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[Illustration: Fig. 13]

We may now continue our account of the fermentation which we were
studying when we made this last digression. On June 17th that
fermentation produced three times as much gas as it did on June
11th, when the residue of hydrogen, after absorption by potash,
was 72.6 per cent.; whilst on the 17th it was only 49.2 per cent.
Let us again discuss the microscopic aspect of the turbid liquid
at this stage. Appended is the sketch we made (Fig. 14) and our
notes on it: "A most beautiful object: vibrios all in motion,
advancing or undulating. They have grown considerably in bulk and
length since the 11th; many of them are joined together in long
sinuous chains, very mobile at the articulations, visibly less
active and more wavering in proportion to the number that go to
form the chain, of the length of the individuals." This
description is applicable to the majority of the vibrios which
occur in cylindrical rods and are homogeneous in aspect. There
are others, of rare occurrence in chains, which have a clear
corpuscle, that is to say, a portion more refractive than other
parts of the segments, at one of their extremities. Sometimes the
foremost segment has the corpuscle at one end, sometimes the
other. The long segments of the commoner kind attain a length of
from 10 to 30 and even 45 thousandths of a millimetre. Their
diameter is from 1 1/2 to 2, very rarely 3, thousandths of a
millimetre. [Footnote: 1 millimetre = 0.039 inch: hence the
dimensions indicated will be--length, from 0.00039 to 0.00117, or
even 0.00176 in.; diameter, from 0.000058 to 0.000078, rarely
0.000117 in.--D. C. R.]

[Illustration: Figure 14.]

On June 28th, fermentation was quite finished; there was no
longer any trace of gas, nor any lactate in solution. All the
infusoria were lying motionless at the bottom of the flask. The
liquid clarified by degrees, and in the course of a few days
became quite bright. Here we may inquire, were these motionless
infusoria, which from complete exhaustion of the lactate, the
source of the carbonaceous part of their food, were now lying
inert at the bottom of the fermenting vessel--were they dead
beyond the power of revival? [Footnote: The carbonaceous supply,
as we remarked, had failed them, and to this failure the absence
of vital action, nutrition, and multiplication was attributable.
The liquid, however, contained butyrate of lime, a salt
possessing properties similar to those of the lactate. Why could
not this salt equally well support the life of the vibrios? The
explanation of the difficulty seems to us to lie simply in the
fact that lactic acid produces heat by its decomposition, whilst
butyric acid does not, and the vibrios seem to require heat
during the chemical process of their nutrition.] The following
experiment leads us to believe that they were not perfectly
lifeless, and that they might behave in the same manner as the
yeast of beer, which, after it has decomposed all the sugar in a
fermentable liquid, is ready to revive and multiply in a fresh
saccharine medium. On April 22nd, 1875, we left in the oven at a
temperature of 25 degrees C. (77 degrees F.) a fermentation of
lactate of lime that had been completed. The delivery tube of the
flask A, (Fig. 15), in which it had taken place, had never been
withdrawn from under the mercury. We kept the liquid under
observation daily, and saw it gradually become brighter; this
went on for fifteen days. We then filled a similar flask, B, with
the solution of lactate, which we boiled, not only to kill the
germs of vibrios which the liquid might contain, but also to
expel the air that it held in solution. When the flask, B, had
cooled, we connected the two flasks, avoiding the introduction of
air, [Footnote: To do this it is sufficient, first, to fill the
curved ends of the stop-cocked tubes of the flasks, as well as
the india-rubber tube C C which connects them, with boiling water
that contains no air.] after having slightly shaken the flask, A,
to stir up the deposit at the bottom. There was then a pressure
due to carbonic acid at the end of the delivery tube of this
latter flask, at the point A, so that on opening the taps R and
S, the deposit at the bottom of flask A was driven over into
flask B, which in consequence was impregnated with the deposit of
a fermentation that had been completed fifteen days before. Two
days after impregnation the flask B began to show signs of
fermentation. It follows that the deposit of vibrios of a
completed butyric fermentation may be kept, at least for a
certain time, without losing the power of causing fementation. It
furnishes a butyric ferment, capable of revival and action in a
suitable fresh fermentable medium.

[Illustration: Fig. 15]

The reader who has attentively studied the facts which we have
placed before him cannot, in our opinion, entertain the least
doubt on the subject of the possible multiplication of the
vibrios of a fermentation of lactate of lime out of contact with
atmospheric oxygen. If fresh proofs of this important proposition
were necessary, they might be found in the following
observations, from which it may be inferred that atmospheric
oxygen is capable of suddenly checking a fermentation produced by
butyric vibrios, and rendering them absolutely motionless, so
that it cannot be necessary to enable them to live. On May 7th,
1862, we placed in the oven a flask holding 2.580 litres (4 1/2
pints), and filled with the solution of lactate of lime and
phosphates, which we had impregnated on the 9th with two drops of
a liquid in butyric fermentation. In the course of a few days
fermentation declared itself: on the 18th it was active; on the
30th it was very active. On June 1st it yielded hourly 35 cc.
(2.3 cubic inches) of gas, containing ten per cent, of hydrogen.
On the 2nd we began the study of the action of air on the vibrios
of this fermentation. To do this we cut off the delivery-tube on
a level with its point of junction to the flask, then with a 50
cc. pipette we took out that quantity (1 3/4 fl. oz.) of liquid
which was, of course, replaced at once by air. We then reversed
the flask with the opening under the mercury, and shook it every
ten minutes for more than an hour. Wishing to make sure, to begin
with, that the oxygen had been absorbed we connected under the
mercury the beak of the flask by means of a thin india-rubber
tube filled with water, with a small flask, the neck of which had
been drawn out and was filled with water; we then raised the
large flask with the smaller kept above it. A Mohr's clip, which
closed the india-rubber tube, and which we then opened, permitted
the water contained in the small flask to pass into the large
one, whilst the gas, on the contrary, passed upwards from the
large flask into the small one. We analyzed the gas immediately,
and found that, allowing for the carbonic acid and hydrogen, it
did not contain more than 14.2 per cent. of oxygen, which
corresponds to an absorption of 6.6 cc., or of 3.3 cc. (0.2 cubic
inch) of oxygen for the 50 cc. (3.05 cubic inches) of air
employed. Lastly, we again established connection by an india-
rubber tube between the flasks, after having seen by
microscopical examination that the movements of the vibrios were
very languid. Fermentation had become less vigorous without
having actually ceased, no doubt because some portions of the
liquid had not been brought into contact with the atmospheric
oxygen, in spite of the prolonged shaking that the flask had
undergone after the introduction of the air. Whatever the cause
might have been, the significance of the phenomenon is not
doubtful. To assure ourselves further of the effect of air on the
vibrios, we half filled two test tubes with the fermenting liquid
taken from another fermentation which had also attained its
maximum of intensity, into one of which we passed a current of
air, into the other carbonic acid gas. In the course of half an
hour, all the vibrios in the aerated tube were dead, or at least
motionless, and fermentation had ceased. In the other tube, after
three hours' exposure to the effects of the carbonic acid gas,
the vibrios were still very active, and fermentation was going
on.

There is a most simple method of observing the deadly effect of
atmospheric air upon vibrios. We have seen in the microscopical
examination made by means of the apparatus represented in FIG.
13, how remarkable were the movements of the vibrios when
absolutely deprived of air, and how easy it was to discern them.
We will repeat this observation, and at the same time make a
comparative study of the same liquid under the microscope in the
ordinary way, that is to say, by placing a drop of the liquid on
an object-glass, and covering it with a thin glass slip, a method
which must necessarily bring the drop into contact with air, if
only for a moment. It is surprising what a remarkable difference
is observed immediately between the movements of the vibrios in
the bulb and those under the glass. In the case of the latter, we
generally see all movement at once cease near the edges of the
glass, where the drop of liquid is in direct contact with the
air; the movements continue for a longer or shorter time about
the centre, in proportion as the air is more or less intercepted
by the vibrios at the circumference of the liquid. It does not
require much skill in experiments of this kind to enable one to
see plainly that immediately after the glass has been placed on
the drop, which has been affected all over by atmospheric air,
the whole of the vibrios seem to languish and to manifest
symptoms of illness--we can think of no better expression to
explain what we see taking place--and that they gradually recover
their activity about the centre, in proportion as they find
themselves in a part of the medium that is less affected by the
presence of oxygen.

Some of the most curious facts are to be found in connection with
an observation, the correlative and inverse of the foregoing, on
the ordinary aerobian bacteria. If we examine below the
microscope a drop of liquid full of these organisms under a
coverslip, we very soon observe a cessation of motion in all the
bacteria which lie in the central portion of the liquid, where
the oxygen rapidly disappears to supply the necessities of the
bacteria existing there; whilst, on the other hand, near the
edges of the cover-glass the movements are very active, in
consequence of the constant supply of air. In spite of the speedy
death of the bacteria beneath the centre of the glass, we see
life prolonged there if by chance a bubble of air has been
enclosed. All round this bubble a vast number of bacteria collect
in a thick, moving circle, but as soon as all the oxygen of the
bubble has been absorbed they fall apparently lifeless, and are
scattered by the movement of the liquid. [Footnote: We find this
fact, which we published as long ago as 1863, confirmed in a work
of H. Hoffman's, published in 1861 under the title of Memoire sur
les bacteries, which has appeared in French (Annales des Sciences
naturelles, 5th series, vol. ix.). On this subject we may cite an
observation that has not yet been published. Aerobian bacteria
lose all power of movement when suddenly plunged into carbonic
acid gas; they recover it, however, as if they had only been
suffering from anaesthesia, as soon as they are brought into the
air again.]

We may here be permitted to add, as a purely historical matter,
that it was these two observations just described, made
successively one day in 1861, on vibrios and bacteria, that first
suggested to us the idea of the possibility of life without air,
and caused us to think that the vibrios which we met so
frequently in our lactic fermentations must be the true butyric
ferment.

We may pause to consider an interesting question in reference to
the two characters under which vibrios appear in butyric
fermentations. What is the reason that some vibrios exhibit
refractive corpuscles, generally of a lenticular form, such as we
see in FIG. 14. We are strongly inclined to believe that these
corpuscles have to do with a special mode of reproduction in the
vibrios, common alike to the anaerobian forms which we are
studying, and the ordinary aerobian forms in which also the
corpuscles of which we are speaking may occur. The explanation of
the phenomenon, from our point of view, would be that, after a
certain number of fisiparous generations, and under the influence
of variations in the composition of the medium, which is
constantly changing through fermentation as well as through the
active life of the vibrios themselves, cysts, which are simply
the refractive corpuscles, form along them at different points.
From these gemmules we have ultimately produced vibrios, ready to
reproduce others by the process of transverse division for a
certain time, to be themselves encysted, later on. Various
observations incline us to believe that, in their ordinary form
of minute, soft, exuberant rods, the vibrios perish when
submitted to desiccation, but when they occur in corpuscular or
encysted form they possess unusual powers of resistance and may
be brought to the state of dry dust and be wafted about by winds.
None of the matter which surrounds the corpuscle or cyst seems to
take part in the preservation of the germ, when the cyst is
formed, for it is all re-absorbed, gradually leaving the cyst
bare. The cysts appear as masses of corpuscles, in which the most
practiced eye cannot detect anything of an organic nature, or
anything to remind one of the vibrios which produced them;
nevertheless, these minute bodies are endowed with a latent vital
action, and only await favourable conditions to develop long rods
of vibrios. We are not, it is true, in a position to adduce any
very forcible proofs in support of these opinions. They have been
suggested to us by experiments, none of which, however, have been
absolutely decisive in their favour. We may cite one of our
observations on this subject.

In a fermentation of glycerine in a mineral medium--the glycerine
was fermenting under the influence of butyric vibrios--after we
had determined the, we may say, exclusive presence of lenticular
vibrios, with refractive corpuscles, we observed the
fermentation, which for some unknown reason had been very
languid, suddenly become extremely active, but now through the
influence of the ordinary vibrios. The gemmules with brilliant
corpuscles had almost disappeared; we could see but very few, and
those now consisted of the refractive bodies alone, the bulk of
the vibrios accompanying them having undergone some process of
re-absorption.

Another observation which still more closely accords with this
hypothesis is given in our work on silk-worm disease (vol. 1, p.
256). We there demonstrated that, when we place in water some of
the dust formed of desiccated vibrios, containing a host of these
refractive corpuscles, in the course of a very few hours large
vibrios appear, well-developed rods fully grown, in which the
brilliant points are absent; whilst in the water no process of
development from smaller vibrios is to be discerned, a fact which
seems to show that the former had issued fully grown from the
refractive corpuscles, just as we see colpoda issue with their
adult aspect from the dust of their cysts. This observation, we
may remark, furnishes one of the best proofs that can be adduced
against the spontaneous generation of vibrios or bacteria, since
it is probable that the same observation applies to bacteria. It
is true that we cannot say of mere points of dust examined under
the microscope, that one particular germ belongs to vibrio,
another to bacterium; but how is it possible to doubt that the
vibrios issue, as we see them, from an ovum of some kind, a cyst,
or germ, of determinate character, when, after having placed some
of those indeterminate motes of dust into clean water, we
suddenly see, after an interval of not more than one or two
hours, an adult vibrio crossing the field of the microscope,
without our having been able to detect any intermediate state
between its birth and adolescence?

[Illustration: Fig. 16]

It is a question whether differences in the aspect and nature of
vibrios, which depend upon their more or less advanced age, or
are occasioned by the influence of certain conditions on the
medium in which they propagate, do not bring about corresponding
changes in the course of the fermentation and the nature of its
products. Judging at least from the variations in the proportions
of hydrogen, and carbonic acid gas produced in butyric
fermentations, we are inclined to think that this must be the
case; nay, more, we find that hydrogen is not even a constant
product in these fermentations. We have met with butyric
fermentations of lactate of lime which did not yield the minutest
trace of hydrogen, or anything besides carbonic acid. Fig. 16
represents the vibrios which we observed in a fermentation of
this kind. They present no special features. Butyl alcohol is,
according to our observations, an ordinary product, although it
varies and is by no means a necessary concomitant of these
fermentations. It might be supposed, since butylic alcohol may be
produced and hydrogen be in deficit, that the proportion of the
former of these products would attain its maximum when the latter
assumed a minimum. This, however, is by no means the case; even
in those few fermentations that we have met with in which
hydrogen was absent, there was no formation of butylic alcohol.

From a consideration of all the facts detailed in this section we
can have no hesitation in concluding that, on the one hand, in
cases of butyric fermentation, the vibrios which abound in them
and constitute their ferment, live without air or free oxygen;
and that, on the other hand, the presence of gaseous oxygen
operates prejudicially against the movements and activity of
those vibrios. But how does it follow that the presence of minute
quantities of air brought into contact with a liquid undergoing
butyric fermention would prevent the continuance of that
fermentation or even exercise any check upon it? We have not made
any direct experiments upon this subject; but we should not be
surprised to find that, so far from hindering, air may, under
such circumstances, facilitate the propagation of the vibrios and
accelerate fermentation. This is exactly what happens in the case
of yeast. But how could we reconcile this, supposing it were
proved to be the case, with the fact just insisted on as to the
danger of bringing the butyric vibrios into contact with air? It
may be possible that LIFE WITHOUT AIR results from habit, whilst
DEATH THROUGH AIR may be brought about by a sudden change in the
conditions of the existence of the vibrios. The following
remarkable experiment is well-known: A bird is placed in a glass
jar of one or two litres (60 to 120 cubic inches) in capacity
which is then closed. After a time the creature shows every sign
of intense uneasiness and asphyxia long before it dies; a similar
bird of the same size is introduced into the jar; the death of
the latter takes place instanteously, whilst the life of the
former may still be prolonged under these conditions for a
considerable time, and there is no, difficulty even in restoring
the bird to perfect health by taking it out of the jar. It seems
impossible to deny that we have here a case of the adaptation of
an organism to the gradual contamination of the medium; and so it
may likewise happen that the anaerobian vibrios of a butyric
fermentation, which develop and multiply absolutely without free
oxygen, perish immediately when suddenly taken out of their
airless medium, and that the result might be different if they
had been gradually brought under the action of air in small
quantities at a time.

We are compelled here to admit that vibrios frequently abound in
liquids exposed to the air, and that they appropriate the
atmospheric oxygen, and could not withstand a sudden removal from
its influence. Must we, then, believe that such vibrios are
absolutely different from those of butyric fermentations? It
would, perhaps, be more natural to admit that in the one case
there is an adaptation to life with air, and in the other case an
adaptation to life without air; each of the varieties perishing
when suddenly transferred from its habitual condition to that of
the other, whilst by a series of progressive changes one might be
modified into the other. [Footnote: These doubts might be easily
removed by putting the matter to the test of direct experiment.]
We know that in the case of alcoholic ferments, although these
can actually live without air, propagation is wonderfully
assisted by the presence of minute quantities of air; and certain
experiments which we have not yet published lead us to believe
that, after having lived without air, they cannot be suddenly
exposed with impunity to the influence of large quantities of
oxygen.

We must not forget, however, that aerobian torulae and anaerobian
ferments present an example of organisms apparently identical, in
which, however, we have not yet been able to discover any ties of
a common origin. Hence we are forced to regard them as a distinct
species; and so it is possible that there may likewise be
aerobian and anaerobian vibrios without any transformation of the
one into the other.

The question has been raised whether vibrios, especially those
which we have shown to be the ferment of butyric and many other
fermentations, are in their nature, animal or vegetable. M. Ch.
Robin attaches great importance to the solution of this question,
of which he speaks as follows: [Footnote: ROBIN, Sur la nature
des fermentations, &c. (Journal de l'Academie et de la
Physiologie, July and August, 1875, P. 386).] "The determination
of the nature, whether animal or vegetable, of organisms, either
as a whole or in respect to their anatomical parts, assimilative
or reproductive, is a problem which has been capable of solution
for a quarter of a century. The method has been brought to a
state of remarkable precision, experimentally, as well as in its
theoretical aspects, since those who devote their attention to
the organic sciences consider it indispensable in every
observation and experiment to determine accurately, before
anything else, whether the object of their study is animal or
vegetable in its nature, whether adult or otherwise. To neglect
this is as serious an omission for such students as for chemists
would be the neglecting to determine whether it is nitrogen or
hydrogen, urea or stearine, that has been extracted from a
tissue, or which it is whose combinations they are studying in
this or that chemical operation. Now, scarcely any one of those
who study fermentations, properly so-called, and putrefactions,
ever pay any attention to the preceding data. ... Among the
observers to whom I allude, even M. Pasteur is to be found, who,
even in his most recent communications, omits to state definitely
what is the nature of many of the ferments which he has studied,
with the exception, however, of those which belong to the
cryptogamic group called torulaceae. Various passages in his work
seem to show that he considers the cryptogamic organisms called
bacteria, as well as those known as vibrios, as belonging to the
animal kingdom (see Bulletin de l'Academie de Medecine, Paris,
1875, pp. 249, 251, especially 256, 266, 267, 289, and 290).
These would be very different, at least physiologically, the
former being anaerobian, that is to say, requiring no air to
enable them to live, and being killed by oxygen, should it be
dissolved in the liquid to any considerable extent."

We are unable to see the matter in the same light as our learned
colleague does; to our thinking, we should be labouring under a
great delusion were we to suppose "that it is quite as serious an
omission not to determine the animal or vegetable nature of a
ferment as it would be to confound nitrogen with hydrogen or urea
with stearine." The importance of the solutions of disputed
questions often depends on the point of view from which these are
regarded. As far as the result of our labours is concerned, we
devoted our attention to these two questions exclusively: 1. Is
the ferment, in every fermentation properly so called, an
organized being? 2. Can this organized being live without air?
Now, what bearing can the question of the animal or vegetable
nature of the ferment, of the organized being, have upon the
investigation of these two problems? In studying butyric
fermentation, for example, we endeavoured to establish these two
fundamental points; 1. The BUTYRIC FERMENT IS A VIBRIO. 2. THIS
VIBRIO MAY DISPENSE WITH AIR IN ITS LIFE, AND, AS A MATTER OF
FACT, DOES DISPENSE WITH IT IN THE ACT OF PRODUCING BUTYRIC
FERMENTATION. We did not consider it at all necessary to
pronounce any opinion as to the animal or vegetable nature of
this organism, and, even up to the present moment, the idea that
vibrio is an animal and not a plant is in our minds, a matter of
sentiment rather than of conviction.