
the culture eventually becomes alkaline.

Indol is produced by B. coli, and may be demonstrated by adding to
10 c.c. of the culture, 1 c.c. of a 1/50 per cent. solution of pure
potassium nitrite; then adding a few drops of concentrated sulphuric
acid, when, if indol be present, a red coloration (nitroso-indol) is
produced. This bacterium reduces nitrates to nitrites. Cultivated
in a 1 per cent. solution of peptone, to which 1/10000 per cent. of
potassium nitrate has been added, after four hours at 37° C., the
presence of nitrite may be shown; after the growth has continued for
seventeen hours, the nitrite is further reduced to ammonia. Among other
products of B. coli, Harden found lactic, formic, acetic, and succinic
acids, ethyl-alcohol, CO_{2} and hydrogen.

In Germany, W. Lembke and H. Becker have specially investigated the
bacterial flora of the dog’s intestines. Lembke, in 1896[79] cultivated
the bacteria from the fæces of the dog, fed in various ways--bread,
meat, and fat diet--and found B. coli constantly present, although the
form of the individuals, as well as the colonies, and the intensity of
the indol and gas formation, showed great variations.

  [79] “Beitrag zur Bakterienflora des Darmes,” Archiv f. Hygiene, Bd.
  26, p. 293.

The other species of bacteria present varied with the kind of food;
this has a great influence on the flora of the intestines, which was
found to be very different when the dogs were fed on bread to what it
was when they were fed on meat.

Lembke describes two other species of bacteria closely resembling B.
coli, one of which he calls B. coli anindolicum, which, as the name
implies, gives no indol reaction; the other, B. coli anaerogenes, is
non-motile, possesses no flagellæ, and differs from B. coli by the
absence of gas production in the fermentation of sugars.

Besides B. coli, there are several species of bacteria which liquefy
gelatin, and a number of facultative organisms, whose presence is
more or less accidental. By changing the food, and introducing with
it quantities of foreign organisms, the composition of the intestinal
flora may be changed. By introducing for a considerable period B. coli
anindolicum, Lembke succeeded in entirely suppressing B. coli commune.
On returning to normal feeding, the foreign organisms in some cases
entirely disappeared.

The researches of Dr. H. Becker[80] were applied more directly to
the use of bacterial cultures for the bating of skins, and to the
elucidation of the bacterial action of dog-dung infusions. He isolated
54 varieties of bacteria from dog-dung, and tried the action of pure
cultures of many of them on a skin.

  [80] Zeit. f. Offt. Chem., Heft. xxiii. Jahr. X. p. 447.

A list of the various bacteria isolated by Becker is given in tabular
form on pp. 98–101.

[Illustration: Fig. 16.--B. Erodiens (Becker).]

[Illustration: Fig. 17.--Plate Culture from Fresh Puer.]

Professor Becker’s Bacterium No. 12, which he has named Bacillus
erodiens (Fig. 16), is undoubtedly a variety of B. coli, but has a
more rapid motion, and does not coagulate milk, although it renders it
somewhat thick. Cultivated in broth it gives off much gas, consisting
of 12 per cent. carbon dioxide, 85 per cent. hydrogen, 3 per cent.
oxygen. If glucose be added, the quantity of carbon dioxide rises to 40
per cent., and acid is produced. The most rapid growth is at 37° C.,
and at this temperature a broth culture has a distinct reducing action
on skin. According to the medium in which it is grown, it produces
acid or alkali, and thus comes under the heading of mixed bacteria. In
sugar[81] solutions acid is produced, and in proteid solutions ammonia
compounds, indol, and evil-smelling gases are given off. Thus, by
varying the medium, the effect produced may also be varied.

  [81] Dr. A. C. Houston has been kind enough to make an examination
  of B. erodiens, and to ascertain its action on various sugars. It
  produces fluorescence in neutral-red broth cultures, acid and gas
  in lactose cultures, indol, and acid, and clot in milk cultures.
  It ferments dulcite with production of acid, but _not_ cane sugar,
  adonite, inulin, inosite, salicin, or raffinose.

B. erodiens does not secrete any tryptic enzymes, hence its action
on the skin is to be attributed either to an intracellular enzyme,
or to its chemical products, which, being secreted _in situ_, have
a more favourable and powerful action than if merely added to the
bating liquid. It was for this reason that I proposed to use a mixed
culture of bacteria, especially bacteria from the sweating process (see
p. 105), which secrete a mild form of proteolytic ferment, capable
of dissolving the more easily soluble portion of the skin fibres (or
certain constituents), but not capable of attacking the hyaline layer.

  --------+-------+-------------+--------+---------------------------+------------
   No. of |       |    Shape    |        |                           |  Growth
  the Bac-| Where |     and     |Motility|     Growth on Gelatin     |    on
   terium | Found | Arrangement |        |                           | Agar-agar
  --------+-------+-------------+--------+-----------------------------------+----
      1   |Dog    |Small rods   |Lively  |In the gelatin stab-culture|A white
          |dung   |of the size  |        |the bacteria show a good   |surface
          |       |of the _Bac. |        |growth in the depth. At the|layer is
          |       |prodigiosus_.|        |surface it forms a small   |formed on
          |       |             |        |white button. The gelatin  |inclined
          |       |             |        |is not liquefied. The col- |stiffened
          |       |             |        |onies which have reached   |agar-agar.
          |       |             |        |the surface of the gelatin-|
          |       |             |        |plate spread in the shape  |
          |       |             |        |of a leaf, with a mother-  |
          |       |             |        |of-pearl-like gloss.       |
          |       |             |        |                           |
      2   | Do.   |Small rods of| Do.    |Stab-culture: The germs    |A yellowish-
          |       |the size of  |        |develop along the entire   |white layer
          |       |the hay      |        |track. Small arms extend   |is formed
          |       |bacillus.    |        |sideways into the gelatin. |along the
          |       |             |        |A white layer is formed on |inoculating
          |       |             |        |the surface. The gelatin is|stab.
          |       |             |        |slowly liquefied.          |
          |       |             |        |                           |
          |       |             |        |Gelatin-plate: When they   |
          |       |             |        |reach the surface, the col-|
          |       |             |        |onies spread out in the    |
          |       |             |        |shape of a leaf and then   |
          |       |             |        |are slightly fluorescent.  |
          |       |             |        |                           |
      3   | Do.   |Very small   | Do.    |Stab-culture: Very good    |A white
          |       |rods rounded |        |growth in depth. Very many |deposit is
          |       |at the       |        |arms extend laterally from |formed on
          |       |ends.        |        |the track of the stab into |inclined
          |       |             |        |the gelatin. Small knots   |agar-agar.
          |       |             |        |are formed at the ends of  |
          |       |             |        |the arms. A thin white     |
          |       |             |        |coating is formed on the   |
          |       |             |        |surface. Gelatin is not    |
          |       |             |        |liquefied.                 |
          |       |             |        |                           |
          |       |             |        |Gelatin-plates: The colon- |
          |       |             |        |ies located deeper down    |
          |       |             |        |appear as pale-yellow small|
          |       |             |        |round disks, which gradual-|
          |       |             |        |ly work up toward the sur- |
          |       |             |        |face and there form circu- |
          |       |             |        |lar disks which show larger|
          |       |             |        |dots in the middle.        |
          |       |             |        |                           |
      4   | Do.   |Small rods as|Slow    |The gelatin stab-culture   |Heavy white
          |       |large as hay |movement|resembles that of the hay  |deposit on
          |       |bacilli.     |        |bacillus, while the growth |the entire
          |       |             |        |in the gelatin-plate more  |surface.
          |       |             |        |resembles that of the an-  |
          |       |             |        |thrax bacillus. Threads ex-|
          |       |             |        |tend from the liquid colon-|
          |       |             |        |ies which have been let in,|
          |       |             |        |which threads are at first |
          |       |             |        |braided and twisted, and   |
          |       |             |        |later on extend straight   |
          |       |             |        |into the gelatin.          |
          |       |             |        |                           |
      7   | Do.   |Small rods   |Lazy    |Stab-culture: Strongly li- |White
          |       |similar to   |        |quefying. A white skin     |unevenly
          |       |the hay      |        |forms on the surface. Along|thin layers
          |       |bacillus.    |        |the liquefied prick are ra-||withspurs.
          |       |             |        |diations into the solid    |
          |       |             |        |gelatin.                   |
          |       |             |        |                           |
          |       |             |        |Gelatin-plate: Quickly li- |
          |       |             |        |quefying colonies which    |
          |       |             |        |form a white skin at the   |
          |       |             |        |top.                       |
          |       |             |        |                           |
     11   | Do.   |Medium-sized |Motile  |Stab-culture: A white coat-|A white
          |       |small rods.  |        |ing is formed on the sur-  |deposit
          |       |             |        |face. The gelatin is not   |along the
          |       |             |        |liquefied. The bacteria    |puncture.
          |       |             |        |grow well in the depth.    |
          |       |             |        |                           |
          |       |             |        |The colonies which come to |
          |       |             |        |the surface spread out     |
          |       |             |        |leaf-like with a mother-of-|
          |       |             |        |pearl-like gloss.          |
          |       |             |        |                           |
     12   |Dog    |Medium-sized |Lively  |Stab-culture: Grows evenly |Heavy white
          |dung   |small rods.  |        |along along the track. Ge- |deposit;
          |       |             |        |latin is not liquefied. A  |glossy.
          |       |             |        |thin glossy deposit on the |
          |       |             |        |surface.                   |
          |       |             |        |                           |
          |       |             |        |Gelatin-plates: The lower- |
          |       |             |        |lying colonies appear as   |
          |       |             |        |pale-yellow circular small |
          |       |             |        |disks. Braids are noticed  |
          |       |             |        |in some colonies similar to|
          |       |             |        |the superficial colonies of|
          |       |             |        |_Proteus_. Strong decaying |
          |       |             |        |smell.                     |
          |       |             |        |                           |
     13   | Do.   | Do.         |Motile  |Stab-culture: A thin coat- |White
          |       |             |        |ing forms on the surface.  |deposit
          |       |             |        |The bacteria grow downward |along the
          |       |             |        |bristle-like. Small buttons|puncture.
          |       |             |        |are formed at the ends of  |
          |       |             |        |the bristles.              |
          |       |             |        |                           |
          |       |             |        |Gelatin-plates: Leaf-like, |
          |       |             |        |mother-of-pearl glossy,    |
          |       |             |        |spreading.                 |
          |       |             |        |                           |
     38   |Pigeon |Small rods   |Lively  |Stab-culture: Bag-shaped   |Yellow
          |and    |             |        |liquefying of the gelatin, |spreading
          |poultry|             |        |thesame being coloured     |over the
          |dung.  |             |        |yellow.                    |surface of
          |       |             |        |                           |the culture
          |       |             |        |Gelatin-plates: The deep-  |medium.
          |       |             |        |seated colonies are gran-  |
          |       |             |        |ular, yellow. Those that   |
          |       |             |        |have forced their way to   |
          |       |             |        |the surface form white     |
          |       |             |        |glistening small buttons.  |
          |       |             |        |                           |
     40   | Do.   | Do.         | Do.    |Stab-culture: A white heavy|White
          |       |             |        |deposit is formed on the   |irregular
          |       |             |        |surface of the culture me- |deposit.
          |       |             |        |dium; grows very well along|
          |       |             |        |the track.                 |
          |       |             |        |                           |
          |       |             |        |Gelatin-plates: Leaf-like  |
          |       |             |        |deposits with line system. |
          |       |             |        |                           |
     42   | Do.   | Do.         | Do.    |Stab-culture: A white heavy| Do.
          |       |             |        |Spreading. Very good growth|
          |       |             |        |along the prick.           |
          |       |             |        |                           |
          |       |             |        |Gelatin-plates: Leaf-shaped|
          |       |             |        |deposits with line system. |
          |       |             |        |                           |
     43   | Do.   |Large,       |Not     |Stab-culture: The gelatin  |White
          |       |grouped,     |motile  |is slowly liquefied. Only  |puncture
          |       |grape-like.  |        |slight growth in the depth.|in yellow.
          |       |             |        |The culture medium is col- |
          |       |             |        |oured chamois colour.      |
          |       |             |        |                           |
          |       |             |        |Gelatin-plates: Yellow     |
          |       |             |        |disks.                     |
          |       |             |        |                           |
     44   |Pigeon |Small rods,  |Lively  |Stab-culture: A heavy white|White
          |and    |quite        |        |spreading on the surface.  |irregular
          |poultry|different    |        |Very good growth along the |deposit.
          |dung.  |in size.     |        |prick.                     |
          |       |             |        |                           |
          |       |             |        |Gelatin-plates Leaf-shaped |
          |       |             |        |deposits with the line sys-|
          |       |             |        |tem.                       |
          |       |             |        |                           |
     45   | Do.   |Small rods   | Do.    |Stab-culture: A heavy white|Very thin
          |       |             |        |spreading on the surface.  |deposit.
          |       |             |        |Slight growth only in      |
          |       |             |        |depth.                     |
          |       |             |        |                           |
          |       |             |        |Gelatin-plates: Leaf-shaped|
          |       |             |        |depositwith clear line sys-|
          |       |             |        |tem. The entire colonies   |
          |       |             |        |appear much thinner than   |
          |       |             |        |those of the preceding     |
          |       |             |        |numbers.                   |
  --------+-------+-------------+--------+---------------------------+------------
  [table continued]
  --------+-----------+----------+------+-------------------+---------------------
   No. of |  Growth   |  Growth  | Best |  Development      |
  the Bac-|    on     |    in    |Growth|      of           |  Special Remarks
   terium | Potatoes  |   Milk   | at-- |     Gas           |
  --------+-----------+----------+------+-------------------+---------------------
      1   |The bacter-|Milk is   |37° C | Only slight       |Without doubt a var-
          |ium shows  |not       |      |                   |iety of bacterium
          |only a weak|changed   |      |                   |_Coli commune_.
          |growth on  |          |      |                   |
          |potatoes.  |          |      |                   |
          |It forms a |          |      |                   |
          |yellow     |          |      |                   |
          |layer.     |          |      |                   |
          |           |          |      |                   |
      2   |Dirty      |Milk      |Room  |Does not occur     |  --
          |yellowish  |remains   |temp. |                   |
          |layer.     |unchanged.|      |                   |
          |           |          |      |                   |
      3   |Yellowish  |Milk is   |37° C |Very pronounced.   |Culture-medium to
          |deposit    |caused to |      |From fifty cubic   |which blue litmus
          |at the     |curdle    |      |centimeters of     |is added turns red.
          |place of   |only after|      |broth 6·5 cubic    |
          |inocula-   |it has    |      |centimeters gas    |
          |tion.      |been in   |      |were produced in   |
          |           |the incu- |      |fifteen hours, of  |
          |           |bator for |      |which 3·5 per cent.|
          |           |four days.|      |was oxygen, 10·7   |
          |           |          |      |per cent. carbonic-|
          |           |          |      |acid gas, and 85·8 |
          |           |          |      |per cent. nitrogen.|
          |           |          |      |                   |
      4   |White, dry,|Milk is   | Do.  |Does not occur     |   --
          |spreading. |changed.  |      |                   |
          |           |Serum is  |      |                   |
          |           |separated |      |                   |
          |           |out.      |      |                   |
          |           |          |      |                   |
      7   |White, dry.|Strong    | Do.  | Do.               |   --
          |           |serum     |      |                   |
          |           |formation.|      |                   |
          |           |          |      |                   |
     11   |Yellowish  |Milk      | Do.  |Weak               |Strongly resembles
          |glossy     |curdles   |      |                   |the bacterium_Coli
          |deposit.   |          |      |                   |commune_ and differs
          |           |          |      |                   |therefrom only in
          |           |          |      |                   |that the milk
          |           |          |      |                   |curdles quicker.
          |           |          |      |                   |
     12   |Yellowish  |Milk      |37°C  |Considerable. Five |If 0·25 cubic centi-
          |glossy     |becomes   |      |cubic centimeters  |meters of broth-
          |covering.  |pappy.    |      |of gas will be de- |culture are injected
          |           |          |      |veloped from fifty |under the epigastri-
          |           |          |      |cubic centimeters  |um, the animal is
          |           |          |      |of common broth in |taken violently ill.
          |           |          |      |an incubator during|After four hours
          |           |          |      |the first fifteen  |violent diarrhea
          |           |          |      |hours and six cubic|occurs. Soon the
          |           |          |      |centimeters during |mouse can hardly move;
          |           |          |      |the first forty-   |looks bristly. On the
          |           |          |      |eight hours, con-  |third day the animal
          |           |          |      |sisting of 12·12   |dies. On opening the
          |           |          |      |per cent. carbonic |body a strong putrid
          |           |          |      |acid, 3 per cent.  |smell is noticed.
          |           |          |      |oxygen, and 84·9   |Some of the injected
          |           |          |      |per cent. hydrogen.|bacteria are found in
          |           |          |      |The gas obtained   |the blood. The intes-
          |           |          |      |from a culture-    |tines are coloured
          |           |          |      |medium containing  |green and black.
          |           |          |      |grape-sugar        |The other organs are
          |           |          |      |contains about 40  |pale.
          |           |          |      |per cent. carbonic |
          |           |          |      |acid.              |
          |           |          |      |                   |
     13   |Glistening,|Strong    | Do.  |Weak               |In old stab-cultures
          |yellowish. |curdling. |      |                   |a brown discoloration
          |           |          |      |                   |of the culture-bed is
          |           |          |      |                   |noticed along the
          |           |          |      |                   |length of the stab.
          |           |          |      |                   |Differs in the gelatin
          |           |          |      |                   |stab-culture from the
          |           |          |      |                   |common bacterium _Coli
          |           |          |      |                   |commune_likewise in the
          |           |          |      |                   |curdling of the milk.
          |           |          |      |                   |
     38   |Yellow     |Milk      | Do.  |Not noticed        |    --
          |glistening |is not    |      |                   |
          |deposits.  |changed.  |      |                   |
          |           |          |      |                   |
     40   |White,     | Do.      | Do.  |Weak. Is only      |    --
          |glistening.|          |      |noticed in culture-|
          |Only very  |          |      |bed containing     |
          |slight     |          |      |grape-sugar.       |
          |growth.    |          |      |                   |
          |           |          |      |                   |
     42   |Sulphur    |No change | Do.  |Strong only in     |    --
          |yellow,    |of the    |      |media containing   |
          |glistening.|milk.     |      |grape-sugar.       |
          |           |          |      |                   |
     43   |No growth  | Do.      | Do.  |None               |    --
          |           |          |      |                   |
     44   |Weak devel-|Curdling  | Do.  |Very weak. Only in |
          |opment. The|          |      |media containing   |
          |culture is |          |      |grape-sugar.       |
          |sulphur    |          |      |                   |
          |yellow.    |          |      |                   |
          |           |          |      |                   |
     45   |Weak       |Milk is   | Do.  |Not noticed        |    --
          |yellowish  |not       |      |                   |
          |deposit.   |changed.  |      |                   |
  --------+-----------+----------+------+-------------------+---------------------

The practical difficulty is to keep such cultures uniform during
propagation, and so far this has prevented their introduction in
practice. Similar difficulties have influenced the use of pure cultures
of yeast in the brewing of English beers, although the use of a single
species of yeast is common in the low fermentation breweries on the
Continent.

I found in studying the bacteria of dog dung, that the species existing
in the _fresh_ dung, which developed in ordinary plate cultures,
appeared to belong to four or five species only, mostly bacilli. At
the end of two or three weeks, the original species had given place to
others, mostly cocci, in a very similar way to the change which takes
place in putrefaction. In fact, many of the organisms are identical
with those which cause putrefaction. It will be seen, therefore, that
no single species produces the complex chemical and physiological
changes which take place, or the bodies necessary for the bating of
skin, as some observers have supposed; but the various species succeed
one another as the medium changes its reaction and composition,
until finally the organic portion is resolved into the simplest
bodies such as carbon dioxide, ammonia, and hydrogen. There is thus
a moment when the dung is at its best so far as the bating action is
concerned, and this moment is due to the vital activity of bacteria,
and consequently varies according to the temperature and some other
influences (electrical condition of the atmosphere, etc.). One may say
it is at its best at about fifteen days in summer, and one month or
more in winter. Puer which has been dried, is not so powerful in its
action as that which is immediately made into a paste with water. It
appears to lose its “nature,” partly owing to irreversible dehydration
processes, and partly because some of the bacteria are killed. Plate
cultures on agar from fresh puer (Fig. 17), and from a puer wheel in
use (Fig. 18), show the number of bacteria in the puer wheel to be much
greater than in the fresh puer. Whence it is evident, that the bacteria
continue to develop in the puer and to produce their various products,
enzymes, etc. We have already considered the action of the chemical
products, and in Chapter V. we propose to discuss the action of enzymes.

[Illustration: Fig. 18.--Plate Culture from Puer Wheel.]

[Illustration: Fig. 19.--B. Putrificus.]

*Pigeon-Dung Bate as used for Hides.*--The bacteria contained in the
intestines of birds and in bird dung have not been studied to the same
extent as those of mammals, so that it is not possible to give anything
but a meagre account of them. A microscopical examination of fresh
pigeon dung, collected on a sterile Petri dish, showed debris of food,
cellulose, etc., among the debris, a large number of dumb-bell bacteria
(_b_) (Fig. 20), and a few motile pairs (_c_); no bacilli were seen.
Cells of a saccharomyces (_a_) were also observed. From this pigeon
dung attenuations were made by a modification of Soyka’s method,[82]
and from the fourth attenuation a plate culture was made in ordinary
nutrient gelatin. The colonies from this plate were principally of
two varieties (both non-liquefying organisms), corresponding to the
bacteria observed in the original dung. Large cultures were made in a
Carlsberg vessel, as described in Chapter VI., and the effect of these
cultures tried upon skin. No particular reducing effect was obtained.

  [82] The modification of Soyka’s method of making attenuations of
  bacteria is that of Günther (Bakteriologie, 1898, p. 204), and
  is carried out as follows:--On the inner surface of the cover of
  a sterile Petri dish (which is to be used for making the plate
  culture), place four drops of sterile broth, or sterile water;
  inoculate the first drop, by means of a platinum needle, with the
  material to be examined; heat the needle in the flame, dip it into
  the first drop, and, with the liquid adhering, inoculate the second
  drop. Proceed in this way, each time heating the needle, to the
  fourth drop. From this fourth drop a tube of nutrient gelatin is
  inoculated, and poured on to the plate. The cover is then put on, and
  the plate put into the incubator. The drops on the cover do not in
  any way contaminate the culture.

[Illustration: Fig. 20.--Organisms in Pigeon Dung. × 1000.]

A microscopical examination of a bating pit used for kips, showed an
extraordinary mixture of bacteria, bacilli, vibrios, and monads; some
comparatively large dumb-bell shaped bacteria, very motile, were
present. The difference between the bating liquor and the fresh dung
was very marked, especially in the variety of species present. Cultures
made from several colonies isolated from the above bate, in a nutrient
liquid, consisting of 10 litres water, and 20 grm. gelatin, peptonized
by heating under pressure with 10 c.c. sulphuric acid, afterwards
neutralizing with ammonia, and adding the soluble matter from 200 grm.
bone-meal, had no action on skin.

It would be unsafe to say from these two experiments that the bacterial
effect of the pigeon-dung bate is negligible, but we may assume that it
is different and not so great as with the dog-dung bate or puer.

A complete research as to the various species of bacteria developing in
the bird-dung bate is necessary before this question can be answered.

*General Considerations on the Growth of Bacteria in Various
Media.*--Since the publication of Further notes on the action of the
dung bate (Chapter VI.), I have found that the bating organisms grow
better in the special medium, when it is neutralized with ammonia, than
when it is neutralized with sodium carbonate, i.e. the presence of
organic ammonium salts is more favourable to the growth of the bacteria
than the corresponding sodium salts.

I also found that bacteria obtained from other sources than dung,
viz. from the roots of wool just beginning to “slip” in a sweating
stove, were equally effective in causing the skin to fall. Now these
bacteria produce ammonia, and it seems clear that they are essential
to the chemical part of the process. They also produce proteoclastic
enzymes, which act upon the skin fibre (see chapter on Enzyme Action).
The products of the bacteria depend very much upon the composition of
the nutrient medium. Many organisms grown in media containing sugar
or other carbohydrates produce acids, but, grown in proteids free
from sugars, they produce alkaline compounds. Villon (“The Leather
Industry,” 1901, p. 408) describes a bacterium which he considers to be
the special micro-organism concerned in the depilation of skins, which
resembles Bacillus _d_ (Wood) (Fig. 21), but he does not describe the
appearance of the cultures; he states, however (p. 410), that this is
the only bacterium which can develop in the limes,[83] and that it is
the cause of the unhairing in this case also. Since the production of
ammonia in limes is known to be due to bacterial action, it is very
probable that this bacterium, which is ubiquitous, is also of use in
the bate, and a research in this direction would be interesting.[84]

  [83] Villon found that Bacterium pilline developed 0·142 per cent.
  ammonia in ten days in lime liquors used for unhairing skin.

  [84] Cultures made from old limes have, however, not been found
  effective in bating.

Some of the fermentations taking place in the dung come under the
heading of putrefactive processes (see p. 116). Tyrosin is formed in
considerable quantities during putrefactive fermentation, but is soon
further decomposed, according to Nencki, with formation of indol,
CO_{2} and hydrogen. Leucin gives valerianic acid, ammonia, CO_{2} and
hydrogen; nitrogenous bodies of the aromatic series are also produced.

[Illustration: Fig. 21.--Bacillus _d_.]

[Illustration: Fig. 22.--Bacillus _e_.]

Bacillus ureaæ, B. prodigiosus, and B. fluorescens putridus, evolve
trimethylamine (Herfeldt), and, as the writer has shown, this amine has
an important action in the puering process. In combination with organic
acids, it removes lime from the skin, and in addition it favours the
growth of bacteria, such as bacillus _d_ and _e_ (Figs. 21 and 22) and
B. coli.

The albumens and peptones of the dung are pretty well decomposed
and absorbed before evacuation; the bacteria subsequently split up
the amido acids into fatty acids and ammonia. The fatty acids are
then decomposed generally in the form of the calcium salts, in the
manner shown in the table (p. 108), for which I am indebted to Dr. E.
Herfeldt, of Bonn.

We have already treated of the action of these various products in
Chapter II., but it will be seen from what has been said in the present
chapter that the chemistry and bacteriology of the puer overlap, and
that it is difficult, if not impossible, to separate them entirely.
The bacteria are continually manufacturing chemical compounds, and
decomposing others.

In this respect it is interesting and instructive to note that Nencki,
in his classical work “The Chemical Mechanism of Putrefaction,”[85]
considers the processes by which the putrefaction of proteids is
brought about by bacteria, to be analogous to those taking place by
melting the bodies with potash, and he holds the view that in the
hydration processes brought about by bacteria, the water plays the same
part as the potash.

  [85] Journ. f. Prakt. Chem., Bd. xvii., 1878; see also Stoklasa,
  Cent. Bl. f. Bakt. vi., p. 526.

  ---+-------------+--------------+---------------------+----------------
  No.| Fermenting  |   Cause of   |    Fermentation     |   Authors.
     | Substance.  |Fermentation. |     Product.        |
  ---+-------------+--------------+---------------------+----------------
   1 |Calcium      |Bacteria from |_Calcium carbonate,  |Hoppe-Seyler,
     |formate      |sewer slime.  |CO_{2} and H_        |Archiv f. d. g.
     |             |              |                     |Physiol. xii.
     |             |              |                     |
   2 |Calcium      |   "   "      |_Calcium carbonate,  |  "   "
     |acetate      |              |CO_{2} and CH_(4)_   |
     |             |              |                     |
   3 |Calcium      |Thin bacillus |1. _Propionic acid_, |Fitz, nine
     |lactate      |              |and, as by-products, |papers in
     |Undergoes    |              |_acetic acid_,       |the “Berichte
     |four         |              |_succinic acid_ and  |der Deutsch.
     |different    |              |_alcohol_.           |Chem.
     |fermentations|              |                     |Gesellschaft,”
     |             |Other species |2. _Propionic acid_  |1876–1884.
     |             |of bacteria:  |and _valerianic      |
     |             |short aerobic,|acid_.               |
     |             |butyric       |                     |
     |             |bacteria      |3. _Butyric acid_ and|
     |             |(Fitz).       |_propionic acid_.    |
     |             |              |                     |
     |             |              |4. _Butyric acid_,   |
     |             |              |according to Pasteur |
     |             |              |(Comptes rend. 1861) |
     |             |              |                     |
   4 |Calcium      |Bacteria      |1. Chief product,    |Schützenberger,
     |malate       |(not          |_propionic acid_;    |“Fermentation,”
     |             |described).   |and, as by-product,  |1876.
     |             |Thin bacilli. |_acetic acid_.       |
     |             |              |                     |
     |             |              |2. Chief product,    |
     |             |              |_succinic acid_;     |
     |             |              |and, as by-product,  |
     |             |              |some _acetic acid_.  |
     |             |              |                     |
     |             |              |3. _Butyric acid_    |
     |             |              |and _H_.             |
     |             |              |                     |
     |             |Bacteria      |4. _Lactic acid_     |
     |             |              |and _CO_{2}_.        |
     |             |              |                     |
   5 |Calcium      |Different     |1. Chief product,    |  "   "
     |tartrate     |species of    |_propionic acid_;    |
     |             |bacteria.     |by-product, _acetic  |
     |             |              |acid_.               |
     |             |              |                     |
     |             |              |2. _Butyric acid_.   |
     |             |              |                     |
     |             |              |3. Chief product,    |
     |             |              |_calcium acetate_;   |
     |             |              |by-products, _ethyl  |
     |             |              |alcohol_, _butyric_  |
     |             |              |and _succinic acids_.|
     |             |              |                     |
   6 |Calcium      |Small, thin   |_Acetic acid_ in     |Fitz.
     |citrate      |bacilli       |large quantities,    |
     |             |              |along with small     |
     |             |              |quantities of _ethyl |
     |             |              |alcohol_ and         |
     |             |              |_succinic acid_.     |
     |             |              |                     |
   7 |Calcium      |Micrococci    |1. _Calcium acetate_,|  "   "
     |glycerate    |              |along with small     |
     |             |              |quantities of        |
     |             |              |_succinic acid_      |
     |             |              |and _ethyl alcohol_. |
     |             |              |                     |
     |             |Medium-sized  |2. _Formic acid_,    |
     |             |bacilli       |with some _methyl    |
     |             |              |alcohol_ and _acetic |
     |             |              |acid_.               |
  ---+-------------+--------------+-------------------+----------------

Nencki explains, for example, the metamorphosis of leucin by
putrefaction in this way: The bacteria decompose the water into
hydrogen and hydroxyl, which act upon the leucin as follows:--

    CH_{3}
          \                         OH
           CH - CH_{2} - CH - COOH + H = NH_{3}
          /               |
    CH_{3}                |
             Leucin     NH_{2}

         + OH - CH_{2} (CH_{2})_{4} COOH (oxycaproic acid)

The resulting oxycaproic acid is then split up by the second water
molecule into methylenglycol and valerianic acid:--

    OH
     |
    CH_{2}     OH
     |        + H = CH_{2} (OH)_{2} + CH_{3} (CH_{2})_{3} COOH
    (CH_{2})_{4}
     |
    COOH

The methylenglycol, which changes into formaldehyde and water, is now
split up into CO_{2} and hydrogen, as it would be by melting with
caustic alkali.

                      OH
     H     H.OH      /
    C O  +       = CO     + 2H_{2}
     H     H.OH      \
                      OH

As we shall see in the chapter on the action of enzymes, the phenomena
are of a catalytic nature. Any urea present is decomposed, by the
direct action of micrococcus ureæ, into ammonium carbonate and ammonium
carbamate, so that it does not play any part in the bating process as
usually carried out with dung which has been kept for some time, but
the ammonia produced plays an important part in the chemical action of
the bate, as we have already seen.

If, however, dung containing the urinary products be used in a fresh
condition, the urea has indirectly a very important influence on the
bating, as it favours the permeability of the skin fibre. (See p. 72.)

The fermentation of the cellulose in the dung has not been studied from
the bating standpoint, but it is well known that it is fermented by
various species of bacteria, which have been grouped together under the
generic name of Amylobacter.

Deherain and Gayon first showed that the solution and fermentation of
cellulose in the form of dead vegetable matter, which had previously
been observed, also took place in dung. Van Tieghem, in 1879, showed
that the solution of cellulose is caused by bacteria, whose properties
correspond with those described by him as Amylobacter. Tappeiner was
able to ferment cellulose by mixed cultures of bacteria from the
intestines of oxen--in neutral solution, CO_{2}, methane, H_{2}S,
aldehyde, butyric acid, and acetic acid, were all recognized. In
alkaline solutions, the principal products were CO_{2} and hydrogen,
together with the same by-products as before.

From the researches of Van Sennis, in 1890, it seems pretty certain
that the fermentation of cellulose is due to the symbiotic action of
at least two different organisms The decomposition of the cellulose
may be explained by considering that first a sugar-like carbohydrate
is formed by hydrolysis, and that this is then split up into equal
volumes of CO_{2} and CH_{4}. It may be noted that the fermentation
is anaerobic, and no doubt, so far as bating is concerned, the chief
products are the organic acids produced, principally butyric and acetic
acids. Van Sennis nearly always found Clostridium butyricum associated
with this fermentation.[86]

  [86] Proc. Roy. Soc., lxvii., 1900.

Another group of organisms which have some influence in the bating
process, are the class called by Beijerinck, Granulobacter. They
produce butyric acid, and this acid, combining with the ammonia
compounds of the dung, forms salts which undoubtedly exert an effect on
the lime in the skins, though its action on the fibre is, perhaps, not
so great as the compounds of lactic and propionic acids.

The most common butyric ferment is the old Clostridium butyricum,
now known as B. butyricus, (Prazmowsky), which is anaerobic. It
forms spindle-shaped spores, hence the name Clostridium (from
_κλωστηρ_κλωστηρ, a spindle). Another species (Fig. 23), found in milk
by Hueppe (1884), is aerobic, and ferments lactic acid and its salts
to butyric acid, CO_{2}, and hydrogen; it appears to correspond with
Granulobacter polymyxa of Beijerinck.

Oxalic acid is known to be produced by some bacteria and the moulds
Penicillium and Sclerotinia, and in the white rot of the turnip it is
produced by Pseudomonas; it is also produced by some saccharomycetes,
such as B. Hansenii.[87] There is reason to believe that its
production plays a part in the bate, as we have already mentioned in
Chapter II., but the organisms producing it and their mode of action
still remain to be investigated.

  [87] Reynolds Green, Fermentation, p. 350.

There are, of course, a large number of putrefactive bacteria in the
puer, among these B. putrificus (Fig. 19), isolated by Bienstock; it
is a spore-bearing anaerobic bacillus, and is interesting as specially
attacking fibrin. Now fibrin is extremely resistant to the action of
most putrefactive bacteria, and it is very probable that specific
organisms ferment the different albuminous compounds, in the same
way that the different carbohydrates are each decomposed by specific
ferments.

Very interesting are the various forms of spirilla met with in dung;
Figs. 24 and 25 show Spirillum volutans in the unstained condition, and
also stained to show the flagellæ. It will be noted that the appearance
is so different that, to an inexperienced observer, they might be
taken for different species. The rôle played by these organisms still
requires investigation.

[Illustration: Fig. 23.--B. Butyricus. (Hueppe.)]

[Illustration: Fig. 24.--Spirillum Volutans. (Kutscher.) Stained to
show Flagellae.]

[Illustration: Fig. 25.--Spirillum Volutans. (Kutscher.) Unstained
Preparation.]

I have pointed out previously the importance of the nutrient medium,
or substratum, in which the bacteria grow, on the species surviving.
In it one can see on a small scale the Darwinian process of natural
selection. There is a great struggle for existence between the
various species, and the circumstances determining the survival of
this or that organism are extremely complicated, and we are yet very
much in the dark as to the action of the various chemical compounds
contained in the puer, so that it is unsafe to neglect even those
which are present in only small amounts. Very minute quantities of
certain bodies, almost too small for detection by chemical means,
are sufficient to cause large differences in the growth of certain
organisms. For instance, Raulin found that the addition of a trace
of zinc to his nutrient liquids increased the crop of the mould
Aspergillus niger more than four times the weight of a crop grown in
the same liquid free from zinc.

If we inoculate a nutrient material with a pure culture of bacteria,
and the medium is not exactly adjusted to the needs of the particular
organism, it will not thrive, and will speedily be overgrown by some
other species obtaining access from the air. This fact very much
discounts the use of pure cultures of bacteria which have been proposed
for bating, although in the case of erodin, where the medium has been
adjusted to suit the organism, considerable success has been attained.
The whole of the enzymes and chemical compounds essential for a perfect
bate, are not present in the dung when it leaves the animal’s body, but
these compounds are produced by the continued action of the intestinal
bacteria and other organisms which obtain access from the air. The
production of the enzymes depends, too, upon the composition of the
nutrient medium, since this exerts a selective influence on the species
of bacteria obtaining access to it. Just as in the spontaneous souring
of milk numerous bacteria have free access to it, yet the lactic
ferment is generally so pure that it may be, and is, used as a pure
culture on a large scale in the manufacture of lactic acid.

Coming to the action of the bacteria on the skin fibres, from the
work of Abt and Stiasny,[88] we may conclude that the substance of
the conjunctive fibres is less profoundly decomposed by bacterial
fermentation than by the action of lime. The latter dissolves about 2
per cent. of skin substance from a fresh skin, whereas a puer acting
normally dissolves about 1 per cent.

  [88] Sensibilité de la peau verte, et de la peau après l’échauffe,
  les pelains, et les confits, à l’égard de la chaux du sel, et de
  l’acide acétique. Georges Abt et Edmund Stiasny, Collegium 1910, p. 1
  9.

The nuclein of the skin fibres appears to be all removed by the puer,
since Abt confirms the fact that no nuclei can be seen under the
microscope in a puered skin. The actual solution of the skin substance
is brought about by enzymes of a tryptic character. (See Chapter V.)

While the main lines of the bacteriology of the dung bate are now
pretty well known and understood, it will be seen that much work
still remains to be done as to details, and this principally with
the anaerobic bacteria of the dung, which have been studied by few
investigators.[89] I have suggested[90] that such a research might
well be undertaken by the bacteriological laboratories of our Leather
Industries Schools in Leeds and London.

  [89] See Les Anaerobies, Jungano and Distaso (Masson et Cie Paris,
  1910).

  [90] The Bacteriology of the Leather Industry, J.S.C.I., 1910, p. 666.

*Moulds and Putrefaction.*--In view of the fact that moulds are of
frequent occurrence on dog dung, a brief mention of them is necessary.
So far as our present knowledge goes the researches of Van Tieghem, De
Bary, Rankin, Marshall Ward, V. H. Blackman and others indicate that
their action on the essential bating constituents of the dung is a
destructive one. They grow usually on acid media, and in so doing break
down the acids present into simple inorganic bodies, such as CO_{2} and
water, utilizing the carbon and nitrogen for their own growth. Although
these fungi secrete almost all varieties of enzymes (Bourquelot), yet
we have no evidence that any of the enzymes contained in dog dung are
from this source. In the usual case of dung preserved in pits or casks,
the upper surface only becomes mouldy, since moulds require a free
supply of oxygen. The mycelium penetrates but a very little way into
the body of the dung, and cannot therefore effect any decomposition,
except of the surface layer. The dung exposed to the action of the
mould is generally a bad colour, and is rejected as unsuitable for
puering.

The following species have been noted and classified as growing on dog
dung, though probably not all of them are specific.

    1. Pilaira dimidiata (Grove).

    2. Mucor caninus (a variety of Mucor mucedo).

    3. Circinella simplex (Van Tieghem).

    4. Pilobolus crystallinus (also on cow-dung).

Certain myxobacteria are found on dung, among these Chondromyces,
described as long ago as 1857 by Berkeley, and at that time included
among the Hyphomycetes. It was rediscovered in 1892 by Thaxton, and
owing to his researches the whole class of myxomycetes is now generally
considered as a division of Bacteria. Another myxomycete, Polyangium
primigenum (Quehl), forming a red fructification on dog dung, is
figured in the Encyclopædia Britannica, XI. edition, vol. 3, p. 163.

The following abstract gives some account of putrefaction, and may be
of use in conjunction with the account of the bacteriology of the bate
which has been given. Since it was written Dr. G. Abt (see Bibliography
51) has also given a very full description of putrefactive processes
as affecting leather manufacture. The subject is still occupying the
attention of a large number of bacteriologists, and we may expect more
light to be thrown on the whole question during the next few years.


    ABSTRACT OF PAPER ON RECENT ADVANCES IN THE BACTERIOLOGY OF
    PUTREFACTION. Read before the Nottingham section of the Society of
    Chemical Industry, January 24, 1906.[91]

  [91] Reprinted from the Journal of the Society of Chemical Industry,
  February 15, 1906, No. 3, vol. xxv. The numbers in brackets refer to
  the Bibliography, Chapter XI.

To those who have to do with the manufacture of leather, the changes
which take place in the skin from the time it leaves the animal are of
the utmost interest. The most important of these changes is the natural
process of decomposition known as putrefaction.

Putrefaction may be defined as the decomposition of nitrogenous organic
matter by living organisms, accompanied by the evolution of malodorous
gases. The study of it may be divided into two parts--(1) the
biological, (2) the chemical. The first concerns the organisms which
break down the proteid molecule either directly or by means of enzymes;
the second that of the different products of the action of these
organisms. It is extremely difficult to separate these two studies.

Dr. Sims Woodhead (59) gives a concise account of the earliest
researches on the organisms causing putrefaction by Leeuwenhoek (1692),
Plenciz of Vienna, Müller of Copenhagen (1786), Needham (1749),
Spallanzani (1769), Schwann (1837), Schroeder and Van Dusch (1854),
Tyndall (1870), Lister (1878). These names show that the history of
putrefaction proceeds parallel with the evolution of the microscope and
the development of the comparatively recent science of bacteriology. I
propose to-night briefly to carry it up to the present day.

I need scarcely say that putrefaction is not a specific fermentation
like alcoholic or acetic fermentation, but that it is extremely
complex. In any putrefying matter, such as gelatin or albumin, a large
number of different species of bacteria may be observed as well as
monads and infusoria, and in some cases moulds, all of which take
part in the process. The first stage is a process of oxidation in the
presence of air, in which ærobic bacteria use up the oxygen present and
only simple inorganic compounds are formed, carbon dioxide, nitrates
and sulphates; this part of the process is generally without odour.
The second stage, or true putrefaction, takes place in the absence of
oxygen by anærobic bacteria, and is a process of reduction. It has
been shown that there are no bacteria in healthy tissues, and if a
muscle or any organ is taken from an animal under antiseptic conditions
it may be preserved indefinitely in a sterile vessel to which filtered
air has free access. Solid matter is usually liquefied by organisms
like B. liquefaciens magnus, which are invariably present in the air,
and which prepare the way for more specifically putrefactive bacteria,
such as Proteus vulgaris and B. putrificus, but if one observes a
number of putrefactions of the same kind of matter under natural
conditions, scarcely any two follow the same course. The modern study
of putrefaction dates from Hauser (58), who, in 1885, isolated from
putrefying flesh the three organisms--Proteus vulgaris, P. mirabilis,
and P. zenkeri. He studied the action of these in pure cultures, and
came to the following conclusions:--

That Bacterium termo (Ehr.) is not a single definite species; various
forms and stages of other organisms have been described under this
name. The various species of Proteus go through a wide range of forms
during their development in which cocci, short and long rods, thread
forms, vibrios, spirilli, and spirochætæ occur. Under special nutritive
conditions Proteus goes through a swarm stage, in which condition
it is capable of moving over the surface and in the solid gelatin.
The Proteus bacteria are facultatively anærobic, they all cause
putrefaction; P. vulgaris and P. mirabilis are the commonest and most
active of all putrefactive bacteria. They do not secrete an unorganised
ferment, but decompose albuminous bodies by direct action. They also
produce a powerful poison, of which small quantities injected into
animals produce septicæmia.

Tito Carbone (60) found amongst the products of P. vulgaris, choline,
ethylenediamine, gadinine, and trimethylamine. Macé (61), criticising
Hauser’s work, considers the cocci form of Proteus to be spores.
Bienstock (62) believes the rôle of the Proteus group somewhat
doubtful. He discovered (1884) another widely distributed putrefactive
organism, which he called Bacillus putrificus; it is a spore bearing,
drumstick shaped bacillus found in fæces; it is anærobic and specially
attacks fibrin. Now fibrin is extremely resistent to the action of
most putrefactive bacteria, and it is very probable that specific
organisms ferment the different albuminous compounds in the same way
that the different carbohydrates are each decomposed by specific
ferments. A certain number of species of bacteria are able to decompose
both carbohydrates and proteids. Tissier and Martelly (70) call these
mixed ferments, and divide them further into two groups (1), mixed
proteolytic ferments, including B. perfringens, B. bifermentans
sporogenes, Staphylococcus albus, Micrococcus flavus liquefaciens,
Proteus vulgaris, this group decompose albumin by means of tryptic
enzymes. (2) Mixed peptolytic ferments are only able to attack the
albumin when it has undergone a preliminary decomposition. This group
comprises B. coli, B. filiformis, Streptococcus pyogenes, Diplococcus
griseus non liquefaciens.

The second class of bacteria are those which are without action on
carbohydrates, and only attack proteids; these consist of the true
proteolytic bacteria B. putrificus, and B. putidus gracilis, and the
peptolytic bacteria, Diplococcus magnus anaerobius and Proteus zenkeri,
which can only decompose peptones.

These authors state that B. putrificus is always present in putrefying
albumin, but always accompanied by facultative ærobes which favour the
growth and development of the special putrefactive bacteria.

In the putrefaction of meat the reaction is first acid owing to the
action of the mixed ferments on the sugars present. In the next stage
ammonia is formed by the tryptic enzymes secreted by the ærobic
bacteria, and so the anærobic organisms are enabled to develop. We can
thus understand how it is that putrefaction proceeds more rapidly the
more mixed ferments there are present, although these were formerly
supposed to hinder putrefaction from taking place.

When meat is exposed to air it is first attacked by the mixed ferments,
Micrococcus flavus liquefaciens, Staphylococcus, Bacillus coli,
Bacillus filiformis, Streptococcus and Diplococcus, and becomes acid;
at the same time, the presence of decomposition products of albumin
may be detected, proteoses, amidoacids, amines and ammonia; the
latter quickly neutralise the acids, and in three to four days the
meat is alkaline, and has a faint putrid smell. Bacillus perfringens
and Bacillus bifermentans sporogenes now make their appearance; the
latter of these organisms produces amines, amido-acids and ammonia. In
this stage the simple anærobic ferments are able to begin their work,
and real putrefaction sets in; as this proceeds, the mixed ferments
gradually disappear, and finally the only organisms remaining are
Bacillus putrificus, Bacillus putidus gracilis, and Diplococcus griseus
non liquefaciens.

Another organism, which appears to play an important part in the
decomposition of animal bodies, is described by Klein (63); he found
that in bodies, which had been buried from three to six weeks, bacteria
such as B. coli and B. proteus had almost disappeared, and an anærobic
bacterium, which he calls B. cadaveris sporogenes, was very active. It
is a motile bacillus 2–4 µ long, with flagellæ all over its surface.
Spores are formed at the rounded ends, giving it a drumstick form. It
coagulates milk, the clot gradually dissolving. It grows on all the
usual nutritive media, but only under strictly anærobic conditions.

In a paper, entitled “Fermentation in the Leather Industry,”[92] I
gave a short account of the progress of putrefaction as it takes
place in the animal skin, and also described some of the organisms
I had observed in putrefying skin. A small piece of skin was placed
in water and allowed to stand at room temperature. During the first
two days there was little change, but on the third day a number of
swiftly moving darting monads made their appearance. Some of these
were propelled by flagellæ, but a few had assumed amœboid forms. A
slowly moving bacillus consisting of a long straight rod, apparently
broken up into cells exactly like the Vibrio subtilis, illustrated
in the “Micrographic Dictionary,” was observed, accompanied by some
species of spirillum. Higher organisms present were a Paramœcium and a
colourless transparent piece of protoplasm, shaped like a dumb-bell,
with a slow rotating motion. On the fifth day the number of vibrios
and spirilli had greatly increased, some with a swifter motion than
others. There were also many large infusoria present; one of a peculiar
double form, which appeared to be a development of the dumb-bell shaped
piece of protoplasm seen on the third day. On the seventh day the most
striking feature was the great increase in the number of vibrios; the
field of the microscope was crowded; masses of the bacilli could be
seen clustered round small particles of the disintegrating skin as
if feeding upon it; there were more infusoria, many of them short,
boat-shaped monads, with a trembling motion, refracting light strongly;
these evidently accompany the putrefaction bacteria, and assist in the
final disintegration. On the ninth day the piece of skin was entirely
dissolved.

  [92] J.S.C.I., 1894, 218.

Procter calls attention to the relative putrescibility of the different
constituents of skin, and especially to the rapid putrescence of the
lymph and serum. So far as I know, this part of the subject has not
been studied at all thoroughly, and there is a considerable field open
to workers in our research laboratories.

Pure fat is not decomposed by bacteria, but if albuminous matter is
present, the fat is split up by several species of bacteria and moulds.
Schreiber (73) has shown that the presence of oxygen is necessary. As
this subject scarcely comes within the category of putrefaction, I
refer you to Schreiber’s paper, and also to an important paper by Otto
Rahn (74) recently published.

In the putrefaction of vegetable matter the cellulose is attacked
by specific organisms, which have been thoroughly investigated by
Omeliansky (75). He has shown that the fermentation of cellulose is
an anærobic process, caused by two species of bacteria belonging to
the class of butyric ferments. Morphologically the organisms closely
resemble one another, but one of them decomposes the cellulose with
evolution of hydrogen, the other with evolution of methane; in both
cases considerable amounts of acetic acid and normal butyric acid are
produced.

I have previously stated that monads and infusoria take part in the
process of putrefaction, but I do not know that their action has
been studied in the same way as that of bacteria. The life history
and morphology of some of these monads was studied in 1871 to 1875
by Dallinger and Drysdale (76). These authors, in their researches
into the life history of the monads found in a putrefying infusion of
cod’s head, came to the conclusion that “bacteria are not the only or
even (in the end) the chief organic agents of putrefaction, for most
certainly in the later stages of a disintegration of dead organic
matter the most active agents are a large variety of flagellate monads.”

Dallinger cultivated some of the monads in Cohn’s fluid, and found
that they lived and multiplied in it. Their spores were killed at a
temperature of 250° F. There is a big field of research open in this
direction.

The consideration of the chemical aspect of putrefaction is a vast
subject, and would demand a special treatise. I shall only call your
attention to one or two points of interest.

Taking the simpler bodies first, sulphuretted hydrogen is formed in
putrefying liquids in two ways: (1) by reduction of the sulphates in
the liquid by an anærobic organism Spirillum desulfuricans; (2) by
bacteria capable of growing in the presence of oxygen such as B. coli
commune and B. lactis ærogenes, which ferment glucoses with formation
of lævorotatory lactic acid and evolution of CO_{2} and hydrogen, and
if at the same time the material contains albumin or sulphur, H_{2}S
is given off; these organisms are incapable of reducing sulphates.
Beijerinck (64) has investigated this process, and found a variety of
different forms intermediate between the two above-mentioned, but all
possessing the same characteristics so far as their chemical action is
concerned, so that they may be classed as one order, which he calls
Aerobacter.

Stich (65) found phosphorus pentoxide in the residue from the
putrefaction of casein, nuclein, lecithin, and protagon; and in the
putrefaction of certain organs of animals and plants, gases containing
phosphorus are evolved. The nucleic acid of yeast yielded phosphoric
acid along with hypoxanthin and xanthin.

Vitali (66) found in the putrefaction of muscle, which had been freed
from sugars and fat, that some alcohol was produced. He considers that
a hexose is split off from the albumin in a similar manner to the
splitting off of a fermentable sugar from the glucoproteids (compounds
of simple proteins with carbohydrates). The formation of alcohol in the
putrefaction of muscle occurs in the alkaline stage. Thus alcoholic
fermentation is caused not alone by saccharomyces, but also by certain
putrefactive bacteria.

Lermer (77) finds that the putrefaction of barley resembles butyric
fermentation. An analysis of the gases given off during the later
stages of the process gave the following result: nitrogen, 58·88;
hydrogen, 37·43; methane, 3·15. In the residue from the putrefaction he
found acetic, butyric and valerianic acids, but not caproic or caprilic
acid. In the normal steeping process employed for barley the gases
given off consisted almost entirely of carbon dioxide and nitrogen.
This observation is interesting to compare with the evolution of
nitrogen in the fermentation of bran shown by Wood and Willcox.[93]

  [93] J.S.C.I., 1893, 442.

The action of putrefactive bacteria has been found capable of
transforming hexoses into pentoses. Salkowski and Neuberg (78)
inoculated a solution of _d_-glukuronic acid with putrefying meat, and
showed that it was changed into _l_-xylose with evolution of CO_{2}
according to the following formula:--

    COH(CHOH)_{4}COOH = CO_{2} + COH[CHOH]_{3}CH_{2}OH.

This is an interesting fact, especially as, according to Neuberg, the
pentose contained in animal nucleo-proteids is _l_-xylose.

I wish to express my indebtedness to Dr. Alfred Koch’s “Jahresbericht
über Gärungs-organismen” for some of the abstracts.

The following is a list of putrefactive bacteria which have been
studied in pure cultures:--

    1. Proteus Vulgaris (Hauser). 2. Proteus mirabilis. 3. Proteus
    Zenckeri. 4. Bacillus Oedematis maligni (Kerry, Nencki, Bovet).
    5. Bacillus Chauvæi = B. sarcophyematos bovis. 6. B. Liquefaciens
    magnus. 7. B. spinosus. 8. B. putrificus (Bienstock). 9. B. pseudo
    œdematicus (Liborius). 10. B. enteritidis sporogenes (Klein). 11.
    B. tetani. 12. Clostridium fœtidum. 13. B. cadaveris sporogenes
    (Klein). 14. Spirillum desulfuricans (Beijerinck). 15. B. coli
    commune. 16. B. lactis ærogenes. 17. B. fermentationis cellulosæ.
    18. Micrococcus flavus liquefaciens (Flügge). 19. Diplococcus
    griseus non liquefaciens (n. sp.). 20. Streptococcus pyogenes. 21.
    Staphylococcus pyogenes albus. 22. Bacillus filiformis ærobius
    (n. sp.). 23. Diplococcus magnus anærobius (n. sp.). 24. Bacillus
    putidus gracilis (n. sp.). 25. B. perfringens (Frankel). 26. B.
    bifermentans sporogenes (n. sp.).

Moulds taking part in putrefaction, principally of fruit and vegetable
matter:--

    1. Penicillium glaucum. 2. Mucor mucedo. 3. Mucor piriformis
    (Fischer, possibly identical with 2). 4. Mucor stolonifer
    (Ehrenberg). 5. Botrytis cinerea (Pers). 6. Mucor racemosus
    (Fres). 7. Monilia fructigena (Pers). 8. Fusarium putrefaciens
    (Osterwalder). 9. Cephalothecium roseum.




CHAPTER V.

ACTION OF ENZYMES.


    “The living organism is enabled by the use of enzymes to bring
    about, under ordinary conditions of temperature and moderate
    concentrations of acid or alkali, many chemical reactions
    which would otherwise require a high temperature or powerful
    reagents.”--W. M. BAYLISS.

In a recent work,[94] Dr. Bayliss defines enzymes as the “catalysts
produced by living organisms.” A catalyst is a body which greatly
accelerates the rate of reaction in chemical processes, without
apparently taking part in the process. For instance, peroxide of
hydrogen is decomposed into oxygen and water by mere contact with
finely divided platinum, while the latter remains unaltered in the
process. In this case the platinum black is the catalyst.[95]

  [94] “The Nature of Enzyme Action,” by Dr. W. M. Bayliss, FRS.

  [95] The important discoveries of MM. Paul Sabatier and Senderens
  on the catalytic action of finely divided metals, notably nickel
  and copper, have recently been extended to a study of the catalytic
  action of various metallic oxides. In the _Comptes rendus_, MM. Paul
  Sabatier and A. Mailhe give an account of a new synthetic method,
  based on the catalytic effect of titanium oxide, which would appear
  to possess many practical applications. They show that if a column
  of titanium dioxide is maintained at a temperature of 280°–300° C.,
  and a mixture of the vapours of a primary alcohol and a fatty acid
  (other than formic acid) is led over it, the corresponding ester
  is formed. The same limit is here reached instantaneously as was
  found by Berthelot after prolonged contact. An excess of either
  constituent favours the limit of combination of the other. Following
  this method, the methyl, ethyl, propyl, butyl, isobutyl, and isoamyl
  esters of acetic, propionic, butyric, isobutyric, isovaleric and
  caproic acids have been prepared. Esters of benzyl alcohol have also
  been readily obtained by this method. The inverse action--the direct
  hydrolysis of esters by water--is also easily effected, and the use
  of titanium dioxide reduces any secondary reactions to a negligible
  amount. See Nature, March 9, 1911, p. 54. See also Dr. Sand’s
  paper--bibliography.

In natural processes the best known type of an enzyme is diastase
(amylase), the enzyme contained in malt, and which enables the malt
to convert starch into dextrin and sugar (maltose). It is capable of
transforming more than 2000 times its own weight into sugar, which
fact is quite sufficient to show that its action differs from that of
an ordinary chemical reaction. Another enzyme, sucrase, according to
O’Sullivan and Thompson, will hydrolyze 100,000 times its weight of
cane sugar to invert sugar. Rennet will coagulate 250,000 times its
own weight of casein in milk. The list of enzymes grows longer almost
daily, as some new one is separated having a specific action, until
one is almost led to believe that the mechanism of life itself, as
manifested in the cell, is due to enzymes.

It has been found that enzymes act very much in the same way as
inorganic catalysers. As an example, the velocity of the reaction of
invertase (the enzyme of yeast which hydrolyses cane sugar to grape
sugar) has been compared with the same hydrolysis brought about by
heating a solution of cane sugar with a mineral acid. In both cases
the reaction is in accordance with the law of mass action (Guldberg
and Waage) that the amount of sugar transformed will decrease as less
remains to be transformed. In the diagram (Fig. 26) the curve A is for
invertase (Jas. O’Sullivan, “Journ. Inst. of Brewing,” vol. v. p. 168);
curve B is for the hydrolysis by acid (Wilhelmy), from which it will
be seen that the manner in which the hydrolysis proceeds is practically
the same in both cases.[96]

  [96] There are apparent exceptions and complications of this law
  which we shall not here enter into, except to say that they may be
  explained by the fact that the action of some enzymes is reversible
  (see p. 141, under lipase.)

[Illustration: Fig. 26.--Curves showing Rate of Hydrolysis.]

In the case of fermentation by the living organism, the fermentation
rises rapidly, and then gradually slows down and comes to an end before
the whole of the fermentable matter is used up; the curve, therefore,
is of a hyperbolic character, C in the diagram, which represents, in a
general way, the fermentation of glucose by B. furfuris. The ordinates
represent the amount of acid produced by the bacteria. The time in this
case would be more nearly represented by hours instead of minutes on
the abscissa.

The mathematical expression for the velocity of the reaction is

    _dx_/_dt_ = _k_(_a_ − _x_)

where

    _a_ = original concentration of solution

    _x_ = the quantity transformed in time t

    _k_ = coefficient of velocity of the reaction

By integrating the above equation, it may be shown that

    1/_t_ log(_a_/(_a_ − _x_)) = _k_

For the experiment of J. O’Sullivan with invertase, _k_ has a mean
value of 0·0013; for Wilhelmy’s experiment with acid, _k_ has a mean
value of 0·001377.

Enzymes are produced by the living cell, with other secretions, and
as a consequence are found in all plants and animals. Certain organs,
however, produce enzymes in large quantities, or appear to be specially
set apart for their production. In plants, the seeds are the chief
seat of enzyme activity; in animals, certain glands, such as the
salivary glands and the pancreas. The mucous membrane of the stomach
and intestines also secrete enormous quantities.

The production of enzymes by bacteria was observed by Wortmann in
1882. It has been found that the secretion of the enzymes depends
upon the composition of the nutrient medium in which the bacteria are
grown. For instance, Pfeffer found that the secretion of diastase by
Bacillus megatherium depended upon the amount of cane sugar in the
nutrient medium. The cane sugar checked the secretion of the diastase,
and the same effect was observed in the case of the common mould
Penicillium glaucum. In Bacillus mesentericus vulgatus, diastase has
been found to exist side by side with four other enzymes. Passini,[97]
in studying the putrefactive anaerobic bacteria of the normal human
intestines, succeeded in separating from B. putrificus a proteolytic
enzyme filtered free from bacteria, and which caused proteolysis in
media which were too acid to permit of the bacteria growing. The enzyme
easily dissolved, without previous neutralization, the coagulated
casein caused in milk by old coli cultures. Acid gelatin media were
also liquefied by the enzyme.

  [97] “Zeit. f. Hygiene,” Bd. xlix. p. 135.

From the evidence we have at present it seems probable that every
variety of enzyme, hydrolysing, oxidizing, ammoniacal, etc., can be
produced by bacteria.

The following useful classification of enzymes is due to Effront.[98]

  [98] Les Enzymes, Dr. Jean Effront, Paris, 1899.

  ----------------------------+-------------------+---------------------
         Name of Enzyme       |Substance on which |  Products of the
                              |  the Enzyme acts  |      Reaction
  ----------------------------+-------------------+---------------------
  A. HYDROLYZING ENZYMES      |                   |
                              |                   |
   1. _Fermenting Carbo-      |                   |
         hydrates_:           |                   |
       Sucrase or invertin    |Cane sugar         |Invert sugar
       Diastase or amylase    |Starch and dextrin |Maltose
       Maltase or glucase     |Dextrin and maltose|Glucose
       Lactase                |Milk sugar         |Glucose and galactose
       Trehalase              |Trehalose          |Glucose
       Inulase                |Inulin             |Levulose
       Cytase                 |Cellulose          |Sugars
       Pectase                |Pectin             |Pectates and sugars
       Caroubinase            |Carobin            |Carobinose
                              |                   |
   2. _Fermenting Glucosides_:|                   |
       Emulsin                |Amygdalin and      |Glucose, oil of
                              |  other glucosides |  bitter almonds,
                              |                   |  and prussic acid
       Myrosin                |Myronate of potash |Glucose and allyl
                              |                   |  isosulphocyanate
       Betulase               |Gaultherin         |Oil of gaultheria,
                              |                   |  glucose
       Rhamnase               |Xanthoramin        |Rhamnetine,
                              |                   |  isodulcite
                              |                   |
   3. _Fermenting Fats_       |                   |
         (Lipolytic):         |                   |
       Steapsin               |Fats             } |Glycerin and
       Lipase                 |Fats             } |  fatty acids
                              |                   |
                              |                   |
   4. _Fermenting Proteids_:  |                   |
       Rennet                 |Casein             |Caseuin
       Plasmase               |Fibrinogen         |Fibrin
       Casease                |Casein           { |
       Pepsin                 |Albuminoids      { |Proteoses, peptones
       Trypsin}               |Ditto            { |Proteose, peptones,
       Papain }               |Ditto            { |  amides
                              |                   |
   5. _Fermenting Urea_:      |                   |
       Urease                 |Urea               |Ammonium carbonate
                              |                   |
  B. OXIDIZING FERMENTS       |                   |
       Laccase                |Uruschic acid,     |Oxyuruschic acid,
                              |  tannin, anilin,  |  various oxidation
                              |  etc.             |  products
       Oxydin                 |Colouring matters  |Ditto
                              |  of cereals       |
       Malase                 |Ditto, of fruits   |Ditto
       Olease                 |Olive oil          |Ditto
       Tyrosinase             |Tyrosin            |Ditto
       Oenoxydase             |Colouring matter   |Ditto
                              |  of wine          |
                              |                   |
  C. FERMENT WHICH SPLITS     |                   |
       UP THE MOLECULE        |                   |
       Zymase                 |Various sugars     |Alcohol and
                              |                   |  carbon-dioxide
  ----------------------------+-------------------+---------------------

A more recent classification based on chemical properties is that of
Kossel and Dakin.[99] They divide ferments into two classes:--

    (1) Oxylytic ferments capable of breaking the O-link by which the
    radicals are held together in fats and carbohydrates.

    (2) Imino-lytic ferments, including the amino-lytic ferments which
    act on the amino groups of urea.

Group 2 is sub-divided into--

    (_a_) Trypsin and erepsin, which separate the imide NH from the
    neighbouring carbonyl CO.

    (_b_) Arginase, which separates off urea from arginin.

  [99] Zeit. f. Physiol. Chem. xli. f. 153 (1904). See also the
  articles by Mr. A. Seymour Jones, B.Sc., writing under the pseudonym
  of “Heof Joppa,” in the Leather Trades’ Review, July 19, 1911,
  p. 540; and Aug. 16, 1911, p. 625.

Although it is somewhat doubtful whether the enzymes contained in
dog dung are of glandular origin,[100] it is quite certain that other
enzymes are secreted by bacteria developing in the dung while it is
kept prior to being used for puering. These enzymes may be separated
by the following method. About 150 c.c. of puer is well mixed with
an equal quantity of glycerin, and allowed to stand for seven days.
It is then filtered through paper by means of a pump, and yields a
clear filtrate of a deep golden-brown colour; the filtrate is poured
in a thin stream into a tall vessel containing about 1500 c.c. of 98
per cent. alcohol. A copious flocculent precipitate of the albuminous
matter and enzymes is thrown down, the solution is filtered and the
precipitate washed on the filter with absolute alcohol, and then dried
over sulphuric acid in vacuo. The resulting powder is, of course, a
mixture of all the albumins in solution, and probably only a small
portion of it consists of the pure enzymes. We have merely succeeded
by this method in concentrating them. The property of albuminous
bodies in the act of coagulation to carry down soluble matter is well
known, and this also renders the preparation of any pure proteid
extremely difficult. It may be mentioned here that recent evidence
goes to show that enzymes are not of a proteid nature, since, by
repeated purification, the proteid matter may be almost entirely got
rid of, while the activity of the residue containing the enzyme becomes
considerably greater.

  [100] See, however, the recent paper by Eberle and Krall, Ueber den
  Nachweis des Trypsins im Hundekot, Collegium 1911, p. 201, in which
  the authors endeavour to show the presence of unchanged trypsin in
  dog dung. Their proof depends upon the action of an antipancreatic
  serum on infusions of dog dung based on the work of Achalme, Ann.
  de l’Inst. Pasteur, 1901, p. 737. See also the criticism on this
  paper by Dr. Otto Röhm and Dr. Max Goldman, Collegium, 1911, p. 265.
  Hammarsten (Physiol. Chemistry, 1911, p. 494) states that “among
  the secretions which undergo putrefaction in the intestine, the
  pancreatic juice, which putrefies most readily, takes place first.”

Krawkov’s method of preparing diastase from saliva (Green, p. 46)
may also be mentioned, as it is of general application. It consists
in salting out the enzymes, by saturating the clear solution (in
this case saliva diluted with an equal volume of water) with neutral
ammonium sulphate. The precipitate which is caused by the saturation is
collected on a filter, and washed for a short time with strong alcohol.
It is then allowed to stand under absolute alcohol for one or two
days, and finally dried at 30° C. On extraction with water it yields
a solution which is strongly diastatic, and which gives no proteid
reactions.

There are several other methods for the preparation and purification of
enzymes, but up to the present it may safely be stated that no one has
succeeded in preparing an enzyme in a state of purity.

In considering the mechanism by which enzymes act, it must be
remembered in the first place that they are colloids, and, as such,
will form absorption compounds with the substrate, or body, upon which
they are acting. It is difficult to understand how an enzyme can
exert an action on the substrate, unless it enters into some kind of
combination with it, although this may be only a temporary one. The
action of some enzymes has been found to be due to extremely small
amounts of certain metals, e.g. in the case of the oxidizing enzyme
laccase, the metal is manganese. In the purest samples of this enzyme
prepared, 0·16 per cent. Mn was found, and it has been supposed by
some observers that the whole of the action of the enzyme may be
attributed to the _physical state_ of the manganese which it contains.
According to this hypothesis,[101] the active part of the enzyme is the
ion Mn. This ion may exist in the solution in two conditions, differing
by the electric charge which they carry. One of them Mn_{++} carries
two positive charges, the other Mn_{+++} carries three. In the first
phase, Mn_{++} is transformed into Mn_{+++} by absorbing the charge of
one ion of hydrogen (H_{+}), and two hydrogen ions thus discharged, in
the _nascent state_, unite with the oxygen dissolved in the liquid to
form water.

  [101] Duclaux. See Bibliography.

    2Mn_{++} + 2H_{+} + 0 = 2Mn_{+++} + H_{2}O

In the second phase the ion Mn_{+++} with three charges will be
transformed into the ion with two charges, by decomposing a molecule of
water, of which the nascent oxygen will attach itself to the oxidizable
body R yielding the oxide RO.

    2Mn_{+++} + H_{2}O + R = 2Mn_{++} + 2H_{+} + RO

and the same cycle of operations will begin again, and continue
indefinitely.

In the above illustration the enzyme action was an oxidizing one.
In the case of puering it is a hydrolytic action, in other words a
molecule of water is added to the skin substance. The fibre or some
portion of it is converted first into proteoses, and finally into
peptones, and simpler bodies. The active metal in these cases appears
to be calcium, but by what mechanism it brings about the hydrolysis is
at present unknown.

Pozerski[102] found that the pancreatic juice, which is secreted
after injections of certain sera (anti-pancreatic action), and as
a consequence has no pancreatic action, contains no calcium, but
pancreatic juice secreted under the influence of pilo-carpin is more
or less rich in calcium, and its proteolytic action increases about
equally with the amount of calcium contained in it. The same probably
holds good for the intestinal juice.

  [102] Koch’s Jahresbericht über Gärhungs Organismen, 1908, 635.

Victor Henri has shown that the power of metals in the colloidal state
to bring about these catalytic actions varies with the metal employed,
and is in inverse ratio to the size of the particle. There is a very
interesting and wide field of research open here in order to determine
the conditions under which the various metals act. To this end the
ashes from the purest enzyme preparations might be studied, and methods
devised for producing these metals in the colloidal state, for it seems
evident that it is the _state_ of the body acting which gives it the
properties observed, and not its chemical properties in the usual sense.

The enzymes contained in dog dung which are effective in the puering
process belong to several groups, principally the proteolytic and
lipolytic groups, but indirectly enzymes of the first group (see
p. 132), (fermenting carbohydrates) and of the fifth group (fermenting
urea) also play some part by decomposing various compounds (e.g.
cellulose and urea) contained in the puer.

The action of certain enzymes from the animal body upon skin has
been tried by the author.[103] Those selected were pepsin and
pancreatin,[104] as being most likely to be present in dung. Pepsin
only acts in presence of HCl. Two portions of the same skin were
taken, one of them was treated with a 1 per cent. solution of pepsin,
acidified with 0·2 per cent. of hydrochloric acid; the other in a bate
liquor of dogs’ dung (puer), both at a temperature of 40°C. At the end
of one hour the skin in the pepsin solution was considerably “fallen,”
but that in the manure solution was bated nearly away, i.e. the
greater part of it was dissolved. A 1 per cent. solution of pancreatin
(Mercks) was found to act far more rapidly than pepsin; 1·5 per cent.
of chloroform was added to the solution, to prevent the development of
bacteria. The skin was reduced, but had not the peculiar touch of a
puered skin. As will be shown later, this was found to be due to the
absence of any chemical action upon the lime salts in the skin, and
consequently it felt “limey.” This action took place in the puered
skin, but not in the skin treated with pancreatin alone.

  [103] J.S.C.I., vol. xiii., 1894, p. 218.

  [104] The word pancreatin is used throughout in the sense of pancreas
  extract. As is well known, this contains several enzymes, trypsin,
  steapsin, maltase, and also a rennin.

W. J. Salomon (9) has also attributed the activity of the bate to
pepsin and pancreatin, but he does not give any proof of the presence
of these ferments in the bate.

Since it is practically impossible up to the present, to separate these
enzymes from the dung in a state of purity, the method described on
p. 134 was employed.

The enzymes prepared in this way consist of a mixture of all the
enzymes present in the dung, the amount obtained from 1000 grm. of
dung being about 4 grm. The product had a slight diastatic action upon
starch; 0·5 grm. in 100 c.c. of water at 35° C. was found to have a
very considerable reducing action upon skin, and when combined with the
amine compounds prepared from the dung, the action was more powerful,
and more rapid than with puer. Limed skin was puered in thirty minutes
in this solution to a perfect condition, in the absence of bacteria,
and with no evil smelling compounds. The reaction of the solution at
the beginning of the experiment was faintly alkaline; at the end of the
experiment it was considerably alkaline.

This experiment proves that the action of the dung is a complex one,
due to the combined action of enzymes and chemical compounds upon the
skin. These compounds, which are principally amines, and salts of
amino-acids, probably assist the enzymes, and at the same time act
upon the lime remaining in the skin from the previous liming process.
Whether a skin, which has never been submitted to the liming operation,
could be bated by enzymes alone, without the addition of amines, has
not, so far as I know, been tried, but it is highly probable that this
would be the case.

In order to compare the action of the enzymes prepared from dung with
that of the enzymes produced by bacteria, a mixed culture of bacteria
from puer in dextrose gelatin, after seven days’ growth, was taken.
200 c.c. of this was mixed with 200 c.c. of dilute alcohol (65 per
cent.), and well shaken: gelatin and albuminoid bodies are by this
means precipitated. The liquid was filtered and poured into eight times
its volume of 98 per cent. alcohol. The precipitate which came down
was washed with absolute alcohol, and dried in vacuo. The enzymes thus
obtained were re-dissolved in water, and the former experiment with
the skin repeated with this solution, with the addition of the amines.
The skin was brought down in exactly the same way as before, showing
conclusively that it is the enzymes produced by bacteria, acting in
conjunction with the amines, which bate the skin. It would seem that
the action of the enzymes is aided by the presence of amine compounds,
in addition to the chemical action which these latter have upon the
skin. The action is interdependent, i.e. bacterial action alone is
insufficient, and chemical action alone is insufficient, the true
bating action being a combination of the two.

There seems little doubt that it is the enzymes which dissolve the skin
substance, or rather certain parts of the intercellular substance of
the fibres, and the compound of this substance with lime. The action is
a digestive one, and may be compared, as we have shown, to that of the
digestive ferment of the pancreas.

This fact has been made use of in the artificial bate “Oropon” (see
Chapter VII.), in which extract of pancreas is combined with ammonium
chloride, and some inert material.

The effect of puering on the fatty matter in the skin is well known.
The fat and grease are partially emulsified, and set free, so that
they can be removed by scudding. This action is a most important one,
and one in which artificial bates have hitherto been wanting. The
emulsification of the fats is brought about by means of an enzyme
either identical with, or closely resembling, lipase.[105] This enzyme
is found in the pancreatic juice, and in the seeds of many plants. It
brings about the emulsification of the fat by saponifying a portion
of it, i.e. the fat is split up into glycerin[106] and a fatty acid,
according to the equation--

      C_{3}H_{5}(C_{18}H_{35}O_{2})_{3} + 3H_{2}O
                 Stearin.

    = C_{3}H_{5}(OH)_{3} + 3(C_{18}H_{35}OHO)
        Glycerin.             Stearic Acid.

  [105] Also known as steapsin, or pyolin (Allen, p. 357, vol. iv.,
  Comm. Org. Analysis).

  [106] In the digestion of fat by dogs the glycerol produced is all
  absorbed before the ileum is reached, so that none exists in the
  excrement. (Levites, Chem. Soc. Abst. 1907, vol. iv. p. 891.)

Lipase was one of the first enzymes in which the reversibility of
the reaction was shown, i.e. it is not only capable of hydrolysing a
fat, but also of causing the formation of one by the combination of
the fatty acid and glycerin.[107] This explains why the reaction of
such an enzyme is never complete. An equilibrium is reached just as
with ordinary reversible chemical reactions like the precipitation of
magnesium hydroxide by ammonia.

  [107] Another instance of the reversibility of enzyme action is the
  secretion of a peptolytic enzyme, by B. pyocyaneus; i.e. it has the
  power of synthesizing proteins, as well as of decomposing them. (Zak.
  Chem. Soc. Abst. 1907, p. 996.)

Lecithin, and possibly other fatty compounds, are known[108] to be
important auxiliaries in the ferment-like actions produced by toxins;
cholesterol has a similar effect, and as this body is a constituent
of dung, it may play some part in puering. Here, again, is a further
problem awaiting investigation.

  [108] Chem. Soc. Annual Reports IV., 1907, p. 252.

Loevenhart has shown that the bile salts, sodium cholate, and sodium
glycocholate, greatly increase the activity of lipase, and Magnus found
that synthetic bile salts have the same effect; such bodies are known
as co-enzymes. I have shown that bile itself is not favourable to the
bating action, but the bile salts, by stimulating the action of the fat
splitting enzymes, are probably essential to the full action of the
dung bate.

Another enzyme which may be of importance in puering is erepsin, the
enzyme of the intestinal juice, which is responsible for the completion
of the digestive process. The pancreatic enzymes act upon the peptones
produced by the pepsin of the stomach, splitting them up into simpler
compounds, while the erepsin acts further upon these products. It
dissociates albumoses and peptones into amino acids, taking as it
were the last traces of nutriment from the food passing through the
intestine. It acts best in alkaline solutions.

This ferment is very widely distributed in the animal kingdom, and
occurs in other organs and tissues besides the intestines. The quantity
of erepsin in the fresh fæces must be considerable, since a dog
secretes from 400 to 500 c.c. of intestinal juice per day. It remains
to be shown whether this retains its properties after excretion, and
for how long.

A most important point in connexion with the activity of enzymes is
the reaction of the medium, i.e. its acidity or alkalinity, or, more
strictly speaking, its hydrogen ion concentration. A very slight
increase or decrease of the acidity or alkalinity of the liquor will
diminish the rate of action of the enzyme by a large amount, and in
some cases cause the action to cease. In all cases enzymes have an
optimum acidity or alkalinity; in other words, for every enzyme there
is a particular hydrogen ion concentration at which its activity is at
a maximum. The work of Soerensen, to which I have already referred,
gives a very complete account of this aspect of enzyme action and also
of the methods he employed to investigate it. His work should certainly
be carefully studied by anyone wishing to take up this part of the
subject.

The same remarks as to optimum conditions apply to temperature,
although the effect of this is better known. Most tryptic enzymes act
best at the body temperature, viz. 98° to 100° F. (37° to 40° C.), and
hence puering should be conducted at this temperature. In the case of
the hen or pigeon-dung bate the enzymes have not yet been studied, so
far as I am aware, so that it is not possible to give an account of
them. At the same time it seems highly probable that, as these bates
are employed at comparatively low temperatures, the enzyme action is
kept back, and therefore the chief action would be a chemical one.

Enzymes are retarded in their action in the first place by their own
products, in a very similar manner to bacteria, in the second place
the retarding or inhibiting action is brought about by the so-called
_anti-bodies_. Of these the longest known and best studied are those
which are produced by toxins, and which neutralize the action of the
toxins upon the animal organism (anti-toxins).[109] Normal serum also
inhibits the action of trypsin and many other enzymes. Another very
important group of anti-bodies are the _precipitins_. If the serum
of an animal be injected repeatedly into another animal of different
species, a precipitin appears in the serum of the animal treated, which
causes a precipitate when added to the serum of the first animal. The
special importance of this fact is, that it can be utilized as a method
of distinguishing between human blood and that of animals, which is
often of importance in medical juris-prudence.[110]

  [109] Hammarsten, p. 70.

  [110] Gulland, Ency. Britt. iv., p. 83.

This fact has been made use of to distinguish dog dung from other
matters, with which it has been adulterated.[111] A perfectly clear
extract of dog dung filtered free from bacteria was injected into a
rabbit. The serum obtained from the rabbit was found to contain a
precipitin, and on addition of the serum to the dog dung extract a
precipitate was produced. When the serum was added to the extract from
the dung of another animal no precipitate was formed. When added to the
extract of the adulterated dung a very much smaller precipitate was
produced than with the extract from dog dung alone.

  [111] Appelius, Technische Briefe, No. 23, April 1909.

The coli bacteria in the puer also produce an
anti-body--_agglutinin_.[112] If a culture of B. coli be examined under
the microscope, the bacteria are seen moving here and there in the
liquid, evenly distributed. On the addition of a trace of the serum
of an animal which has been previously injected with coli bacteria
the bacteria on the slide cease their movements and collect together
in clumps. They are said to be agglutinated. This property is used to
diagnose bacteria in suspected cases of cholera and some other diseases.

  [112] Harden, see Bibliography.

The wonderful discoveries that have been made in this direction
constitute one of the most marvellous chapters in the history of
science. Serum diagnosis and serum therapeutics are now firmly
established as invaluable aids to the physician in his fight against
disease and death.

The extent of our present knowledge of the action of enzymes in puering
may be summed up as follows. Active enzymes are produced by bacteria
growing in the infusion of dung, in addition to digestive enzymes
which may be originally present in the dung; the bacterial enzymes
are produced more rapidly in a dilute infusion, as employed in the
puer wheel, than in the dung itself. The enzymes are of various kinds,
proteolytic, peptolytic, lipolytic, etc., but the proteolytic and
lipolytic are the most important. These have a solvent action on the
fibres of the skin, but little or no action on the hyaline layer, at
the concentration usually found in the puer liquor. The fatty matters
and soaps in the skin are acted upon by the lipolytic enzymes, and the
fats to some extent emulsified, so that they may be easily removed from
the skin by scudding or pressing.

It must be clearly understood that enzyme action alone is not
sufficient, as has been previously explained, but that the dung
enzymes, acting in conjunction with the chemical compounds present,
produce the specific puering effect.

There is still much work to be done before the action of the enzymes in
dung is fully understood, but from the above short account it will be
seen that the part they play in the bate is of great importance.




CHAPTER VI.

ORIGINAL PAPERS ON BATING.


The reprints of these papers being no longer available, I have thought
it best to print them in the present volume exactly as published,
with the exception of the paper entitled “Fermentation in the Leather
Industry” from which only so much is reproduced as relates to puering
and bating.

    I. PART OF PAPER ENTITLED “FERMENTATION IN THE LEATHER INDUSTRY.”
    Read before the Nottingham Section of the Society of Chemical
    Industry, February 14, 1894.

In the next process, that of “bating,” the bacteria play a more
important part, and may be put in our second category, viz. helpful to
the tanner, though, from the very nature of the material used, it forms
a nidus for various putrefactive ferments, and is therefore dangerous.
The materials usually employed are bird dung and dog dung; the former
of these is sharp and piercing in its action, while the latter has a
more softening action on the skin. In both bates, however, a rapid
solution of the gelatinous and albuminous hide substance (coriin)
takes place. The hide fibres, however, are not attacked until all the
nutrient material in the liquid has been consumed by the bacteria.

Eitner (Der Gerber, xv., 158) states that a sterile bate is without
action on the skins, and appears in that article to attribute the
entire working of the bate to the action of micro-organisms. He
seems to have based his conclusions on experiments with an old bate
sterilised with creolin, which he found had no action on the skins. If,
however, a fresh bate be made and boiled for half an hour, then allowed
to cool to 90°–95° F., it will be found to have a considerable action
on the skins, though not so rapid a one as the unboiled bate. The
boiling kills all organisms, and fresh ones have not time to develop
from spores still remaining before the experiment is at an end.

Lately, the action of unorganised digestive ferments in the dung has
been found to be considerable. Thus bating is an exceedingly complex
process. The action appears to be threefold:--

    (i) A purely chemical action of the soluble salts present in the
    bate on the lime in the skin.[113]

    (ii) An action due to organised ferments.

    (iii) An action due to unorganised ferments.

  [113] _References to bating_:
    Procter. Text-book of Tanning, 1885, 184.
    Der Gerber, x. (1884), 197, Mistbeizen.
    Der Gerber, xv. (1889), 267.
    J. Anal, and Appl. Chem., 1893, vii. 87, 95.
    J.S.C.I., xii. 530, Palmer and Sandford.
    Salomon, W. J., Tech. Quarter, 1892, v. 81, etc.
    J.S.C.I., xii. 774, ix. 27.


The precise amount of influence each of these actions has on the skin
is difficult to assign. The chemical action of the ammonia compounds
dissolves the lime remaining in the skin, but the simple removal of the
lime is not sufficient, as may be shown by removing it completely with
dilute hydrochloric acid or other similar means, and washing perfectly
free from acid in distilled water. When tanned, such a skin is hard and
brittle.

The organised ferments or bacteria, of which there are many species
in the bate, probably act on the skin by secreting soluble ferments,
which have the power of dissolving hide fibre. I have isolated in plate
cultivations several species which liquefy the gelatin.

If skins are allowed to lie in the bate, zoogloæ of these bacteria
collect in the folds and attack the fine “grain,” so that the leather
is covered with lines and markings, or “flaked.” Thus the operation
is a very critical one, requiring careful watching; under certain
atmospheric conditions and at a temperature of 35°–40° C. the skin,
if only left a short time too long, will completely melt away. The
more skin substance is dissolved in the liquid, the more rapid and
pronounced the bacterial action becomes.

So far as is at present known the unorganised ferments in the dung,
besides those which are secreted by the bacteria, are mostly digestive
ferments[114] --pepsin, pancreatin, and trypsin--certain quantities
of which pass out of the animal’s body in an unchanged condition. Of
these, _pepsin_ only acts in an acid solution, and, although fresh
bate liquor is faintly acid to litmus, it quickly becomes neutralised
by the lime in the skins, so that the action of this ferment can only
be limited in extent. _Pancreatin_ will act in a neutral solution, and
has, therefore, a considerable effect on the skin.

  [114] Harris and Gow, Jour. of Physiol., xiii. 469. G. Tammian,
  Zeitschr. f. Physiol. Chem., xv. 271. O. Loew., J. Prak. Chem., 37,
  101. J.S.C.I., 1888, 224.

SOME DUNG ANALYSES.

  --------------------------+--------------------+--------------------------
           Hen Dung         |      Dog Dung      |         Guano
  --------------------------+--------------------+--------------------------
                  Per Cent. |          Per Cent. |                 Per Cent.
   Moisture           60·88 | Ca          43·049 | Urea                 5·00
   Organic matter†    19·22 | Mg           0·087 | K_{2}SO_{4}          7·90
   Phosphates          4·47 | K            0·302 | NaCl                 5·00
   CaCO_{3} and             | Na           0·438 | (NH_{4})_{3}PO_{4}   5·50
     CaSO_{4}          7·85 | Si           0·004 | (NH_{4})_{2}O        0·60
   Alkaline salts      1·09 | PO_{4}       3·446 | SiO_{2}              2·25
   Silica and sand     6·69 | CO_{2}       7·464 | Ca_{3}(PO_{4})_{2}   9·00
                     ------ | Cl           0·037 | MgNH_{4}PO_{4}      15·25
                     100.00 | Fe and loss  0·008 | Ammon. urate        15·25
                            | Organic            | Organic matter
                            |   matter    14·152 |   (17 per cent. N)  41·73
     † Containing           | H_{2}O      31·013 | Moisture             6·52
       N = ammonia 0·74     |            ------- |                    ------
                            |            100.000 |                    100.00
  --------------------------+--------------------+--------------------------

   W. I. Macadam, J.S.C.I., 1888, 80.   _Viollet_, Diction d’Analyses Chim.

  The dog dung actually used in a tannery is from animals fed on a more
  vegetable diet, the one given having an extremely high percentage of
  lime, no doubt owing to the animal having eaten bones. A dung as brought
  from the kennels was found to contain:--

                            Per Cent.
          Mineral matter       4·679
          Organic   "          9·731
          Water     "         85·590
                              ------
                             100·000

I find by experiments with the purified ferments that their action is
very slow compared with the bate itself. Two portions of the same
skin were taken: one of them was treated with a 1 per cent. solution
of pepsin acidified with 0·2 per cent. of hydrochloric acid, the
other in a bate liquor of dog’s dung; both at a temperature of 40° C.
(104° F.). At the end of one hour, the skin in the pepsin solution was
considerably “fallen,” but that in the manure solution was bated nearly
away, i.e. the greater part of it was dissolved.

A 1 per cent. solution of pancreatin (Merck’s) was found to act far
more rapidly than pepsin. At 40° C. in a neutral solution the skin
fell rapidly, and the action continued even in the cold. In this
experiment it was found that in 15 hours the liquid was swarming with
minute bacteria. In order to guard against the influence of bacteria
a similar experiment, at the suggestion of Mr. H. R. Procter, of the
Yorkshire College, Leeds, was tried with the addition to the pancreatic
solution of 1·5 per cent. of chloroform; this prevents the development
of bacteria, while it does not interfere with the action of the
pancreatin. The skin was reduced as before, but in neither case had it
the peculiar touch of a “puered” skin, nor were the characteristics of
the leather the same.


    II. NOTES ON THE CONSTITUTION AND MODE OF ACTION OF THE DUNG BATE
    IN LEATHER MANUFACTURE. Read before the Nottingham section of the
    Society of Chemical Industry, Oct. 26, 1898.

In a paper read before the Society of Chemical Industry (J.S.C.I.,
1894, 219), the author gave an outline of the bating process as far
as he had then investigated it. Further researches have shown that,
although the views then put forward must be somewhat modified, in the
main they are correct.

In 1895–96 a series of experiments was undertaken with a view of
ascertaining how much of the reducing action of the bate was due to:
(1) the chemical constituents; (2) the bacteria; (3) the digestive
ferments and enzymes.

The puer used was dog dung obtained freshly every week from hunting
kennels. It contained on an average: water, 85 per cent.; organic
matter, 10 per cent.; mineral matter, 5 per cent.; about 3 per cent. of
the organic is soluble and about 1 per cent. of the mineral matter.

According to Gamgee (Phys. Chem. 2), a dog fed on flesh diet excretes
from 27 to 40 grm. of fæces in 24 hours, of which 12·9 grm. are solids.
On a bread diet the amount of fæces is much more.

An ultimate analysis gave the following results:--

    ---------------+------------+----------
                   | Bread Diet | Meat Diet
    ---------------+------------+----------
    C              |   47·39    |   43·44
    H              |    6·59    |    6·47
    N              |    2·92    |    6·50
    O              |   36·08    |   13·58
    Mineral matter |    7·02    |   30·01
    ---------------+------------+----------

These figures alone are sufficient to show what variable quantity is
the puer employed in a tannery.

The mineral matter is composed as follows:--

    --------------------+----------------------+-------+------
    Soluble in water   {|NaCl and Na_{2}SO_{4} | 1·37} | 4·00
                       {|Sodium phosphate      | 2·65} |
                        |                      |       |
                       {|Earthy phosphates     |80·37} |
    Insoluble in water {|Ferric phosphate      | 2·09} |94·93
                       {|CaSO_{4}              | 4·52} |
                       {|Silicic acid          | 7·94} |
    --------------------+----------------------+-------+------

The ammonia compounds combine with the lime remaining in the skin, but
the constitution of these compounds was unknown.

The puer was examined for ammonia by Schlösing’s method, 50 c.c. being
mixed with 50 c.c. milk of lime placed under an air-tight bell-glass,
together with an open vessel containing a measured quantity of standard
acid, for 36 hours. No NH_{3} was given off.[115]

  [115] This is not invariably the case: other samples of puer examined
  have given off NH_{3} when similarly treated, but only in small
  quantity. A similar instance has been observed by Rideal and Orchard
  in the liquefaction and decomposition of gelatin by B. fluorescens
  liquefaciens (The Analyst, October 1897), where the quantity of
  ammonia produced was insignificant, amounting even after 16 days’
  incubation to only 0·168 grm. of N per 100 c.c., corresponding to
  0·204 grm. of ammonia.

The mixture of dung and Ca(OH)_{2} was filtered and a perfectly clear
filtrate of a golden-yellow colour obtained. This contained free
amines, calcium salts of volatile and non-volatile acids.

It was distilled until the distillate was no longer alkaline, and made
up to 500 c.c. 50 c.c. required for neutralisation 5·7 c.c. of (N/100)
H_{2}SO_{4}, equivalent to 0·1938 grm. of NH_{3} per litre of original
puer, being the amines set free by Ca(OH)_{2} in cold solution.

A further 50 c.c. was distilled with NaOH, the amines given off
neutralised 8 c.c. of (N/1) HCl, equivalent to 2·72 grm. of NH_{3} per
litre. The neutralised liquid was evaporated to dryness; the residue,
consisting of amine hydrochlorates, amounted 0·27 grm. A qualitative
test showed both primary and secondary amines to be present.

The action of these amine hydrochlorides was tried upon skin, pieces of
limed sheep grain previously washed in water being used. The solution
was maintained at 35°C. In the first experiment 0·27 grm. was dissolved
in 100 c.c. of water. In two hours the skin was considerably reduced,
but had not the touch of a “puered” skin. It tanned well and was a good
colour, showing that the lime had been removed, but the leather was not
sufficiently soft.

With a strength of 1 grm. per 100 c.c. the action was hastened, the
same result being obtained in 1-1/4 hours, but the coriin of the skin
was not dissolved. A comparison piece of the same skin in dung was
bated too much.

With a view of comparing the action of analogous bodies, skin was
treated in a solution of anilin hydrochloride, 1 grm. per 100 c.c. for
1-1/4 hours at 35° C. The solution was acid, lime was removed, and the
skin felt very similar to that in the previous experiment. Coriin was
not dissolved, and the skin did not “come down.”

Of the mineral constituents mentioned above, only the chlorides[116]
have a reducing action on skin, the other compounds being inert, e.g.
silicates, or merely supply food for the bacteria.

  [116] Procter, Text Book of Tanning, p. 85.

For a general outline of the decompositions taking place in dung,
see Dr. Herfeld’s paper (J.S.C.I., May 1895). We need only consider
the action of the chief organic compounds, as the amount of chlorine
is too small (in one case 0·053 per cent. on the dry dung) to be of
importance. The principal compounds are:--

_Organic Acids._--Formic, acetic, butyric, valerianic, lactic, malic,
tartaric, citric, and glyceric.

_Amido Compounds._--Leucin, tyrosin, glutamic acid, glutamine,
asparagin, glycocol.

_Bases_, consisting of amines, skatol, and indol, and varying amounts
of ammonia, according to the age of the dung.

The organic acids exist partly as salts of the alkaline earths, partly
combined with amines. A distillation of “puer,” after acidifying with
H_{2}SO_{4}, gave 2·2 grm. per litre of volatile acids reckoned as
acetic. No HCl came over. On decomposing the sodium salts of the acids
with H_{2}SO_{4}, the smell of butyric acid predominated. I have also
shown the presence of lactic acid in the dung, but have been unable to
determine the amount.

_Action of Amido Compounds._--A mixture of glycocol and leucin[117] was
prepared by boiling 8 grm. of gelatin in 400 c.c. of water, acidified
with 1 c.c. of HCl for two hours with inverted condenser. The acid was
then neutralised with ammonia. The solution had a very considerable
reducing action on skin at 35° C. in 90 minutes, but not sufficient for
practical purposes. It appears to dissolve a little coriin in a similar
way to dilute acids. It was found on further investigation that the
action was largely due to the NH_{4}Cl present.

  [117] According to the text books; but for later researches see Paal
  and Schilling, Chem. Zeit. 1895, 1487; also J.S.C.I., 1898, 589.

_Action of Dilute Acids._--Although the bate in practice is almost
always alkaline, yet a fresh bate is _acid_, and it may be as well,
before proceeding, to consider briefly the action of weak and dilute
acids on skin.

The fibres of the skin have only a limited capacity for holding acids,
and soon begin to swell abnormally and partially dissolve. Reimer[118]
has found that the material may be reprecipitated by lime water. It
forms a fibrous mass, which has not the sticky feel of gelatin, but is
at once converted into that body by boiling.

  [118] H. K. Procter, “Text Book of Tanning,” 1885, p. 18.

The author has examined the action of dilute H_{2}SO_{4} on sheep
skins, and this may be considered typical of the action of acids on
skin generally.

When puered and drenched sheep skin is treated for 20 minutes with a
very dilute solution of H_{2}SO_{4} (1/280), it swells up considerably
and becomes soft and semi-transparent, The fibres of the skin, which
under normal conditions have a diameter of 4µ–6µ become 20µ or more; on
staining with picro-carmin there are seen to be certain fibres which
are unaffected or only slightly affected by the acid. These are the
elastic fibres[119] and the capillary blood vessels.

  [119] See Procter, “Text Book of Tanning,” pp. 8 and 21.

On filtering a solution of the above strength in which skin in excess
has been treated for 20 minutes, and evaporating 100 c.c. to dryness
in a platinum dish, a residue of 0·4992 grm. was obtained. A second
experiment gave 0·496 grm. of which 61 per cent. was organic matter.

By Kjeldahling another portion of the residue, the N in 0·12 grm.
amounted to 0·0122 grm. equivalent to 0·073 grm. of skin substance;
i.e. 60 per cent. of the soluble matter is skin. It is evident that
the matter in solution is a compound of skin substance and H_{2}SO_{4}
together with a little soluble mineral matter contained in the skin.
The amount of substance dissolved depends upon the condition and
previous treatment of the skin.

In the presence of NaCl, which prevents the swelling of the fibres,
puered sheep grains take up 7·0 c.c. of (N/1) H_{2}SO_{4} from
a solution containing 17 c.c. of (N/1) H_{2}SO_{4} (0·833 grm.
H_{2}SO_{4}) and 7·8 grm. of NaCl per 100 c.c. About 300 grm. wet skin
per litre were used. The skin substance dissolved by the acid in the
time named does not appear to be more than that dissolved by the weak
acids of a drench, although an equivalent weight of H_{2}SO_{4} has a
much more powerful swelling action on skin than these acids.

A piece of puered and drenched sheep skin was carefully washed in
distilled water and then dried _in vacuo_ until it ceased to lose
weight; the absolute dry weight was 4·2670 grm. The skin was soaked
down in distilled water and again drenched for 30 hours in a clean bran
drench, developing approximately 0·8 grm. lactic acid, and 0·5 grm.
acetic acid per litre (see this Journal, May 1893). It was again washed
and dried _in vacuo_. It weighed 4·12 grm., a loss of 0·14 grm. = 3·44
per cent.

The action, therefore, of the acids is a reducing one, inasmuch as skin
is dissolved, but as the fibres hold a certain amount of acid, the skin
appears to be plumped. Where the acid is a mineral one the skin tans
plump, but with a brittle and inelastic fibre, weak organic acids make
a plump, soft leather with a moderately elastic fibre, giving to the
skin a somewhat india-rubber feel. We thus see that weak acids alone do
not produce a similar result to a dung bate, which causes the skin to
fall and produces a flat soft leather with a stretch in it, which will
not spring back.

I have also tried the action of the following Na and NH_{3} salts upon
skin:--

_Sodium lactate_, C_{3}H_{5}O_{3}Na, prepared by neutralising 2 grm.
lactic acid with sodium carbonate in 1000 c.c. water. The solution
was used under the same conditions as the other bating experiments,
viz. at 37° C. the reducing action was nil, but on prolonging the
digestion, the medium was found to be very suitable for the development
of putrefactive organisms, and, as a consequence, the skin was attacked
and partly peptonised.

_Ammonium lactate_, prepared in a similar way, had an almost identical
action on the skin, but was more effective in removing the last traces
of lime.

_Ammonium butyrate_, prepared as above, removed lime, but instead of a
reducing action it appeared to plump the skin slightly.


_Action of Bile._

Amongst other compounds in the dung which might have some action on
skin were the bile salts and bile colouring matters. (For a full
description of these, see Gamgee, Phys. Chem. ii.)

Experiments were made with ox bile, 25 c.c. being diluted with 250 c.c.
of water. Pieces of sheep grain previously washed in water were
digested in this solution at 37° C. for 1–4 hours. The bile had no
reducing action on the skin and in fact hardened it, at the same time
staining it a dirty yellowish brown, the colour differing from that of
the colouring matter of dung (hydrobilirubin). Even after continued
standing there was little or no development of bacteria, the bile
appearing to have an antiseptic action.


_Action of Bacteria._

Having ascertained the action of the chief chemical constituents of the
dung, that of the bacteria was next examined. In the first experiment,
eliminating all but bacterial action, a tube of nutrient gelatin was
inoculated from an active bate. In two days the gelatin had liquefied
along the needle track.

The culture was now transferred to the following solution:--

    Gelatin          4  grm.
    Dextrose         4   "
    K_{2}HPO_{4}     1·0 "
    MgSO_{4}         0·2 "
    NaCl             0·4 "
    Water           2000 c.c.

The growth in this was fairly rapid and the smell very slight at the
end of a week. A piece of washed sheep grain worked in this culture
at 37° C. for four hours was considerably reduced, the grain was
not attacked, the action differing in this respect from a prolonged
puering. This mixed culture was grown in various other nutrient media,
gelatin, gelatin and mineral salts, broth made from sheep and calf
fleshings, etc., with practically the same results. In no case did the
bating action equal that of dung, and was insufficient for practical
purposes.

A cursory bacteriological examination of dung will show that the number
of species of bacteria it contains is very large, but on making a
plate cultivation from a sufficiently dilute infusion of fresh dog
dung, most of the colonies developing appear to belong to four or five
species. The following is a list of all the organisms which, so far as
my knowledge goes, have been isolated from different kinds of dung:--

    LIST OF KNOWN BACTERIA IN DUNG.

                                             {These organisms have
     1. _Bacillus fluorescens putridus_      {been formerly de-
     2.     "          "      _liquefaciens_ {scribed as Bacterium
     3. _Proteus vulgaris_.                  {termo by various
     4.     "    _mirabilis_.                {authors.
     5. _Bacillus subtilis_.     }J. T. Wood, J.S.C.I.,
     6. _Micrococcus fulvus_.    }  1890, 28.
     7. _Bacterium ureæ_. (Cohn.)}
     8. _Bacillus saprogenes, I., II., III_. _Vide_ Herfeld, J.S.C.I.,
                                                    May 1895.
     9. _Bacillus butyricus_.
    10. _B. putrificus coli_.
    11. _B. pyocyaneus_.
    12. _B. janthinus_.
    13. _B. coprogenes fœtidus_.
    14. _B. pyogenes fœtidus_.
    15. _B. prodigiosus_.
    16. _B. zenkeri_.
    17. _B. magnus_.
    18. _B. spinosus_.
    19. _B. liquefaciens_.
    20. _B. coli_.
    21. _B. duclauxii = B. ureæ_.
    22. _Micrococcus ureæ_.
    23.       "        "  _liquefaciens_.
    24. _Proteus sulphureus_.
    25. _Bacterium sulphureum_.
    26. _Bacillus amylobacter_.
    27. _Clostridium butyricum_.
    28. _B. acidi paralactici_.
    29. _Bacillus I_.  }Isolated from horse manure by
    30.     "    _II_.  }S. A. Severin, Centr.-Bl. f. Bakt. ii.
    31.     "    _III. }  [1], 97.
    32. _Spirillum serpens_. }
    33.     "     _tenue_.   }
    34.     "     _undula_.  }Isolated by Kutscher, Zeit. f.
    35.     "     _volutans_.}  Hyg. xx. 45.
    36. _Vibrio I_.          }
    37.    "   _II_.         }
    38.    "   _III_.        }
    39. _Spirillum_ from pig dung. Smith. Centr.-Bl. f. Bakt.
           xvi. [1], 124.
    40. _Streptococcus liquefaciens coli_. Gamgee. Phys.
           Chem. ii.
    41. _Bacillus mycoides_.[120]

  [120] Since the above was written, Severin (Centr.-Bl. iii. [2],
  628) has described seven other species of bacteria, isolated from
  horse dung; for the description and property of these the original
  memoir must be consulted. Popp and Becker, German Patent 86,335, 28,
  describe fourteen species which they isolated from dog and pigeon
  dung, of which three have considerable bating action on skins, and
  which form the subject of the patent. Some of these are probably
  identical with organisms in the above list.

There are besides many unnamed species of bacteria in dung, among which
are the two following isolated by me from puer--

(_a_) _Plate Cultures._--Small yellowish colonies, slightly
fluorescent, liquefy the gelatin slowly. Rods resembling B. subtilis
when at rest, but moving with a rapid undulatory motion. Cultures in
nutrient gelatin have a considerable reducing action on skin.

(_b_) _Plate Cultures._--Bluish colonies liquefying gelatin rapidly,
micro-bacteria in pairs. Not so marked an action on skin as (_a_). The
organism resembles Proteus vulgaris, but swarming islets not observed.

Of the species mentioned above, a pure culture of B. subtilis in meat
broth, maintained for one week at 35° C., had _no_ reducing action on
skin. B. fluorescens liquefaciens had a moderate reducing action.

The species developing on the plates vary according to the age of the
dung. In horse manure, according to Severin,[121] bacilli predominate
in the early stages. At the end of two to three weeks micro-bacteria,
cocci and diplococci make their appearance, while the bacillar form
becomes scarce. In three months cocci predominate, forming zoogloæ;
strepto- and staphylo-cocci and spirilli were seldom found. Yeasts and
sarcina were never observed. From observations extending over several
years it is evident that a similar cycle occurs in dog dung. When quite
fresh it contains comparatively few bacteria; then certain groups of
species take possession, causing decompositions in the dung; these in
their turn give place to other species, which decompose the products
formed by the first, so that no one species produces or can produce the
complex chemical and physiological changes which take place, or the
bodies necessary for the bating of skin, as has been supposed by some
observers.

  [121] Centr.-Bl. ii. [1], 97.

None of the experiments, either with mixed or pure cultures of
bacteria, had a perfect reducing action on skin, although the action
was considerably greater than that of the chemical solutions. It was
found, however, that by adding a small quantity of the amines above
mentioned to the bacterial cultures, the reducing action became nearly
as rapid and effective as with dung itself; and it was, therefore,
clear that the principal bating effect was due to a combination of two
things, viz. an active growth of bacteria in the presence of amine
compounds.

It was now necessary to ascertain the mode of action of the bacteria on
the skins.

I have previously shown[122] the action of digestive ferments or
enzymes to be notable in the case of pancreatin. A careful study of the
behaviour of the digestive enzymes in the animal body shows, however,
pretty conclusively, that they are all destroyed before the fæcal
matter is discharged,[123] and, therefore, the enzymes in the dung are
not from this source.

  [122] J.S.C.I., 1894, March 31, 218–221.

  [123] See Gamgee, Phys. Chem. 2.

An attempt was made to filter the dung diluted with water through a
Berkefeld filter, and thus obtain a clear filtrate containing all the
enzymes and free from bacteria; it was found impossible to filter the
liquid in this way.

The method of Claudio Fermi[124] was also tried, but a clear filtrate
could not be obtained.

  [124] Ann. de Micrograph, ii. [6], March 20, 1896.

The author has succeeded in obtaining the enzymes best in the following
manner:--About 150 c.c. of the above-mentioned “puer” was well
mixed with an equal quantity of glycerin and allowed to stand for
seven days; it was then capable of being filtered on a filter pump,
although very slowly, and yielded a clear filtrate of a deep golden
brown colour; the filtrate was poured in a thin stream into a tall
vessel containing about 1500 c.c. of 98 per cent. alcohol. A copious
flocculent precipitate of the albuminous matters and enzymes was thrown
down, the alcohol was filtered off and the precipitate washed on
the filter with absolute alcohol, and dried over sulphuric acid. The
resulting amorphous body was of light brown colour, and became darker
when exposed to the air. The amount obtained from 150 c.c. of dung was
0·55 grm. (3·66 grm. per litre). The body consists of a mixture[125]
of all the enzymes existing in the dung along with other albuminous
bodies. It has a very slight diastatic action on starch. 0·5 grm. in
100 c.c. of water at 35° C. had a very considerable reducing action
on skin. A further experiment with 0·5 grm. of amine hydrochlorides,
0·5 grm. of enzymes and 100 c.c. of water, at 35° C. brought down
a piece of limed sheep grain in 30 minutes exactly like puer; the
reaction of the solution at the commencement of the experiment was
faintly alkaline; at the end of the experiment it was considerably
alkaline.

  [125] I did not consider it necessary to purify the enzymes, the
  object being to examine the action of the bacterial products apart
  from the living organisms.

It was noted that the fæcal odour of the glycerin solution disappeared
on standing for two or three weeks, and the solution smelt strongly of
ethyl butyrate, the enzymes being still present.

These enzymes were prepared from dung, and it now remained to prepare
them in the laboratory by the action of bacteria alone. For this
purpose 200 c.c. of a mixed culture of dung bacteria in the solution
above described, seven days old, was mixed with 200 c.c. of dilute
alcohol (alcohol 65, water 100) and well shaken. Gelatin and albuminoid
bodies are by this means precipitated. The liquid was filtered and
poured into eight times its volume of 98 per cent. alcohol. The
precipitate which came down was washed with absolute alcohol and dried
in the usual way. The enzymes thus obtained were redissolved in water
and the former experiment with skin repeated with this solution. The
same effect was produced, showing conclusively that it is the enzymes
produced by the bacteria, acting in conjunction with the amines, which
bate the skin. It would seem that the special action of the enzymes is
aided by the presence of amine compounds, in addition to the chemical
action which these latter have upon the skin. The action is shown to
be interdependent, i.e., bacterial action alone is insufficient, and
chemical action alone is insufficient, the true bating action being a
combination of the two.

In conclusion the author has pleasure in acknowledging the valuable
assistance of Mr. H. S. Shrewsbury in carrying out some of the
experiments.


III. FURTHER NOTES ON THE ACTION OF THE DUNG BATE.[126]

  [126] Reprinted from the Journal of the Society of Chemical Industry,
  November 30, 1899, No. 11, vol. xviii.

The present notes are a continuation of those published in 1898. It has
been impossible for me to follow up the subject except in a desultory
manner, but I think it desirable to put further results, however
meagre, before you with the object of throwing more light on the
complex process of bating hides and skins.

Since the previous notes were written, Dr. Th. Körner[127] has called
attention to the physics of the process, a matter which I had not
previously mentioned. He explains part of the action by differences
of osmotic pressure. The ammonium salts and salts of organic bases
contained in the dung, possess a smaller osmotic pressure than the
solution of calcium hydrate contained in the skins. The ammonium ion
endeavours to unite with the hydroxyl ion of the calcium hydrate to
form undissociated ammonium hydroxide. In consequence of the osmotic
pressure and the electrostatic attraction of the oppositely charged
ions, the calcium hydrate solution is withdrawn from the skin and the
latter falls.

  [127] 10 Jahresber. d. Deutsch. Gerberschule zu Freiberg, 1898–99:
  Beiträge zur Kentniss der wissenschaftlichen Grundlagen der Gerberei,
  p. 32.


_Experiments with Bacteria on a Large Scale._

I have shown[128] that simple cultures of bacteria in certain nutrient
media, in the absence of amines and other bodies, have an imperfect
bating action on skin. This was the case, whether the cultures were
pure or mixed, but the mixed cultures have a better action than
the pure cultures of any of the organisms I have hitherto used.
Bacteriological processes in nature are usually carried on by a
mixture of species of bacteria; still there are fermentations like the
spontaneous souring of milk, the formation of nitrites from nitrates,
and the ammoniacal fermentation of urea, which may be considered
as natural pure cultures. These are examples of what I may call the
selective influence of the nutrient medium. The nutrient medium used
in my former experiments was a solution of gelatin and mineral salts,
such as is used in general bacteriological work, modified in various
ways. On careful consideration, this did not appear to be a medium at
all corresponding to the natural bate. The albuminoids in the process
of digestion are peptonised, and dung contains only those bodies which
the animal is incapable of assimilating. It is from these bodies that
the necessary enzymes are produced by bacteria. Of the numerous trials
with pure cultures of different organisms from dung, I found that
most of those having the property of secreting bating enzymes, were
non-liquefying bacteria. Popp and Becker have also pointed out that
peptonising bacteria do not exert a favourable reducing action on skin.
It is impracticable to grow these non-peptonising organisms on a large
scale in a solid medium, and it occurred to me that gelatin, previously
peptonised by chemical means, would be a favourable medium. Experiments
with hydrochloric and sulphuric acids gave hopeful results. The acid
was neutralised with ammonia after the chemical action was complete,
and the bacteria cultivated in this solution.

  [128] J.S.C.I., 1898, p. 1011, see previous paper, p. 152.

A better result was obtained by digesting 10 grm. gelatin, 5 grm.
lactic acid (anhydrous by calculation), 100 c.c. of water, in a closed
vessel on the water bath for three hours. A slight black precipitate of
melanoidic acid (humic acid) is formed. The liquid is of a clear brown
colour, and contains a large number of nitrogenous bodies resulting
from the breaking down of the gelatin molecule. It has been impossible
for me to ascertain its exact composition, but I have partially
examined it with the following results:--

1. A small quantity of the liquid poured into absolute alcohol, gives
a white precipitate which re-dissolves on the addition of about 30 per
cent. water. This shows absence of gelatin.

2. On saturating with ammonium sulphate, a brownish precipitate is
thrown down which is perfectly soluble in cold water. It consists of
gelatose lactates which are evidently formed in an analogous manner,
to the gelatose hydrochlorides prepared by C. Paal.[129] The gelatoses
correspond to the protoses formed during the digestion of albumin. In
one sample 1·3 grm. gelatose lactates per 100 c.c. was found.

  [129] Ber. 25, 1202; also Allen, “Comm. Org. Analysis,” iv., 466. F.
  Marpmann, Centralblatt f. Bakt. 2, 5, 67.

3. The filtrate from (2) was dialysed against running water for
15 hours to get rid of the ammonium sulphate. The resulting
gelatin-peptones, or gelatones, amounted to 9·1 grm. per 100 c.c. In
other words, the heating under pressure had transformed 87 per cent. of
the original gelatin into true peptones.

It may be presumed that the other and simpler nitrogenous bodies
formed, are the same as those produced by the treatment of gelatin
solutions by dilute mineral acids, and that the bases combine with the
free lactic acid. An excellent list of these and a very interesting
account of the protamines and hexones which go to form the complex
albumin molecule is given in a paper by Dr. A. Kossel.[130]

  [130] Rev. Gen. des Sciences, 1899, p. 380.

The solution of gelatone lactates and free lactic acid prepared as
above, was neutralised with sodium carbonate and diluted to 1000 c.c.;
it was found to be a very good medium for the growth of the bating
organisms after the addition of a small quantity of potassium phosphate.

In looking round for suitable means of testing the action of both pure
and mixed cultures of bacteria on a practical scale, it occurred to me
that the Carlsberg vessels, as used by Hansen for the pure cultivation
of yeast,[131] would answer for the cultivation of bacteria also. I was
unable to obtain any information on the subject, and was surprised to
find that the apparatus is very little used in England. I procured two
of these vessels from Jensen of Copenhagen, and found they answered
very well. The bacteria were transferred from the test tube in which
the original inoculation had been made to a Pasteur flask of 250 c.c.
capacity containing the nutrient solution; when the growth in this
flask was sufficiently vigorous the Carlsberg vessel was inoculated
from it in the manner described by Hansen, and with all the usual
precautions.

  [131] Jorgensen, the “Micro-organisms of Fermentation,” p. 20.

After three days at a temperature of 37° C. the whole of the 10
litres was used for inoculating 100 litres of the nutrient medium
above mentioned contained in a clean barrel standing in a room,
the temperature of which was maintained at 37° C. By using this
comparatively large volume of pure culture for pitching, if I may be
allowed to use a familiar brewing term, the large culture was kept
practically pure, although it could not be supplied with germ-free air,
as in the case of the Carlsberg vessel.

Using a nutrient medium containing 1 per cent. original gelatin, the
maximum bating effect was obtained with the majority of organisms in
three days.

In my previous notes I showed[132] that a mixture of digestive enzymes
and amine hydrochlorates was effective in bating skin. The liquid in
this case was free from bacteria. It was found, however, that the
action of the bate was hastened when an active growth of bacteria was
going on at the same time in the liquid, even though the quantity of
enzyme present was smaller. This is what one might infer from a careful
study of the process. It appears better for small quantities of enzymes
to be produced in the liquid as required, than to use larger quantities
of stale enzymes.

  [132] J.S.C.I., 1898, p. 1012.

Mr. Loxley Meggitt was kind enough to concentrate for me about 170
litres of a culture of bating organisms. The concentration was
conducted _in vacuo_ at a temperature below 50° C., at which the
enzymes were certainly not injured, and it was found that when this
concentrated culture was diluted to the same strength as the original,
the action was considerably diminished. This diminution of activity was
due in part to a loss of volatile products in the evaporation, but more
especially to the absence of an active fermentation going on in the
liquid.

The nutrient medium above described, besides acting in a selective
manner on a mixture of bacteria, and providing them with food stuff,
contains amido compounds and other bodies, which are eminently
favourable to the proteolytic action of the enzymes, and therefore act
in the same way as the amine hydrochlorates which I used in former
experiments.

The action of the following bacteria grown in this medium has been
tried on skin. The pure cultures are lettered in continuation of
previous descriptions (see previous paper, p. 162).

1. Bacillus _c_, isolated from pigeon dung bate. Small, round,
bluish-white colonies, standing up slightly above the surface of the
gelatin, but not spreading out on the surface, non-liquefying pairs of
micro-bacteria. This was the principal organism found in a pigeon dung
bate as used for the bating of E.I. kips. Grown in nutrient broth or in
the special medium it had no reducing effect on skin.

2. Bacillus pyocyaneus from blue skin, i.e. skin in an early stage of
putrefaction. No action.

3. Mixed culture from fresh pigeon dung, collected in a sterile vessel.
This contained very few bacteria capable of developing in the special
medium. The action on skin was no more than that due to the chemical
compounds present.

4. Mixed culture from fresh dog dung. This contained remarkably few
species of bacteria which came to a good development in the special
medium. The principal organisms were a jointed bacillus with extremely
rapid vibrionic movement, and a small bacterium in pairs; the action on
skin was perceptible but slight.

5. Mixed culture from dog dung one month old, as used in the bate. The
action of this in a certain medium has already been described (see
p. 160). In the special medium it was distinctly better and almost
equal to that of the dung bate.

6. Mixed culture from fresh fæces gave a similar result to (3). A
microscopic examination showed pairs of small bacteria and micrococci,
but no bacillar forms.

7. Mixed culture from fresh horse dung had a moderate but distinct
action on skin, about equal to (4).

8. Bacillus _d_, isolated from wool infusion. Very slight action.

9. Bacillus _e_, isolated from wool infusion. Similar result to (8).

10. Mixed culture of bacilli _d_ and _e_ only. Very powerful bating
action, skin bated more rapidly than with dung.

These experiments tend to confirm the conclusion I previously arrived
at, viz. that no single species of bacteria produces the complex
chemical and physiological changes which take place in the dung, and
which result in the production of the bodies necessary for the proper
bating of skin. It is a well-known fact that dog dung requires keeping
for at least a month before it gives the best result. During this time
it undergoes a kind of fermentation and continues to improve up to two
months, after which it deteriorates. The rate of fermentation depends
upon the season.

It is evident from these facts that the bacteria present in the dung
when it leaves the animal’s body do not produce the required enzymes
and chemical compounds, and that these are produced by bacteria which
obtain access from the air. It is also evident that the production of
these enzymes depends upon the composition of the nutrient medium,
since this exerts a selective influence on the species of bacteria
obtaining access to it. In the spontaneous souring of milk numerous
bacteria in the air have free access to the milk, yet the lactic
ferment is generally so pure that it may be and is used as a pure
culture on a large scale in the manufacture of lactic acid. Dog dung
is a favourable medium for the bacteria secreting the bating enzymes;
but if dextrose or other carbohydrate be added to fresh dung an acid
fermentation is set up which effectually prevents the development of
the bating organisms.

Reasoning along these lines I was led to try some cultures of air
bacteria in my special medium, and I found that a good source of such
organisms likely to have a useful effect was a sweating stove as used
for the depilation of skins. The hair roots are loosened by bacterial
action; the wool, when it slips, brings away with it the epidermis.
The root portions of the wool were cut off and digested in water at
35° C., the liquid strained off and used for making plate cultures
in the usual way. In making the attenuations for the plate cultures,
the fourth was found to be practically a pure culture of the organism
I have called bacillus _d_, or the sweating bacillus. It forms large
whitish colonies, spreading on the surface, with irregular contour. The
bacilli are very small, mostly in pairs, but sometimes joining together
in thread-like forms. (Fig. 21.)

Grown in the special medium it had little or no action on skin. At the
same time I found that a culture made from the original liquid, i.e.,
a mixed culture of the sweating organisms, had an exceedingly powerful
bating action; indeed, the skin was bated more rapidly than with dung.

All the experiments carried out so far tend to prove that mixed
cultures of suitable bacteria possessed the required action, whereas
pure cultures do not. A further examination of the infusion of wool
roots showed that the bacteria contained in it, consisted practically
of two species only. The first of these I have already described,
the second one, bacillus _e_ (Fig. 22), forms small brownish-yellow
boat-shaped colonies on gelatin plates, very similar to one of the dung
bacteria. They consist of plump cells, two or three times the size of
bacillus _d_, united in pairs and short chains and surrounded by a
capsule; the cells appear to vary considerably in size. Cultivated in
the same way as the others it has very little action on skin. It is
evident from these facts that the growth of the bacteria is a symbiotic
one; separately they exert little or no action, whereas used together
the action is most remarkable.

The author wishes to express his indebtedness to Mr. J. Golding,
F.I.C., of University College, Nottingham, who made the first pure
cultures of these “wool” bacteria.


_The Influence of Solid Matter in the Bate._

In making comparative tests of my artificial bates and dog dung, I
usually took the latter from the paddles in which it was diluted
ready for use, and maintained both solutions at exactly the same
temperature. Parts of the _same_ skin were then digested in each
solution, and the results noted after 1-1/2 hours. In order to make
them more strictly comparable I filtered the dung bate, so as to employ
the matter actually in solution when the bate was made up. It was found
that the filtered bate had far less action than when it was used in an
unfiltered condition. That this is not wholly due to the passing into
solution of some of the solid matter during the bating was shown by
adding an inert solid, viz., kaolin, to the filtered bate, when the
action was greatly hastened. On adding kaolin to the artificial bate
and keeping the liquid agitated, the same result was obtained. It seems
that the finely-divided solid matter acts as a carrier for the enzymes,
perhaps by a kind of mass action, each particle offering a surface
vastly greater than the molecules of dissolved solids. In the case of
“puer,” the organic insoluble matter is gradually brought into solution
by bacterial action as the bating proceeds, though I do not believe
this occurs to any great extent.

The following quantities of soluble and insoluble matter were found in
puer wheels in actual practice per 100 c.c.:--

    -------------+-------+------+------+------
       ----      |   1   |  2   |  3   |  4
    -------------+-------+------+------+------
    Total solids | 10·20 | 8·63 | 8·64 | 3·26
    Soluble      |  6·00 | 4·57 | 6·19 | 2·14
    Insoluble    |  4·20 | 4·06 | 2·45 | 1·12
    -------------+-------+------+------+------

    1. Wheel in constant use for a week.
    2. Wheel freshly made up.
    3. The same wheel after one lot of skins.
    4. From pigeon dung bate pit.

On comparing 2 and 3 it will be seen that apparently 1·61 grm. of
the insoluble matter has passed into solution. This is not entirely
the case, for part of the additional soluble matter is lime and hide
substance from the skins, while part of the insoluble at the same time
adheres to the skins and is not estimated.

In conclusion, it will be seen that it is now quite possible to
produce an artificial dung bate, or rather a bate having the essential
properties of a dung bate, by producing the required enzymes by
fermentation and then adding to them the amine compounds. Such a bate
I am able to show you here. The chief difficulty in its practical
application in the tannery is the question of cost. If this can be
overcome, and efforts are being made to overcome it, there is no doubt
that the tanners of the next generation will be supplied with a bate
which will do the same work as the dung bate, and which will have a
definite and uniform composition and properties, and a regular and
certain action. The tannery will become cleaner and healthier, and
what is perhaps of equal importance, the sewage effluents will be
comparatively harmless.




CHAPTER VII.

ARTIFICIAL BATES.


As may be supposed, inventors have from very early times turned their
attention to the substitution of some other material in place of the
unappetizing one so long in use. But as I pointed out in a short
article entitled “Rationale of Bating”[133] many of them looked upon
bating and puering as a process for removing lime from the skins,
and nothing more; whereas we have seen that it is a very complicated
process in which, not only is lime removed more or less effectively,
but the skin fibres are acted upon, and portions of them rendered
soluble, and the skin thus got into the necessary condition.

  [133] Leather Industries, Sept. 1898.

This misconception of the object of bating or puering prevented the
success of most inventions of artificial bates up to quite recent
times. Procter states that the use of ammonium chloride as a bate was
patented by Zollikoffer in 1838. Such agents as this, and various
acids, which have been proposed are merely chemical deliming agents,
and, as such, we do not propose to discuss their use as artificial
bates. Procter[134] gives an excellent account of these processes,
including one of his own, in which ammonium chloride is used in
conjunction with metabisulphite of soda, and for successive packs
sufficient sulphuric acid is added to neutralize the ammonia formed.

  [134] Principles of Leather Manufacture, p. 160.

The same author[135] also mentions an American invention for bating
by the use of old lime liquors neutralized with sulphuric acid. This
method is much more scientific than would at first sight appear, and
more nearly approximates to the conditions of the dung bate than any of
the early inventions. Old lime liquors contain much ammonia and weak
organic acids, such as caproic, amido-caproic (leucin) and tyrosin.
On adding sulphuric acid, the lime forms an inert sulphate, and the
sulphate of ammonia and the weak organic acids which remain dissolved
are just what are required in a chemical bate. The author has used this
bate with success on certain classes of goods.

  [135] Text Book of Tanning, 1885, p. 185.

*Tiffany’s bate*, which is used in America, is composed of glucose and
glue, and bates of this composition are also in use in England. As an
example, 10 lb. glucose and 1/2 lb. of glue dissolved in 1000 gallons
water, at a temperature of 75° F., and allowed to ferment for a few
hours, may be used for the bating of bellies, and if used in a paddle
or latticed drum will bring them “down” very rapidly.

Davis[136] gives a list of twenty-nine patents for bating issued by the
U.S.A. Government from 1790 to 1883 inclusive. I do not know whether
any of these are in use at the present day.

  [136] The Manufacture of Leather, Philadelphia, 1885.

*Erodin.*--Since the publication of the authors researches on the
constitution and mode of action of the dung bate, renewed attention has
been given to the construction of bates, or puers, having an identical
action. One of these has been worked out by Professor Dr. H. Becker, of
Frankfort o/M, in conjunction with the author under the name of Erodin
(Latin, _erodere_, the equivalent of the German _beizen_--to bate).

As this was the first artificial bate in which a pure culture of
bacteria has been applied to the treatment of skins, I propose to
describe it in some detail.

Erodin[137] consists essentially of a nutrient material in the form of
powder and of a pure culture of _B. erodiens_ which is added to the
powder mixed in a definite quantity of warm water (40° C.). On the
average about 10 grm. of this material is sufficient to bate one kilo
of wet prepared skin.

  [137] At the third Conference of the I.A.L.T.C., held in Copenhagen
  Aug., 1899, F. Kathreiner (Worms) brought forward communications from
  Wood (Nottingham) and Dr. Becker (Frankfort-on-Main) on artificial
  bates, from which it is shown by experiments and trials on a large
  scale at the works of Messrs. Doerr and Reinhart that both these
  gentlemen had worked out similar processes quite independently. Wiss.
  Techn. Beilage des Ledermarkt, 1899–1900, p. 8. Bemerkungen über die
  Wirkung der Kothbeizen, J. T. Wood. Idem, p. 43.

  Popp and Becker’s und Wood’s Ersatzmittel für Köthbeizen. Franz
  Kathreiner. Idem, p. 50.

  Einiges über die Anwendung von Erodin einem Ersatzmittel für
  Hundekoth u. dergl. in der Leder industrie. Bericht von Dr. H.
  Becker, Frankfort-on-Main. Wiss. Techn. Beilage des Ledermarkt. Bd.
  i., p. 39.

  Further particulars about the bate and its construction will be found
  in Chapter VIII.

In practice it is found that this material acts far more energetically
on the skins when living bacteria are present in the liquid; we
suppose that they penetrate the skin and form their products in the
intercellular spaces. It was found that, in the evaporation of the
cultures to dryness, the bacteria were mostly killed or so far reduced
in activity, that on preparing the bate after a certain lapse of time
it was liable to become infected by foreign organisms.

In order to ensure uniform results a pure culture of the special bating
bacteria is supplied with every 5 kilos of the erodin powder. The
weighed amount of erodin is placed in a perfectly clean mashing-tub,
with 50 times the weight of water at a temperature of 40° C. a pure
culture of Bacillus erodiens is added to the mixture, and the whole
covered with a clean cover and allowed to stand for two or three days.
A little steam is admitted morning and evening so as to maintain the
temperature at about 40° C., or some other suitable device for heating
is applied. Several tubs may be kept in a small stove heated to the
right temperature by steam pipes. In this way the bating bacteria
develop rapidly, and if the proper precautions as to cleanliness and
temperature have been observed, there is no fear of any adventitious
germs getting the upper hand. As soon as a vigorous growth is attained,
usually on the third day, the bate is ready for use.

Erodin is in use on a fairly large scale, and, as is usually the case,
difficulties have arisen on a manufacturing scale which do not occur
in the laboratory. In the practical application of the material the
mashing-tub may be 3 ft. 9 in. in diameter and 3 ft. 3 in. in height
and of a capacity of 200 galls. Steam is admitted directly by means of
a copper pipe fitted with a boiling jet.

The goods are bated in the ordinary bating paddle or puer wheel
(Fig. 3, p. 13).

During the year 1901 about 9000 dozens of sheep “grains” were puered
with Erodin at Trent Bridge Works (Messrs. Turney Brothers, Ltd.)
Nottingham, and considerable experience gained in its use.

The mode of operation was as follows:--On Fridays 30 kilos of Erodin
powder was mashed in a wooden vessel in 1600 litres water at 40° C. and
inoculated with a sufficient quantity of a pure culture of B. erodiens.
The temperature was kept at 35°–40° C. by admission of a little steam
first thing in the morning, again at noon, and in the evening, and the
liquid so prepared was ready for use on Monday. One wheel or paddle was
kept going for the work. To start the operation, 200 litres of stock
liquor was put into the wheel, and sufficient water added for the goods
to turn freely. The skins previously washed, as described in Chapter
I., were paddled until sufficiently “fallen” or “down.”

For the following lots, the liquor was strengthened by the addition
of 100 to 140 litres of the stock liquor, the wheel being run off and
cleaned out once a week. The general result showed that “high-limed”
skins and previously salted skins were more refractory to this bate
than to dung.

As in the English system of collecting pelts there are all stages of
liming, skins containing from 1.5 to 9 per cent. CaO on the dry skin,
it is not surprising that considerable difficulty was experienced in
keeping the puering uniform. Skins containing over 3 per cent. CaO
on the dry skin, seem to require a real putrefactive action in order
to “bring them down” sufficiently, or it may be that their alkalinity
is too great to allow the bating enzymes to act properly. It may here
be said that if the fibres of skin are actually damaged by excessive
action of lime or any other chemical, no subsequent treatment is of
avail to remedy the defects.

Shearling pelts were excellent when puered with erodin, a further proof
that the alkalinity is of importance, since these pelts do not require
so much lime as “old fells” to bring them into proper condition for
splitting.

For sheep and calf skins treated from the market condition and
consequently containing a uniform amount of lime--2–3 per cent.--the
process is quite reliable, and is in practical use on a large scale.
Goat skins have been successfully bated with erodin by treating them
in a concentrated liquor in a drum,[138] instead of a paddle, the
mechanical effect of the drumming enabling the bate to penetrate the
hard and compact skin, while the more concentrated solution of the
enzymes attacks the so-called “beard,” and renders the skins as supple
as sheep skins bated in a paddle.

  [138] One litre of erodin stock liquor (1 part erodin, 40 parts
  water) were used per kilo drained washed pelt, in the drum for 1-1/2
  hours. In some cases less than 1 litre sufficed.

It may be of interest to give here some of the results of the early
trials of erodin in a tabular form, due to the late Franz Kathreiner.
They are a further proof of the thoroughness with which he carried out
all his work.


LEATHER, N.F. AND KALOCHROM. Date of Trial, Nov. 22, 1898.

  ---+---------------------+--------+-----------+----------+------------
  No.|         Test        |  E 47  |    B 3    |    B 4   |    B 5
  ---+---------------------+--------+-----------+----------+------------
   1 |Erodin, mark and date|Erodin M|   Wood    |   Wood   |   Wood
     |                     | 21/9/98|  1/11/98  |  1/11/98 |  1/11/98
     |                     |        |           |          |
   2 |  "     reaction     |Slightly|           |          |
     |                     |  acid  |   Acid    |   Acid   |   Acid
     |                     |        |           |          |
   3 |  "    total quantity| 500 gr.|   26 L.   | 13 L. +  |
     |                     |        |           |  700 gr. |   13 L.
     |                     |        |           |  kaolin  |
     |                     |        |           |          |
   4 |  "   quant. per kilo|        |           |          |
     |       scudded weight|        |           |          |
     |                     |        |           |          |
   5 |  "   quant. per kilo|        |           |          |
     |       slated        |        |           |          |
     |                     |        |           |          |
   6 |  "   quant. per kilo|        |           |          |
     |       tumbled       |  3 gr. |  160 c.c. |As for B3 |260 c.c. not
     |                     |        |neutralized|          |neutralized
     |                     |        |           |          |
   7 |                     |        |           |          |
     |                     |        |           |          |
   8 |                     |        |           |          |
     |                     |        |           |          |
   9 |                     |        |           |          |
     |                     |        |           |          |
  10 |Water used           | 260 l. |   210 l.  |  140 l.  |   125 l.
     |                     |        |           |          |
  11 |Temperature of bate  |        |           |          |
     |           at 10.25  | 34° R. |   34° R.  |  33° R.  |   32° R.
     |                     |        |           |          |
  12 |    "       of bate  |        |           |          |
     |           at 10.30  |   28°  |    27°    |    28°   |    26°
     |                     |        |           |          |
  13 |    "       of bate  |        |           |          |
     |           at  2.30  |   26°  |    25°    |    25°   |  24-1/2°
     |                     |        |           |          |
  14 |Time run             | 4 hrs. |3-1/4 hrs. |3-1/4 hrs.|3-1/4 hrs.
     |                     |        |           |          |
  15 |  "  standing        |  --    |  1/2 hr.  |  1/2 hr. |  1/2 hr.
     |                     |        |           |          |
  16 |Mode of motion       |Paddle 1|  Paddle 2 | Paddle 3 | Paddle 4
     |                     |        |           |          |
  17 |Acidity of 50 c.c.   |0·2 N/10| 1·8 N/10  |1·15 N/10 | 2·7 N/10
     |  before neutralizing|  soda  |   soda    |  soda    |   soda
     |                     |        |           |          |
  18 |     "     50 c.c.   |        |           |          |
     |         after kaolin|   --   |    --     |1·15 N/10 |    --
     |                     |        |           |  soda    |
     |                     |        |           |          |
  19 |     "    50 c.c.    |        |           |          |
     |         after kaolin|        |           |          |
     |                     |        |           |          |
  20 |Alkalinity of 50 c.c.|1·2 N/10| 2·2 N/10  | 1·4 N/10 | 1·3 N/10
     |  after 2 hrs        |  acid  |   acid    |   acid   |   acid
     |                     |        |           |          |
  21 |     "        50 c.c.|        |           |          |
     |                     |        |           |          |
  22 |     "        50 c.c.|        |           |          |
     |                     |        |           |          |
  23 |No. of skins and size|  45 X  |  46 X 3   |   23 X   |   23 X
     |                     |        |goat skins |          |
     |                     |        |           |          |
  24 |Weight after         |        |           |          |
     |                     |        |           |          |
  25 |  "      "           |        |           |          |
     |                     |        |           |          |
  26 |  "      "  tumbling |100 kilo| 100 kilo  | 50 kilo  | 50 kilo
     |                     |        |2-1/2 goat |          |
     |                     |        |           |          |
  27 |  "      "     "     |        |           |          |
     |                     |        |           |          |
  28 |  "      "  striking |        |           |          |
     |               out   |   77   |    77     |    36    |    36
     |                     |        |           |          |
  29 |Invoice              |        |           |          |
     |                     |        |           |          |
  30 |Date of soaks        |   --   |  5/11/98  |    --    |    --
     |                     |        |           |          |
  31 |Mark                 |   --   |   T. 71   |    --    |    --
     |                     |        |           |          |
  32 |                     |        |           |          |
     |                     |        |           |          |
  33 |                     |        |           |          |
     |                     |        |           |          |
  34 |                     |        |           |          |
  ---+---------------------+--------+-----------+----------+-----------

_Remarks._--22 skins of E 47 after 3 hours running had 250 c.c. 70 per
cent. lactic acid; they were marked with a hole in the tail; were more
fallen than the others.

_Judgment after Scudding._--All lots well down; E 47 whitest colour;
all smooth grain, E 47 and B 3 smoothest. The 3 goat were not low
enough.

It was observed both in laboratory experiments[139] and in practical
work that the presence of solid matter in the puering liquor had a
considerable influence on the speed at which the goods fell. The
clearer the liquor the slower the action. The explanation of this
fact is not quite clear. Possibly the finely divided solid matter
acts as a carrier for the enzymes, each particle offering a very
extended surface, or it may be that the action of the solid matter is
mechanical, like that observed in bran drenching.

  [139] J.S.C.I., 1899, p. 910. See also p. 73.

The goods puered with erodin, whether split or unsplit skins, should be
scudded on the grain. Sheep splits are scudded with a machine of Sir
John Turney’s type. (Fig. 6, p. 17.) Roans and goat may be scudded with
any of the spiral knife machines (Turner, Mœnus, etc.) in general use.

The following figures, p. 187, giving the reactions of an erodin bate
in c.c. N/5 alkali or acid required to neutralize 50 c.c. filtered
bate, are of interest as being taken from actual work. They were kindly
communicated to me by Herr Karl Schorlemmer, chemist to Messrs. Doerr
and Reinhart, of Worms.

Erodin, tested by the electrometric method, was found to have the
following hydrogen ion concentrations:--Paddle before goods, π = 0·672
volt, equivalent to a hydrogen ion concentration p_{H+} 6·6. The same
liquor after goods, π = 0·710, equivalent to p_{H+} 7·3. Compare puer
liquors, p. 79.

    ---------------+--------------+-------------
         Bate      | Before Goods | After Goods
    ---------------+--------------+-------------
     1. Old        |  0·05 alkali |  0·4 acid
     2. Fresh      |  0·15   "    |   neutral
     3. Old        |  0·05   "    |  0·55 acid
     4. Fresh      |  0·2    "    |  0·05 "
     5. Old        |  0·1    "    |  0·1 "
     6. Fresh      |  0·45   "    |  0·15 alk.
     7. Old        |  0·15   "    |  0·3 acid
     8. Fresh      |  0·5    "    |  0·1 alk.
     9. Old        |  0·1    "    |  0·3 acid
    10. Fresh      |  0·3    "    |   neutral
    11. Old        |  0·1    "    |  0·7 acid
    12. Fresh      |  0·2    "    |  0·05 alk.
    13. Old.       |  0·1    "    |  0·25 acid
    14. Fresh      |  0·4    "    |  0·15 alk.
    15. Old.       |  0·05   "    |  0·3 acid
    16. Fresh      |  0·45   "    |  0·1 alk.
      Mean of old  |  0·087  "    |  0·31 acid
      Mean of fresh|  0·33   "    |  0·051 alk.
    ---------------+--------------+-------------

*Oropon.*--An interesting application of the theory of the bating
process which was put forward by me[140] is that of Dr. Otto Röhm[141]
in 1908, in which, instead of a bacterial culture, he employs an
infusion of pancreas in conjunction with ammonium salts. This bate has
been put upon the market in the form of a powder under the name of
Oropon.

  [140] J.S.C.I., 1898, p. 1013.

  [141] See Chapter VIII.

The approximate composition is:--

                          Per cent.
    Ammonium chloride           65
    Wood fibre                  31
    Dry pancreas           about 3·5

The nitrogen unaccounted for by ammonium chloride is 0·32 per cent.

Its chief advantage is the simplicity of application, no previous
fermentation being required. About 5 oz. to 9 oz. are required per
100 lb. wet pelt, and it is recommended to soften the water by adding
a certain quantity of clear lime water to it previous to making up the
puer.

Its disadvantages are, that unless a previous bath is used, the
quantity of ammonia given off is detrimental to a proper bating action,
so that for good working a preliminary bating is required. Another
disadvantage is that the whole of the enzymes being in the bate their
action is more superficial and the grain may be puered too much while
the interior of the skin is not penetrated by the enzymes.

The author has made some experiments with an enzyme bate using ammonium
butyrate instead of ammonium chloride, and castor oil seed meal in
place of the wood fibre.

It was found that the amount of ammonium salts in solution in the most
effective bate was approximately one gram per litre. The bating liquid
was therefore made up as follows:--

    Ammonium butyrate        1 grm.
    Castor oil seed meal     2  "
    Dry pancreas             0·01
    Water                    1000 c.c.

This liquid at 40° C. bated a skin well in one hour. On increasing
the quantity of castor oil seed meal to 4 grm. per litre, the bating
effect was not so good. Substituting 0·5 c.c. liquor pancreaticus
(Benger) for the solid pancreas, the bating effect was too powerful,
the thinner skins were attacked by the bate, and the grain destroyed
in patches in a very similar manner to over-puered skins. Further
experiments showed that a little more than one-tenth of this amount of
pancreatic solution per litre would be sufficient (70/100 c.c. liquor
pancreaticus per 1000 litres).

Castor oil seed contains a lipatic enzyme, and when shaken up with
olive oil will emulsify the oil. 75 grm. castor oil seed meal, and
10 c.c. of olive oil were shaken up with 490 c.c. of water and formed
a perfect emulsion, no fat being visible on the surface of the liquid.
The mixture was diluted to 7 litres, and a grain treated in it at
40° C. for half an hour; it was fairly well bated in this time, and
had a smooth slippery feel, such as skins have in the puer, but was
perceptibly “higher” than the skins bated in the new bate containing
pancreatin.

This experiment appears to confirm the fact that a mixture of enzymes
is necessary, or, at any rate, that a lipatic enzyme alone is
insufficient for complete puering.

*Dermiforma.*[142]--This is an American preparation, put on the
market by the Nowak Manufacturing Co. of Chicago, and is described as
a natural bacterial liquor of organic acidity. For 1200 lb. scudded
calf skins, 9 gallons of the liquid Dermiforma are used at 100° F.,
one-third being put in to start with and the remainder added gradually;
time required, about 3 hours. For sheep skin a less quantity is
required. This bate was found to be a preparation of whey, and contains
a certain quantity (18–20 per cent.) of lactic and other organic acids,
produced by fermentation; it is therefore to be classed as a drench,
and is not a true bate or puer, since tryptic ferments or bacteria are
not present.

  [142] See Chapter VIII.; also J.S.C.I. 1906, p. 647.

*Puerine.*--Another bate, manufactured in America is advertised
under the name of Puerine, “the long-looked-for bacterial bate.” The
manufacturers give the following directions for use. To start a bating
liquor, 10 lb. of the dry powdered puerine is put into a half barrel of
warm water (100° F.), and allowed to stand for 3 or 4 days, maintaining
the temperature by occasionally warming the infusion with a steam pipe.
This amount is sufficient for each 100 galls. water in the bating vat
or paddle wheel, and will bate as many skins as can be turned in 100
galls. When the warm infusion of puerine has stood the required length
of time, in order to generate the necessary bacterial ferments, the
bating vat is run up with warm water say, 100 degrees in warm weather
and 110 in cold weather, and the infusion of puerine, including the
undissolved portion, is added to the bating vat, and the skins entered
into the bating liquor. As the action of the puerine on the skins is
exactly the same as a dung bate, the judgment of the operator must
determine when the skins are “low enough.” It is not necessary to
make a fresh bating liquor for each pack of skins, because the bating
liquor improves with age. With each succeeding pack of skins, enough
warm water is added to the bating vat to make up for the loss of liquor
which is occasioned by the removal of the previous pack, and from 3
to 5 lb. of puerine for each 100 galls. added directly to the bating
liquor; it is then in condition immediately to use again.

The bate was found to consist of

    Sugar.
    Gelatin.
    Sodium          }
    Ammonium        }   Chlorides and sulphates.
    Bisulphite of lime.
    Meal.

As it is allowed to ferment without the addition of bacteria, it will
be seen that it acts in quite a different manner to erodin, being more
of a modified drench than a true bate, and is not in any way a perfect
substitute for the dung bate.

*Sucanine.*--This is a French bate, put on the market by Messrs. Muller
and Co., Rouen; it is described as “concentrated dog puer, chemically
clean, i.e., all foreign matters have been eliminated and it contains
only that which is really necessary.” The directions state that the
quantity to be used is the same as of dog puer prepared ready for use.
The paddle is kept at a temperature of 30° C.

This material was found to be a liquid containing ammonium sulphide,
phosphates and chlorides of sodium in solution, a considerable quantity
of solid matter in suspension consisted of calcium phosphate and
calcium sulphide. There was sufficient organic matter to permit of
bacteria growing in the diluted liquid.

It had no harmful effect on skins and “brought down” most sheep skins
in an effective manner; some resistant skins appeared to require
further puering.

*Purgatol* is a patent bate put on the market by Dr. G. Eberle and
Co., of Stuttgart. 1-3/4 lb. is said to be sufficient to bate 100 lb.
wet pelt in 1–2 hours at 38°–40° C. It is a dark brown liquid with an
unpleasant smell. When boiled with potash it gives volatile alkaline
products.

Distilled with sulphuric acid it gives an acid distillate consisting
of organic acids mostly of the fatty series. It contains amines and
volatile nitrogenous bases, but no enzymes or bacteria. It must
therefore be classed as a deliming agent and not as a true bate or puer.

*“Esco.”*--This recent artificial bate is of an acid character, and
is said to have an action intermediate between the bran drench and
pigeon dung; it contains no animal enzymes, but appears to contain some
vegetable enzymes of a lipatic character; it also contains 18 per cent.
of ammonium chloride in which respect it resembles “Oropon.” It is
manufactured by E. Stickelberger and Co., Haltigen, near Basle.

Other artificial bates are treated of in the next chapter under the
respective patents. There is also an interesting article on new bates
by Eitner in Der Gerber, Nos. 878 and 879, 1911. See also Collegium,
1911, p. 402.




CHAPTER VIII.

PATENTS.


In Chapter VII. we have described a few of the many artificial bates
which have been proposed, and for which in many cases patents have been
taken out. It would be impossible within the limits of a single chapter
to give an adequate account of all these patents, and therefore it is
not proposed to deal with those which are merely deliming processes,
but solely those in which the use of bacteria and enzymes is directly
applied. We mention, however, one or two of the more interesting
deliming patents.[143]

  [143] A most useful source of information is Section XV. (Journal
  and Patent Literature) in the Journal of the Society of Chemical
  Industry, published fortnightly at Palace Chambers, Westminster.
  Abstracts of all patents relating to bates will be found under the
  above heading.


*Amend, Otto Paul*, New York (_U.S. Pat._ 763,347, 1904; _Eng. Pat._
18,514 _and_ 18,514A, 1904).--Improved Process for Bating and Puering
Skins, and Improvements relating to the Bating, De-acidifying and
Oxidizing of Hides and Skins.

The skins are treated with a solution of an ammoniacal salt, small
quantities of an acid such as hydrochloric acid being added at
intervals, so as to reform the salts. Pickled skins are de-acidified
and oxidized by treatment in a neutral or slightly alkaline solution of
a nitrite of an alkali or alkaline earth such as sodium nitrite.


*Nowak, O. H.*, Chicago (_French Pat._ 360,854, 1905; _Eng. Pat._
26,771, 1905).--Substance for Treating Hides and Process for Making
same. This is the “dermiforma” described in Chapter VII. Although
bacterial action is used to prepare the bate, the essential action is
an acid one, and, as we have said, resembles the drench.


*Norris, W. M.*, New Jersey (_U.S. Pat._ 840,794, 1907; _Eng. Pat._
29,661, 1906).--Improved Process of Treating Hides and Skins. In this
process the skins are subjected to treatment with a dilute solution
of hyposulphite of soda, which is said to “set” the gelatinous fibres
of the skin. The deliming is completed with hydrochloric acid in the
presence of salt.


*Boehringer Sohn, C. H.* (_Eng. Pat._ 3,140, 1911) Proposes to use in
place of the free organic acid, in this case lactic, the sodium salt,
and then to add slowly the amount of hydrochloric or sulphuric acid
requisite for deliming. The mineral acid combines with the sodium salt,
liberating an equivalent amount of the weaker organic acid, which then
acts upon the lime in the skins. For deliming 100 kilos. of pelt, 0·25
kilo. of sodium lactate (50 per cent.) and 0·56 kilo. hydrochloric acid
(20° Bé.) are used.

The advantage of the method is that a considerable saving is effected
in comparison to the quantity of the free organic acid which would be
required. In the above example 1 kilo. of lactic acid (43 per cent.)
would be required to neutralize a corresponding amount of lime.

In addition to these deliming processes, there are a number of bates in
which various nutritive media are allowed to ferment naturally, without
the addition of any specific bacteria. Among these may be mentioned:--


*Dr. Nördlinger’s Bate*, in which a paste of potatoes is allowed to
ferment at 30°–37° C. for several days; it is then dried at 40°–50° C.,
and used in the same way as dog dung.


*H. E. Simons’, Phosphorbutyralin*, consists of the beetroot residues
in sugar manufacture which have undergone an acid fermentation, and
contain butyrates and phosphates of ammonia.


*Oakes* (_US. Pat._ 798,070, 1905; _Eng. Pat._ 24,488, 1905).--In this
patent an albuminous body, such as casein is allowed to putrefy, at a
temperature of 100° F., for several days; an equal weight of flowers
of sulphur is then added, and the mixture used as a bate or puer. The
sulphur is said to play an essential part in the bate.

A similar process, by the same author (_U.S. Pat._ 798,293), is
described by Dr. Allen Rogers[144] in which 5 per cent. of glucose
syrup and 10 per cent. of sulphur, computed on the weight of the skins,
is fermented with yeast for twenty-four hours, after which it is used
as a bate. From a chemical point of view, it is difficult to express
the reactions taking place.

  [144] J.S.C.I. 1906, p. 103.

When the bath is first prepared, it is neutral in reaction, but, after
standing, gradually assumes a slight acid reaction. On introducing the
skins, it becomes alkaline, owing to diffusion of the lime. After a
short time, it becomes neutral again, and remains thus until the skins
are in a perfect condition, when a slight acid reaction is observed.
These changes are caused by the fermentation of the glucose, carbon
dioxide and alcohol being generated. Owing to the presence of sulphur,
a quantity of hydrogen sulphide is produced, which, in its nascent
condition, combines with the alcohol to form mercaptans, which in their
turn are slowly oxidized to thioacids. These mercaptans and thioacids,
being of weak acid character, act in conjunction with any excess of
hydrogen sulphide upon the lime, converting it into soluble compounds,
which are readily removed by diffusion.


*Lederer, L.* (_French Pat. No._ 404,926 _of_ 1909).--Method of
Preparing a Substitute for Animal Excrement (dog dung) in Puering
Skins for Glacé Leather. In this process a mixture of 10 parts finely
powdered bone meal, and 1 part of crushed lupin seeds or beans is
formed into a paste with water and allowed to undergo spontaneous
fermentation from 3–4 weeks. The mixture is said to form a perfect
culture medium for the micro-organisms indispensable in bating. The
removal of the lime is facilitated by the addition of 2 per cent. of
fat, 0·75 per cent. sodium polysulphide and 1-1/2 per cent. common salt
reckoned on the weight of the bone meal.

In this patent the researches of Wood, Andreasch and Eitner are
referred to, and also Benker’s process of bating with Peruvian guano to
which soda has been added.


*Turney, Sir J. and Wood, J. T.*--Coming now to bacterial and enzymatic
bates, the author in conjunction with Sir John Turney applied for a
patent for a new and improved method of bating skins (No. 25,894,
1896), in which cultures of suitable bacteria were to be used in place
of infusions of dog or pigeon dung. It was found however that this
patent had been anticipated by the patent 21,720 of 1895, of Dr. Popp
and Dr. Becker of Frankfort o/M, and the application was therefore not
proceeded with. As has been related in Chapter VII. the late Franz
Kathreiner, who knew of the work which was being done both by Dr. H.
Becker and the author, made us personally acquainted, and most of our
later work was done in conjunction. Like many other new ideas, it was
arrived at quite independently by Dr. H. Becker and the author. (See
Report of Third Conference of I.A.L.T.C., Copenhagen, 1899, Wiss.
Techn. Beilage des Ledermarkt, 1899–1900, p. 8.)

We shall therefore give the original patents in full, and afterwards
the translation of Dr. Otto Röhms’ patent, and the recent patent of Dr.
Eberle, as these cover the whole ground of the bacterial and enzymatic
bates up to the present time.


*Dr. Popp and Dr. Becker’s English Patent* (No. 21,720, 1895).
Improvements in Lyes or Liquors for Preparing Hides for Tanning. An
important role in the process of manufacture of many kinds of leather
is played by the dung liquor or lye, which in particular for calf skins
and skivers constitutes one of the necessary preparatory operations for
the subsequent actual tanning process, and which has for its purpose
to bring the skins or hides, which have been previously swelled by the
liming process, into a condition which allows the easy and thorough
removal of the lime and lime soap as well as the remaining hairs by the
subsequent mechanical treatment, and to obtain a uniform shrinking of
the swelled hides.

The process hitherto employed consists in treating the hides or skins
for some time (such as for 4 or 5 hours) in a vat provided with a reel,
and containing a lye or brine formed of solutions of dog or pigeon or
poultry dung.

Generally speaking, the dog dung acts best in some cases, and pigeon or
poultry dung in other cases, but no satisfactory and sound explanation
has as yet been given as to the nature of the action.

My correspondents have in the first place ascertained that the action
of these lyes or brines is the result of bacteriological processes,
especially of the products created by the action of certain bacteria,
and is certainly not, as it has hitherto been generally assumed, the
result of the action of phosphoric acid or other acids.

The examination of the dog, pigeon and poultry dung in bacteriological
respect, and of the industrial applicability of all the different
results obtained, shows in the first instance that, even after removal
of all other ingredients, which, according to the hitherto ruling
views, enter into reaction, the result of the treatment remains the
same as it had been before the removal. The continued research led to
the second result that a great number of micro-organisms play a role in
the lye, partially an advantageous, and partially a detrimental one.

Generally speaking, the non-peptonizing bacteria act advantageously,
while the peptonizing ones generally have a detrimental effect. In
connexion herewith two methods of utilizing these discoveries have been
found.

One can either, according to the methods of bacteriology (plate
process), remove all the detrimental or even the less useful bacteria,
and effect the treatment of the hide or skins by applying a pure
culture of one or at least a mixture of the different most effective
bacteria; or one can, without employing such a refined process, but
still utilizing the said discoveries, apply the dog dung, and in
particular the white descriptions which are most effective, or, for
certain kinds of leather, the pigeon or poultry dung, subjecting the
said dungs to a certain treatment, which, by the use of a suitable
cultivating bed, will develop the useful bacteria, while on the other
hand, the increase of detrimental bacteria may be prevented or retarded
by the addition of materials hindering the development of the latter.

In every case the employment of the process secures greater rapidity
of execution, and more reliable and better results than the process
heretofore applied.

The leathers obtained are tough and yet softer, and in any case any
discolouring, such as is often unavoidable with dog dung, is avoided.

An advantage not to be underrated with the use of germ cultures, is
that liquors or cakes having a definite percentage of really effective
substance can be prepared, whereas it was heretofore necessary to
proceed by way of trials, the result being subject to great uncertainty.

In addition, the dung liquors are subject to changes overnight
under the influence of atmospheric changes (thunder-storms) in a
most unaccountable manner, owing, as has now been discovered by my
correspondents, to the growth of detrimental bacteria which are mixed
with the effective ones, and which gain the upper hand over the latter
under the change of atmospheric conditions.

The features of the newly discovered useful bacteria are fully
described in the following, and experiments have shown that the
particular kinds designed with 3, 7 and 12 in the table of bacteria,
set forth in the Specification to German Patent No. 86,335 and
hereinafter fully described, are those which are of value for the
purpose in question; in particular No. 12 is the most effective, and 3
the least effective of these three descriptions.

All three are bacteria to be found in the dog dung, and bacteria No.
3 consists of very small rods rounded at the ends and are exceedingly
mobile, while bacteria No. 7, which are similar to the hay bacillus,
are slowly-moving rods, and bacteria No. 12 are medium-sized rods
having rapid motion.

The prick-culture gives with bacteria No. 3 a very good growth in the
depth; from the inoculating prick extend laterally many arms into the
gelatin. At the ends of the arms are formed small knots. On the surface
is formed a thin white covering. The gelatin is not liquefied.

On gelatin plates, the deep-seated colonies consist of pale yellow
round disks, which gradually penetrate to the surface, and there they
form circular disks which show at the centre larger spots. On inclined
agar-agar there is formed a white superstratum.

With bacteria No. 7, the gelatin is strongly liquefied in the
prick-culture. On the surface of the zone of liquefaction there is
formed a white skin.

Along the liquefied prick are formed radiations into the solid gelatin.
On gelatin plates there are formed rapidly liquefying colonies forming
at top a white skin, agar-agar gives a white unequal thin superstratum
with branchings.

Bacteria No. 12 grow with prick-culture equally well along the prick;
the gelatin is not liquefied. On the surface there is formed a thin
shining covering.

On gelatin plates, the deep lying colonies consist of pale yellow
circular disks.

Many colonies have tails similar to the surface colonies of the
Proteus. During the growth of this bacterium on gelatin plates a strong
putrid smell is developed.

On agar-agar this bacterium forms a strong white shining superstratum.

These bacteria grow easily in any decoction of meat, and the flesh
particles from the flesh sides of the hides, which otherwise form a
waste product of little or no value, may be advantageously employed
for this purpose; bran, and other materials known to offer good
culture-beds, may be employed instead.

For this purpose a vessel provided with a cover is charged with equal
parts of waste flesh, such as is obtained in the tannery, and water,
and this is boiled for about half an hour by the direct introduction
of steam, after which concentrated carbonate of soda solution is
introduced until the reaction is slightly alkaline, after which the
decoction is cooled down. As soon as the temperature has sunk to about
35° C. there is added the pure culture obtained by the gelatin plate
process (with dilution), described in the works on bacteriology of
Koch, Fraenkel, Hueppe and others, and also by the further treatment
corresponding to the characteristic peculiarities of the bacteria, and
the whole is allowed to stand covered up for a period of from twelve
hours to five days, depending on the time of year and the temperature.

The hides or skins are then dipped into the concentrated decoction for
a short time, according to the state of development of the bacteria; or
the decoction can be diluted and the hides be then immersed therein for
a greater length of time.

The further treatment is then carried out in exactly the same way as
though the hides or skins had been treated in dog-dung lye, with the
exception that the duration is shorter.

In case the pure culture of the bacteria is to be dispensed with, and
it is only desired to obtain an advantage over the existing methods in
so far as to obtain, on the one hand an artificial increase of the ooze
medium viz. the number of useful bacteria and their products existing
in the dog dung, by providing a suitable culture bed, and on the other
hand to be in a position to limit oneself to the best initial material,
the process can be carried out by starting with a practically proved
dog dung, therefore in particular the white kinds, and the bacteria are
caused to propagate in a decoction such as above described. Afterwards
it is advantageous to effect the killing of the detrimental bacteria
by a small addition of acid, such as 0·5 per cent. of dilute sulphuric
acid or equivalent materials, as in this case the useful bacteria are
also the most enduring.

The more useful bacteria show, in particular in the presence of
alkalis, the same behaviour.

For example, the more useful bacteria arrive at development equally
with the detrimental ones, if to the described culture bed be added 0·5
per cent. of carbonate of soda, while the useful bacteria preponderate
to a considerable extent, and the detrimental ones are practically
unimportant, if the additions of carbonate of soda be raised to about
one per cent.

The pure cultures of the bacteria may be preserved in a suitable liquid
or solid material, and brought on the market in a suitable packing.

In order to obtain a product which shall both be durable, and capable
of being delivered in definite doses, sterilized bran, flour, etc., can
be inoculated with definite quantities of the pure cultures, and the
percentage of water be reduced, by drying, pressing, etc., to under a
certain percentage (10 to 12 per cent.) which will not allow of, or
will retard the evolution of germs.

The bacteria may also, after having been properly developed, especially
after having created a sufficient quantity of their products, be
killed, for instance by heating, and the resulting stuff be brought
on the market in liquid or solid form, as an effectual lye or brine,
it having been found that the liquids containing the said bacteria do
not lose their effect if they are sterilized and the bacteria thereby
killed, provided that a sufficient number of bacteria has been produced.

_Claim._--1. The process for the production of a lye for treating hides
or skins preparatory to tanning, consisting in the propagation of the
bacteria of dung, in particular dog and bird dung, by means of suitable
culture beds substantially as described.

2. As a new article of manufacture a lye containing a pure culture of
one or more of the described bacteria.

3. In the process of producing a lye referred to in the first claim,
the elimination of the detrimental agents substantially as described.

4. In the process referred to in the first claim, the addition of acids
or alkalis for the purpose set forth and substantially as described.

5. The process for the production of a lye for treating hides,
consisting in the separation from the dung of the bacteria herein
designated as 3, 7 and 12, producing pure cultures thereof, and
introducing the same into a decoction preferably freed by boiling from
other bacteria and containing a suitable culture bed, substantially as
described.

6. A process for the production of cakes, for the treatment of hides,
consisting in the inoculation of sterilized bran, flour, etc., with
cultures having a definite percentage of bacteria and the removal of
the proportion of water that is necessary for the development of the
bacteria, substantially as described.

7. As a new article of manufacture sterilized cakes, containing a
certain quantity of the described bacteria, substantially as described.

8. As a new article of manufacture the solid or liquid material
containing the products of the decomposition, as generated by the pure
cultures of the said bacteria, whether such material does or does not
also contain the said bacteria, either in the active condition or
killed by sterilization, substantially as described.


*Wood’s English Patent* (12,549, 1898).--Improvements in and relating
to the means used for the Bating of Hides and Skins. This invention
has reference to improvements in and relating to the means, liquid or
bate, used for the bating or puering of hides and skins previous to the
process of tanning the same.

At the present time skins are usually bated by means of a liquid or
bate prepared from dog or pigeon dung, and although the bating action
or actions have not hitherto been thoroughly understood, it is a well
known fact that fermentations going on in the dung play an essential
part. It was also pointed out in 1885 by H. R. Procter, in his “Text
Book on Tanning,” that the action of the bate was in a great measure
due to bacteria. It is also a known fact to practical tanners that a
dung bate, properly applied, produces the best quality of leather.

Owing, however, to the varying composition of the dung used, the
bate made therefrom possesses varying properties, its bating action
is uncertain, its properties are readily affected by atmospheric
influences, and the skins treated are liable to putrefaction.

As a substitute for dung bate, it has been proposed, as set forth in
the Specification of Patent No. 21,720, A.D. 1895, to use a bate made
from a pure culture of bacteria. Such bate, however, does not possess
all the essential properties of a dung bate; and although bacterial
action is necessary, such action alone is not sufficient, as was
pointed out by the present inventor in 1894 in the “Journal of the
Society of Chemical Industry.”

The present inventor has now discovered that the bating action of a
dung bate is due to the combined action of enzymes or unorganized
ferments, and to certain amine compounds in the dung. These compounds
consist of amines (i.e. ammonia in which one or more atoms of hydrogen
has or have been replaced by alcohol radicles), in combination with
lactic, acetic and other organic acids contained in the dung, the acids
named being the principal ones.

The present inventor has also discovered that the bating action of
the enzymes alone, or the action of the chemical compounds alone, are
inefficient, and that the enzymes exert their bating action in the
presence of the chemical compounds, while these latter in addition have
an independent action.

The object of this invention is the production, according to scientific
methods, of a bate having similar properties to those of a dung bate,
such properties in the improved bate being defined and definite, but
capable of being modified according to the requirements of the skins to
be bated and the leather to be produced.

According to this invention, the liquid or bate is made by producing by
fermentation an enzyme or enzymes of the same character as the enzymes
contained in dog dung or produced from bacteria obtained from dung,
and to the liquid thus obtained there is added an organic acid and an
alkali.

The inventor has discovered that for an efficient bate a pure culture
of bacteria is insufficient, and that no single organism hitherto
isolated will give results equal to dung. He has also discovered that
the production of these enzymes depends more upon the composition of
the nutrient medium in which the bacteria are cultivated than upon the
kind of bacteria, although these latter must be capable of producing
the required enzymes, the production of which appears to be due to a
symbiotic growth of bacteria, at present little understood.

It is desirable that the nutrient medium should be without sugars or
starchy matters, and, although the composition of the nutrient medium
may be varied, good results can be obtained according to ordinary
bacteriological methods by using a medium composed of--

    Gelatin                          50 parts
    Potassium or sodium phosphate     1 do.
    Water                          2500 do.

This medium is maintained at a temperature of about 37° C. (the most
favourable temperature for the fermentation) until the required
bacterial action is completed, the time necessary being about seven
days, and to each litre of the liquid thus obtained there are added
from 2 to 6 grs. of lactic acid. Acetic or other similar organic acid
may be used instead of the lactic acid, but sulphuric or mineral acids
must not be used. The acid liquid is afterwards neutralized by ammonia,
which is added when the bate is to be used, and there are then formed
certain chemical compounds similar to those found in a dung bate.

The addition of the acid to the nutrient medium will immediately stop
the growth of bacteria, but the acid is not added for that particular
reason, but for the particular purpose set forth.

Instead of the enzymes obtained from the bacteria of dung, other
enzymes of a similar character may be used, such as the enzymes
obtained from the bacteria found on skins during the sweating process.

For bating skins in a practical manner with the improved bate herein
set forth, the method of using the same is preferably varied according
to the skins to be treated and the kind of leather it is required to
produce. Thus for kid skins, skivers and other skins for which dog dung
has hitherto been used, the skins, after the liming process, are first
well washed in water in the usual way to remove the bulk of the lime.
The skins are then placed in a wheel or paddle with the bate prepared
as before described, with the acid exactly neutralized by the ammonia
or so nearly neutralized that the alkaline action of the skins will
complete the neutralization, all skins being alkaline when introduced
into the bate.

The bate is diluted according to the amount of liming the skins have
undergone, and when in the paddle or wheel the skins and bate are
maintained at a temperature of about 37° C., and the skins paddled
until they have “fallen” considerably. The skins are then removed
from the wheel or paddle and scudded preferably on the “grain” side,
after which they are returned to the wheel or paddle and the paddling
continued until the bating action is completed, this being judged in
the usual way by a practical tanner according to the feel of the skins.
The strength of the bate after the scudding process may be varied if
found desirable.

After the bating process is completed the skins are treated in the
usual manner.

When the improved bate is to be used for light hides and similar
skins which have hitherto been treated with pigeon dung, the skins,
after the liming process, are washed in the usual manner, heated to a
temperature of about 37° C., and placed in a wheel or paddle containing
by preference the improved bate in an acid condition, that is before
the ammonia has been added, the bate being previously diluted according
to the amounts of liming the skins have undergone. The skins are then
paddled until the tanner, by the feel, judges that the whole of the
lime has been removed. The acid bate is then neutralized by ammonia,
with or without removing the skins, and the paddling is continued until
the bating process is completed, this being judged of by the feel of
the skins.

The bated skins are then scudded, washed, and treated in the usual
manner.

The strength of the bate, into which the skins are placed for both
methods, is varied according to the amount of liming the skins have
undergone; that is to say, for “high” limed skins, a stronger bate is
required than for skins which have been subjected to a moderate liming.

_Claim._--1. A bate having the essential properties of a natural dung
bate, but prepared according to scientific methods from the means
and in the manner substantially as set forth in the accompanying
specification.

2. The bate described and set forth in the accompanying specification.


*Wood’s American Patent* (638,828, _Dec. 12, 1899_) *Bate.*--This
invention has reference to improvements in and relating to the means,
liquid, or bate, used for the bating or puering of skins previous to
the process of tanning the same.

At the present time, skins are usually bated by means of a liquid
or bate prepared from dog or pigeon dung; and, although the bating
action or actions have not hitherto been thoroughly understood, it is
a well-known fact that fermentations which the dung undergoes play an
essential part. It was also pointed out, by H. E. Procter in 1885, in
his “Text Book of Tanning,” that the action of the bate was in a great
measure due to bacteria. It is also a known fact to practical tanners
that a dung bate properly applied produces the finest and most supple
leather. Owing, however, to the varying compositions of the dung used,
the bate made therefrom possesses varying properties: its bating action
is uncertain, its properties are readily affected by atmospheric
influences, and the skins treated are liable to putrefaction.

As a substitute for a dung bate, it has been proposed, as set forth
in the specification of the British Patent No. 21,720, A.D. 1895, and
the specification of the German Patent No. 86,335, Class 28, to use a
bate made from a pure culture of bacteria. Such bate, however, does
not possess all the essential properties of a dung bate, and, although
bacterial action is necessary, such action alone is not sufficient,
as was pointed out by me in 1894 in the “Journal of the Society of
Chemical Industry.”

I have now discovered that the bating action of a dung bate is due to
the combined action of enzymes, or unorganized ferments, and certain
chemical compounds, which compounds consist principally of amines--i.e.
ammonia in which one or more atoms of hydrogen has or have been
replaced by alcohol radicles--in combination with organic acids usually
found in dung, and of the compounds so formed the lactates and acetates
are the most important. I have also discovered that the bating action
of the enzymes alone, or the action of the above-referred-to chemical
compounds alone, is inefficient, and that the enzymes exert their
bating action in the presence of the chemical compounds, while these
latter, in addition, have an independent action on the limes in the
skins, and on the skin fibre.

The object of this invention is the production, according to scientific
methods, of a bate having similar properties to those of a dung bate,
such properties in the improved bate being modified according to the
requirements of the skins to be bated and the leather to be produced.

According to this invention, the liquid or bate is made by producing by
fermentation, an enzyme or enzymes of the same character as the enzymes
contained in dog dung, or produced from bacteria contained in the dung,
and to the liquid thus obtained there is added an organic acid and an
alkali.

I have discovered that for an efficient bate a pure culture or single
species of bacteria is insufficient, and that no single organism
hitherto isolated will give results equal to dung. I have also
discovered that the production of enzymes of the character above
referred to, depends more upon the composition of the nutrient medium
in which the bacteria are cultivated, than upon the kind of bacteria,
although these latter must be capable of producing the required
enzymes, the production of which appears to be due to a symbiotic
growth of bacteria at present little understood. The bacteria used for
the purpose of this invention may be the bacteria found in dog dung, or
the bacteria obtained from the roots of the hairs of skins during the
sweating process, these latter being used, by preference, on account
of the ease with which they are obtained, compared with the difficulty
of isolating from the dog dung the particular mixture of species most
desirable, there being so many undesirable species present. Of the
bacteria found on the roots of the hair during the sweating process,
and hereinafter referred to as “sweating” bacteria, I have found that
two species predominate, and, so far as I am aware, these have not been
previously isolated. The principal organism, which I call “bacillus
_d_,” in continuation of a series of previous investigations described
in the “Journal of the Society of Chemical Industry,” A.D. 1898, forms
large whitish colonies, with irregular contour spreading on the surface
of the gelatin, the bacilli being very small, mostly occurring in
pairs, but sometimes joined together in thread-like forms. The second
organism, called by me “bacillus _e_,” forms small brownish-yellow
boat-shaped colonies on gelatin plates. The bacteria consist of plump
cells two or three times the size of bacillus _d_, united in pairs and
chains, but the cells vary considerably in size, and are surrounded
by a capsule. I have discovered that the above bacteria, when used
separately as pure cultures, exert little or no action on the skin, but
when used together the action is very remarkable.

The gelatin and gelatin plates referred to in the above description are
the gelatin and gelatin plates used for the cultivation of bacteria,
according to the ordinary bacteriological methods as described in
Günther’s “Bakteriologie,” published by George Thieme, Leipzig, in
1898, and in other text-books.

In the accompanying drawings, made from photographs which are of record
in this case, the bacillus _d_ is shown in Fig. 21, and the bacillus
_e_ in Fig. 22, both photographs showing the bacilli magnified 1000
diameters.

A culture may be prepared from the sweating bacteria by taking the
hair from the skin when it “slips”--that is, when it can be removed by
simply pulling it. The root portion of the hairs are then cut off, and
about 10 grm. carefully digested in 100 c.c. of water at a temperature
of 32° to 35° centigrade for several hours. The liquid portion is then
poured off, and used for inoculating 10,000 c.c. of a nutrient medium
of the character hereinafter described. The inoculated nutrient medium
is then maintained for three days at a temperature of 37° centigrade,
and may then be used for the inoculation of a larger quantity of
nutrient medium.

Theoretically, a single cell of a bacterium is sufficient to inoculate
any quantity of any suitable fluid; but in practice it is found
necessary or expedient to employ 5 or 10 per cent. of inoculating
matter to the volume of the liquid to be inoculated. The inoculation of
the 10,000 c.c. of nutrient medium may be done in a Carlsberg vessel,
as described by Hansen in his “Untersuchungen aus der Praxis der
Gärungsindustrie.”

It is desirable that the nutrient medium should be without sugars
or carbohydrates; and, although its composition may be varied, good
results can be obtained by ordinary bacteriological methods, by using
a medium composed of gelatin, 50 parts or 20 grm.; potassium or sodium
phosphate, 1 part or 0·4 grm.; sodium chloride, 1 part or 0·4 grm.;
water, 2500 parts or 1000 c.c. This medium, after inoculation, is
maintained at a temperature of 37° centigrade, until the required
bacterial action is completed, i.e., until the whole of the nutriment
for the bacteria is exhausted, at which time the maximum quantity of
the required enzymes has been produced. The time required for this
action is not less than three days, nor more than seven days. The
exhaustion of the nutriment may be ascertained scientifically by a
microscopic examination of the liquid, the bacilli at this period
commencing to form spores. To each litre of this liquid there is then
added from 2 to 6 grm. of lactic acid, and the acid liquid is afterward
neutralized by ammonia, which is added when the liquid is to be used as
a bate, the addition of the ammonia forming certain chemical compounds
similar to those found in the dung. Instead of lactic acid, acetic
or other similar organic acid may be used, but sulphuric or mineral
acids must not be used. The addition of the acid to the liquid will
immediately stop the growth of the bacteria; but the acid is not added
for that particular purpose, but for the formation of the chemical
compounds above referred to.

The above-described method is most suitable, when the bate has to be
kept for a time before being used, or when it is desired to concentrate
it for transit or export; but if the bate is to be used immediately,
it may be prepared as follows: 100 parts of gelatin are dissolved in
1000 parts of water, and there is then added 50 parts of commercial
lactic acid. The mixture is then heated, in a closed vessel, to 100°
centigrade, by which operation the gelatin is partially peptonized,
and the subsequent bacterial action is hastened. The acid liquid is
afterward neutralized with ammonia, or other alkali, and the whole
diluted with water to 20,000 parts. The dilution may conveniently take
place in the wheel or paddle in which the bating is to be done, and
to the liquid thus obtained there is added from 5 to 10 per cent. of
the active culture from the Carlsberg vessel, as above described. The
liquid is then allowed to stand for 15 to 20 hours at a temperature of
37° centigrade, and is then ready for use.

For bating skins in a practical manner with the improved bate herein
set forth, the method of using the same is varied, according to the
skins to be treated and the kind of leather it is required to produce,
and the strength of the bate is varied in exactly the same manner as
the strength of a dung bate, and such variation is made according to
the judgment of the tanner, the improved bate acting in the same manner
as a dung bate.

For kid-skins, calf-skins, skivers, and other skins for which dog
dung has hitherto been used, the skins, after the liming process, are
first well washed in water in the usual way, to remove the bulk of the
lime. The skins are then placed in a wheel or paddle, with the bate
prepared, as above described, with the acid exactly neutralized, or so
nearly neutralized, that the alkaline action of the skins will complete
the neutralization, all skins being alkaline when introduced into the
bate. The bate is maintained at a temperature of 37° centigrade, and
the skins are kept in motion until they have “fallen” considerably,
i.e., until the swollen condition of the fibres due to the liming has
disappeared. The skins are then removed from the wheel or paddle and
scudded by hand or machine, preferably on the grain side, and the skins
not sufficiently reduced are returned to the paddle, and the bating
continued until the action is completed, this being judged in the
usual way by the tanner by the feel of the skins. The strength of the
bate, when skins are returned thereto, may be varied from the original
strength, if desired, and after the bating is completed the skins are
treated in the usual manner.

The strength of the bate into which the skins are placed, is varied
according to the amount of liming the skins have undergone--that
is to say, for a high-limed skin a strong bate is required. For
moderately-limed skins the bate may be of such a strength that each
litre as prepared for use contains five grm. of the original gelatin
contained in the nutrient medium, which is equal to one part of liquid
in which the culture has taken place to three parts of water. When
the bate is prepared for immediate use, its strength is varied in
the paddle as required. For very hard skins, such as goat-skins, the
culture may be used as a bate undiluted or it may be diluted with an
equal quantity of water.

For light-dressing hides and similar skins, which have hitherto
been treated with pigeon dung, the skins are washed in the usual
manner after the liming process, heated to 37° C., and placed in a
wheel or paddle containing the improved bate, preferably in an acid
condition--that is, before the ammonia or alkali has been added--the
strength of the bate being equivalent to five grm. of original gelatin
contained in the nutrient medium to each litre of the bate used. When
the lime has been removed from the skins by the bate, which will be
in about one hour, a quantity of the original culture previously
neutralized with ammonia or other alkali and equal to about one-half
of the quantity of the original culture first added is placed in the
wheel and the bating continued until the process is completed, this
being judged by the feel of the skins. If in the above method the
chemical reaction of the bate be examined before the second portion
of culture be added, it will be found neutral, and the action of
the enzymes takes place in a neutral or alkaline solution, as in the
first-described method.

The character of the principal enzyme in dung and in the improved
bate is a proteolytic one; i.e. it resembles the trypsin contained in
pancreatic juice.

Instead of adding an organic acid and neutralizing it by an alkali,
as described with reference to both processes, there may be added an
organic salt of the same character as that formed by the combination of
the acid and the alkali.

_Claim._--1. A bating liquor or bate containing enzymes of the same
character as the enzymes contained in dog dung, an organic acid,
and an alkali, or their chemical equivalent, i.e. an organic salt
substantially as herein set forth.

2. A bating liquor or bate containing enzymes of the same character
as the enzymes contained in dog dung and produced by fermentation an
organic acid and an alkali, or their chemical equivalent substantially
as herein set forth.

3. A bating liquor or bate containing one or more enzymes of the
character of the enzymes contained in dog dung, an organic acid and an
alkali, or their chemical equivalent substantially as herein set forth.

4. A bating liquor or bate containing one or more enzymes of the
character of the enzymes contained in dog dung, or produced from
bacteria obtained from dog dung, an organic acid, and an alkali or
their chemical equivalent substantially as herein set forth.

5. A bating liquor or bate containing enzymes of the same character as
the enzymes contained in dog dung, two to six grm. of an organic acid
to each litre of the liquid and neutralized by an alkali substantially
as herein described.

6. A bating liquor or bate containing enzymes of the same character as
the enzymes contained in dog dung, from two to six grm. of lactic acid
to each litre of liquor and neutralized by ammonia substantially as
herein described.

7. A bating liquor or bate containing enzymes of the same character as
the enzymes contained in dog dung and obtained from bacteria found on
the roots of hairs during the sweating process, from two to six grm.
of lactic acid, and neutralized by ammonia substantially as herein set
forth.

8. A bating liquor or bate containing enzymes produced from bacteria
obtained from dog dung and cultivated in a medium composed of fifty
parts of gelatin, one part of potassium or sodium phosphate, one part
of sodium chloride and two thousand five hundred parts of water, two to
six grm. of lactic acid to each litre of liquor and neutralized by the
addition of ammonia substantially as herein described.


*Oropon* (German Patent 200,519, July 21, 1908).--Process for Bating
Hides. Dr. Otto Röhm, Esslingen a/Neckar. In the present process, a
watery extract of the pancreas in combination with ammonia or alkaline
salts or mixtures of these salts is used as a bate liquor. The object
of the process is to replace the dung bate. From the researches of
Wood[145] also Jettmar[146] it appears that the peculiar behaviour
of the dog dung bate is to be attributed to the action of enzymes in
combination with organic amine compounds and ammonia salts. Starting
from the assumption that other enzymes, known to have an action on hide
substance, might have an analogous action to the dog dung bate, the
author discovered that the trypsin of the pancreas had exactly the same
bating action as the enzymes of dog dung. The action is aided by the
fat-splitting enzyme of the pancreas, steapsin, which saponifies the
fat in the hides.

  [145] J.S.C.I. 1898, pp. 1010 to 1013, and 1899, pp. 990 to 993.

  [146] An abstract of Wood’s work. 1901, p. 148, section 4, p. 149.

A good bating action is obtained by a watery extract of pancreas, by
the addition of an ammonium salt, which changes the caustic lime (from
the limed pelt) into a soluble lime salt, and gradually reduces the
alkalinity of the bate liquor caused by the lime in the skins, since
the ammonia liberated by the lime escapes to a great extent from the
liquor. The favourable action of the ammonium salt on the skins causes
them to fall and become thinner, nor do they become rough when placed
in clean water after bating, as a strongly alkaline skin will if placed
in water containing calcium carbonate.

For example, the process may be conducted by extracting a pancreas
weighing about 250 grm. with 1 litre of water, and adding 10 c.c.
of such extract to 990 c.c. of a solution containing 0·15 per
cent. ammonium hydrosulphide and 0·3 per cent. sodium chloride.
Such a solution forms a very active bate. In place of the ammonium
hydrosulphide, any other ammonium salt yielding a soluble lime salt
may be used, e.g., ammonium chloride.

The pancreas extract used in the above process must be used in a fresh
condition or preserved from putrefaction by the addition of some
suitable preservative; if putrefied, and consequently acid, it will not
work. The preservation of the pancreas may also be brought about by
drying, and the bate liquor made from the dry material by putting it in
water. No putrefaction occurs during the bating process. The action of
the enzymes is favoured by the alkaline reaction of the hide.

_Claim_.--Process for bating hides, characterized by the use of a
watery extract of the pancreas, with the addition of an ammonium or
alkali salt which forms soluble lime salts, or mixtures of such salts
to the liquid.

In an addition to the above patent, Dr. Röhm proposes to soften the
water used by adding the proper amount of clear lime water to it.
Starch paste can also be used for the same purpose, or a mixture of
lime water and starch paste.

In a further addition, a weak acid mixture, consisting of 0·05 to 0·1
per cent. lactic acid and 0·05 to 0·1 per cent. ammonium chloride, is
proposed in place of the ammonium salt alone, since the evolution of
free ammonia had been found to be detrimental to the skins.


*Eberle’s English Patent* (21,202, 1909).--Improved Process for Bating
Hides. Efforts have been made for years to replace dogs’ excrement
used in many cases in tanning as a bate, by other simpler products
and methods which in particular shall have the advantage of greater
uniformity and thereby act with greater certainty.

Now it has been ascertained that substances which are only solvents of
lime, such as acids of inorganic or organic nature and their salts,
form an incomplete substitute. It has been shown by Eitner, Wood and
others that there are enzymes and bacteria to which dogs’ dung owes its
specific or bating properties. The action of dogs’ dung is explained
as one which liquefies or dissolves the skin substance, such as is
otherwise only done by enzymes and bacteria.

Experiments in the laboratory and in practice have shown that it is
not the dissolving action on the skin substance in the narrow sense
which is desired in bating the derm with dogs’ dung, but in order to
prepare the hide for the manufacture of a soft and elastic leather, the
albuminous intercellular substances and more particularly the layers
of fat lying at the roots of the hair, in the corium and in many cases
also on the epidermis, must be dissolved and thereby the fibrils of the
skin loosened. The highly solvent action of the dung bate on the skin
substance means in very many cases a direct disadvantage by reason of
the loss of leather substance.

Extensive experiments with the separate constituents of dogs’ dung have
shown that with none of them employed alone can a satisfactory action
be obtained. For the experiments, the organic and inorganic salts
contained in dog’s dung were employed. Pure cultures of intestinal
bacteria were also examined and also the intestinal juices, secretions
of the gall bladder, of the pancreatic gland and secretions of the
intestinal mucous membrane. The relatively best bating action was
obtained with the secretion of the gall bladder; only a moderate bating
action could be obtained with the juices of the pancreatic gland and
the secretions of the intestinal mucous membrane. It was therefore
very surprising to find and could not be foreseen that mixtures of
these three secretions of the digestive apparatus alone exerted an
excellent bating action, and that such mixtures not only formed a
perfect substitute for the bate of dogs’ dung, but that they acted far
better than the latter, without it being necessary, as was recommended
elsewhere,[147] to add lime-dissolving salts to the bate, or to take
measures in any other manner for the removal of the lime from the
water.[148]

  [147] 1. See German Patent No. 200,519 or British Patent No.
  5377^{08} Roehm, Esslingen.

  [148] 1. See German Patent of Addition No. 203,889 Roehm, Esslingen.

The explanation of the effective action of such mixtures is evident
from the following statements:--

Pancreatic juice contains three ferments, of which for our purpose
trypsin, which decomposes albumen, and steapsin which decomposes fat,
are the most important. Both ferments are contained as zymogens, that
is to say in an inactive form, in the pancreatic juice. The latter
therefore only gives an incomplete bating action. Gall contains
ferments which have properties which are mainly fat decomposing,
but are also albumen dissolving. Gall also contains the salts of
gallic acids which have the power in a high degree of dissolving and
emulsioning greases or fats, and among others have a great solvent
action on the chloresterin contained in the skin, more particularly in
the roots of the hair. As regards the fat-decomposing and dissolving
action, gall is far superior to pancreatic juice.

The ferments of gall are, however, also capable of energising the
action of the steapsin in the pancreatic juice as a fat-decomposing
ferment, that is to say of imparting to the pancreatic juice, which in
a pure condition has a very slight action on fat, a high capacity for
decomposing fat.[149]

  [149] 2. See Abderhalden, “Text-book of Physiological Chemistry,”
  1906, page 555 and following.

Finally gall in bating the derms may be of great importance inasmuch
as its acids form easily soluble lime salts. Extensive experiments
have shown that gall, as such, is adapted to dissolve a comparatively
large quantity of lime. In hard water an addition of gall prevents the
precipitation of calcium carbonate, and the roughening of the derm,
which, for instance, has been noticed when pure bacteria cultures are
employed, is prevented by the addition of gall.

The intestinal juice contains in addition to carbonates and alkalis,
enzymes which again exert a slighter bating action on the hide than
those of gall. The intestinal juice alone has a similar action to
pancreatic juice, but if intestinal juice be added to pancreatic
juice, the most powerful enzyme of pancreatic juice, namely the
albumen-decomposing trypsin is energised, and only in this way
does the pancreatic juice fully acquire the property of dissolving
albuminous substances.[150]

  [150] 1. See Abderhalden, “Text-book of Physiological Chemistry,”
  1906, page 556.

The proportions in which such mixtures best develop their actions, are
those which obtain in animal organisms; the temperature also especially
plays a great part. The maximum temperature lies about 40° C.

From the above statements it is evident that it is possible to
produce various actions with the juices of the pancreas, gall and
the intestines, quite dependent on which are considered the most
important in the bating. Similarly, as the proteolytic action of the
mixture of the three juices may be increased or diminished by using
larger or smaller quantities of intestinal juice and pancreas, the
fat-decomposing and fat-dissolving action of the mixture may also be
increased or diminished by using larger or smaller quantities of gall,
and of gall and pancreas respectively. Finally the quantity of the gall
to be added is decided according to the amount of lime contained in the
water employed in the bating. Experience has shown that a relatively
small addition of gall will act very advantageously.

It must be especially remarked that artificial bates made in this
way are extremely cheap as compared with the natural dogs’ dung, as
gall and intestinal juice may be procured at very low prices and
a considerably smaller quantity of the energised, more expensive,
pancreatic juice is necessary than, for instance, would be necessary if
this juice were employed in a pure condition.

The advantage of this invention also consists in its being possible to
make better bates than that which is made, for instance, from dogs’
dung because, as hereinbefore stated, it is a disadvantage of this
latter bate that it in many cases dissolves too large a quantity of
the albuminous substance. Otherwise, the composition of the improved
bate quite coincides qualitatively with the composition of dogs’ dung,
as the latter actually consists in addition to the unabsorbed portions
of food, of secretions of gall, the pancreatic gland and the mucous
membrane of the intestines. The trypsin and steapsin respectively as
well as being derived from the pancreatic juice, may also be derived
from the vegetable kingdom, for instance, there are considerable
quantities of trypsin in the sap of the fig. Steapsin, in not
inconsiderable quantity, is found in the saps of flax, hemp, maize, etc.

It must also be remarked that the colouring matters in gall are of
quite subordinate importance, and may be disregarded. The view formerly
adopted that the colouring matters in gall stain the leather is
incorrect.

_Example_:--200 c.cm. of gall juice in 1 litre of water are mixed with
the watery extract of 200 grm. of chopped small intestine in 1 litre
of water on the one hand, and 100 grm. of the pancreatic gland in 1
litre of water, on the other hand. This watery solution is added to the
bating bath, according to the bating action desired. It can also, in
order to allow of being more easily handled, be obtained in solid form
by absorption in sawdust or kieselguhr and drying at 30° to 40° C.

_Claim._--A process for bating hides, characterised by the use of
mixtures of gall, the secretion of the pancreatic gland and intestinal
juice, and by the use of mixtures of the active enzymes in these, or
active enzymes derived from the vegetable kingdom, substantially as
described.


*Prof. Dr. H. Becker’s English Patent* (24,982, 1910).--Improvements
in Puering and Bating Hides and Skins.--It has been shown by Wood, of
Nottingham, and by Dr. Popp and Dr. Becker, of Frankfort-on-the-Main,
that the action of a good puer or bate on hides, is connected with the
development of certain species of bacteria.

The preparation of pure cultures of these bacteria (which are derived
from dog dung) has enabled what may be properly termed a safe puer to
be obtained, inasmuch as by its use overpuering the hides is entirely
prevented. Furthermore, the leathers are fuller and do not lose any
excess of hide substance, so that a dozen sheepskins that have been
suitably puered by the bacteriological method, will weigh about 9
ounces more than a dozen skins of the same kind and origin, that have
been treated in exactly the same way in other respects, but puered with
dog dung.

The method of action of the bacterial puer has been elucidated as
follows. The bacteria present in the puer are microscopically small and
pass easily into the skin through the pores of the latter, while on the
surface and in the substance of the skin they produce their metabolic
products and enzymes. These substances are partly proteolytic, partly
fat decomposing, and also diastatic in character, so that the highly
proteid intermediate substance of the skin is degraded. By means of
the ultimately formed ammonia, amine bases and ammonium bases assisted
by the action of organic acids produced by the bacteria, the lime soaps
are decomposed. Lime and fat are partially dissolved, and partially
further decomposed or converted into a form which enables them to be
worked out by mechanical means. At the same time the roots of the hairs
are also loosened so that the hairs also can be readily removed by
mechanical treatment.

Another essential factor is the liberation of gases formed within
the skin by the action of the micro-organisms and exerting a very
gentle pressure on their environment. The structure of the fibres is
loosened, the fibrils are straightened individually without sustaining
any important loss of corporeal matter, and in this way a firm, tough
and yet soft handling leather is obtained. Attempts have been made to
improve this puer by adding, for instance, organic acids and salts to
the bacterial cultures.

Other methods of puering, based on the direct weakening of the skin
fibres from the outside, may also effect the pulling down of the hides,
and moreover, in consequence of that attack on the skin fibres without
straightening the fibrils, an immoderate amount of substance is lost,
and therefore a hard, stiff, thin and light leather is obtained.

To this class belong also the purely digestive puers such as pancreatic
juice, intestinal juice and the like, or purified enzymes. These
enzymes are colloidal and therefore incapable of diffusion, and
consequently they digest the hide mainly from the surface, that is to
say, the grain side and inner side of the hide. The fibres, in-so-far
as they are accessible at all to the action of the colloidal enzymes,
are removed from outside without the fibrils being loosened and
straightened. Hence they are weakened by the digestive puer to such an
extent that the hides seem to be gradually pulled down; but the skin
will by that time have lost too much substance, so that, _inter alia_,
even the grain appears to have been corroded. In this way the leather
is rendered thinner and lighter than that from the dung or bacterial
puer--a defect manifest to every expert.

This danger is particularly great when, by the artificial admixture
of corporeal juices, complications are set up, that intensify the
destructive action of the digestive enzymes on the material, and when
their supplementary action is not suitably controlled.

These animal or digestive enzymes and fat decomposing substances
originate, in-so-far as they are not of vegetable origin, for the
most part in the upper portions of the digestive tract. The use of
these substances effects an imitation of the purely enzymatic and fat
decomposing processes that go on in the upper part of the intestines,
but omits the processes--not less important for digestion and the
formation of excrement--occurring in the large intestine and the
adjoining portions of the digestive tract. Here the bacteria and the
vegetable enzymes secreted thereby, play the chief part. They decompose
not only the food constituents--partly by putrefaction--but also the
albuminoid digestive secretions and the gall, so that only small
quantities thereof are left. Hence when applied to the puering process,
the use of bacteria, together with their specific enzymes and metabolic
products, is equivalent to a restriction of the immoderate digestive
action of the pancreatic and digestive juices, i.e. of the animal
enzymes.

Digestion therefore, with the resulting formation of excrement, is
carried on with the accompaniment of a mutual control, excitation and
restriction on the part of the digestive secretions and the bacterial
phenomena.

These latter form the real basis of the puering of hides in dung
preparations.

This is also evident from the purely practical experience that dog
dung puer, e.g. for glove leather, is essentially improved by leaving
the dung to ferment for several weeks or rather months. During this
process an increase occurs in the micro-organisms present, and also in
their metabolic products and enzymes, but not in the digestive juices
originating in the organs of the animal body--the term “digestive
juices” being restricted to indicate only such juices as are produced
in the course of the digestive tract by the organs and cells of the
animal body, in contradistinction to the juices produced by bacteria.

According to the present invention, the bacterial cultures, together
with their metabolic products or products of digestion and enzymes,
are used in combination with one or more of the substances that are
excreted in the digestive tract by the animal body itself in order
that not only each of these substances may exert its special action on
the skin, but also that the aforesaid mutual influencing, and more
particularly regulating of the action may take place.

On this principle, in preparing an artificial puer for hides, and
in accordance with the nature and preliminary treatment of the raw
material, and the requirements put forward in respect of the finished
product, the bacterial puer, nutrient medium or culture is mixed
with the corresponding digestive juices, or with purified animal or
vegetable enzymes, as well as with fat decomposing media, if necessary,
such as ground castor-oil seeds, hemp seed or the like, or with
extracts of same, or with gall. Where it seems necessary, substances
capable of dissolving lime may also be added, for example, sugar,
molasses, salts of ammonia, organic acids, or the corresponding salts
or the like.

Finally, also, the several operations may be carried out independently
of one another, it being in such case again advisable to adhere to the
processes going on in the intestines.

If, for instance, specially hard goat skins are to be pulled down
strongly by puering, it is advisable to add to the nutrient medium
of the bacteria a little papayotin or papain, either--according as
rendered necessary by the local conditions--in the preparation of the
bacterial puering liquor, or during the propagation of the bacteria.
If the fells are less hard so that less pulling down is required, the
hides are first treated with a weak solution of the digestive ferments,
and then with the bacterial liquor. In this way excessive digestive
action is counteracted in good time.

Sheep and goat skins that are particularly rich in fat are immersed
in a bacterial puering liquor, containing ground castor-oil seed or
castor-oil cake in suspension. The fat-decomposing ferment contained
in this meal then comes into immediate action, and the liberated fatty
acids form readily soluble soaps with the ammonia, originating from the
degradation of the proteid substances, and with the allied bases.

Extensive practical experiments have also shown that, in the case of
very fat skins, the action of the bacterial puer is increased by mixing
a suitable salt of sulpho-ricinoleic acid with the puering liquor, or
by washing the skins with solutions of similar soaps either before they
are immersed in the puering liquor, or subsequently.

Local conditions, and the requirements laid on the finished leather,
must decide which of these methods is specially advisable.

In any event, however, the combinations of the bacterial puer afford
a means of obtaining an increased puering effect in many cases where
puering with the separate auxiliaries mentioned does not produce a
sufficient result.

_Claim._--In the method of puering hides wherein bacterial cultures,
their metabolic products and enzymes are employed, the combined use
with these agents of digestive juices, mixtures of digestive enzymes
or pure digestive enzymes (and, if desired, of fat-decomposing
substances), either simultaneously with said agents or subsequently
thereto.




CHAPTER IX.

DRENCHING.


In the manufacture of many kinds of light leathers, skins, after
bating, are drenched. The process usually consists in placing the
skins in a mixture of bran and water, half to one per cent. of bran
being used (5 to 10 grm. per litre, or 1/2 to 1 lb. per 10 gall.) at
a temperature of 29° to 35° C. This ferments vigorously for 18 to
24 hours, with evolution of a considerable quantity of gas, and the
formation of weak organic acids.

The acids neutralize any lime which has not been neutralized in the
bate, and they do this in a very gradual and mild way, being produced
by bacterial fermentation exactly as required, in a similar manner to
fermentations taking place in presence of calcium carbonate. The gases
evolved bring the skins to the surface of the drench, and distend the
fibres; the workman attending to the drenches puts the skins down
under the liquor with a pole, but a better way is to have the drench
vat fitted with a wooden paddle, which may be pulled round by hand
when necessary; in this way the skins are put under the liquor without
danger of tearing or trapping them; in the case of grain splits the
paddle is a great advantage over the pole.

The skins are allowed to rise two or three times, according to the
condition required. For many kinds of alum leather the bate is omitted
and the drench only is used.

In English practice the bran is mashed in the vat, in which the
drenching is to take place, at a temperature of about 95° F. (35° C.);
the skins are then put in and the drench well stirred or paddled until
the whole is uniform, the temperature is then usually down to 85° to
83° F. (29·5° to 28·2° C.), at which temperature the fermentation
begins. The temperature allowed varies with the state of the goods
and the prevailing exterior temperature; in winter it may be higher
than in summer, but never usually higher than 90° F. (32·2° C.). In
some continental works the bran is mashed in boiling water, allowed to
stand for some hours to cool, and only the bran-water is used; in this
case the fermentation is different to that in which the bran is mashed
at a lower temperature. In the first process the starch of the bran
undergoes changes to dextrin and glucoses, which are then fermented by
bacteria with the production of acids and gases (see Chapter X.), in
the latter case the starch, which has been gelatinized is fermented by
other organisms, which probably secrete diastatic enzymes. Less gas is
given off in this fermentation than where the bran is mashed at 95° to
100° F. The temperature of fermentation is kept low, usually 25° C.
(77° F.). Bran contains a sufficient amount of nitrogenous matter,[151]
to furnish the bacteria with nutriment, and until this is exhausted
there is no fear of the skins being attacked.

  [151] Bran examined by Kjeldahl’s process contained 2·2 per cent. N,
  equivalent to about 13–14 per cent. proteids.

There is also a “sweet” bran drench used on the continent, in which
fermentation is not allowed to take place, the skins being paddled in
an infusion of bran in water for a short time only, two to three hours
at the most. In addition to the mechanical cleaning action, which the
fresh bran has upon the skins, there is also a softening effect due to
some constituent of the bran, which is at present not understood. One
reason for this, is that the usual drench employed being an acid one,
research has practically been confined to the latter.

Another method of preparing the bran drench, consists in allowing
the required quantity of bran to soak in cold water for several
hours; a sufficient quantity of hot water is then added to bring the
temperature up to 50° C.; at this temperature the diastatic enzymes
in the bran act rapidly on the starch and transform it into easily
fermentable sugars. The temperature is allowed to fall to 34° to
35° C., and then a quantity of an old drench is added to start the
fermentation. The liquor becomes acid, and in two days usually reaches
its maximum acidity, when it is ready for use. The bran is strained
off and only the milky liquor used. Such a drench is largely employed
in the manufacture of chamois leather, the goods (sheep fleshes) being
paddled in it for some hours previous to pressing. The French also use
a similar process in the preparation of suede leather. The bran is
strained off and only the sour partly fermented and fermenting liquor
used. The straining off of the bran also saves working over the beam
which would otherwise be required to get rid of the bran adhering to
the skins.

It has recently been proposed to employ the bacillus bulgaricus, the
organism which causes the fermentation of milk in the preparation of
Bulgarian and Turkish “Yoghurt” (Bibliography 124) for drenching.
Bacillus bulgaricus is capable of producing up to 2·5 per cent. of
lactic acid, or about three times the quantity of that produced by
ordinary lactic bacteria. Dr. Hugo Kühl (loc. cit.) proposes to
cultivate the bacillus in the mother liquors, which are a by-product
in the manufacture of milk sugar, and to use such liquor as a drench.
Where skim milk or butter milk is to be had, this may also be used.
A similar process is already in use in America, where the evaporated
product is put upon the market under the name of Dermiforma (see
pp. 189 and 194).

As a substitute for the drench, and in order to further remove lime
after erodin, the late Franz Kathreiner employed a solution of
anticalcium, 1 in 500 water, in which the skins remained overnight.
Anticalcium is a mixture of sulphonic acids derived from cresols,
and has antiseptic properties, so that its action is quite distinct
from that of the bran drench. It is put on the market by J. Hauff,
Feuerbach, near Stuttgart.

*Drench Damage.*--Drenching is a very important and useful process in
leather manufacture, but, like other fermentive processes, requires
great care and experience in its application. Although not so risky as
puering, a good deal of serious damage may occur in the drench. The
writer knows of no fuller discussion of all the various forms of drench
damage than the articles of Eitner and others which appeared in “Der
Gerber” during a long series of years (1882–1900). In these articles
perhaps every known form of drench damage is described. Procter[152]
also gives a concise account of drenching. It will suffice to mention
briefly the chief accidents to which the drench is liable; these are
generally known as the “turning” of the drench.

  [152] Principles, pp. 166–170.

1. _Acid “turning.”_--This usually occurs when the atmosphere is
charged with electricity--during thunderstorms. It appears to be an
extremely rapid form of butyric fermentation, which has not been fully
investigated. The skins become swollen, transparent and tender, and,
unless the fermentation is stopped, soon dissolve to a jelly. The only
way to save the skins from destruction is to add salt to the drench;
this reduces the swelling and, in fact, pickles the skin. I have
pointed out elsewhere that this is really the origin of the modern
pickling process, which is still known by the name of “rising.” A very
acid drench will swell the skins almost the same as dilute sulphuric
acid, and if they are then placed in a salt solution, or salt is added
to the sour liquor, it is absorbed by the skins, and they are preserved
almost as effectually as by the use of mineral acids.

2. _Putrid “turning.”_--This occurs under very similar atmospheric
conditions to 1, and may be almost as rapid. Instead of becoming acid,
the liquor turns slightly alkaline, frequently becoming bluish black,
due to the presence of certain chromogenic bacteria. The goods fall
as in a puer. Both the chromogenic and other bacteria present are
peptonizing organisms, which obtain the upper hand owing to favourable
conditions, and rapidly digest the skin unless the action is stopped.
The best remedy is to remove the skins from the drench and pickle them
without delay by the usual acid and salt process.

3. _“Pinholey” drench._--This is the same as the Germans call
“pikiren,” and is generally due to a too rapid evolution of gas both in
the drench and in the capillary spaces of the skin itself. The gases
form under the hyaline layer, and finally burst through in small holes.
A damage very similar in appearance is also caused by colonies of
gelatin liquefying bacteria developing on the grain. Each colony forms
a small hole. The trouble is usually due to the setting of the drench
at too high a temperature.

4. _Spongy leather_ may be produced by leaving the goods too long in
a sound drench, if they are allowed to rise too often. In this case
experience alone can determine the proper duration of the drenching
process.

A special case in which the hyaline layer is slightly attacked and
its brilliance destroyed has been described by Eitner.[153] A slimy
film was formed on the surface of the drench and also on the grain of
the skins. This film was found to consist of Bacillus megaterium (the
potato bacillus), and where it was growing on the hyaline layer the
latter was etched as it were, by the peptonizing enzyme secreted by the
bacillus, so that when tanned the grain was covered with dull patches
very much resembling “blast.”

  [153] Der Gerber, 1898, p. 204.

*Fermentations in the Drench.*--Compared with the dung bate, or puer,
the fermentation taking place in the bran drench is a simple one; as
will be shown in detail in the next chapter, about half the weight of
the bran consists of starch; this becomes hydrolysed by the enzymes in
the bran (and in some cases secreted by the bacteria) into glucoses.
According to the following equation:--

    (C_{6}H_{10}O_{5})_{n} + H_{2}O = n C_{6}H_{12}O_{6}
          starch                            glucose

This expresses the change in its simplest form; in reality it is much
more complex, various dextrins and sugars being formed simultaneously.

The sugars are then fermented by a variety of bacteria with formation
of various organic acids, in accordance with the equation

    C_{6}H_{12}O_{6} = 2 CH_{3}CH(OH)COOH

which represents the formation of lactic acid.

In addition, formic and butyric acids are produced along with small
quantities of other bodies, as will be shown. It is pretty certain
that the butyric acid is formed by the further fermentation of the
lactic acid and lactic salts in the liquid; at the same time CO_{2} and
hydrogen are evolved as follows:--

    2 C_{3}H_{6}O_{3} = C_{4}H_{8}O_{2} + 2 CO_{2} + H_{2}

[Illustration: Fig. 27.--Organisms in Bran Drench × 1000.]

Fig. 27 is a drawing of the organisms seen in the drench, under a
magnification of 1000 diameters. This was taken from a drench which
had been set about 3 hours, and was just beginning to ferment; the
bacteria form chains of varying length: the short chains have a slow
motion in the liquor. As the fermentation proceeds, these form longer
chains (Fig. 28), some of which are very beautiful objects, especially
when viewed by dark ground illumination. The other organisms present
are bacteria and bacilli from the puer, and at a later stage, as the
nutrient material for the bran ferment becomes less, they begin to
develop, first causing butyric and finally putrefactive fermentation.

[Illustration: Fig. 28.--Chains of Bran Bacteria × 1000.]

[Illustration: Fig. 29.--Bran Fermentation. Advanced Stage.]

A fairly pure culture may be obtained by inoculating a tube of
bran infusion, previously sterilized, by means of a freshly made
capillary pipette, which is pushed into the tube without removing the
cotton-wool plug. The tube is placed in the incubator at 30° C., and
the organisms causing the fermentation develop rapidly; as soon as
the tube is observed to become cloudy, indicating the development of
the bacteria, another tube is inoculated in the same manner, using
a very minute quantity of liquid; this second “attenuation” is then
carried on to five or six more tubes. In the fifth tube will usually
be found a pure culture of the bran fermenting organisms, which I have
called Bacterium furfuris. The bacteria (see Fig. 30) are mostly in
the form of dumb-bells or pairs, each cell 0·75 µ × 1·25 µ: they vary
slightly in size, some forming chains; those on the surface become
surrounded by a kind of jelly, and form an iridescent pellicle on the
surface of the liquid, the zoogloae form. When the nutriment of the
liquid is exhausted or the acidity becomes too great,[154] this film
sinks to the bottom and the liquid above becomes comparatively clear.
There is no spore formation, and, consequently, the culture soon dies
unless inoculated into a fresh nutrient medium. The liquor, after the
fermentation both in the drenches and tubes of pure cultivations, is
always slightly ropy.

  [154] Claflin (92) gives the limits of acidity for lactic bacteria
  from 0·02 to 0·5 per cent. Reynolds Green (97, p. 346) gives the
  upper limit for B. acidi lactici as 8 per cent. lactic acid.

B. furfuris probably belongs to the group of coli bacteria, some of
which ferment milk and other sugars with production of lactic acid and
gases. This explains the ease with which the fermentation takes place,
since the skins bring in immense numbers of bacteria from the bate,
among which are the organisms described.

Eitner[155] states that the action of the bran drench cannot be
considered a chemical one, and that the chief action is a dynamical
one, resulting from the gases generated in the liquid and in the skin.
While I do not deny that the gas has such action--indeed I pointed out
in 1893[156] that the gas acted by floating and distending the skins,
and so enabling them better to take up the acids--I maintain, however,
that the chief action is a chemical one and due to the weak organic
acids produced; this was confirmed experimentally. The researches on
bran drenching commenced in 1887 by myself, and later in conjunction
with Dr. W. H. Willcox, prove conclusively that the amount of acid
produced, and especially the lactic acid, is sufficient to produce a
very decided action on skin. In actual working we found from 1·07 to
2·34 grm. total acids per 1000 c.c., while in artificial cultures,
with addition of calcium carbonate, inoculated with pure cultures of
bacteria, obtained from drenches, we obtained from 4·53 to 11·44 grm.
total acids per 1000 c.c. Now traces of lime in the skins act in just
the same way as the calcium carbonate; they neutralize a portion of
the acid produced by the bacteria, and so allow a greater amount of
acid to be produced than if it accumulated in the liquid. By preparing
solutions of the above acids of suitable strength and agitating skins
in them, results were obtained in 1-1/2 hours equal to a drenching of
14 hours. The defect of such artificially prepared liquids in practice
is due to the fact that they do not contain any particles of bran or
flour; these take up dirt from the skin mechanically, and so produce a
better-coloured leather. Where the skin has been bated in a perfectly
clean bate, and then paddled in such an artificial drench, the leather
is in every way equal to that prepared by means of the dung bate,
followed by a bran drench.

  [155] Der Gerber, 24, 570.

  [156] Jour. Soc. Chem. Ind., 1893, p. 426.

By far the most important action for the tanner is the solution of the
last traces of lime not dissolved by the bate, and this is the chief
reason for its use, where colour is a desideratum, and where sweet
liquors are used; for some kinds of leather where it is desirable not
to lose pelt, the drench is used alone for this purpose. A dung bate,
such as most light leather tanners use, is seldom run off more than
once a week, and contains a considerable amount of dissolved lime; the
amount in a freshly made-up puer wheel is from 0·1 grm. to 0·3 grm.
lime (CaO) per litre. After the wheel has been in use for a week, it
rises to 0·5 grm. to 0·8 grm. per litre. It will be readily seen that a
liquid which already contains such considerable quantities of lime is
not an especially good medium to use for its complete removal from the
skins, hence drenching is necessary to get rid of the lime left in the
skins.

Eitner states that the fermentation is alcoholic at the commencement,
and then becomes an acid fermentation with generation of gas. In
tan liquors there is no doubt that this is the course of the acetic
fermentation, as has been shown by Andreasch,[157] but in the usual
type of bran drench the action is certainly a different one. The
acetic acid, as far as we can ascertain, is produced directly from
the dextrose, without the previous production of alcohol, since the
presence of the latter is not shown by tests in any stage of the
fermentation.[158] No yeasts were observed[159] and no alcohol was
found in any stage of the fermentation.[160] J. O’Sullivan remarks
that although it has not been noticed it is possible that in this
case the production of alcohol and its oxidation into acetic acid are
simultaneous, but that there is no _preliminary_ fermentation by yeasts
followed by acetic fermentation by bacteria such as Eitner indicates is
certain.

  [157] Der Gerber, 1895, p. 193, etc.

  [158] J.S.C.I., 1897, p. 513.

  [159] Ibid., 1890, p. 28.

  [160] Ibid., 1893, p. 422.

To sum up the conclusions arrived at from the experiments above quoted.

The starch of the bran or flour is first transformed into glucoses and
dextrin by the action of an unorganized ferment called _cerealin_. The
glucoses are then fermented by certain species of bacteria, Bacillus
furfuris being perhaps the chief, with the formation of lactic,
acetic, formic and butyric acids, and the evolution of carbon dioxide,
hydrogen, nitrogen, and a small quantity of sulphuretted hydrogen.

The principal acid produced is lactic; the acetic acid is produced
directly from the glucoses by the above-mentioned bacteria without any
preliminary alcoholic fermentation by yeasts.

The mode of action of the drench on the skins may be summed up as
follows:--

1. The solution of the last traces of lime which has not been removed
by the bate, by the organic acids produced by the fermentation, and the
subsequent swelling action of these on the skin fibres. The acids also
dissolve a small amount of skin substance.

2. Simultaneously with (1) the distension and floating of the skins by
gases produced by the fermentation, so enabling them better to take up
the acids.

3. The mechanical absorption of dirt by the particles of bran or flour
in the drench.

It is not proposed to enter into further detail, since the next
chapter, consisting of the original papers of Dr. W. H. Willcox and
the author, contains a pretty complete account of the analysis of the
drench and theory of the drenching process.




CHAPTER X.

ORIGINAL PAPERS ON DRENCHING.


1. FURTHER CONTRIBUTION ON THE NATURE OF BRAN FERMENTATION. By J. T.
Wood & W. H. Willcox, B.Sc.(Lond.).

  (Reprinted from the Journal of the Society of Chemical Industry,
  May 31, 1893. No. 5, vol. xii. p. 422.)

The paper which one of us had the honour of reading before this
Section on December 11, 1890,[161] dealt chiefly with methods of
bacteriological research, but especially in connection with the
fermentation of bran as applied in the manufacture of light leathers.
The object of the present paper is to give an account of further
research into the nature of the fermentation and its products, the
former communication being very incomplete. In further investigating
the matter, we endeavoured--

  [161] J.S.C.I., 1890, 27.

1. To obtain a complete knowledge of the products of the actual
fermentation as it takes place in practice.

2. To discover in what way the ferment acts, both on the materials
fermenting and on the skins.

3. To examine in the same way the products of a pure cultivation of the
bacterium causing the fermentation.

1. *The Products of the Actual Fermentation.*--These may be divided
into three groups:--(1) gases; (2) volatile bodies; (3) non-volatile
bodies. It was stated in the former paper that the ferment produced an
inflammable gas along with considerable quantities of CO_{2}, H_{2}S,
etc. The inflammable gas was thought by analogy from the researches
of Tappeiner[162] to be methane; it has, however, proved to be pure
hydrogen. The absence of hydro-carbons was shown by the following
method. Some of the gases were collected, the CO_{2} and SH_{2}
removed by absorption with KOH, and the remaining gases exploded in a
eudiometer tube with oxygen. The gases which remained after explosion
did not diminish in volume after standing over KOH solution, showing
absence of the paraffins and olefines.

  [162] Zeits. für Biologie, xxiv. 105.

For the purpose of analysis, about 1-1/2 litres of the gas were
collected at a time in a large flask, fitted with a caoutchouc stopper
and a funnel having an area of 28 sq. in. This was inverted, filled
with the drench liquid, over the vat. The gas when collected was
transferred immediately in the vat to a glass-stoppered bottle, sealed
with a small quantity of the fermenting liquid and examined at once.
Nine analyses have been made, most of them in duplicate. The following
table, p. 248, gives the results of three of these duplicate analyses,
which have been performed by Hempel’s method, the hydrogen being
estimated by combustion in air over heated palladinised asbestos.

We find that the gases given off during the fermentation are
practically the same, with or without skins.

    ---------------------+------+------+------
             Gases       |  A   |   B  |  C
    ---------------------+------+------+------
    CO_{2} and H_{2}S    | 21·9 | 25·2 | 42·4
    O_{2}                |  1·0 |  2·1 |  3·6
    H_{2}                | 53·1 | 46·7 | 28·2
    N_{2}                | 24·0 | 26·0 | 25·8
    ---------------------+------+------+------

    A is from a vat containing no skins, 1–2 days.
    B from a vat containing skins, 2–3 days.
    C from a vat containing skins, 3–4 days.

The H_{2}S is present only in small quantities from 1–2 per cent. Its
presence was shown by aspirating the gases dissolved in 1 litre of
drench through a dilute solution of lead acetate containing a few drops
of acetic acid. The gases were liberated by heating the liquid, and at
the same time aspirating air through it. The H_{2}S is present both in
the gases evolved from drenches which do not contain skins, and from
those which do, though to a slightly greater extent in the latter. The
amount of CO_{2} given off increases as the fermentation proceeds; the
oxygen also increases, the nitrogen remaining practically constant. We
consider that some of the nitrogen given off is that dissolved in the
water, the oxygen being partly used up by the ferment in its earlier
stages; the remainder of the nitrogen is probably produced from the
decomposition of the nitrogenous bodies contained in the bran. It may
be noted here that the bran fermentation under ordinary circumstances
ceases on the fourth day, and sometimes earlier.

Frankland and Frew, in a paper on a pure fermentation of mannitol
and dulcitol[163] have shown the hydrogen and carbon di-oxide given
off were produced by the decomposition of formic acid, the ferment
producing formic acid and the latter immediately splitting up into an
equal number of molecules of carbonic anhydride and hydrogen. We have
every reason to believe, from experiments which will be included in the
third section of the paper, that the source of the H and CO_{2} is the
same in the fermentation we are considering.

  [163] Jour. Chem. Soc., 1892, Trans. 254.

2. *Volatile Bodies.*--These may be divided into (1) acids, and
(2) amines. We had previously shown the absence of aldehyde by the
rosaniline reaction, and of alcohol by Lieben’s iodoform test.

In the first experiment to determine the acids, 15,670 c.c. of the
liquid from a normally fermenting vat was taken when the fermentation
was at its height; this was submitted to distillation, and to the
last portions distilled water added and 16,200 c.c. distilled over;
the distillate was neutralised with sodium carbonate, and the whole
was then evaporated in a porcelain dish, the residue dried first at
100° C., then over strong sulphuric acid, the weight of the sodium
salts of the volatile acids thus obtained being 18·07 grm.

These salts were treated with 200 c.c. absolute alcohol and 20 c.c,
strong H_{2}SO_{4}; heat was evolved, and there was a strong smell of
ethyl acetate and butyrate.[164] The mixture was allowed to stand for
24 hours and then distilled on the oil-bath, the temperature for a
long time remaining at 81° C., finally rising to 96° C. 219 c.c. of
distillate was obtained, and to this a saturated solution of common
salt was added, but the esters of the volatile acids did not separate
out. The whole was again redistilled with the same result.

  [164] Der Gerber, xvi. (368), 4.

Failing in this way to separate the acids in the form of their esters,
the mixture of esters and alcohol was examined qualitatively; 75 c.c.
was taken and saponified with 80 c.c. N/1 NaOH in a distilling flask,
with inverted condenser, for half an hour, until all the fragrant smell
of the esters had disappeared. 71 c.c. N/1 HCl was then added, and the
apparatus connected with a condenser in the usual way. The distillate
was acid. 800 c.c. was taken off, forming fraction 1. The remainder
of the acid required to neutralise the sodium hydrate was added, and
another 800 c.c. distilled off, forming fraction 2. Fraction 1 smelt
strongly of butyric acid, fraction 2 of acetic acid. The fractions were
then each boiled for half an hour with excess of barium carbonate; this
was filtered off and washed, the filtrate evaporated to dryness, and
dried at 130°.

A portion of barium salt of fraction 1 was heated with alcohol and
sulphuric acid and gave the characteristic pineapple smell of ethyl
butyrate.

The solution of the barium salts of fraction 2 was concentrated by
evaporation, and to a portion excess of H_{2}SO_{4} was added, the
BaSO_{4} filtered off, and the filtrate carefully neutralised with
ammonia and a portion added to a solution of neutral ferric chloride.
It was coloured dark red, and a finely-divided precipitate of basic
acetate of iron came down on boiling. A second portion of the
concentrated solution was heated with sulphuric acid and gave off
a strong smell of _acetic acid_. To a third portion silver nitrate
was added, and a slight excess of acetic acid; a little silver was
deposited in the form of a film, showing a trace of _formic acid_.

Having thus shown the presence of butyric, acetic, and formic acids in
the drench, we proceeded to ascertain the quantities formed in a normal
fermentation of bran without skins. For this purpose a drench was made
in a clean vessel with 10 litres of distilled water and 200 grm. of
bran mashed at a temperature of 38° C., and, after cooling to 33°,
inoculated with bacteria from an actual drench which was fermenting
vigorously; this was kept in the drench house so that the fermentation
and general conditions might be exactly similar. In 48 hours all gases
had ceased to be evolved, and the true fermentation was at an end.
The liquid had an acid, not unpleasant smell, and was acid to litmus
paper. The bran was strained off through muslin, washed with a little
water, and well squeezed; the liquid measured 10 litres. Three litres
of this were taken for the separation and estimation of the volatile
acids, the remainder being set aside for examination and estimation of
the non-volatile acids, etc. Two of the three litres were placed in a
distilling flask with 5 grm. of pure CaCO_{3}, and distilled down to
one litre. The distillate was _alkaline_ to litmus, and had a peculiar
fishy smell; the remaining litre of drench was added, and the liquid
taken down until the distillate ceased to be alkaline and only bad a
faint smell. The alkaline distillate gave a yellow precipitate with
Nessler’s solution, as well as the following reactions:--

    AgNO_{3}     A faint brown precipitate.

    HgCl_{2}     A yellowish-white curdy precipitate.

    PbA          A brownish-white precipitate.

    CuSO_{4}     A dirty blue precipitate, which changed to a
                   brownish turbidity on boiling.

HCl was added to the distillate in slight excess and the liquid
evaporated to a small bulk; to a portion chloroform and alcoholic
potash were added, and the liquid heated; no smell of isocyanides was
given off; the body is, therefore, not a primary amine. Phosphomolybdic
acid gives no precipitate, therefore the body is not an alkaloid. From
the above tests and its characteristic smell, we conclude that the
body is _trimethylamine_. The platinum salt was formed by evaporating
the above concentrated solution of trimethylammonium hydrochloride
with excess of platinum chloride, a precipitate of the platinum salt
insoluble in alcohol being formed. There was not, however, a sufficient
quantity from the three litres to ascertain the molecular weight.

Proceeding with the estimation of the volatile acids, 100 c.c. N HCl
being required to completely neutralise the CaCO_{3} used, 50 c.c.
were first added and the distillation continued; the distillate was
only very faintly acid. Four fractions were now distilled off, using
respectively 10, 10, 10, and 20 c.c. N HCl.[165]

  [165] Method recommended to us by Dr. Frankland. _Vide_ Jour. Chem.
  Soc. Trans. 59, p. 94. Appendix II. Determ. of Vol. Acids by dist.
  with HCl.

Fraction 1 was boiled with excess of barium carbonate, filtered, the
BaCO_{3} washed with hot water, and the filtrate evaporated to dryness,
and the Ba salts dried at 130° C. till the weight was constant; the
salts were then decomposed with strong sulphuric acid, ignited, and the
barium sulphate weighed.

Fractions 2, 3, and 4 were treated in an exactly similar way, the
barium salts obtained and the weight and percentage of barium sulphate
being shown in the following table:--

    ----------+-----------+-----------+-----------
     Fraction | Weight of | Weight of | Per Cent
              |   Salts   |  BaSO_{4} | BaSO_{4}
    ----------+-----------+-----------+-----------
         I.   |  0·5585   | 0·4991    |  89·365
        II.   |  0·5060   | 0·4618    |  91·265
       III.   |  0·31675  | 0·29415   |  92·86
        IV.   |  0·4475   | 0·4252    |  95·02
    ----------+-----------+-----------+-----------

Calculating the fractions 1 and 2 as mixtures of barium butyrate and
acetate, and fractions 3 and 4 as mixtures of barium acetate and
formate, we may summarise the results thus:--

    ----------+-----------+---------+----------+---------
     Fraction | Weight of |   Ba    |    Ba    |   Ba
              |    Salt   | Acetate | Butyrate | Formate
    ----------+-----------+---------+----------+---------
        I.    |  0·5585   | 0·4904  |  0·0681  |   ..
       II.    |  0·5060   | 0·5030  |  0·0030  |   ..
      III.    |  0·31675  | 0·2749  |    ..    | 0·04185
       IV.    |  0·4475   | 0·2629  |    ..    | 0·18460
        Total |  1·82875  | 1·5312  |  0·0711  | 0·22645
    ----------+-----------+---------+----------+---------

or as free acids:--

    ----------+--------+---------+--------
     Fraction | Acetic | Butyric | Formic
    ----------+--------+---------+--------
        I.    | 0·2308 |  0·0385 |  ..
       II.    | 0·2367 |  0·0017 |  ..
      III.    | 0·1293 |    ..   | 0·0170
       IV.    | 0·1237 |    ..   | 0·0748
        Total | 0·7205 |  0·0402 | 0·0918
    ----------+--------+---------+--------

Proceeding now to (3) non-volatile bodies, we find these to consist
of (1) acids, and (2) soluble carbohydrates. For examination, the
remaining 4 litres of experimental drench were evaporated down to 1
litre and filtered; the residue (consisting of starchy matter) was
well washed and the washings added to the filtrate, the whole placed
in a large distilling-flask and distilled with continued addition of
distilled water until the distillate was no longer acid (this required
the addition and distillation of 4 litres of distilled water). A
further deposit of solid matter rendering boiling dangerous, the
liquid was again filtered and the residue washed free from acid. This
residue was found to consist of nitrogenous organic matter and _calcium
phosphate_, together with a trace of _calcium oxalate_, both being
derived from the bran.

The clear liquid containing the non-volatile acids and other bodies
was further concentrated and made up to 500 c.c.; it was dark brown
in colour and was very acid to litmus; owing to a trace of flocculent
matter, it was again filtered.

The presence of lactic acid was shown in the following manner: 10 c.c.
of the liquid were placed in a small distilling-flask along with 2 c.c.
strong H_{2}SO_{4} and about 0·5 grm. potassium chromate in a little
water. This was distilled and the vapours received in a test-tube
surrounded by cold water; on adding magenta solution, decolorised by
SO_{2}, to the liquid in the test-tube, a red colour was produced by
the aldehyde formed from the lactic acid; aldehyde was also recognised
by its smell. We find this an exceedingly delicate test for lactic
acid, and as far as we know it is quite new in this form.

For 10 c.c. of liquid to be examined, we find 2 c.c. strong H_{2}SO_{4}
and 1 grm. of potassium chromate to be the best proportions. Formic,
acetic, propionic, butyric, valerianic, succinic, malic, tartaric, and
citric acids do not give the reaction.

The liquid was now tested for _succinic_ and _malic_ acids. 25 c.c.
was taken and decolorised by treatment with pure animal charcoal for
half an hour; the liquid was then filtered, the charcoal washed free
from acid, and the filtrate concentrated; ammonia was added in slight
excess, the precipitate of calcium phosphate filtered off. To the
filtrate CaCl_{2} was added in slight excess to remove the remainder
of phosphates, the liquid filtered, and the filtrate cautiously
neutralised with HCl; the addition of neutral ferric chloride to a
portion gave no precipitate, showing the _absence of succinic acid_.
To the remainder an equal volume of absolute alcohol was added and the
liquid boiled; there was no precipitate. Twice the volume of absolute
alcohol was then added and gave no precipitate. On the addition of four
times the volume of absolute alcohol, a slight white precipitate came
down, which evidently consisted of dextrin, from the manner in which
it settled round the sides of the tube, and from its insolubility on
adding HCl. We therefore conclude that there is no malic acid present,
and that the only non-volatile acid produced is _lactic acid_.

The acidity was first determined by titrating 10 c.c. of the solution
with N/10 sodium hydrate, using glazed litmus paper to determine the
point of neutralisation. The 10 c.c. required 6·66 c.c. N/10 NaOH,
equivalent to 0·666 of N lactic acid = 0·7481 grm. per litre of
original drench.

In order to separate the lactic acid from the colouring matter
and other bodies (dextrin and soluble starch) present, 100 c.c.
of the concentrated liquid was decolorised with 10 grm. of animal
charcoal, the mixture filtered and the charcoal well washed, the
filtrate evaporated to dryness, and 15 drops of N/10 H_{2}SO_{4}
added to decompose any salts of lactic acid present; this was now
extracted with ether, the ethereal extract of lactic acid placed in a
distilling-flask, the ether distilled off, and the residue boiled with
distilled water and pure calcium carbonate. The small amount of calcium
sulphate present was removed by boiling the filtrate from the CaCO_{3}
with barium carbonate, and filtering. The filtered liquid now consisted
of a solution of calcium lactate; it was evaporated to dryness in a
platinum dish, and the residue dried at 110° C.; it was then washed
first with ether, then absolute alcohol.[166]

  [166] Julius Wladika. _Vide_ Der Gerber, xvi. 28. Zur Kenntniss der
  Organ. Säuren in Fichtenbrühen.

The insoluble residue was dried at 110° C. till the weight was constant.

The total weight of calcium salts obtained was 0·7661 grm. (from
4/5 litre of drench), of which 0·5493 grm. yielded on ignition
0·1398 grm. CaO = 25·45 per cent. Theory requires for calcium lactate
(C_{3}H_{5}O_{3})_{2}Ca = 25·69 per cent. CaO.

0·7661 grm. calcium lactate in the quantity used = 0·9576 grm. per
litre = 0·7907 grm. lactic acid per litre. The difference between
this and the preceding amount of lactic acid found by titration, viz.
0·0426 grm., is probably accounted for by the presence of a small
quantity of salts of lactic acid in the drench.

The second part of the subject which we proposed to consider was “In
what way does the ferment act on the bran and on the skins?” The
average composition of bran is shown in the following table:--

                             Per Cent.

    Water.                      14
    Fibrin, etc.                15
    Starch                      44
    Fat                          4
    Lignose and cellulose       17
    Ash                          6

It will be seen, as stated in the former communication,[167] that the
starch must be the principal body acted upon; but the cellulose is also
an important constituent, and before going further it was necessary
to ascertain if it took part in the fermentation. For this purpose
some pure cellulose was prepared from cotton wool in the usual way,
and small portions placed in tubes containing yeast-water[168] as a
nutrient material. These were sterilised by steaming; two tubes were
inoculated from a pure cultivation of the bacteria obtained in 1889,
two were inoculated from an actual drench, and three left uninoculated;
all of them were placed on the incubator at a temperature of 30°–33°.
On the second day the inoculated tubes were cloudy, but no gas was
given off, nor was any acid formed; in 10 days the cellulose had
not disappeared, nor on examination with the microscope could any
action be detected. The experiment was repeated with peptone as a
nutrient medium, but with the same result. The conclusion is that the
bacterium does not attack the cellulose, which thus takes no part
in the fermentation. The starch and nitrogenous bodies of the bran
are therefore the only bodies acted upon by the bacteria in this
fermentation.

  [167] J.S.C.I. 1890, 27.

  [168] 7 grm. of yeast boiled in 100 c.c. H_{2}O.

From the fact that bran drenches ferment in the same way when mashed
at all temperatures from 20° C. to 40° C., and that in all cases the
starch is decomposed, it was supposed that the ferment was capable of
attacking the starch in its undissolved condition. To ascertain if this
were so, it was necessary to use pure cultivations in the laboratory.

The usual methods employed had thrown no light on this part of
the subject, as in order to sterilise the solutions they had been
repeatedly boiled, and were thus not comparable with the fermentation
as it takes place in the works.

In order to get rid of this difficulty, the starch was sterilised in
a dry condition in the hot-air oven, by heating for several hours on
successive days to 110° C.

This sterile starch was mixed with sterilised water in tubes plugged
with sterile cotton-wool. Eight tubes were taken, as follows:--

1. Sterile starch and water.

2. Sterile starch and water inoculated pure culture.

3. Sterile starch and yeast-water inoculated pure culture.

4. Sterile starch and asparagin inoculated pure culture.

5. Yeast-water alone inoculated pure culture.

6. Dextrin[169] and yeast-water inoculated pure culture.

  [169] Prepared by precipitation with alcohol.

7. Soluble starch[170] and yeast-water inoculated pure culture.

  [170] Prepared by heating starch and water to 50° C. for 18 hours and
  filtering.

8. Starch mucilage and yeast-water inoculated pure culture.

These were allowed to stand on the incubator at 33°–35°C., and examined
for acid each day by the method described in the previous communication
for starch testing. They all remained neutral, although the bacteria
developed in all but No. 1. These experiments were repeated several
times, with the same result in every case. They show that this ferment
is unable to act on starch either in its insoluble or soluble
condition, alone or in the presence of nitrogenous bodies.

Now it has been known for a considerable period that bran contains
an unorganised ferment called _cerealin_,[171] which is capable
of changing starch into dextrin and other carbohydrates; but the
information to be obtained about it was very meagre, and as it appeared
that this body might play an important part in the fermentation, we
proceeded to prepare some pure cerealin and to ascertain its action on
pure starch. The cerealin was prepared by taking a kilo of bran and
extracting it with 2 litres of distilled water at 30° C; the extract
was filtered clear and 2 litres of strong spirits of wine containing 90
per cent. alcohol was added, when a flocculent precipitate separated,
which was washed on a filter with alcohol, dehydrated with absolute
alcohol, and dried over H_{2}SO_{4}. The cerealin thus prepared is an
amorphous substance not quite white, difficultly soluble in water,
though we think this is due to its having been coagulated, and that
it might be prepared in some other way which would show it to be more
soluble than that which you now see.

  [171] Watts’ Dict., Old Ed. Cerealin discovered by Mèje Mouriés,
  Comptes rend. 37, 351; 38, 505; 43, 1122; 48, 431; 50, 467.

To show its action on starch we took 10 grm. of pure starch in 200 c.c.
water at 40° C., and placed equal quantities in two flasks; to No. 1
about 0·1 grm. of the cerealin was added; No. 2 was left blank. These
were kept at 40° C. for 10 hours, the clear liquid filtered off, and
examined with Fehling’s solution. No. 1 reduced it strongly, showing
that _glucoses_ were present in considerable quantity. No. 2 had no
effect whatever.

Addition of alcohol to No. 1 gave a white precipitate; this was thrown
on to a filter, washed with alcohol, and dried at 100°, again dissolved
in water, and inverted by boiling the solution with 1/20 part of strong
sulphuric acid and then neutralising with sodium hydrate. The resulting
solution reduced Fehling’s solution, showing that the body was dextrin.
Strong bran infusion (which of course contains cerealin) acting on a
thick starch mucilage, liquefies it, and forms glucoses and dextrin.

We have thus shown that the cerealin produces _glucoses_ as well as
dextrin, both from solid starch and from starch mucilage. This is
most important, as it has been previously shown by one of us that the
ferment attacks glucose very easily.

The drenches were now examined in order to ascertain the presence of
glucose and dextrin; in the former communication it was stated that
these were absent. We find, however, that by concentrating the liquid,
that both are present in the early stages. Samples were taken one hour
after mashing, and at 3, 6, 12, and 18 hours, while the drench was
working.

These were evaporated to 1/5 bulk, filtered, and divided into two
portions, one of which was examined with Fehling’s solution; to the
other alcohol was added; the white precipitate after addition of
alcohol was filtered off, washed with alcohol, dried at 100° C.,
redissolved in water, and boiled with sulphuric acid to invert it, then
examined with Fehling’s solution.

The following results were obtained:--

    ---------------------+------------+--------+------+------+------
            Hours        |      1     |   3    |   6  |  12  |  18
    ---------------------+------------+--------+------+------+------
    Glucoses or sugars   |Present in  |Present |Absent|Absent|Absent
      reducing Fehling’s |considerable|in small|      |      |
      solution directly  |quantity    |quantity|      |      |
                         |            |        |      |      |
    Dextrins, Fehling’s  |Present in  |Present |Traces|Faint |Absent
      solution reduced   |considerable|        |      |traces|
      after inversion    |quantity    |        |      |      |
                         |            |        |      |      |
    Soluble starch       |Absent      |Absent  |Absent|Absent|Absent
    ---------------------+------------+--------+------+------+------

In a bran infusion kept from fermenting by a little ether or
chloroform, the formation of glucose and dextrin goes on continuously,
the glucose increasing in quantity; the action is, however, much slower
than in the case of diastase; at the end of 12 hours, at a temperature
of 40° C., about half the starch is transformed.

It appears from this and a number of other experiments that glucoses
and dextrin are formed by the cerealin, the former only being
decomposed by the bacteria almost as fast as it is produced, for after
three hours no glucose is found in the drenches.

We have thus shown that the acids and gases are produced from the
starch contained in the bran, the starch being first changed into
glucoses by the action of an unorganised ferment or ferments; and that
the glucoses are decomposed by a specific organism, the nitrogenous
material in the bran serving for its nutriment; that the action is the
same with or without skins although there appears to be a little more
H_{2}S gas given off from drenches containing skins, than from those
containing none.

The ferment has no direct action on the skins. This may be shown by
taking a piece of limed skin, in which a considerable portion of the
lime exists as carbonate, and submitting it to the action of the
ferment; in this case the action goes on much longer than in the
drenches, being complete in about 15 days, but the skin may be left
in the resulting liquid for three months without undergoing further
change than solution of the lime, provided that suitable means be taken
to exclude moulds, which, by destroying the organic salts and acids
produced, enable putrefactive fermentation to begin.

It has been thought that the bran itself exercised some peculiar action
on the skin, and possibly this may be so to a slight extent,[172] as
the sweet bran drench is occasionally used on the continent, but if
skins are placed in a mixture of bran and water (in the proportions for
drenching) which is prevented from working by the addition of a minute
quantity of HgCl_{2} 1/10000, such a drench has no action on them, and
when tanned they are harsh and hard, similar experiments have been made
on a smaller scale, using ether and chloroform to prevent fermentation,
with the same results.

  [172] Vide der Gerber xiv. 257 Süsse Kleienbeize.

In order to show whether the acids alone were the cause of the action
on the skins, an artificial drench was made up of the following
composition--

    0·5 grm. per 1000 glacial acetic acid.
    1·0  "    "   "   lactic acid sp. gr. 1·210.

In this skins were worked intermittently for 1-1/2 – 2 hours, and it
was found that in this time they were in a similar condition to skins
which had been in a drench from 12–16 hours. They were afterwards
tanned, and found to be good leather, and in every way equal to similar
skins which had been “drenched.” A number of experiments have been
tried with sulphuric and hydrochloric acids in order to ascertain if
these had a similar action, but the results have not been satisfactory.

With regard to the third portion of the research, viz. the products
of a pure cultivation of the bacteria, we have obtained a good number
of results; but as the description of the experiments is of a greater
length than we anticipated, and as there is still some work to do in
verifying them, we are obliged to leave this portion for another paper,
which we hope to have ready by the next session.

In conclusion, we may summarise the results obtained up to the present
in the fermentation of bran by the organism we have used; remarking
that there may be other organisms capable of fermenting bran in a
somewhat similar manner.

1. It has been shown that the fermentation investigated is due to a
specific organism, of which we find no account, and which, pending
further experiments, we have therefore provisionally named Bacterium
furfuris.

2. That the starch and nitrogenous bodies in the bran, alone take part
in the fermentation, the starch being first transformed into glucoses
and dextrin by the action of an unorganised ferment or ferments;
the glucoses and nitrogenous bodies only, being decomposed by the
bacteria, with the formation of formic, acetic, butyric, and lactic
acids, and the simultaneous evolution of hydrogen, carbon dioxide,
nitrogen, and a small quantity of sulphuretted hydrogen. The following
table shows the quantities found in an experimental drench per 1000 c.c.

                      Grm.
    Formic acid      0·0306
    Acetic acid      0·2402
    Butyric acid     0·0134
    Lactic acid      0·7907
                     ------
           Total     1·0749

We find in actual work that the quantity of acid produced varies from 1
to 3 grm. per litre.

3. That if these acids are applied to the skins in the same proportions
as they occur in the drench, the action on them is the same, and much
quicker than an ordinary drench.

4. That the gas therefore, has no action on the skins _per se_, with
the exception of floating and distending them, and so enabling them
better to take up the acids.

We are indebted to Mr. H. R. Procter, of the Yorkshire College, Leeds,
and to Dr. Percy F. Frankland for valuable suggestions in carrying out
some of the work.


    II. ON A PURE CULTIVATION OF A BACILLUS FERMENTING BRAN INFUSIONS.
    By J. T. Wood and W. H. Willcox, B.Sc.(Lond.).

    Reprinted from the Journal of the Society of Chemical Industry,
    30th June, 1897, No. 6, vol. xvi. p. 510.

*Isolation of Pure Culture.*--In our previous communication on bran
fermentation as applied in the manufacture of light leathers,[173] we
gave an account of the actual fermentation and its products, together
with the mode of action on the bran and on the skins for which this
fermentation is used, reserving to the present paper an account of the
products of a pure cultivation of the bacteria causing the fermentation.

  [173] J.S.C.I. 1893, 422.

The cultivation used in the first experiments for this purpose was one
isolated in 1889, and used in the cellulose and starch experiments
described in the above-mentioned paper.

This cultivation had not been obtained from a single colony from
gelatin, and in order to make quite sure that the cultures used were
pure, it was decided to make another attempt to isolate the bacillus
by plate cultivation. Previous attempts to do this had failed, bate
organisms and gelatin liquefying bacilli, developing in such numbers
that the plates were spoiled before the organism, which caused the
fermentation, had time to develop; beside which the organisms, as
obtained direct from the drenches, grew with difficulty in the ordinary
nutrient gelatin. A special gelatin was therefore prepared of the
following composition:--

    Gelatin               100 grm.
    Glucose                30   "
    [174]Salt solution    200 c.c.
    Water                 800  "

  [174] Potassium phosphate, 1 grm.; magnesium sulphate, 0·2 grm.;
  calcium chloride, 0·1; water, 1000 c.c. _Vide_ Frankland and Frew,
  Trans. 1892, 255.

Plates of this gelatin in Petri dishes were prepared from the
previously used supposed pure cultures which had been preserved in
sealed tubes. These were found to be dead. A modification of the method
previously described by one of us[175] was adopted.

  [175] J.S.C.I. 1890, 28.

A solution of nutrient glucose was inoculated from a working drench,
and as soon as the liquid was observed to become cloudy, a tube of the
solid glucose gelatin was inoculated from it by plunging in a platinum
needle. In two days the bacteria developed along the needle track.
Fig. 32 shows the appearance of the tube four days after inoculation, a
bubble of gas being formed in the solid gelatin. On the following day,
the tube was broken, and from the portion where gas was given off most
vigorously other tubes of solid and liquid media were inoculated. Acid
was quickly formed in the nutrient glucose solutions. In the gelatin
tubes, the bacteria developed well in the depth. The now purified
culture was passed through three more glucose gelatin tubes, each time
also a glucose tube being inoculated. From the last of these tubes a
very minute quantity was taken 12 hours after inoculation on the point
of a platinum needle, and a streak culture made on glucose gelatin. In
24 hours a growth could be seen on the surface of the gelatin in the
form of minute dots perfectly separated one from another.

[Illustration: Fig. 32.--Cultures of α in Glucose Gelatin, showing
Bubbles of Gas.]

[Illustration: Fig. 30.--B. Furfuris α.

Illustration: Fig. 31.--B. Furfuris β.

ORGANISMS CAUSING BRAN FERMENTATION. PURE CULTURES.]

From one of these dots a tube was inoculated and from this several
plate cultivations were made. The colonies which developed on these
plates were of two kinds, the majority being round, yellowish and
of small size, a smaller number spreading out on the surface of the
gelatin and slightly iridescent. These surface expansion colonies
when examined with a low power appear like a milky drop, with very
fine granular contents, the whole surrounded by wavy lines which
follow exactly the irregular contour of the expansion. The small
round colonies growing in the depth occur in the proportion of about
3 to 1 of the surface expansion colonies. The microscopic appearance
of the bacteria composing the two kinds of colonies, is almost exactly
similar, they are extremely small and regular in size, 0·75 µ × 0·5 µ
to 0·7 µ × 1 µ. When spread upon a slide, they are not readily miscible
with water, and appear greasy. Both colonies inoculated into glucose
tubes produced acid. The existence of these two organisms was confirmed
in the following way:--A glucose tube was inoculated from a drench in
active fermentation; as soon as the liquid became cloudy, a second
tube was inoculated from it by means of a platinum needle; from this
tube the fermentation was carried through two more tubes; a plate
cultivation was made from the last tube 10 hours after inoculation.
Again, the two kinds of colonies developed exactly similar in every
respect to those obtained from the streak cultures.

It seems probable from these results, and also from a comparison of the
fermentations made with the organisms from an actual drench,[176] and
from purified cultures with those from a single organism, which are
described in the present paper, that the action in the drenches is a
symbiotic one in which two or more organisms take part.

  [176] J.S.C.I. 1892, 422.

*The Fermentations.*--During the time occupied by the isolation of pure
cultures of the bacteria, two fermentations were conducted with the
supposed pure cultures. These fermentations (or rather the second of
them, for the first was unfortunately lost through the breakage of a
bottle) may prove of considerable interest as throwing some light on
the symbiotic action of the two organisms.

The first fermentation with pure cultures of the bacillus α (B.
furfuris) obtained from a _single_ colony in glucose gelatin, was
inoculated on September 16, 1894, the composition of the fermenting
liquid being--

    Glucose                     27 grm.
    Peptone                    1·4  "
    Salt solution              140 c.c.
    Water                      860  "
    Pure calcium carbonate      10 grm.

This was contained in a narrow-necked litre flask fitted with a rubber
stopper, and narrow delivery tube dipping under mercury, and sterilised
with all the usual precautions. The fermentation began on the second
day, reached its height from the 6th–8th day, and continued for 39
days, when gas ceased to come off. The examination of the gases will
be described later on. When the fermentation was over, the liquid was
brought to boiling temperature. It was then examined for the volatile
acids in exactly the same manner as we described in our previous paper.

140 c.c. normal HCl was added and distillation commenced; the
distillate was acid. The distillation was continued until the
distillate ceased to be acid, forming fraction I. Three more fractions
were now distilled off using respectively 10, 20, and 17 c.c. N_{1}HCl.

The fractions were boiled with excess of BaCO_{3} filtered, the
BaCO_{3} washed with hot water, the filtrate evaporated to dryness, and
the barium salts dried at 130° C. till the weight was constant.[177]
The salts were then decomposed with strong H_{2}SO_{4}, ignited, and
the barium sulphate weighed. The following is a tabulated statement of
the results:--

  [177] _Vide_ Note on the Estimation of Butyric Acid, W. H. Willcox,
  J. Chem. Soc., Nov. 21, 1895.

    ----------+---------+---------+-------------
     Fraction |Weight of|Weight of|Per Cent. of
              |  Salts  |BaSO_{4} |  BaSO_{4}
    ----------+---------+---------+-------------
        I.    | 1·2420  | 1·0915  |   87·88
       II.    | 0·9915  | 0·9170  |   92·49
      III.    | 1·2155  | 1·1980  |   98·56
       IV.    | 0·6350  | 0·6230  |   98·11
    ----------+---------+---------+-------------

Calculating fraction I. as a mixture of barium acetate and butyrate,
and fractions II., III., and IV. as mixtures of barium acetate and
formate,[178] we get:--

  [178] _Vide_ J. Chem. Soc. (Trans.), lix., 94, App. II.

    ----------+---------+--------+-------+--------
     Fraction |Weight of|   Ba   |  Ba   |  Ba
              |  Salts  |Butyrate|Acetate|Formate
    ----------+---------+--------+-------+--------
        I.    | 1·2420  | 0·2630 |0·9790 |   ..
       II.    | 0·99151 |   ..   |0·89330| 0·0982
      III.    | 0·2155  |   ..   |0·44000| 0·7755
       IV.    | 0·6350  |   ..   |0·2552 | 0·3798
    ----------+---------+--------+-------+--------
      Totals  | 4·0840  | 0·2630 |2·5675 | 1·2535
    ----------+---------+--------+-------+--------

Calculating the barium salts into their respective acids we get:--

    ----------+-----------+-----------+-------------
     Fraction |Formic Acid|Acetic Acid|Butyric Acid
    ----------+-----------+-----------+-------------
        I.    |    ..     |  0·46070  |  0·1488
       II.    |  0·03980  |  0·42040  |    ..
      III.    |  0·3143   |  0·20710  |    ..
       IV.    |  0·1539   |  0·1201   |    ..
    ----------+-----------+-----------+-------------
      Totals  |  0·5080   |  1·2083   |  0·1488
    ----------+-----------+-----------+-------------

The total volatile acids produced amounting to 1·8651 grm.

The residual liquid containing the non-volatile acids was submitted to
the test for lactic acid previously used,[179] and it was found to be
present.

  [179] J.S.C.I. 1893, 424; vide supra, p. 254.

The method employed for estimating lactic acid in our previous
communication proving somewhat difficult, we endeavoured to improve
it by extracting the concentrated solution of the non-volatile acids
on prepared pumice stone with ether in a paper thimble contained in a
Soxhlet fat-extraction apparatus. After repeated trials we found that
this method did not give accurate results. The solution was therefore
titrated with 1/10 N sodium hydrate, using glazed litmus paper to
determine the point of neutralisation. The acidity found corresponded
to 2·438 grm. of lactic acid per 1000 c.c. of the fermented liquid.

We have done several other fermentations with this organism and find
the same acids produced and the same gases evolved, the results just
given being fully confirmed. At the same time the amount of the acids
produced and their proportions vary, that is to say, the quantity
of acid from a given fermentation cannot be predicted with absolute
accuracy, although the conditions under which we carried out the
experiments were made as like as possible.

We give the total acids from four fermentations to show the amount of
variation. I. is a symbiotic fermentation caused by organisms α and β;
the remainder are fermentations by α alone.

    ---------------------------+---------+---------+---------+---------
           Fermentation        |    I    |    II   | III[180]|   IV
    ---------------------------+---------+---------+---------+---------
    Total vol. acids, grm. per |  2·4968 |  1·8651 |  0·9738 |  1·5636
    1000 c.c.                  |         |         |         |
                               |         |         |         |
    Mean percentage BaSO_{4}   | 89·17   | 91·76   | 95·4    | 93·6
    from Ba salts of vol. acids|         |         |         |
                               |         |         |         |
    Lactic acid                |  8·9500 |  2·4380 |  1·4737 |  2·9700
    ---------------------------+---------+---------+---------+---------


COMPARISON OF ACIDS FROM FERMENTATION II. AND III.[180]

    ------+--------+--------+--------+---------
      --  | Lactic | Formic | Acetic | Butyric
    ------+--------+--------+--------+---------
      II. | 2·4380 | 0·5080 | 1·2083 | 0·1488
     III. | 1·4737 | 0·3914 | 0·5593 | 0·0231
    ------+--------+--------+--------+---------

  [180] 2000 c.c. gave only about the same amount of acids as 1000 c.c.
  fermentations. For percentages of acids, see table, p. 281.


FERMENTATION III., 2000 C.C.

    ---------+---------+---------+------------
             |Weight of|Weight of|Per Cent. of
     Fraction|  Salts  | BaSO_{4}|  BaSO_{4}
    ---------+---------+---------+------------
        I.   | 0·7260  | 0·6535  |   90·01
       II.   | 0·8150  | 0·7410  |   90·92
      III.   | 2·1525  | 2·1342  |   99·15
       IV.   | 0·3695  | 0·3627  |   98·16
        V.   | 0·3530  | 0·3500  |   99·15
    ---------+---------+---------+------------


CALCULATION OF BARIUM SALTS AS BARIUM BUTYRATE, ACETATE, AND FORMATE.

    ---------+-----------+----------+-----------
       --    |Ba Butyrate|Ba Acetate|Ba Formate
    ---------+-----------+----------+-----------
        I.   |   0·0597  |  0·6663  |    ..
       II.   |   0·0223  |  0·7927  |    ..
      III.   |     ..    |  0·6662  |  1·4863
       IV.   |     ..    |  0·1469  |  0·2226
        V.   |     ..    |  0·1070  |  0·2440
             +-----------+----------+-----------
      Totals |   0·0820  |  2·3791  |  1·9529
    ---------+-----------+----------+-----------

    Equivalent to 0·0463 grm. butyric acid.
         "        1·1186  "   acetic   "
         "        0·7828  "   formic   "

or one-half of these quantities per litre of the fermented liquid.

    NOTE.--Mr. Adrian J. Brown, of Burton-on-Trent, has been kind
    enough to examine a sample of the glucose used in the above
    fermentations, and found the rotary power to be equivalent to 95·6
    per cent. pure dextrose. The zinc salt of the lactic acid produced
    had no rotary power.

*The Gases.*--In dealing with the gases evolved, we first compare
those given off in the fermentation of glucose with that of bran under
exactly similar conditions. The fermentation was conducted in open
vessels as before described,[181] and the gases were collected and
examined in the same way.

  [181] J.S.C.I. 1893, 423.


MEAN OF THREE ANALYSES.

    -------+----------------+-----------------
      --   | Bran and Skins |Glucose and Skins
    -------+----------------+-----------------
    CO_{2} |      25·2      |      24·5
    O_{2}  |       2·1      |       1·5
    H_{2}  |      46·7      |      49·8
    N_{2}  |      26·0      |      24·2
    -------+----------------+-----------------

The composition of the gases is thus almost exactly similar, and, we
think, fully proves our previous conclusions as to the change of the
starch of the bran into glucoses by means of an unorganised ferment
(cerealin).

In the closed fermentations we had previously collected only small
quantities of gas over mercury, owing to the difficulty of continuously
collecting large quantities which came off during the night.

In the fermentation of September 16, 1894, we collected the whole of
the gas given off, taking samples every day over mercury, the gas
coming off at night being collected over warm water. Of course this
method does not give the total amount of gas evolved with absolute
accuracy, but the exact composition of the gases was known from day to
day, and the amount of CO_{2} absorbed by the water could be calculated
with moderate accuracy.

The fermentation was conducted in a narrow-necked litre flask fitted
with a narrow delivery tube dipping under mercury, and sterilised with
all the usual precautions. The temperature was maintained at 25°–30°,
gas was evolved for 39 days, when it ceased to come off, the total
amount collected being 3435 c.c. One-half of this quantity, however,
came off in seven days. About 300 c.c. of CO_{2} was absorbed by the
water during the whole period. The diagram (Fig. 33) shows the manner
of evolution of the gases, the ordinates representing volume of gas and
the abscissæ lapse of time after inoculation. The following table shows
the composition of the gas at different stages of the fermentation.
(The fermentation (II.) is the one of which the chemical analysis has
been previously given, page 273):--


COMPOSITION OF GASES EVOLVED IN FERMENTATION OF 1000 C.C. GLUCOSE WITH
PURE FERMENT. SEPTEMBER 16, 1894.

    +-------+-----------------------------------------+-----
    |       |                   Day                   |
    +  --   +------+------+------+------+------+------+ Mean
    |       |  5   |  9   |  12  |  19  |26–31 |35–39 |
    +-------+------+------+------+------+------+------+-----
    |CO_{2} | 53·7 | 56·4 | 44·3 | 52·6 | 55·6 | 43·2 | 49·9
    |O_{2}  |  1·8 |  0·5 |  1·7 |  1·9 |  0·8 |  2·8 |  1·8
    |H_{2}  | 35·8 | 34·6 | 41·2 | 30·3 | 34·7 | 31·8 | 34·8
    |N_{2}  |  8·7 |  8·5 | 12·8 | 15·2 |  8·9 | 22·2 | 13·5
    +-------+------+------+------+------+------+------+-----

The total quantity of CO_{2} actually collected = 1563 c.c. =
3·090 grm.; the amount of CO_{2} due to decomposition of the CaCO_{3}
by the acids produced was found to be 667 c.c. = (1·3189 grm). The vol.
of hydrogen collected was 1086 c.c. = 0·973 grm.

[Illustration: Fig. 33.--Evolution of Gases from Pure Cultivation of B.
Furfuris.]

In a second fermentation (III.) we endeavoured to ascertain the exact
amount of CO_{2} evolved, as in the previous fermentation this had not
been done. It was therefore decided to absorb the CO_{2} by means of
potash.

The fermentation in this case was conducted in a narrow-necked flask
of 2000 c.c. capacity, connected by means of a narrow glass tube with
two potash bulbs containing strong caustic potash, and furnished with
a delivery tube dipping under water; the whole apparatus stood upon
an iron plate, and was maintained at a temperature of 25°–30° in the
same manner as the previous fermentation. The gases were evolved for 21
days--a considerably shorter period than the 1000 c.c. fermentation;
but resembling it in that one-half the gas was evolved in eight days.
The diagram shows the curve as in the previous fermentation, which it
resembles for the first 14 days, afterwards however stopping suddenly.
When the fermentation was at an end the flask and contents were heated
to boiling point, at the same time a current of air free from CO_{2}
was drawn through it, and the CO_{2} given off being collected in
potash bulbs as in the fermentation. Unfortunately the estimation of
the CO_{2} was rendered valueless owing to an accident.

The table shows the composition of the gases other than CO_{2} evolved
in this second fermentation.


GASES FROM FERMENTATION OF 2000 C.C. (EXCLUDING CO_{2}) FERMENTATION
III.

    ------+-------------------------------------------+-------
          |                   Days                    |
      --  +------+------+------+------+-------+-------+  Mean
          |  2–4 |  4–5 |  5–6 |  11  | 14–15 | 16–17 |
    ------+------+------+------+------+-------+-------+-------
    O_{2} |  3·4 |  2·2 |  1·5 |  0·42|   0·9 |   2·4 |  1·48
    H_{2} | 81·3 | 83·3 | 82·4 | 79·0 |  71·7 |  72·2 | 77·72
    N_{2} | 15·3 | 14·5 | 16·1 | 20·5 |  27·4 |  25·4 | 20·78
    ------+------+------+------+------+-------+-------+-------

The gas from days 18–21 was unfortunately mixed with air. On comparing
the mean composition of gases other than CO_{2} collected from both
fermentations, we get the following result:--

    ------+-------------+-------------
      --  |Fermentation,|Fermentation,
          |  1000 c.c.  |  2000 c.c.
    ------+-------------+-------------
    O_{2} |      3·57   |     1·48
    H_{2} |     69·4    |    77·72
    N_{2} |     27·0    |    20·78
    ------+-------------+-------------

If now the O and part of the N in the proportion to form air be taken
away, the composition of the gases from the two fermentations is found
to be almost exactly similar:--

    ------+-------------+-------------
      --  |Fermentation,|Fermentation,
          |  1000 c.c.  |  2000 c.c.
    ------+-------------+-------------
    H_{2} |     84·4    |     83·9
    N_{2} |     15·6    |     16·1
          +-------------+-------------
          |    100.0    |    100.0
    ------+-------------+-------------

The gases from a third fermentation were almost exactly similar in
composition, but the total volume was not measured.

A remarkable fact in this fermentation is the evolution of free N,
which seems to be rare, except in the case of putrefactive organisms,
as in the vast number of fermentative decompositions due to bacteria,
almost the only gases found are carbonic anhydride, hydrogen, H_{2}S,
and marsh gas.

Gayon[182] in 1875, in an investigation on the putrefaction of eggs,
collected the gas given off from large ostrich eggs, and found in it
29 per cent. of nitrogen; he adds, however, that its presence may be
due to the accumulation of a certain quantity of air in the air-bubble
before putrefaction.

  [182] Schutzenberger, “Fermentation,” 1876, p. 227.

Béchamp[183] found that yeast cells under suitable conditions, but
sugar being withheld, produced pure nitrogen along with leucin,
tyrosin, a soluble albuminous substance coagulable by heat, an enzyme,
a peculiar gummy substance, phosphates and acetic acid, alcohol and
CO_{2}. These are almost the only instances where observers of repute
have been convinced of the evolution of free N by bacteria. We find
since the above work was carried out that Immendorf[184] has found
certain bacteria in dung which form ammonium nitrate, and this body, as
is known, splits up at a comparatively low temperature into nitrogen
and water.

  [183] Woodhead, “Bacteria and their Products,” p. 125.

[184] Die Stickstoffkonservirung im Stalldünger, Jour. f. Landwirthsh.,
xlii. p. 69.

From the bacteriological as well as the chemical results, it is now
evident that the fermentation as it takes place in practice is a
symbiotic one in which two organisms play the most important part, and
very probably cause the entire fermentation. This is shown by comparing
the acids produced by the fermentation in the works with those produced
by a mixture of the organisms α and β, the relative amounts being
very close, while in all the fermentations with α alone a much less
proportion of lactic acid is produced, as the following table shows:--

    -----------+------------+------------+------------+------------
        --     |Fermentation|Fermentation|Fermentation|Fermentation
               |  in Works  |   α and β  |   α (II.)   |  α (III.)
    -----------+------------+------------+------------+------------
    Formic acid|     2·8    |     0·8    |    11·8    |    16·0
    Acetic  "  |    22·5    |    16·4    |    27·9    |    22·7
    Butyric "  |     1·2    |     4·5    |     3·5    |     0·9
    Lactic  "  |    73·5    |    78·3    |    56·7    |    60·2
    -----------+------------+------------+------------+------------

    NOTE.--Ruge (Sitzungsber. d. Wien. Akad. d. Wiss, Vol. xliv., 1862,
    734) found that the gases of the large intestine contained 57·8 per
    cent. of N, and Gamgee remarks “in part a diffusate from the blood,
    but is certainly in part derived from the bacterial decomposition
    of proteids.” (Gamgee, Phys. Chem. Vol. ii., p. 467.)

The acetic acid, as far as we can ascertain, is produced directly
from dextrose without the previous production of alcohol, since the
presence of the latter is not shown by its tests at any stage of the
fermentation. We have also ascertained that the organism is without
action on dilute solutions of alcohol, in yeast water, no acid being
produced.

We are indebted to Mr. H. S. Shrewsbury for the analysis of some of the
gases and volatile acids, and also for the preparation of the diagrams.
In conclusion we may state that the investigation of this fermentation
in the tannery has been the means of pointing the way to a still more
complicated process, viz., “bating.” It may even be possible in the
future to place these processes on somewhat the same footing as the
accurately understood fermentations in the brewing industry although
the difficulties in the way are much greater.




CHAPTER XI.

BIBLIOGRAPHY.


The following short bibliography of works on tanning and the
bacteriology of leather manufacture, does not profess to be complete,
but is given in the hope that it may prove useful. It includes most of
the works consulted or referred to in this book. For particulars of
some of the earlier works (Nos. 1, 2, 4, 5, 6, 7, and 8), I am indebted
to Dr. E. Stiasny, of Leeds University.

Nos. 1–10 are old works on Tanning generally.

Nos. 11–57 books and papers referring chiefly to bating and the
bacteriology of Leather Manufacture.

Nos. 58–78 deal with putrefaction.

Nos. 79–133 are general works consulted, in order of date.

    1. La Tannerie et la preparation des Cuirs. (MS.) Desbillettes.
    1708.

    2. L’art du tanneur. De la Lande. 1764.

    3. The art of tanning and currying leather, with an account of all
    the different processes made use of in Europe and Asia for dyeing
    leather red and yellow, collected and published at the expense of
    the Dublin Society. To which are added Mr. Philipo’s method of
    dyeing the Turkey leather as approved of by the Society for the
    Encouragement of Arts, etc., and for which he had a reward of £100
    and their gold medal for the secret. Also the new method of tanning
    invented by the late David Macbride, M.D., London, reprinted for J.
    Nourse in the Strand, Bookseller to His Majesty. 1780.

    4. Lohgerberei, Ignatz Bautsch. Dresden, 1793.

    5. Ueber die Bearbeitung der Tierhäute zu allen Gattungen von
    Leder. P. J. Kasteleyn. German translation from the Dutch. Leipzig,
    1797.

    6. Chemisch-technologische Grundsätze der gesammten Ledergerberei.
    2 vols. S. F. Hermbstädt. Berlin, 1805 and 1807.

    7. Dictionary of Chemistry and Mineralogy. 2 vols. A. and C. R.
    Aikin, 1807.

    8. Hand-Encyclopädie für das Gerben, Zurichten, etc., des Leders,
    L. F. Kummer, Berlin, 1830.

    9. Handbuch der Gesammten Lohgerberei. Vom Dr. Ch. H. Schmidt.
    Weimar, 1847.

    10. Lehrbuch der Sohlledergerberei. Dr. G. W. Bichon, Berlin.

    11. “Das Beizen der Glacé-Felle” in Handbuch der Weissgerberei.
    Anton Brüggemann. Quedlinburg and Leipzig, 1857. p. 21.

    12. Erfahrungen auf dem Gebiete der Gerberei. J. C. H. Lietzmann,
    Berlin, 1862.

    13. “Das Behandeln in der Kleienbeize” in Handbuch der
    Weissgerberei. Dr. W. F. Gintl. Weimar, 1873. p. 51.

    14. Mistbeizen. Der Gerber, 1884. p. 197.

    15. The Manufacture of Leather. Davis, Philadelphia, 1885. p. 335,
    etc.

    16. Traité pratique de la Fabrication des cuirs et du Travail des
    peaux. Villon, 1889. p. 407.

    17. Die Englische Methode für die Chevrettengerbung. Beizverfahren,
    Der Gerber, Bd. XV. 1889. p. 267.

    18. Methods of Bacteriological Research, with some account of Bran
    Fermentation, by J. T. Wood. Jour. Soc. Chem. Ind. IX. 1890. p. 27.

    19. The Theory and Practice of Tanning. W. J. Salomon. Tech.
    Quarterly, 1892. 5 (1 and 2), 81–88.

    20. Further contribution on the Nature of Bran Fermentation, by
    J. T. Wood, and W. H. Willcox, B.Sc., London. Jour. Soc. Chem. Ind.
    XII. 1893. p. 422.

    21. The Chemistry of the Grainer Pit, by T. Palmer and P. G.
    Sandford. J. Anal. and Appl. Chem., 1893, 7, 87–95.

    22. Fermentation in the Leather Industry, by J. T. Wood. Jour. Soc.
    Chem. Ind. XIII. 1894. p. 218.

    23. The Bacteria of Stable Manure and their Action. Dr. E.
    Herfeldt, Bonn. Jour. Soc. Chem. Ind. XIV. 1895, pp. 449–452.
    Translated with foreword by J. T. Wood. (This paper contains a
    Bibliography of 21 items.)

    24. Uber die Beziehung der Bakteriologie zur Gerberei. Dr. F. H.
    Haenlein. Cent. Bl. f. Bakt. II. 1895. p. 26.

    25. Die im Miste vorkommenden Bacterien und deren physiologische
    Rolle bei der Zerzetzung desselben. S. A. Severin. Central Bl. f.
    Bakt. (II), Vol. I., 1895, pp. 97, 160, 799. Vol. III. pp. 628, 706.

    26. Gährungserscheinungen in Gerbbrühen. Fr. Andreasch. Der Gerber,
    1895–7.

    27. W. Schmitz Dumont. The Sweating Process in the Tannery. Ding.
    Polyt. Jour. 1896, 300, 139–144. J.S.C.I. 1896, p. 461.

    28. On a pure cultivation of a Bacillus fermenting Bran Infusions,
    by J. T. Wood and W. H. Willcox, B.Sc., London. Jour. Soc. Chem.
    Ind. XVI. 1897. p. 510.

    29. The Rationale of Bating, by J. T. Wood. Leather Industries.
    September, 1898.

    30. Notes on the constitution and mode of action of the Dung Bate
    in leather manufacture. J. T. Wood, Jour. Soc. Chem. Ind. XVII.
    1898. p. 1010.

    31. “Schwitzen” in Gerberei Chemie, Sammlung von Aufsätzen
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    32. Beizen und neue Beizmittel, W. Eitner, Der Gerber. Bd. XXIV.
    No. 563–567, 1898.

    33. The Rationale of Drenching, by J. T. Wood. Leather Trades
    Review, Nov. 15, 1898.

    34. Cantor Lectures on Leather Manufacture, by Prof. H. R. Procter,
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    Wood. Jour. Soc. Chem. Ind. XVIII. 1899. p. 990.

    36. Das Beizen der Blössen in Handbuch der Chromgerbung. Jos.
    Jettmar. Schulze and Co., Leipzig, 1900.

    37. J. Borgmann. Die Feinleder Fabrikation, Berlin, 1901. Das
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    38. Das Beizen der Blössen in Praxis und Theorie der Leder
    Erzeugung, von Josef Jettmar, Julius Springer, 1901.

    39. Micro-organisms and Antiseptics in Tanning. F. Jean, Rev. Chim.
    Ind., 1901.

    40. The Principles of Leather Manufacture, by Professor H. R.
    Procter. E. and F. N. Spon, London, 1903. Chap. XIII.

    41. La Tannerie, par Louis Meunier and Clément Vaney,
    Gauthier-Villars. Paris, 1903. p. 125, etc.

    42. Bakteriologische Vorgänge in der Lederindustrie. 1904, H.
    Becker. Zeitschrift f. Offentliche Chemie, Heft. XXIII. Jahrgang. X.

    43. Untersuchungen über die Bakterien im Verdauungskanal des
    Rindes. P. Ankersmit. Centralbl. f. Bacter. I, O, Bd. 39, 1905. p.
    359.

    44. Über anaerobe Bakterien im Rinderdarm, Neubauer. Archiv f.
    wissensch. u. prakt. Tierheilk. Bd. 31, p. 153, 1905.

    45. Studien über fäulnisserregende anaerobe Bakterien des normalen
    menschlichen Darmes, und ihre Bedeutung. F. Passini. Zeitsch. f.
    Hygiene. Bd. 49, p. 135. 1905.

    46. A new process of puering or bating hides and skins. Allen
    Roger. J.S.C.I., p. 103, 1906.

    47. Recent advances in the Bacteriology of Putrefaction, by J. T.
    Wood. J.S.C.I. XXV. 1906, p. 119. (Contains a bibliography of 21
    items.)

    48. Über den Bakteriengehalt menschlicher und thierischer Fäces.
    M. Lissauer. (Archiv f. Hyg. Bd. 58, p. 136.) See also Koch’s
    Jahresbericht, 1906, p. 195.

    49. Character of the Bacterial Flora of Carnivorous and Herbivorous
    animals. A. Herter. (Science N.S. Vol. 24, p. 859. 1906.)

    50. Leather Trades Chemistry. S. R. Trotman, Griffin, 1908. p. 79.

    51. Le Rôle des Microbes dans la putrefaction des peaux en poils et
    en tripe et dans les confits, par Dr. Gr. Abt. Bull. Mens. du Synd.
    Gen. de cuirs et peaux de France. Nov.-Dec., 1908. p. 416.

    52. Bakterien in der Lederindustrie. H. Becker, Collegium, 1909.
    p. 169.

    53. A new Chromogenic Organism. S. R. Trotman, J.S.C.I., 1909.
    p. 1238.

    54. Problems of the Leather Industry. Prof. H. R. Procter,
    J.S.C.I., 1910. p. 329.

    55. Les Anaerobies, par M. Jungano et A. Distaso. Paris, Masson and
    Cie., 1910.

    56. The Bacteriology of the Leather Industry. J. T. Wood, J.S.C.I.,
    June 15th, 1910. p. 666.

    57. The Seymour-Jones Anthrax Sterilization Method, by Alfred
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    59. Woodhead, G. S. Bacteria and their products. (W. Scott, 1891.)

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    61. Macé, Traité pratique de Bacteriologie. Paris. Baillière.

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    63. Klein. Ein Beitrag zur Kentniss der Leichenverwesung. (Cent.
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    64. Beijerinck, W. Schwefelwasserstoffbildung in den Stadt. gräben
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    65. Stich, C. Ueber die Bildung gasförmiger Phosphorverbindungen
    bei der Fäulniss. (Mitth. a. d. analyt. Laborat, der
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    66. Vitali, D. Bildung von Alcohol bei der Fäulniss von
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    68. Bienstock. Untersuchungen über die Aetiologie der
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    Zweite Abth. XV. 53.)

    75. Omelianski, W. Sur la fermentation forménique de la cellulose.
    (Arch. de sciences biol. St. Petersburg, t. 11, 251. Centr. Bl. f.
    Bakt. II., Bd. 8, 193.)

    76. Dallinger and Drysdale. Monthly Microscopical Journal, vol. X.,
    53, 245; XI., 7, 69; XII., 261; XIII., 185; XIV., 106.

    77. Lermer, K. Ueber die Produkte der Fäulniss der Gerste,
    Zeitschr. f. d. ges. Brauwesen, p. 165.

    78. Salkowski and Neuberg. Die Verwandlung von d. Glukuronsaure in
    1-Xylose Zeitschr. f. physiol. Chemie Bd. 36, p. 261.

    79. Études chimiques sur la Végétation. J. Raulin, Paris, 1870.

    80. Die Spaltpilze. W. Zopf. Breslau, 1885.

    81. A Text Book of Tanning. H. R. Procter. Spon, 1885.

    82. Micro-organisms and Disease. E. Klein. Macmillan, 1886.

    83. Manual of Bacteriology. E. M. Crookshank. Lewis, 1887.

    84. Manures, Natural and Artificial. W. Iveson Macadam. J.S.C.I.,
    1888, p. 79.

    85. Methods and Formulæ used in the Preparation of Animal and
    Vegetable Tissues. P. W. Squire. Churchill, 1892.

    86. Micro-organisms and Fermentation. A. Jörgensen. Macmillan
    (London), 1900.

    87. Micro-organisms in Water. Percy Frankland and Mrs. Frankland.
    Longmans, 1894.

    88. Disinfection and Disinfectants. S. Rideal. Griffin, 1895.

    89. Guide to the British Mycetozoa, Dept. of Botany, British
    Museum. Arthur Lister, F.L.S., 1895.

    90. Elementary Practical Bacteriology. Kanthack and Drysdale.
    Macmillan, 1895.

    91. Les Ferments Solubles. E. Bourquelot. Paris, 1896.

    92. The Manufacture and Applications of Lactic Acid. A. Claflin.
    J.S.C.I., 1897, p. 516.

    93. Bakteriologie. C. Günther. Leipzig, 1898.

    94. Bates and New Bating Materials. Der Gerber 24 [563], [564] and
    [565]. J.S.C.I., 1898, p. 361.

    95. Les Enzymes. J. Effront. Paris, 1899.

    96. Fixerung, Färbung und Bau des Protoplasmas. A. Fischer. Jena,
    1899.

    97. Soluble Ferments and Fermentation. J. Reynolds Green. Cambridge
    University Press, 1899.

    98. Ueber die Zusammensetzungund Wirkungsweise der Mistbeizen. Der
    Gerber, 1899, p. 31.

    99. The Mycetozoa. Sir Edward Fry and Agnes Fry, London,
    “Knowledge” Office, 1899.

    100. Le Proteus Vulgaris. L. Felz. Baillière, Paris, 1900.

    101. Bating Process, Improvements in. Leather Trades Review, 34
    [773], 21. Abstr. in Jour. Soc. Chem. Ind., 1901, p. 263.

    102. Oppenheimer, C. Ferments and their Actions. Trs. C. A.
    Mitchell. Griffin and Co., 1901.

    103. Harden. The Chemical Action of Bacillus Coli communis on
    Carbohydrates and allied Compounds. J. Chem. Soc., Trans. 79, 1901,
    610.

    104. Traité de Technique Microbiologique. Nicolle et Remlinger.
    Doin., Paris, 1902.

    105. Carini. Sull’ Applicazione della Bilancia idrostatica per il
    controllo della concia delle Pelli. Milan, 1903.

    106. Traité d’Electrochimie. Max Le Blanc. Paris, 1904.

    107. The Role of Diffusion during Catalysis by Colloidal Metals and
    similar Substances. Henry J. S. Sand, Ph.D. M.Sc. Proc. Chem. Soc.
    74, 356. 1904.

    108. Chemistry of the Proteids. G. Mann. Macmillan, 1906.

    109. Canning and Preserving Food Products, with Bacteriological
    Technique. E. W. Duckwall, M.S., Bacteriologist to the National
    Canners Laboratory, etc. New York, 1905.

    110. Précis de Coprologie Clinique. R. Gaultier. Baillière, Paris,
    1907.

    111. Allgemeine Chemie der Kolloide. A. Müller. Leipzig, 1907.

    112. Zur Erkenntniss der Kolloide. R. Zsigmondy. Jena, 1905.

    113. The Nature of Enzyme Action. W. M. Bayliss. Longmans, 1908.

    114. Ueber die Interfibrilläre Substanz der Lederhaut bei
    Säugethieren. G. H. B. Van Lier. Coll., 1909, 321.

    115. Practical Causerie on Deliming. Alex. T. Hough. Tanner’s Year
    Book, 1909, p. 83.

    116. Grundriss der Kolloid Chemie. M. Ostwald. Dresden, 1909.

    117. Beizen. W. Appelius. Technische Briefe Monatshefte von “Häute
    und Leder,” No. 23. April 1909.

    118. Kolloid Chemische Beihefte. Ed. Wo. Ostwald. Dresden,
    1909–1911.

    119. Verfahren zum Beizen von Häuten. Otto Röhm. Coll., 1909, p. 58.

    120. A Brief Review of Bacteriological Research in Phytopathology,
    with Bibliography. M. C. Potter, Sc.D., M.A. Science Progress, Oct.
    1910, p. 191.

    121. La Chimie de la Matière Vivante. J. Duclaux. Felix Alcan,
    Paris, 1910.

    122. Sensibilité de la peau verte, et de la peau après l’échauffe,
    les pelains, et les confits, à l’égard de la chaux, du sel, et
    de l’acide acétique. Georges Abt et Edmund Stiasny. Coll., 1910,
    p. 189.

    123. Bennett, H. G. The Analytical Examination of Bating. Leather
    Trades Review, 1911, pp. 697, 972.

    124. Der Bacillus bulgaricus des Yoghurt in der Gerberei.
    Leder-technische Rundschau, No. 25, 1911. Collegium, 1911, p. 459.

    125. Physiological Chemistry. Olaf. Hammarsten. Trans. J. A.
    Mandel, Wiley, 1911.

    126. Précis de Biochemie. E. Lambling. Paris, 1911.

    127. Bacteriological and Enzyme Chemistry, an Introduction to.
    Gilbert J. Fowler. Edward Arnold, 1911.

    128. Microbes et Toxines. E. Burnet. Bibliothèque de Philosophie
    Scientifique. Flammarion. Paris, 1911.

    129. The Physical Chemistry of the Proteins. T. B. Robertson,
    Professor at the Berkeley University, California, 1911.

    130. Contributions to the History of the Enzyme Bate. Collegium,
    1911. Otto Röhm, H. Becker, G. Eberle, J. T. Wood, pp. 271, 276,
    278, 281, 312, 324.

    131. The Fungi of Excreta. Jas. Scott. The Leather World, Jan. 11,
    1912, p. 21.

    132. Microbiology, by various Contributors. Edited by Charles E.
    Marshall, East Lancing, Michigan, Professor of Bacteriology and
    Hygiene, Michigan Agricultural College. J. and A. Churchill.

    133. Technical Mycology. Lafar. C. Griffin and Co., 1911.

    134. Manual of Bacteriology. R. T. Hewlett (Churchill), 1912.

    135. Taschenbuch für den bakteriologischen Praktikanten. Dr. Rudolf
    Abel. Würzburg. A. Stuber’s Verlag.


LIST OF AUTHORS.

_The numbers refer to the Bibliography._

    Abel, R., 135

    Abt, G., 51, 122

    Aikin, A. and C. R., 7

    Andreasch, F., 26

    Ankersmit, P., 43

    Appelius, W., 117


    Bayliss, W. M., 113

    Bautsch, I., 4

    Becker, H., 42, 52, 130

    Behrens, J., 67

    Bennett, H. G., 123

    Beijerinck, W., 64

    Bichon, G. W., 10

    Bienstock, 62, 68

    Borgmann, J., 37

    Bourquelot, E., 91

    Brüggemann, A., 11

    Burnet, E., 128


    Carbone Tito, 60

    Carini, 105

    Claflin, A., 92

    Crookshank, E. M., 83


    Davis, 15

    Dallinger, 76

    De la Lande, 21

    Desbillettes, 1

    Distaso, A., 55

    Drysdale, 76, 90

    Duckwall, E. W., 109

    Duclaux, J., 121


    Eberle, G., 130

    Eitner, W., 14, 17, 32, 94, 98

    Effront, J., 95

    Felz, L., 100

    Fischer, A., 96

    Fischer, H., 71

    Fowler, Gilbert J., 127

    Frankland, P. and Mrs., 87

    Fry, Sir Edward and Agnes, 99


    Gaultier, R., 110

    Gintl, W. F., 13

    Green, J. Reynolds, 97

    Günther, C., 93


    Haenlein, F. H., 24

    Hammarsten, O., 125

    Harden, A., 103

    Hauser, G., 58

    Herter, A., 49

    Herfeldt, E., 23

    Hermbstädt, 6

    Hewlett, R. T., 134

    Hough, A. T., 115


    Jean, F., 39

    Jettmar, J., 36, 38

    Jörgensen, A., 86

    Jungano, M., 55


    Kanthack, 90

    Kasteleyn, K. T., 5

    Klein, E., 63, 82

    Kummer, L. F., 8


    Lafar, 133

    Lambling, E., 126

    Le Blanc, Max, 106

    Lermer, K., 77

    Lietzmann, J. C. H., 12

    Lissauer, M., 48

    Lister, Arthur, 89


    MacBride, 3

    Macé, E., 61

    Macadam, W. Iveson, 84

    Mann, G., 108

    Marshall, C. E., 132

    Martelly, 70

    Meunier, L., 41

    Müller, A., 111


    Neubauer, 44

    Neuberg, 78

    Nicolle, M., 104


    Omelianski, W., 75

    Oppenheimer, C., 102

    Osterwalder, A., 72

    Ostwald, Wo., 116, 118


    Palmer, T., 21

    Passini, F., 45

    Potter, M. C., 120

    Procter, H. R., 34, 40, 54, 81


    Rahn, O., 74

    Raulin, J., 79

    Remlinger, P., 104

    Rideal, S., 88

    Röhm, O., 119, 130

    Robertson, T. B., 129

    Roger, A., 46


    Salomon, W. J., 19

    Salkowski, 78

    Sandford, P. G., 21

    Sand, H. J. S., 107

    Schneider, Ph., 71

    Schreiber, K., 73

    Schmidt, Dr. Ch. H., 9

    Schmitz-Dumont, 27

    Schröder, J. von, 31

    Scott, J., 131

    Severin, S. A., 25

    Seymour-Jones, 57

    Squire, P. W., 85

    Stich, C., 65

    Stiasny, 122


    Taylor, A. E., 69

    Tissier, H., 70

    Trotman, S. R., 50, 53


    Vaney, C., 41

    Van Lier, G. H. B., 114

    Villon, 16

    Vitali, D., 66


    Willcox, W. H., 20, 28

    Wood, J. T., 18, 20, 22, 28, 29, 30, 33, 35, 47, 56, 130

    Woodhead, G. Sims, 59

    Wohltmann, F., 71


    Zopf, W., 80

    Zsigmondy, R., 112




CONCLUSION.


                    “What is a man,
    If his chief good and market of his time,
    Be but to sleep and feed? A beast--no more.
    Sure, He that made us with such large discourse,
    Looking before, and after, gave us not
    That capability and godlike reason
    To fust in us unused.”

Professor Procter, in his paper, entitled “Problems of the Leather
Industry,”[185] says: “There is, however, no reason that all the
necessary effects both of puering and bating should not ultimately be
attained by purely chemical treatment without the risk and uncertainty
which must always attach to bacterial and ferment action.”

  [185] J.S.C.I. 1910, p. 331.

While I agree with him in this, still it is well to remember that in
the case of one of the very oldest of the fermentation industries, that
of the production of alcohol, a comparatively simple body, the natural
process has not yet been replaced by a chemical one, and I believe this
applies also to the manufacture of vinegar.

Chemical methods of bating may be used for leathers like chrome and
alum leather, but even here natural processes like drenching, in which
acids are produced gradually during the working of the skins, give more
beautiful results. With vegetable tanning materials, the advantage is
still on the side of the natural processes when these are conducted in
a proper manner.

Finally, the author regrets that the subject of the work is not an
inspiring and lofty one, such as is astronomy for example.[186] Limited
in extent, imperfect in execution, and in parts only suggestive in
character, this little book may perhaps serve as a foundation, on which
students of the science of tanning may raise the superstructure of
their own experience and eventually perfect the processes touched upon,
so that the use of such filthy materials may be entirely avoided. In
that case the author’s intention will be fulfilled.

  [186] Celestial Objects, Webb, 1881.


    FINIS




INDEX.


    Absorption phenomena, 70

    Abt, Dr. G., 51, 72

    Acid “turning” of drench, 237

    Acids, action of, 156

    ---- fractionating, 270

    ---- deliming with, 9

    ---- in drench, 265

    ---- to remove lime, cost of, 11

    Adulteration in dog-dung, detecting, 144

    Agglutinin, 144

    Alum, a term for puer, 3

    “Alumed” calf skins, 3

    Amend’s bate, 193

    Amido compounds, 156

    Amines, 163, 206

    ---- in dung, 154

    Ammonia in puer liquors, 41

    Ammonium phosphate, 45

    Analyses, bird excreta, 53

    ---- dog dung, 26, 150, 152

    ---- drench, 249

    ---- gas, 247

    ---- grains, 7

    ---- guano, 150

    ---- hen dung, 150

    ---- puer, 26

    ---- ultimate, of dung, 153

    Andreasch, F., 244

    Anti-bodies, 143

    Anti-calcium, 236

    Arginase, 133

    Artificial drench, 263

    Ash of skin, 39

    Authors, list of, 290


    _Bacillus butyricus_, 112

    ---- _c_, 172

    ---- _Coli commune_, 94

    ---- _d_, 106, 173, 174

    ---- _e_, 107, 173

    ---- _erodiens_, 97

    ---- from pigeon dung bate, 172

    Bacteria, action of, 160

    ---- bate, examination of, 87

    ---- bran, pure culture of, 269

    ---- dung, 89, 98, 102, 161

    ---- growth in various media, 105

    ---- pigeon dung, 103

    ---- putrefactive, 125

    ---- “Sweating,” 105, 213

    Bacterial diastase, 131

    _Bacterium furfuris_, 241, 264, 269

    ---- _termo_, 118

    Barium salts from pure cultures of _B. furfuris_, 271

    ---- ---- of volatile acids, 253

    Bate, Amend’s, 193

    ---- Becker’s patent, 227

    ---- bird dung, 52

    ---- Boehringer’s patent, 197

    ---- concentration of salts in, 47

    ---- Dermiforma, 189

    ---- Eberle’s patent, 221

    ---- Erodin, 179

    ---- Esco, 192

    ---- examination of bacteria in, 87

    ---- glucose, 179

    ---- Lederer’s, 196

    ---- Nordlinger’s, 195

    ---- Norris’s, 194

    Bate, Nowak’s patent, 194

    ---- Oake’s, 195

    ---- Oropon, 140, 186, 219

    ---- phosphates in, 43

    ---- pigeon dung, 103

    ---- Phosphorbutyralin, 195

    ---- Popp and Becker’s patent, 197

    ---- Puerine, 190

    ---- Purgatol, 192

    ---- reducing action of, 152

    ---- Röhm’s patent, 219

    ---- Simon’s, 195

    ---- solid matter, influence of, 176

    ---- soluble matter of, 176

    ---- Sucanine, 191

    ---- Tiffany’s, 179

    ---- Wood’s American patent, 210

    ---- ---- English patent, 205

    ---- Zollikoffer’s, 178

    Bayliss, Dr. W. M., 127

    Béchamp, 280

    Betulase, 132

    Becker’s patent, 227

    Bibliography, 282

    Bile, 31

    ---- action of, 159

    ---- colouring matters, 30

    ---- salts, effect on puer, 46

    ---- ---- ---- ---- lipase, 142

    Bird dung bate, 52

    ---- excreta, composition of, 53

    Boehringer’s patent, 194

    Bone meal, 196

    Books of reference, 89, 282

    Bran bacteria, 240

    ---- ---- pure cultures of, 269

    ---- composition of, 257

    ---- drench, 233

    ---- ---- non-volatile bodies in, 254

    ---- ---- products of fermentation, 247

    ---- ---- sweet, 263

    ---- ---- volatile bodies in, 249

    ---- ferments, action on starch, 258

    Brown, Adrian J. Prof., 274

    Butyric acid, 11

    ---- ---- fermentation, 111


    Calcium in enzymes, 136

    ---- phosphate, 45

    Carlsberg vessel, 170, 214

    Caroubinase, 132

    Casease, 132

    Castor-oil seed, 188, 232

    Catalysts, 127

    Cellulose fermentation, 110

    Cerealin, 245, 260

    Chemical composition of puer, 24

    ---- deliming, 5

    Chloroform, use of, in pancreatin solution, 151

    Cholesterol, 30

    Collagen, 56

    Colloidal state, 57, 137

    Concentration of salts in bate, 47

    Condenser, Abbe’s, 87, 88

    ---- dark ground, 87

    Conductivity of puer liquors, 80, 81

    Copenhagen conference, 180

    Cultivation of bating bacteria, 199

    Culture medium, 105, 207

    Cytase, 132


    Deliming, chemical, 5

    Density of skin, 60

    Dermiforma, 189

    Dextrin, 259, 261

    Diastase, 128, 131, 132

    ---- Krawkov’s method of preparing, 135

    Diastatic enzymes, 235

    Digestive ferments, 148

    ---- juices, 230

    Dog dung, analyses of, 26, 150, 152

    ---- bacteria in, 89, 98

    ---- detecting adulteration of, 144

    ---- fresh, bacteria in, 102

    ---- influence of food on, 28, 95, 153

    ---- method of analysis, 25

    Dog dung, separating enzymes from, 134

    ---- trypsin in, 134

    Doerr and Reinhardt’s laboratory, 32

    Drench, acid “turning” of, 237

    ---- acids in, 156

    ---- action of, 233, 242, 245

    ---- artificial, 263

    ---- bacteria in, 239

    ---- damage, 236

    ---- fermentations of, 238

    ---- gases from, 242, 248

    ---- glucoses in, 261

    ---- “pinholey,” 238

    ---- putrid “turning” of, 237

    ---- sweet bran, 235

    Dressing hides, 19

    Drop culture, 87

    Dung analyses, 26, 150, 152, 153

    ---- mineral matter of, 153


    Eberle, G., 13

    Eberle’s patent, 221

    Effront, Dr., 11

    Eitner, 242, 244

    Electrometric apparatus, 76

    ---- method, 33, 49

    Emulsin, 132

    Enzymatic bates, 197

    Enzymes, action of, 145

    ---- ---- of serum on, 144

    ---- ---- on skin, 140

    ---- calcium in, 136

    ---- defined, 127

    ---- diastatic, 235

    ---- from bacteria, 131

    ---- hydrolysing, 132

    ---- in dog’s dung, 137

    ---- lipolytic, 145

    ---- manganese in, 136

    ---- preparation of, 134, 164

    ---- proteolytic, 145

    Erepsin, 133, 142

    Erodin, 179

    Erodin, stock liquor, 182

    Esco, 192

    Esters, synthesis of, 127

    Ethyl butyrate, 165

    Ethylamine butyrate, 42

    ----  lactate, 42

    ----  propionate, 42


    Fahrion, Dr., 52

    Falling, 67

    ----  apparatus for measuring degree of, 82

    Fat in puer, 25, 30

    ----  in skins, 16

    Feeding of dogs, 28, 95

    Fermentation in the leather industry, 147

    ----  nitrogen from, 279

    Ferments, digestive, 148

    ----  gall, 224

    ----  hydrolysing, 132

    ----  imino-lytic, 133

    ----  oxidizing, 133

    ----  oxylytic, 133

    Flagellæ, staining of, 94

    “Flaked” grain, 149

    Formic Acid, 9

    Fractionating acids, 250

    Frankland and Frew, 249


    Gall ferments, 224

    Gamgee, 281

    Gas analysis, 247

    Gases from drench, 242, 248

    ----  from pure cultures, 275

    ----  intestinal, 281

    Gayon, 280

    Gelatin, 56

    ----  density of, 63

    ----  glucose, 267

    ----  molecular weight of, 56

    ----  peptonized, 168

    Glucose bate, 179

    ----  gelatin, 267

    Glucoses in drench, 261

    Glycerin, 141

    Goat skins, 183

    ---- ---- bating of, 231

    Guano, analysis of, 150

    Golding, J., 175

    “Grains,” analysis of, 7


    Harness backs, 17

    Hauff, J., 236

    Hen dung, analysis of, 150

    Hexoses, 125

    Hide-powder, density of, 66

    Hides, bating of, 17

    Houston, Dr., 93, 94, 97

    Hydrogen from drench, 239

    Hydrogen-ion concentration, 74, 79

    Hydrochloric acid, use in deliming, 11

    Hydrolysis, 128

    ----  velocity of, 130

    Hydrolysing enzymes, 132


    Immendorf, 280

    Imino-lactic ferments, 133

    Indol, 95, 106

    Infusoria, 123

    Intestinal gases, 281

    ---- juice, 225, 228

    Inulase, 132


    Jean, F., 41

    Jones, Arnold Seymour, 75, 133


    Kaolin, 176

    Kathreiner, F., 46, 236

    ----  bate trials by, 183, 184

    Koch’s Jahresbericht, 125

    Körner, Dr. Th., 67, 167

    Kossel, Dr. A., 170

    ----  and Dakin, 133

    Kral, Prof., xv

    Kühl, Dr. H., 236


    Laccase, 133, 135

    ----  manganese in, 135

    Lactase, 132

    Lactic acid, 10

    ---- ---- from _B. furfuris_, 272

    Lamb, M. C., 9

    Law’s volumenometer, 61

    Lecithin, 141

    Lederer’s bate, 196

    Lime in sheep grains, 23, 37

    ----  in puer liquors, 36, 40, 44

    ----  soaps, 16

    Limed skin, 5, 7

    Lion’s dung, 29

    Lipase, 132, 141

    ----  effect of bile salts on, 142

    Lipolytic enzymes, 145

    Liquor pancreaticus, 189


    Malase, 133

    Maltase, 132

    Manganese in enzymes, 136

    Mastering, a term for puer, 3

    Mercaptans, 46

    Measuring truck, 8

    Meggitt, Loxley, 171

    Metchnikoff, 93

    Mineral matter of dung, 153

    Minot, Prof., 81

    Mixed cultures, 173

    Molecular weight of gelatin, 56

    Monads, 123

    Morocco leather, 3

    Moulds, 114, 126

    Müller, 72

    Muscle, contraction of, 73

    Myrosin, 132

    Myxomycetes, 116


    Nencki, 107

    Nitrogen from fermentation, 279

    ----  in bran, 234

    ----  in puer liquors, 48

    Nitrites, use of, in bating, 193

    Non-electrolytes, influence of, 72

    Nördlinger’s bate, 194

    Nowak’s patent, 194

    Nuclein, 114


    Oake’s bate, 195

    Oenoxydase, 133

    Olease, 133

    Organic acids, use in deliming, 13

    ---- ---- in puer, 155

    Oropon, 140, 186, 219

    Osmosis, 58

    Osmotic pressure, 59, 167

    O’Sullivan, J., 244

    ---- and Thompson, 128

    Oxalic acid fermentation, 111

    “Oxidizing” skins, 193

    Oxydin, 133

    Oxylytic ferments, 133


    Paddle, 14

    Pancreas, 220

    Pancreatin, action on skin, 138, 151

    Papain, 132

    Parker, Dr. Gordon, xv, 11

    Pectase, 132

    Pentoses, 125

    Pepsin, 132

    ----  action on skin, 138

    Peptolytic ferments, 119

    Peptonized gelatin, 168

    Phosphates in the bate, 43

    Phosphorbutyralin, 195

    Pigeon dung, 103

    “Pinholey” drench, 238

    Plasmase, 132

    Plate cultures, 103

    Popp and Becker’s patent, 197

    Precipitin, 144

    Pribram, Dr., 73

    Procter, Prof. H. R., 10, 55, 205, 210

    Proteolytic enzymes, 145

    ----  ferments, 119

    _Proteus_ bacteria, 118

    Puer, amido compounds in, 156

    ----  chemical composition of, 24

    ----  fat in, 25, 30

    ----  organic acids in, 155

    ----  quantity used, 15

    ----  reactions of, 31

    ----  wheel, 13

    Puerine, 190

    Puering, solution of skin substance in, 47

    Pure cultures of bran bacteria, 269

    Purgatol, 192

    Purin bases, 30

    Putrefaction, 116

    ----  _See_ Nos. 58 to 78 of Bibliography, 282

    ----  mechanism of, 107

    ----  of meat, 120

    Putrefactive bacteria, 107

    Putrid “turning” of drench, 237


    Reaction of puer, 31

    Rennet, 128, 132

    Rhamnase, 132

    Rogers, Dr. Allen, 195

    Röhm’s patent, 219

    Ruge, 281


    Sabatier and Senderens, 127

    Salt solution, physiological, 87

    Salts solution, 267

    Sand, Dr., xv, 33, 81

    Schorlemmer, K., 186

    Scud, 51

    Scudding, 16

    ----  machine, 17

    Serum, action of, on enzymes, 144

    Shearling pelts, 183

    Shrewsbury, H. S., 166, 281

    Simon’s bate, 195

    Skatol, 46

    Skin substance, ash of, 39

    Skin substance, density of, 60

    ---- ---- fat in, 16

    ---- ---- solution of, 47, 51

    Soerensen, Prof., 43, 80

    Solid matter in bate, 73

    ---- ---- influence of, 175

    Soluble matter of bate, 176

    Soyka’s dilution method, 103

    _Spirillum volutans_, 112

    ---- _delsulfuricans_, 124

    Spongy leather, 238

    Staining and mounting, books on, 89

    ---- of flagellæ, 94

    Starch, action of bran ferments on, 259

    Steapsin, 132

    Stiasny, Prof. E., 282

    Sucanine, 191

    Sucrase, 128, 132

    Sulphuric acid, use in deliming, 11

    Sulphuretted hydrogen, 46

    “Sweating” bacteria, 105, 213

    Sweet bran drench, 235

    Swelling, 67

    ----  alkaline, 72

    Symbiosis, 269

    Synthesis of esters, 127

    Syntonin, 32


    Thio-acids, 196

    Tiffany’s bate, 179

    Toxins, 143

    Trehalase, 132

    Trent bridge laboratory, 107, 252

    Trimethyl-amine, 107, 252

    ----  butyrate, 42

    Trypsin, 132, 133

    Trypsin, in dog dung, 134

    Turney, Sir John, scudding machine, 17

    “Turning” of drench, 287

    Tyrosinase, 133


    Urea, 29

    Urease, 132

    Uric Acid, 29


    Velocity of hydrolysis, 130

    Volumenometer, 61

    ---- air, 67

    ---- Law’s, 61


    Wladika, J., 256

    Willcox, Dr. W. H., 243, 245

    Wood’s American patent, 210

    ---- English patent, 205

    Wool bacteria, 174


    Xylose, 125


    Yeast water, 259

    Yoghurt, 236


    Zinc, effect on growth of moulds, 113

    Zollikoffer’s bate, 178

    Zoogloæ, 242

    Zymase, 133


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