But what is the nature of these vibrios? We have already said

that we believe that they are nothing but the ordinary vibrios of

putrefaction, reduced to a state of extreme tenuity by the

special conditions of nutrition involved in the fermentable

medium used; in a word, we think that the fermentation in

question might be called putrefaction of tartrate of lime. It

would be easy enough to determine this point by growing the

vibrios of such fermentation in media adapted to the production

of the ordinary forms of vibrio; but this is an experiment which

we have not ourselves tried.

One word more on the subject of these curious beings. In a great

many of them there appears to be something like a clear spot, a

kind of bead, at one of their extremities. This is an illusion

arising from the fact that the extremity of these vibrios is

curved, hanging downwards, thus causing a greater refraction at

that particular point, and leading us to think that the diameter

is greater at that extremity. We may easily undeceive ourselves

if we watch the movements of the vibrio, when we will readily

recognize the bend, especially as it is brought into the vertical

plane passing over the rest of the filament. In this way we will

see the bright spot, THE HEAD, disappear, and then reappear.

The chief inference that it concerns us to draw from the

preceding facts is one which cannot admit of doubt, and which we

need not insist on any further--namely that vibrios, as met with

in the fermentation of neutral tartrate of lime, are able to live

and multiply when entirely deprived of air.

V.--ANOTHER EXAMPLE OF LIFE WITHOUT AIR--FERMENTATION OF LACTATE

OF LIME

As another example of life without air, accompanied by

fermentation properly so called, we may lastly cite the

fermentation of lactate of lime in a mineral medium.

In the experiment described in the last paragraph, it will be

remembered that the ferment liquid and the germs employed in its

impregnation came in contact with air, although only for a very

brief time. Now, notwithstanding that we possess exact

observations which prove that the diffusion of oxygen and

nitrogen in a liquid absolutely deprived of air, so far from

taking place rapidly, is, on the contrary, a very slow process

indeed; yet we were anxious to guard the experiment that we are

about to describe from the slightest possible trace of oxygen at

the moment of impregnation.

We employed a liquid prepared as follows: Into from 9 to 10

litres (somewhat over 2 gallons) of pure water the following

salts [Footnote: Should the solution of lactate of lime be

turbid, it may be clarified by filtration, after previously

adding a small quantity of phosphate of ammonia, which throws

down phosphate of lime. It is only after this process of

clarification and filtration that the phosphates of the formula

are added. The solution soon becomes turbid if left in contact

with air, in consequence of the spontaneous formation of

bacteria.] were introduced successively, viz:

Pure lactate of lime. ... . ... . ... . ... . .. 225 grammes

Phosphate of ammonia. ... . ... . ... . ... . .. 0.75 grammes

Phosphate of potassium. ... . ... . ... . ... .. 0.4 grammes

Sulphate of magnesium. ... . ... . ... . ... ... 0.4 grammes

Sulphate of ammonia. ... . ... . ... . ... . ... 0.2 grammes

(1 gramme = 15.43 grains.)

[Illustration with caption: Fig. 11]

On March 23rd, 1875, we filled a 6 litre (about 11 pints) flask,

of the shape represented in FIG. 11, and placed it over a heater.

Another flame was placed below a vessel containing the same

liquid, into which the curved tube of the flask plunged. The

liquids in the flask and in the basin were raised to boiling

together, and kept in this condition for more than half-an-hour,

so as to expel all the air held in solution. The liquid was

several times forced out of the flask by the steam, and sucked

back again; but the portion which re-entered the flask was always

boiling. On the following day when the flask had cooled, we

transferred the end of the delivery tube to a vessel full of

mercury and placed the whole apparatus in an oven at a

temperature varying between 25 degrees C. and 30 degrees C. (77

degrees F. and 86 degrees F.) then, after having refilled the

small cylindrical tap-funnel with carbonic acid, we passed into

it with all necessary precautions 10 cc. (0.35 fl. oz) of a

liquid similar to that described, which had been already in

active fermentation for several days out of contact with air and

now swarmed with vibrios. We then turned the tap of the funnel,

until only a small quantity of liquid was left, just enough to

prevent the access of air. In this way the impregnation was

accomplished without either the ferment-liquid or the ferment-

germs having been brought in contact, even for the shortest

space, with the external air. The fermentation, the occurrence of

which at an earlier or later period depends for the most part on

the condition of the impregnating germs, and the number

introduced in the act, in this case began to manifest itself by

the appearance of minute bubbles from March 29th. But not until

April 9th did we observe bubbles of larger size rise to the

surface. From that date onward they continued to come in

increasing number, from certain points at the bottom of the

flask, where a deposit of earthy phosphates existed; and at the

same time the liquid, which for the first few days remained

perfectly clear, began to grow turbid in consequence of the

development of vibrios. It was on the same day that we first

observed a deposit on the sides of carbonate of lime in crystals.

It is a matter of some interest to notice here that, in the mode

of procedure adopted, everything combined to prevent the

interference of air. A portion of the liquid expelled at the

beginning of the experiment, partly because of the increased

temperature in the oven and partly also by the force of the gas,

as it began to be evolved from the fermentative action, reached

the surface of the mercury, where, being the most suitable medium

we know for the growth of bacteria, it speedily swarmed with

these organisms. [Footnote: The naturalist Cohn, of Breslau, who

published an excellent work on bacteria in 1872, described, after

Mayer, the composition of a liquid peculiarly adapted to the

propagation of these organisms, which it would be well to compare

for its utility in studies of this kind with our solution of

lactate and phosphates. The following is Cohn's formula:

Distilled water. ... . ... . ... . ..20 cc. (0.7 fl. oz.)

Phosphate of potassium. ... . ... ...0.1 gramme (1.5 grains)

Sulphate of magnesium. ... . ... . 0.1 gramme (1.5 grains)

Tribasic phosphate of lime. ... ... 0.01 gramme (0.15

grain)

Tartrate of ammonia. ... . ... . ... 0.2 gramme (3 grains)

This liquid, the author says, has a feeble acid reaction and

forms a perfectly clear solution.] In this way any passage of

air, if such a thing were possible, between the mercury and the

sides of the delivery-tube was altogether prevented, since the

bacteria would consume every trace of oxygen which might be

dissolved in the liquid lying on the surface of the mercury.

Hence it is impossible to imagine that the slightest trace of

oxygen could have got into the liquid in the flask.

Before passing on we may remark that in this ready absorption of

oxygen by bacteria we have a means of depriving fermentable

liquids of every trace of that gas with a facility and success

equal or even greater than by the preliminary method of boiling.

Such a solution as we have described, if kept at summer heat,

without any previous boiling, becomes turbid in the course of

twenty-four hours from a SPONTANEOUS development of bacteria; and

it is easy to prove that they absorb all the oxygen held in

solution. [Footnote: On the rapid absorption of oxygen by

bacteria, see also our Memoire of 1872, sur les Generations dites

Spontanees, especially the note on page 78.] If we completely

fill a flask of a few litres capacity (about a gallon) (Fig. 9)

with the liquid described, taking care to have the delivery-tube

also filled, and its opening plunged under mercury, and, forty-

eight hours afterwards by means of a chloride of calcium bath,

expel from the liquid on the surface of the mercury all the gas

which it holds in solution, this gas, when analyzed, will be

found to be composed of a mixture of nitrogen and carbonic acid

gas, WITHOUT THE LEAST TRACE OF OXYGEN. Here, then, we have an

excellent means of depriving the fermentable liquid of air; we

simply have completely to fill a flask with the liquid, and place

it in the oven, merely avoiding any addition of butyric vibrios,

before the lapse of two or three days. We may wait even longer;

and then, if the liquid does become impregnated spontaneously

with vibrio germs, the liquid, which at first was turbid from the

presence of bacteria, will become bright again, since the

bacteria, when deprived of life, or, at least, of the power of

moving, after they have exhausted all the oxygen in solution,

will fall inert to the bottom of the vessel. On several occasions

we have determined this interesting fact, which tends to prove

that the butyric vibrios cannot be regarded as another form of

bacteria, inasmuch as, on the hypothesis of an original relation

between the two productions, butyric fermentation ought in every

case to follow the growth of bacteria.

We may also call attention to another striking experiment, well

suited to show the effect of differences in the composition of

the medium upon the propagation of microscopic beings. The

fermentation which we last described commenced on March 27th and

continued until May 10th; that to which we are now to refer,

however, was completed in four days, the liquid employed being

similar in composition and quantity to that employed in the

former experiment. On April 23, 1875, we filled a flask of the

same shape as that represented in Fig. 11, and of similar

capacity, viz., 6 litres, with a liquid composed as described at

page 69. This liquid had been previously left to itself for five

days in large open flasks, in consequence of which it had

developed an abundant growth of bacteria. On the fifth day a few

bubbles, rising from the bottom of the vessels, at long

intervals, betokened the commencement of butyric fermentation, a

fact, moreover, confirmed by the microscope, in the appearance of

the vibrios of this fermentation in specimens of the liquid taken

from the bottom of the vessels, the middle of its mass, and even

in the layer on the surface that was swarming with bacteria. We

transferred the liquid so prepared to the 6 litre flask arranged

over the mercury. By evening a tolerably active fermentation had

begun to manifest itself. On the 24th this fermentation was

proceeding with astonishing rapidity, which continued during the

25th and 26th. During the evening of the 26th it slackened, and

on the 27th all signs of fermentation had ceased. This was not,

as might be supposed, a sudden stoppage due to some unknown

cause; the fermentation was actually completed, for when we

examined the fermented liquid on the 28th we could not find the

smallest quantity of lactate of lime. If the needs of industry

should ever require the production of large quantities of butyric

acid, there would, beyond doubt, be found in the preceding fact

valuable information in devising an easy method of preparing that

product in abundance. [Footnote: In what way are we to account

for so great a difference between the two fermentations that we

have just described? Probably it was owing to some modification

effected in the medium by the previous life of the bacteria, or

to the special character of the vibrios used in impregnation. Or,

again, it might have been due to the action of the air, which,

under the conditions of our second experiment, was not absolutely

eliminated, since we took no precaution against its introduction

at the moment of filling our flask, and this would tend to

facilitate the multiplication of anaerobian vibrios, just as,

under similar conditions, would have been the case if we had been

dealing with a fermentation by ordinary yeast.]

Before we go any further, let us devote some attention to the

vibrios of the preceding fermentations.

On May 27th, 1862, we completely filled a flask capable of

holding 2.780 litres (about five pints) with the solution of

lactate and phosphates. [Footnote: In this case the liquid was

composed as follows: A saturated solution of lactate of lime, at

a temperature of 25 degrees C. (77 degrees F.), was prepared,

containing for every 1OO cc. (3 1/2 fl. oz.) 25.65 grammes (394

grains) of the lactate, C6 H5 O5 Ca O (NEW NOTATION, C6 H10 Ca

O6) This solution was rendered very clear by the addition of 1

gramme of phosphate of ammonia and subsequent filtration. For a

volume of 8 litres (14 pints) of this clear saturated solution we

used (1 gramme = 15.43 grains):

Phosphate of ammonia. ... . ... . ... . ... 2 grammes

Phosphate of potassium. ... . ... . ... . ... 1 gramme

Phosphate of magnesium. ... . ... . ... . ... 1 gramme

Sulphate of ammonia. ... . ... . ... . ... 0.5 gramme]

We refrained from impregnating it with any germs. The liquid

became turbid from a development of bacteria and then underwent

butyric fermentation. By June 9th the fermentation had become

sufficiently active to enable us to collect in the course of

twenty-four hours, over mercury, as in all our experiments, about

100 cc. (about 6 cubic inches) of gas. By June 11th, judging from

the volume of gas liberated in the course of twenty-four hours,

the activity of the fermentation had doubled. We examined a drop

of the turbid liquid. Here are the notes accompanying the sketch

(Fig. 12) as they stand in our note-book: "A swarm of vibrios, so

active in their movements that the eye has great difficulty in

following them. They may be seen in pairs throughout the field,

apparently making efforts to separate from each other. The

connection would seem to be by some invisible, gelatinous thread,

which yields so far to their efforts that they succeed in

breaking away from actual contact, but yet are, for a while, so

far restrained that the movements of one have a visible effect on

those of the other. By and by, however, we see a complete

separation effected, and each moves on its separate way with an

activity greater than it ever had before."

[Illustration with caption: Fig. 12]

One of the best methods that can be employed for the

microscopical examination of these vibrios, quite out of contact

with air, is the following. After butyric fermentation has been

going on for several days in a flask, (Fig. 13), we connect this

flask by an india-rubber tube with one of the flattened bulbs

previously described, which we then place on the stage of the

microscope (Fig. 13). When we wish to make an observation we

close, under the mercury, at the point B, the end of the drawn-

out and bent delivery-tube. The continued evolution of gas soon

exerts such a pressure within the flask, that when we open the

tap R, the liquid is driven into the bulb LL, until it becomes

quite full and the liquid flows over into the glass V. In this

manner we may bring the vibrios under observation without their

coming into contact with the least trace of air, and with as much

success as if the bulb, which takes the place of an object glass,

had been plunged into the very centre of the flask. The movements

and fissiparous multiplication of the vibrios may thus be seen in

all their beauty, and it is indeed a most interesting sight. The

movements do not immediately cease when the temperature is

suddenly lowered, even to a considerable extent, 15 degrees C.

(59 degrees F.) for example; they are only slackened.

Nevertheless, it is better to observe them at the temperatures

most favourable to fermentation, even in the oven where the

vessels employed in the experiment are kept at a temperature

between 25 degrees C. and 30 degrees C. (77 degrees F. and 86

degrees F.).

[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.

M. Robin, however, would have no difficulty in determining the

limits of the two kingdoms. According to him, "every variety of

cellulose is, we may say, insoluble in ammonia, as also are the

reproductive elements of plants, whether male or female. Whatever

phase of evolution the elements which reproduce a new individual

may have reached, treatment with this reagent, either cold or

raised to boiling, leaves them absolutely intact under the eyes

of the observer, except that their contents, from being partially

dissolved, become more transparent. Every vegetable whether

microscopic or not, every mycelium and every spore, thus

preserves in its entirety its special characteristics of form,

volume and structural arrangements; whilst in the case of

microscopic animals, or the ova and microscopic embryos of

different members of the animal kingdom, the very opposite is the

case."

We should be glad to learn that the employment of a drop of

ammonia would enable us to pronounce an opinion with this degree

of confidence on the nature of the lowest microscopic beings; but

is M. Robin absolutely correct in his assumptions? That gentleman

himself remarks that spermatozoa, which belong to animal

organisms, are insoluble in ammonia, the effect of which is

merely to make them paler. If a difference of action in certain

reagents, in ammonia, for example, were sufficient to determine

the limits of the animal and vegetable kingdoms, might we not

argue that there must be a very great and natural difference

between moulds and bacteria, inasmuch as the presence of a small

quantity of acid in the nutritive medium facilitates the growth

and propagation of the former, whilst it is able to prevent the

life of bacteria and vibrios? Although as is well known, movement

is not an exclusive characteristic of animals, yet we have always

been inclined to regard vibrios as animals, on account of the

peculiar character of their movements. How greatly they differ in

this respect from the diatomacae, for example! When the vibrio

encounters an obstacle it turns, or after assuring itself by some

visual effort or other that it cannot overcome it, it retraces

its steps. The colpoda--undoubted infusoria--behave in an exactly

similar manner. It is true one may argue that the zoospores of

certain cryptogamia exhibit similar movements; but do not these

zoospores possess as much of an animal nature as do the

spermatozoa? As far as bacteria are concerned, when, as already

remarked, we see them crowd round a bubble of air in a liquid to

prolong their life, oxygen having failed them everywhere else,

how can we avoid believing that they are animated by an instinct

for life, of the same kind that we find in animals? M. Robin

seems to us to be wrong in supposing that it is possible to draw

any absolute line of separation between the animal and vegetable

kingdoms. The settlement of this line however, we repeat again,

no matter what it may be, has no serious bearing upon the

questions that have been the subject of our researches.

In like manner the difficulty which M. Robin has raised in

objecting to the employment of the word GERM, when we cannot

specify whether the nature of that germ is animal or vegetable,

is in many respects an unnecessary one. In all the questions

which we have discussed, whether we were speaking of fermentation

or spontaneous generation, the word GERM has been used in the

sense of ORIGIN OF LIVING ORGANISM. If Liebig, for example, said

of an albuminous substance that it gave birth to ferment, could

we contradict him more plainly than by replying "No; ferment is

an organized being, the germ of which is always present, and the

albuminous substance merely serves by its occurrence to nourish

the germ and its successive generations"?

In our Memoir of 1862, on so-called SPONTANEOUS generations,

would it not have been an entire mistake to have attempted to

assign specific names to the microscopic organisms which we met

with in the course of our observations? Not only would we have

met with extreme difficulty in the attempt, arising from the

state of extreme confusion which even in the present day exists

in the classification and nomenclature of these microscopic

organisms, but we should have been forced to sacrifice clearness

in our work besides; at all events, we should have wandered from

our principal object, which was the determination of the presence

or absence of life in general, and had nothing to do with the

manifestation of a particular kind of life in this or that

species, animal or vegetable. Thus we have systematically

employed the vaguest nomenclature, such as mucors, torulae,

bacteria, and vibrios. There was nothing arbitrary in our doing

this, whereas there is much that is arbitrary in adopting a

definite system of nomenclature, and applying it to organisms but

imperfectly known, the differences or resemblances between which

are only recognizable through certain characteristics, the true

signification of which is obscure. Take, for example, the

extensive array of widely different systems which have been

invented during the last few years for the species of the genera

bacterium and vibrio in the works of Cohn, H. Hoffmann, Hallier,

and Billroth. The confusion which prevails here is very great,

although we do not of course by any means place these different

works on the same footing as regards their respective merits.

M. Robin is, however, right in recognizing the impossibility of

maintaining in the present day, as he formerly did, "That

fermentation is an exterior phenomenon, going on outside

cryptogamic cells, a phenomenon of contact. It is probably," he

adds, "an interior and molecular action at work in the innermost

recesses of the substance of each cell." From the day when we

first proved that it is possible for all organized ferments,

properly so called, to spring up and multiply from their

respective germs, sown, whether consciously or by accident, in a

mineral medium free from organic and nitrogenous matters other

than ammonia, in which medium the fermentable matter alone is

adapted to provide the ferment with whatever carbon enters into

its composition, from that time forward the theories of Liebig,

as well of Berzelius, which M. Robin formerly defended, have had

to give place to others more in harmony with facts. We trust that

the day will come when M. Robin will likewise acknowledge that he

has been in error on the subject of the doctrine of spontaneous

generation, which he continues to affirm, without adducing any

direct proofs in support of it, at the end of the article to

which we have been here replying.

We have devoted the greater part of this chapter to the

establishing with all possible exactness the extremely important

physiological fact of life without air, and its correlation to

the phenomena of fermentations properly so called--that is to

say, of those which are due to the presence of microscopic

cellular organisms. This is the chief basis of the new theory

that we propose for the explanation of these phenomena. The

details into which we have entered were indispensable on account

of the novelty of the subject no less than on account of the

necessity we were under of combating the criticisms of the two

German naturalists, Drs. Oscar Brefeld and Traube, whose works

had cast some doubts on the correctness of the facts upon which

we had based the preceding propositions. We have much pleasure in

adding that at the very moment we were revising the proofs of

this chapter, we received from M. Brefeld an essay, dated Berlin,

January, 1876, in which, after describing his later experimental

researches, he owns with praiseworthy frankness that Dr. Traube

and he were both of them mistaken. Life without air is now a

proposition which he accepts as perfectly demonstrated. He has

witnessed it in the case of Mucor racemosus and has also verified

it in the case of yeast. "If," he says, "after the results of my

previous researches, which I conducted with all possible

exactness, I was inclined to consider Pasteur's assertion as

inaccurate and to attack them, I have no hesitation now in

recognizing them as true, and in proclaiming the service which

Pasteur has rendered to science in being the first to indicate

the exact relation of things in the phenomenon of fermentation."

In his later researches, Dr. Brefeld has adopted the method which

we have long employed for demonstrating the life and

multiplication of butyric vibrios in the entire absence of air,

as well as the method of conducting growths in mineral media

associated with fermentable substance. We need not pause to

consider certain other secondary criticisms of Dr. Brefeld. A

perusal of the present work will, we trust, convince him that

they are based on no surer foundation than were his former

criticisms.

To bring one's self to believe in a truth that has just dawned

upon one is the first step towards progress; to persuade others

is the second. There is a third step, less useful perhaps, but

highly gratifying nevertheless, which is, to convince one's

opponents.

We therefore, have experienced great satisfaction in learning

that we have won over to our ideas an observer of singular

ability, on a subject which is of the utmost importance to the

physiology of cells.

VI. REPLY TO THE CRITICAL OBSERVATIONS OF LIEBIG, PUBLISHED IN

1870.

[Footnote: LIEBIG, Sur la fermentation et la source de la force

musculaire (Annales de Chimie et de Physique, 4th series, t.

xxiii., p. 5, 1870).]

In the Memoir which we published, in 1860, on alcoholic

fermentation, and in several subsequent works, we were led to a

different conclusion on the causes of this very remarkable

phenomenon from that which Liebig had adopted. The opinions of

Mitscherlich and Berzelius had ceased to be tenable in the

presence of the new facts which we had brought to light. From

that time we felt sure that the celebrated chemist of Munich had

adopted our conclusions, from the fact that he remained silent on

this question for a long time, although it had been until then

the constant subject of his study, as is shown by all his works.

Suddenly there appeared in the Annales de Chimie et de Physique a

long essay, reproduced from a lecture delivered by him before the

Academy of Bavaria in 1868 and 1869. In this Liebig again

maintained, not, however, without certain modifications, the

views which he had expressed in his former publications, and

disputed the correctness of the principal facts enunciated in our

Memoir of 1860, on which were based the arguments against his

theory.

"I had admitted," he says, "that the resolution of fermentable

matter into compounds of a simpler kind must be traced to some

process of decomposition taking place in the ferment, and that

the action of this same ferment on the fermentable matter must

continue or cease according to the prolongation or cessation of

the alteration produced in the ferment. The molecular change in

the sugar, would, consequently, be brought about by the

destruction or modification of one or more of the component parts

of the ferment, and could only take place through the contact of

the two substances. M. Pasteur regards fermentation in the

following light: The chemical action of fermentation is

essentially a phenomenon correlative with a vital action,

beginning and ending with it. He believes that alcoholic

fermentation can never occur without the simultaneous occurrence

of organization, development, and multiplication of globules, or

continuous life, carried on from globules already formed. But the

idea that the decomposition of sugar during fermentation is due

to the development of the cellules of the ferment, is in

contradiction with the fact that the ferment is able to bring

about the fermentation of a pure solution of sugar. The greater

part of the ferment is composed of a substance that is rich in

nitrogen and contains sulphur. It contains, moreover, an

appreciable quantity of phosphates, hence it is difficult to

conceive how, in the absence of these elements in a pure solution

of sugar undergoing fermentation, the number of cells is capable

of any increase."

Notwithstanding Liebig's belief to the contrary, the idea that

the decomposition of sugar during fermentation is intimately

connected with a development of the cellules of the ferment, or a

prolongation of the life of cellules already formed, is in no way

opposed to the fact that the ferment is capable of bringing about

the fermentation of a pure solution of sugar. It is manifest to

any one who has studied such fermentation with the microscope,

even in those cases where the sweetened water has been absolutely

pure, that ferment-cells do multiply, the reason being that the

cells carry with them all the food-supplies necessary for the

life of the ferment. They may be observed budding, at least many

of them, and there can be no doubt that those which do not bud

still continue to live; life has other ways of manifesting itself

besides development and cell-proliferation.

If we refer to the figures on page 81 of our Memoir of 1860,

Experiments D, E, F, H, I, we shall see that the weight of yeast,

in the case of the fermentation of a pure solution of sugar,

undergoes a considerable increase, even without taking into

account the fact that the sugared water gains from the yeast

certain soluble parts, since in the experiments just mentioned,

the weights of solid yeast, washed and dried at 100 degrees C.

(212 degrees F.), are much greater than those of the raw yeast

employed, dried at the same temperature.

In these experiments we employed the following weights of yeast,

expressed in grammes (1 gramme=15.43 grains):

(1) 2.313

(2) 2.626

(3) 1.198

(4) 0.699

(5) 0.326

(6) 0.476

which became, after fermentation, we repeat, without taking into

account the matters which the sugared water gained from the

yeast:

grammes. grains.

(1) 2.486 Increase 0.173 = 2.65

(2) 2.963 Increase 0.337 = 5.16

(3) 1.700 Increase 0.502 = 7.7

(4) 0.712 Increase 0.013 = 0.2

(5) 0.325 Increase 0.009 = 0.14

(6) 0.590 Increase 0.114 = 1.75

Have we not in this marked increase in weight a proof of life,

or, to adopt an expression which may be preferred, a proof of a

profound chemical work of nutrition and assimilation?

We may cite on this subject one of our earlier experiments, which

is to be found in the Comptes rendus de l'Academie for the year

1857, and which clearly shows the great influence exerted on

fermentation by the soluble portion that the sugared water takes

up from the globules of ferment:

"We take two equal quantities of fresh yeast that have been

washed very freely. One of these we cause to ferment in water

containing nothing but sugar, and, after removing from the other

all its soluble particles--by boiling it in an excess of water

and then filtering it to separate the globules--we add to the

filtered liquid as much sugar as was used in the first case along

with a mere trace of fresh yeast insufficient, as far as its

weight is concerned, to affect the results of our experiment. The

globules which we have sown bud, the liquid becomes turbid, a

deposit of yeast gradually forms, and, side by side with these

appearances, the decomposition of the sugar is effected, and in

the course of a few hours manifests itself clearly. These results

are such as we might have anticipated. The following fact,

however, is of importance. In effecting by these means the

organization into globules of the soluble part of the yeast that

we used in the second case, we find that a considerable quantity

of sugar is decomposed. The following are the results of our

experiment; 5 grammes of yeast caused the fermentation of 12.9

grammes of sugar in six days, at the end of which time it was

exhausted. The soluble portion of a like quantity of 5 grammes of

the same yeast caused the fermentation of 10 grammes of sugar in

nine days, after which the yeast developed by the sowing was

likewise exhausted."

How is it possible to maintain that, in the fermentation of water

containing nothing but sugar, the soluble portion of the yeast

does not act, either in the production of new globules or the

perfection of old ones, when we see, in the preceding experiment,

that after this nitrogenous and mineral portion has been removed

by boiling, it immediately serves for the production of new

globules, which, under the influence of the sowing of a mere

trace of globules, causes the fermentation of so much sugar?

[Footnote: It is important that we should here remark that, in

the fermentation of pure solution of sugar by means of yeast, the

oxygen originally dissolved in the water, as well as that

appropriated by the globules of yeast in their contact with air,

has a considerable effect on the activity of the fermentation. As

a matter of fact, if we pass a strong current of carbonic acid

through the sugared water and the water in which the yeast has

been treated, the fermentation will be rendered extremely

sluggish, and the few new cells of yeast which form will assume

strange and abnormal aspects. Indeed this might have been

expected, for we have seen that yeast, when somewhat old, is

incapable of development or of causing fermentation even in a

fermentable medium containing all the nutritive principles of

yeast if the liquid has been deprived of air; much more should we

expect this to be the case in pure sugared water, likewise

deprived of air.]

In short, Liebig is not justified in saying that the solution of

pure sugar, caused to ferment by means of yeast, contains none of

the elements needed for the growth of yeast, neither nitrogen,

sulphur nor phosphorus, and that, consequently, it should not be

possible, by our theory, for the sugar to ferment. On the

contrary, the solution does contain all these elements, as a

consequence of the introduction and presence of the yeast.

Let us proceed without examination of Liebig's criticisms:

"To this," he goes on to say, "must be added the decomposing

action which yeast exercises on a great number of substances, and

which resembles that which sugar undergoes. I have shown that

malate of lime ferments readily enough through the action of

yeast, and that it splits up into three other calcareous salts,

namely, the acetate, the carbonate and the succinate. If the

action of yeast consists in its increase and multiplication, it

is difficult to conceive this action in the case of malate of

lime and other calcareous salts of vegetable acids."

This statement, with all due deference to the opinion of our

illustrious critic, is by no means correct. Yeast has no action

on malate of lime, or on other calcareous salts formed by

vegetable acids. Liebig had previously, much to his own

satisfaction, brought forward urea as being capable of

transformation into carbonate of ammonia during alcoholic

fermentation in contact with yeast. This has been proved to be

erroneous. It is an error of the same kind that Liebig again

brings forward here. In the fermentation of which he speaks (that

of malate of lime), certain spontaneous ferments are produced,

the germs of which are associated with the yeast, and develop in

the mixture of yeast and malate. The yeast merely serves as a

source of food for these new ferments without taking any direct

part in the fermentations of which we are speaking. Our

researches leave no doubt on this point, as is evident from the

observations on the fermentation of tartrate of lime previously

given.

It is true that there are circumstances under which yeast brings

about modifications in different substances. Doebereiner and,

Mitscherlich, more especially, have shown that yeast imparts to

water a soluble material, which liquefies cane-sugar and produces

inversion in it by causing it to take up the elements of water,

just as diastase behaves to starch or emulsin to amygdalin.

M. Berthelot also has shown that this substance may be isolated

by precipitating it with alcohol, in the same way as diastase is

precipitated from its solutions. [Footnote: DOEBEREINER, Journal

de Chimie de Schweigger, vol. xii., p. 129, and Journal de

Pharmacie, vol. i., p. 342.

MITSCHERLICH, Monatsberichte d. Kon. Preuss. Akad. d. Wissen, eu

Berlin, and Rapports annuels da Berzelius, Paris, 1843, 3rd year.

On the occasion of a communication on the inversion of cane-sugar

by H. Rose, published in 1840, M. Mitscherlich observed: "The

inversion of cane-sugar in alcoholic fermentation is not due to

the globules of yeast, but to a soluble matter in the water with

which they mix. The liquid obtained by straining off the ferment

on a filter paper possesses the property of converting cane-sugar

into uncrystallizable sugar."

BERTHELOT, Comptes rendus de l'Academie. Meeting of May 28th,

1860, M. Berthelot confirms the preceding experiment of

Mitscherlich, and proves, moreover, that the soluble matter of

which the author speaks may be precipitated with alcohol without

losing its invertive power.

M. Bechamp has applied Mitscherlich's observation, concerning the

soluble fermentative part of yeast, to fungoid growths, and has

made the interesting discovery that fungoid growths, like yeast,

yield to water a substance that inverts sugar. When the

production of fungoid growths is prevented by means of an

antiseptic, the inversion of sugar does not take place.

We may here say a few words respecting M. Bechamp's claim to

priority of discovery. It is a well-known fact that we were the

first to demonstrate that living ferments might be completely

developed if their germs were placed in pure water together with

sugar, ammonia, and phosphates. Relying on this established fact,

that moulds are capable of development in sweetened water in

which, according to M. Bechamp, they invert the sugar, our author

asserts that he has proved that "living organized ferments may

originate in media which contain no albuminous substances." (See

Comptes rendus, vol. ixxv., p. 1519.) To be logical, M. Bechamp

might say that he has proved that certain moulds originate in

pure sweetened water without nitrogen or phosphates or other

mineral elements, for such a deduction might very well be drawn

from his work, in which we do not find the least expression of

astonishment at the possibility of moulds developing in pure

water containing nothing but sugar without other mineral or

organic principles.

M. Bechamp's first note on the inversion of sugar was published

in 1855. In it we find nothing relating to the influence of

moulds. His second, in which that influence is noticed, was

published in January, 1858, that is, subsequently to our work on

lactic fermentation, which appeared in November, 1857. In that

work we established for the first time that the lactic ferment is

a living, organized being, that albuminous substances have no

share in the production of fermentation, and that they only serve

as the food of the ferment. M. Bechamp's note was even subsequent

to our first work on alcoholic fermentation, which appeared on

December 21st, 1857. It is since the appearance of these two

works of ours that the preponderating influence of the life of

microscopic organism in the phenomena of fermentation has been

better understood. Immediately after their appearance M. Bechamp,

who from 1855 had made no observation on the action of fungoid

growths on sugar, although he had remarked their presence,

modified his former conclusions. (Comptes rendus, January 4th,

1858.)] These are remarkable facts, which are, however, at

present but vaguely connected with the alcoholic fermentation of

sugar by means of yeast. The researches in which we have proved

the existence of special forms of living ferments in many

fermentations, which one might have supposed to have been

produced by simple contact action, had established beyond doubt

the existence of profound differences between those

fermentations, which we have distinguished as fermentations

proper, and the phenomena connected with soluble substances. The

more we advance, the more clearly we are able to detect these

differences. M. Dumas has insisted on the fact that the ferments

of fermentation proper multiply and reproduce themselves in the

process whilst the others are destroyed. [Footnote: "There are

two classes of ferments; the first, of which the yeast of beer

may be taken as the type, perpetuate and renew themselves if they

can find in the liquid in which they produce fermentation food

enough for their wants; the second, of which diastase is the

type, always sacrifice themselves in the exercise of their

activity." (DUMAS, Comptes rendus de l'Academie, t. lxxv., p.

277, 1872.)] Still more recently M. Muntz has shown that

chloroform prevents fermentations proper, but does not interfere

with the action of diastase (Comptes rendus, 1875). M. Bouchardat

had already established the fact that hydrocyanic acid, salts of

mercury, ether, alcohol, creosote, and the oils of turpentine,

lemon, cloves, and mustard destroy or check alcoholic

fermentations, whilst in no way interfering with the glucoside

fermentations (Annales de Chimie et de Physique. 3rd series, t.

xiv., 1845). We may add in praise of M. Bouchardat's sagacity,

that that skilful observer has always considered these results as

a proof that alcoholic fermentation is dependent on the life of

the yeast-cell, and that a distinction should be made between the

two orders of fermentation.

M. Paul Bert, in his remarkable studies on the influence of

barometric pressure on the phenomena of life, has recognized the

fact that compressed oxygen is fatal to certain ferments, whilst

under similar conditions it does not interfere with the action of

those substances classed under the name of SOLUBLE FERMENTS, such

as diastase (the ferment which inverts cane sugar) emulsin and

others. During their stay in compressed air, ferments proper

ceased their activity, nor did they resume it, even after

exposure to air at ordinary pressures, provided the access of

germs was prevented.

We now come to Liebig's principal objection, with which he

concludes his ingenious argument, and to which no less than eight

or nine pages of the Annales are devoted.

Our author takes up the question of the possibility of causing

yeast to grow in sweetened water, to which a salt of ammonia and

some yeast-ash have been added--a fact which is evidently

incompatible with his theory that a ferment is always an

albuminous substance on its way to decomposition. In this case

the albuminous substance does not exist; we have only the mineral

substances which will serve to produce it. We know that Liebig

regarded yeast, and, generally speaking, any ferment whatever, as

being a nitrogenous, albuminous substance which, in the same way

as emulsin, for example, possesses the power of bringing about

certain chemical decompositions. He connected fermentation with

the easy decomposition of that albuminous substance, and imagined

that the phenomenon occurred in the following manner: "The

albuminous substance on its way to decomposition possesses the

power of communicating to certain other bodies that same state of

mobility by which its own atoms are already affected; and through

its contact with other bodies it imparts to them the power of

decomposing or of entering into other combinations." Here Liebig

failed to perceive that the ferment, in its capacity of a living

organism, had anything to do with the fermentation.

This theory dates back as far as 1843. In 1846 Messrs. Boutron

and Fremy, in a Memoir on lactic fermentation, published in the

Annales de Chimie et de Physique, strained the conclusions

deducible from it to a most unjustifiable extent. They asserted

that one and the same nitrogenous substance might undergo various

modifications in contact with air, so as to become successively

alcoholic, lactic, butyric, and other ferments. There is nothing

more convenient than purely hypothetical theories, theories which

are not the necessary consequences of facts; when fresh facts

which cannot be reconciled with the original hypothesis are

discovered, new hypotheses can be tacked on to the old ones. This

is exactly what Liebig and Fremy have done, each in his turn,

under the pressure of our studies, commenced in 1857. In 1864

Fremy devised the theory of hemi-organism, which meant nothing

more than that he gave up Liebig's theory of 1843, together with

the additions which Boutron and he had made to it in 1846; in

other words, he abandoned the idea of albuminous substances being

ferments, to take up another idea, that albuminous substances in

contact with air are peculiarly adapted to undergo organization

into new beings--that is, the living ferments which we had

discovered--and that the ferments of beer and of the grape have a

common origin.

This theory of hemi-organism was word for word the antiquated

opinion of Turpin. * * * The public, especially a certain section

of the public did not go very deeply into an examination of the

subject. It was the period when the doctrine of spontaneous

generation was being discussed with much warmth. The new word

hemi-organism, which was the only novelty in M. Fremy's theory,

deceived people. It was thought that M. Fremy had really

discovered the solution of the question of the day. It is true

that it was rather difficult to understand the process by which

an albuminous substance could become all at once a living and

budding cell. This difficulty was solved by M. Fremy, who

declared that it was the result of some power that was not yet

understood, the power of "organic impulse." [Footnote: FREMY,

Comptes rendus de l'Academie, vol. lviii., p. 1065, 1864.]

Liebig, who, as well as M. Fremy, was compelled to renounce his

original opinions concerning the nature of ferments, devised the

following obscure theory (Memoir by Liebig, 1870, already cited):

"There seems to be no doubt as to the part which the vegetable

organism plays in the phenomenon of fermentation. It is through

it alone that an albuminous substance and sugar are enabled to

unite and form this particular combination, this unstable form

under which alone, as a component part of the mycoderm, they

manifest an action on sugar. Should the mycoderm cease to grow,

the bond which unites the constituent parts of the cellular

contents is loosened, and it is through the motion produced

therein that the cells of yeast bring about a disarrangement or

separation of the elements of the sugar into molecules."

One might easily believe that the translator for the Annales has

made some mistake, so great is the obscurity of this passage.

Whether we take this new form of the theory or the old one,

neither can be reconciled at all with the development of yeast

and fermentation in a saccharine mineral medium, for in the

latter experiment fermentation is correlative to the life of the

ferment and to its nutrition, a constant change going on between

the ferment and its food-matters, since all the carbon

assimilated by the ferment is derived from sugar, its nitrogen

from ammonia and phosphorus from the phosphates in solution. And

even all said, what purpose can be served by the gratuitous

hypothesis of contact-action or communicated motion? The

experiment of which we are speaking is thus a fundamental one;

indeed, it is its possibility that constitutes the most effective

point in the controversy. No doubt Liebig might say, "but it is

the motion of life and of nutrition which constitutes your

experiment, and this is the communicated motion that my theory

requires." Curiously enough, Liebig does endeavour, as a matter

of fact, to say this, but he does so timidly and incidentally:

"From a chemical point of view, which point of view I would not

willingly abandon, a VITAL ACTION is a phenomenon of motion, and,

in this double sense of LIFE M. Pasteur's theory agrees with my

own, and is not in contradiction with it (page 6)." This is true.

Elsewhere Liebig says:

"It is possible that the only correlation between the

physiological act and the phenomenon of fermentation is the

production, in the living cell, of the substance which, by some

special property analogous to that by which emulsin exerts a

decomposing action on salicin and amygdalin, may bring about the

decomposition of sugar into other organic molecules; the

physiological act, in this view, would be necessary for the

production of this substance, but would have nothing else to do

with the fermentation (page 10)." To this, again, we have no

objection to raise.

Liebig, however, does not dwell upon these considerations, which

he merely notices in passing, because he is well aware that, as

far as the defence of his theory is concerned, they would be mere

evasions. If he had insisted on them, or based his opposition

solely upon them, our answer would have been simply this: "If you

do not admit with us that fermentation is correlated with the

life and nutrition of the ferment, we agree upon the principal

point. So agreeing, let us examine, if you will, the actual cause

of fermentation;--this is a second question, quite distinct from

the first. Science is built up of successive solutions given to

questions of ever increasing subtlety, approaching nearer and

nearer towards the very essence of phenomena. If we proceed to

discuss together the question of how living, organized beings act

in decomposing fermentable substances, we will be found to fall

out once more on your hypothesis of communicated motion, since

according to our ideas, the actual cause of fermentation is to be

sought, in most cases, in the fact of life without air, which is

the characteristic of many ferments."

Let us briefly see what Liebig thinks of the experiment in which

fermentation is produced by the impregnation of a saccharine

mineral medium, a result so greatly at variance with his mode of

viewing the question. [Footnote: See our Memoir of 1860 (Annales

de Chimie et de Physique, vol. lviii, p. 61, and following,

especially pp. 69 and 70, where the details of the experiment

will be found).] After deep consideration he pronounces this

experiment to be inexact, and the result ill-founded. Liebig,

however, was not one to reject a fact without grave reasons for

doing so, or with the sole object of evading a troublesome

discussion. "I have repeated this experiment," he says, "a great

number of times, with the greatest possible care, and have

obtained the same results as M. Pasteur, excepting as regards the

formation and increase of the ferment." It was, however, the

formation and increase of the ferment that constituted the point

of the experiment. Our discussion was, therefore, distinctly

limited to this: Liebig denied that the ferment was capable of

development in a saccharine mineral medium, whilst we asserted

that this development did actually take place, and was

comparatively easy to prove. In 1871 we replied to M. Liebig

before the Paris Academy of Sciences in a Note, in which we

offered to prepare in a mineral medium, in the presence of a

commission to be chosen for the purpose, as great a weight of

ferment as Liebig could reasonably demand. [Footnote: PASTEUR,

Comptes rendus de l'Academie des Sciences, vol. lxxiii., p. 1419.

1871.] We were bolder than we should, perhaps, have been in 1860;

the reason was that our knowledge of the subject had been

strengthened by ten years of renewed research. Liebig did not

accept our proposal, nor did he even reply to our Note. Up to the

time of his death, which took place on April 18th, 1873, he wrote

nothing more on the subject. [Footnote: In his Memoir of 1870,

Liebig made a remarkable admission: "My late friend Pelouze," he

says, "had communicated to me nine years ago certain results of

M. Pasteur's researches on fermentation. I told him that just

then I was not disposed to alter my opinion on the cause of

fermentation, and that if it were possible, by means of ammonia,

to produce or multiply the yeast in fermenting liquors, industry

would soon avail itself of the fact, and that I would wait to see

if it did so; up to the present time, however, there had not been

the least change in the manufacture of yeast. "We do not know

what M. Pelouze's reply was; but it is not difficult to conceive

so sagacious an observer remarking to his illustrious friend that

the possibility of deriving pecuniary advantage from the wide

application of a new scientific fact had never been regarded as

the criterion of the exactness of that fact. We could prove,

moreover, by the undoubted testimony of very distinguished

practical men, notably by that of M. Pezeyre, director of

distilleries, that upon this point also Liebig was mistaken.]

When we published, in 1860, the details of the experiment in

question, we pointed out at some length the difficulties of

conducting it successfully, and the possible causes of failure.

We called attention particularly to the fact that saccharine

mineral media are much more suited for the nutrition of bacteria,

lactic ferment, and other lowly forms, than they are to that of

yeast, and in consequence readily become filled with various

organisms from the spontaneous growth of germs derived from the

particles of dust floating in the atmosphere. The reason why we

do not observe the growth of alcoholic ferments, especially at

the commencement of the experiments, is because of the

unsuitableness of those media for the life of yeast. The latter

may, nevertheless, form in them subsequent to this development of

other organized forms, by reason of the modification produced in

the original mineral medium by the albuminous matters that they

introduce into it. It is interesting to peruse, in our Memoir of

1860, certain facts of the same kind relating to fermentation by

means of albumens--that of the blood for example, from which, we

may mention incidentally, we were led to infer the existence of

several distinct albumens in the serum, a conclusion which, since

then, has been confirmed by various observers, notably by M.

Bechamp. Now, in his experiments on fermentation in sweetened

water, with yeast-ash and a salt of ammonia, there is no doubt

that Liebig had failed to avoid those difficulties which are

entailed by the spontaneous growth of other organisms than yeast.

Moreover, it is possible that, to have established the certainty

of this result, Liebig should have had recourse to a closer

microscopical observation than from certain passages in his

Memoir he seems to have adopted. We have little doubt that his

pupils could tell us that Liebig did not even employ that

instrument without which any exact study of fermentation is not

merely difficult but well-nigh impossible. We ourselves, for the

reasons, mentioned, did not obtain a simple alcoholic

fermentation any more than Liebig did. In that particular

experiment, the details of which we gave in our Memoir of 1860,

we obtained lactic and alcoholic fermentation together; an

appreciable quantity of lactic acid formed and arrested the

propagation of the lactic and alcoholic ferments, so that more

than half of the sugar remained in the liquid without fermenting.

This, however, in no way detracted from the correctness of the

conclusion which we deduced from the experiment, and from other

similar ones; it might even be said that, from a general and

philosophical point of view--which is the only one of interest

here--the result was doubly satisfactory, inasmuch as we

demonstrated that mineral media were adapted to the simultaneous

development of several organized ferments instead of only one.

The fortuitous association of different ferments could not

invalidate the conclusion that all the nitrogen of the cells of

the alcoholic and lactic ferments was derived from the nitrogen

in the ammoniacal salts, and that all the carbon of those

ferments was taken from the sugar, since, in the medium employed

in our experiment, the sugar was the only substance that

contained carbon. Liebig carefully abstained from noticing this

fact, which would have been fatal to the very groundwork of his

criticisms, and thought that he was keeping up the appearance of

a grave contradiction by arguing that we had never obtained a

simple alcoholic fermentation. It would be unprofitable to dwell

longer upon the subject of the difficulties which the propagation

of yeast in a saccharine mineral medium formerly presented. As a

matter of fact, the progress of our studies has imparted to the

question an aspect very different from that which it formerly

wore; it was this circumstance which emboldened us to offer, in

our reply to Liebig before the Academy of Sciences in 1871, to

prepare, in a saccharine mineral medium, in the presence of a

commission to be appointed by our opponent, any quantity of

ferment that he might require, and to effect the fermentation of

any weight of sugar whatsoever.

Our knowledge of the facts detailed in the preceding chapter

concerning pure ferments, and their manipulation in the presence

of pure air, enables us completely to disregard those causes of

embarrassment that result from the fortuitous occurrence of the

germs of organisms different in character from the ferments

introduced by the air or from the sides of vessels, or even by

the ferment itself.

Let us once more take one of our double-necked flasks, which we

will suppose is capable of containing three or four litres (six

to eight pints).

Let us put into it the following:

Pure distilled water.

Sugar candy. ... . ... . ... . ... . ... . 200 grammes

Bitartrate of potassium. ... . ... . 1.0 grammes

Bitartrate of ammonia. ... . ... ... 0.5 grammes

Sulphate of ammonia.,. ... . ... ... 1.5 grammes

Ash of yeast. ... . ... . ... . ... . ... 1.5 grammes

(1 gramme = 15.43 grains)

Let us boil the mixture, to destroy all germs of organisms that

may exist in the air or liquid or on the sides of the flask, and

then permit it to cool, after having placed, by way of extra

precaution a small quantity of asbestos in the end of the fine

curved tube. Let us next introduce a trace of ferment into the

liquid, through the other neck, which, as we have described, is

terminated by a small piece of india-rubber tube closed with a

glass stopper.

Here are the details of such an experiment:--

On December 9th, 1873, we sowed some pure ferment--saccharomyces

pastorianus. From December 11, that is, within so short a time as

forty-eight hours after impregnation, we saw a multitude of

extremely minute bubbles rising almost continuously from the

bottom, indication that at this point the fermentation had

commenced. On the following days, several patches of froth

appeared on the surface of the liquid. We left the flask

undisturbed in the oven, at a temperature of 25 degrees C. (77

degrees F.) On April 24, 1874, we tested some of the liquid,

obtained by means of the straight tube, to see if it still

contained any sugar. We found that it contained less than two

grammes, so that 198 grammes (4.2 oz. Troy) had already

disappeared. Some time afterwards the fermentation came to an

end; we carried on the experiment, nevertheless, until April 18,

1875.

There was no development of any organism absolutely foreign to

the ferment, which was itself abundant, a circumstance that,

added to the persistent vitality of the ferment, in spite of the

unsuitableness of the medium for its nutrition, permitted the

perfect completion of fermentation. There was not the minutest

quantity of sugar remaining. The total weight of ferment, after

washing and drying at 100 degrees C. (212 degrees F.), was 2.563

grammes (39.5 grains).

In experiments of this kind, in which the ferment has to be

weighed, it is better not to use any yeast-ash that cannot be

dissolved completely, so as to be capable of easy separation from

the ferment formed. Raulin's liquid [Footnote: M. Jules Raulin

has published a well-known and remarkable work on the discovery

of the mineral medium best adapted by its composition to the life

of certain fungoid growth; he has given a formula for the

composition of such a medium. It is this that we call here

"Raulin's liquid" for abbreviation.

Water . . . . . . . . . . . . . . . . . . 1,500

Sugar candy . . . . . . . . . . . . . . . 70

Tartaric acid . . . . . . . . . . . . . . 4

Nitrate of ammonia . . . . . . . . . . . 4

Phosphate of ammonia . . . . . . . . . . 0.6

Carbonate of potassium . . . . . . . . . 0.6

Carbonate of magnesia . . . . . . . . . . 0.4

Sulphate of ammonia . . . . . . . . . . . 0.25

Sulphate of zinc . . . . . . . . . . . . 0.07

Sulphate of iron . . . . . . . . . . . . 0.07

Silicate of potassium . . . . . . . . . . 0.07

--J. Raulin, Paris, Victor Masson, 1870. These pour le

doctorat.] may be used in such cases with success.

All the alcoholic ferments are not capable to the same extent of

development by means of phosphates, ammoniacal salts, and sugar.

There are some whose development is arrested a longer or shorter

time before the transformation of all the sugar. In a series of

comparative experiments, 200 grammes of sugar-candy being used in

each case, we found that whilst saccharomyces pastorianus

effected a complete fermentation of the sugar, the caseous

ferment did not decompose more than two-thirds, and the ferment

we have designated NEW "HIGH" FERMENT not more than one-fifth:

and keeping the flasks for a longer time in the oven had no

effect in increasing the proportions of sugar fermented in these

two last cases.

We conducted a great number of fermentations in mineral media, in

consequence of a circumstance which it may be interesting to

mention here. A person who was working in our laboratory asserted

that the success of our experiments depended upon the impurity of

the sugar-candy which we employed, and that if this sugar had

been pure--much purer than was the ordinary, white, commercial

sugar-candy, which up to that time we had always used--the

ferment could not have multiplied. The persistent objections of

our friend, and our desire to convince him, caused us to repeat

all our previous experiments on the subject, using sugar of great

purity, which had been specially prepared for us, with the utmost

care, by a skilful confectioner, Seugnot. The result only

confirmed our former conclusions. Even this did not satisfy our

obstinate friend, who went to the trouble of preparing some pure

sugar for himself, in little crystals, by repeated

crystallizations of carefully selected commercial sugar-candy; he

then repeated our experiments himself. This time his doubts were

overcome. It even happened that the fermentations with the

perfectly pure sugar instead of being slow were very active, when

compared with those which we had conducted with, the commercial

sugar-candy.

We may here add a few words on the non-transformation of yeast

into penicillium glaucum.

If at any time during fermentation we pour off the fermenting

liquid, the deposit of yeast remaining in the vessel may continue

there, in contact with air, without our ever being able to

discover the least formation of penicillium glaucum in it. We may

keep a current of pure air constantly passing through the flask;

the experiment will give the same result. Nevertheless, this is a

medium peculiarly adapted to the development of this mould,

inasmuch as if we were to introduce merely a few spores of

penicillium an abundant vegetation of that growth will afterwards

appear on the deposit. The descriptions of Messrs. Turpin,

Hoffmann, and Trecul have, therefore, been based on one of these

illusions which we meet with so frequently in microscopical

observations.

When we laid these facts before the Academy, [Footnote: PASTEUR,

Comptes rendus de l'Academie, vol. lxxviii., pp. 213-216.] M.

Trecul professed his inability to comprehend them: [Footnote:

TRECUL, Comptes rendus de l'Academie, vol. lxxviii., pp. 217,

218.] "According to M. Pasteur," he said, "the yeast of beer is

ANAEROBIAN, that is to say, it lives in a liquid deprived of free

oxygen; and to become mycoderma or penicillium it is above all

things necessary that it should be placed in air, since, without

this, as the name signifies, an aerobian being cannot exist. To

bring about the transformation of the yeast of beer into

mycoderma cerevisiae or into penicillium glaucum we must accept

the conditions under which these two forms are obtained. If M.

Pasteur will persist in keeping his yeast in media which are

incompatible with the desired modification, it is clear that the

results which he obtains must always be negative."

Contrary to this perfectly gratuitous assertion of M. Trecul's we

do not keep our yeast in media which are calculated to prevent

its transformation into penicillium. As we have just seen, the

principal aim and object of our experiment was to bring this

minute plant into contact with air, and under conditions that

would allow the penicillium to develop with perfect freedom. We

conducted our experiments exactly as Turpin and Hoffmann

conducted theirs, and exactly as they stipulate that such

experiments should be conducted--with the one sole difference,

indispensable to the correctness of our observations, that we

carefully guarded ourselves against those causes of error which

they did not take the least trouble to avoid. It is possible to

produce a ready entrance and escape of pure air in the case of

the double-necked flasks which we have so often employed in the

course of this work, without having recourse to the continuous

passage of a current of air. Having made a file-mark on the thin

curved neck at a distance of two or three centimetres (an inch)

from the flask, we must cut round the neck at this point with a

glazier's diamond, and then remove it, taking care to cover the

opening immediately with a sheef of paper which has been passed

through the flame, and which we must fasten with a thread round

the part of the neck still left. In this manner we may increase

or prolong the fructification of fungoid growths, or the life of

the aerobian ferments in our flasks.

What we have said of Penicillium glaucum will apply equally to

Mycoderma cerevisiae. Notwithstanding that Turpin and Trecul may

assert to the contrary, yeast, in contact with air as it was

under the conditions of the experiment just described, will not

yield Mycoderma vini or Mycoderma cerevisiae any more than it

will Penicillium.

The experiments described in the preceding paragraphs on the

increase of organized ferments in mineral media of the

composition described, are of the greatest physiological

interest. Amongst other results, they show that all the proteic

matter of ferments may be produced by the vital activity of the

cells, which, apart altogether from the influence of light or

free oxygen (unless indeed, we are dealing with aerobian moulds

which require free oxygen), have the power of developing a

chemical activity between carbohydrates, ammoniacal salts,

phosphates, and sulphates of potassium and magnesium. It may be

admitted with truth that a similar effect obtains in the case of

the higher plants, so that in the existing state of science we

fail to conceive what serious reason can be urged against our

considering this effect as general. It would be perfectly logical

to extend the results of which we are speaking to all plants, and

to believe that the proteic matter of vegetables, and perhaps of

animals also, is formed exclusively by the activity of the cells

operating upon the ammoniacal and other mineral salts of the sap

or plasma of the blood, and the carbo-hydrates, the formation of

which, in the case of the higher plants, requires only the

concurrence of the chemical impulse of green light.

Viewed in this manner, the formation of the proteic substances,

would be independent of the great act of reduction of carbonic

acid gas under the influence of light. These substances would not

be built up from the elements of water, ammonia, and carbonic

acid gas, after the decomposition of this last; they would be

formed where they are found in the cells themselves, by some

process of union between the carbo-hydrates imported by the sap,

and the phosphates of potassium and magnesium and salts of

ammonia. Lastly, in vegetable growth, by means of a carbo-hydrate

and a mineral medium, since the carbo-hydrate is capable of many

variations, and it would be difficult to understand how it could

be split up into its elements before serving to constitute the

proteic substances, and even cellulose substances, as these are

carbo-hydrates. We have commenced certain studies in this

direction.

If solar radiation is indispensable to the decomposition of

carbonic acid and the building up of the primary substances in

the case of higher vegetable life, it is still possible that

certain inferior organisms may do without it and nevertheless

yield the most complex substances, fatty or carbo-hydrate, such

as cellulose, various organic acids, and proteic matter; not,

however, by borrowing their carbon from the carbonic acid which

is saturated with oxygen, but from other matters still capable of

acquiring oxygen, and so of yielding heat in the process, such as

alcohol and acetic acid, for example, to cite merely carbon

compounds most removed from organization. As these last

compounds, and a host of others equally adapted to serve as the

carbonaceous food of mycoderms and the mucedines, may be produced

synthetically by means of carbon and the vapour of water, after

the methods that science owes to Berthelot, it follows that, in

the case of certain inferior beings, life would be possible even

if it should be that the solar light was extinguished. [Footnote:

See on this subject the verbal observations which we addressed to

the Academy of Sciences at its meetings of April 10th and 24th,

1876].

THE GERM THEORY AND ITS APPLICATIONS TO MEDICINE AND SURGERY

[Footnote: Read before the French Academy of Sciences, April

29th, 1878. Published in Comptes Rendus de l' Academie des

Sciences, lxxxvi., pp. 1037-43.]

The Sciences gain by mutual support. When, as the result of my

first communications on the fermentations in 1857-1858, it

appeared that the ferments, properly so-called, are living

beings, that the germs of microscopic organisms abound in the

surface of all objects, in the air and in water; that the theory

of spontaneous generation is chimerical; that wines, beer,

vinegar, the blood, urine and all the fluids of the body undergo

none of their usual changes in pure air, both Medicine and

Surgery received fresh stimulation. A French physician, Dr.

Davaine, was fortunate in making the first application of these

principles to Medicine, in 1863.

Our researches of last year, left the etiology of the putrid

disease, or septicemia, in a much less advanced condition than

that of anthrax. We had demonstrated the probability that

septicemia depends upon the presence and growth of a microscopic

body, but the absolute proof of this important conclusion was not

reached. To demonstrate experimentally that a microscopic

organism actually is the cause of a disease and the agent of

contagion, I know no other way, in the present state of Science,

than to subject the microbe (the new and happy term introduced by

M. Sedillot) to the method of cultivation out of the body. It may

be noted that in twelve successive cultures, each one of only ten

cubic centimeters volume, the original drop will be diluted as if

placed in a volume of fluid equal to the total volume of the

earth. It is just this form of test to which M. Joubert and I

subjected the anthrax bacteridium. [Footnote: In making the

translation, it seems wiser to adhere to Pasteur's nomenclature.

Bacillus anthracis would be the term employed to-day.--

Translator.] Having cultivated it a great number of times in a

sterile fluid, each culture being started with a minute drop from

the preceding, we then demonstrated that the product of the last

culture was capable of further development and of acting in the

animal tissues by producing anthrax with all its symptoms. Such

is--as we believe--the indisputable proof that ANTHRAX IS A

BACTERIAL DISEASE.

Our researches concerning the septic vibrio had not so far been

convincing, and it was to fill up this gap that we resumed our

experiments. To this end, we attempted the cultivation of the

septic vibrio from an animal dead of septicemia. It is worth

noting that all of our first experiments failed, despite the

variety of culture media we employed--urine, beer yeast water,

meat water, etc. Our culture media were not sterile, but we

found--most commonly--a microscopic organism showing no

relationship to the septic vibrio, and presenting the form,

common enough elsewhere, of chains of extremely minute spherical

granules possessed of no virulence whatever. [Footnote: It is

quite possible that Pasteur was here dealing with certain

septicemic streptococci that are now know to lose their virulence

with extreme rapidity under artificial cultivation.--Translator.]

This was an impurity, introduced, unknown to us, at the same time

as the septic vibrio; and the germ undoubtedly passed from the

intestines--always inflamed and distended in septicemic animals--

into the abdominal fluids from which we took our original

cultures of the septic vibrio. If this explanation of the

contamination of our cultures was correct, we ought to find a

pure culture of the septic vibrio in the heart's blood of an

animal recently dead of septicemia. This was what happened, but a

new difficulty presented itself; all our cultures remained

sterile. Furthermore this sterility was accompanied by loss in

the culture media of (the original) virulence.

It occurred to us that the septic vibrio might be an obligatory

anaerobe and that the sterility of our inoculated culture fluids

might be due to the destruction of the septic vibrio by the

atmospheric oxygen dissolved in the fluids. The Academy may

remember that I have previously demonstrated facts of this nature

in regard to the vibrio of butyric fermentation, which not only

lives without air but is killed by the air.

It was necessary therefore to attempt to cultivate the septic

vibrio either in a vacuum or in the presence of inert gases--such

as carbonic acid.

Results justified our attempt; the septic vibrio grew easily in a

complete vacuum, and no less easily in the presence of pure

carbonic acid.

These results have a necessary corollary. If a fluid containing

septic vibrios be exposed to pure air, the vibrios should be

killed and all virulence should disappear. This is actually the

case. If some drops of septic serum be spread horizontally in a

tube and in a very thin layer, the fluid will become absolutely

harmless in less than half a day, even if at first it was so

virulent as to produce death upon the inoculation of the smallest

portion of a drop.

Furthermore all the vibrios, which crowded the liquid as motile

threads, are destroyed and disappear. After the action of the

air, only fine amorphous granules can be found, unfit for culture

as well as for the transmission of any disease whatever. It might

be said that the air burned the vibrios.

If it is a terrifying thought that life is at the mercy of the

multiplication of these minute bodies, it is a consoling hope

that Science will not always remain powerless before such

enemies, since for example at the very beginning of the study we

find that simple exposure to air is sufficient at times to

destroy them.

But, if oxygen destroys the vibrios, how can septicemia exist,

since atmospheric air is present everywhere? How can such facts

be brought in accord with the germ theory? How can blood, exposed

to air, become septic through the dust the air contains?

All things are hidden, obscure and debatable if the cause of the

phenomena be unknown, but everything is clear if this cause be

known. What we have just said is true only of a septic fluid

containing adult vibrios, in active development by fission:

conditions are different when the vibrios are transformed into

their germs, [Footnote: By the terms "germ" and "germ

corpuscles," Pasteur undoubtedly means "spores," but the change

is not made, in accordance with note 3, above.--Translator.] that

is into the glistening corpuscles first described and figured in

my studies on silk-worm disease, in dealing with worms dead of

the disease called "flacherie." Only the adult vibrios disappear,

burn up, and lose their virulence in contact with air: the germ

corpuscles, under these conditions, remain always ready for new

cultures, and for new inoculations.

All this however does not do away with the difficulty of

understanding how septic germs can exist on the surface of

objects, floating in the air and in water.

Where can these corpuscles originate? Nothing is easier than the

production of these germs, in spite of the presence of air in

contact with septic fluids.

If abdominal serous exudate containing septic vibrios actively

growing by fission be exposed to the air, as we suggested above,

but with the precaution of giving a substantial thickness to the

layer, even if only one centimeter be used, this curious

phenomenon will appear in a few hours. The oxygen is absorbed in

the upper layers of the fluid--as is indicated by the change of

color. Here the vibrios are dead and disappear. In the deeper

layers, on the other hand, towards the bottom of this centimeter

of septic fluid we suppose to be under observation, the vibrios

continue to multiply by fission--protected from the action of

oxygen by those that have perished above them: little by little

they pass over to the condition of germ corpuscles with the

gradual disappearance of the thread forms. So that instead of

moving threads of varying length, sometimes greater than the

field of the microscope, there is to be seen only a number of

glittering points, lying free or surrounded by a scarcely

perceptible amorphous mass. [Footnote: In our note of July 16th,

1877, it is stated that the septic vibrio is not destroyed by the

oxygen of the air nor by oxygen at high tension, but that under

these conditions it is transformed into germ-corpuscles. This is,

however, an incorrect interpretation of facts. The vibrio is

destroyed by oxygen, and it is only where it is in a thick layer

that it is transformed to germ-corpuscles in the presence of

oxygen and that its virulence is preserved.] Thus is formed,

containing the latent germ life, no longer in danger from the

destructive action of oxygen, thus, I repeat, is formed the

septic dust, and we are able to understand what has before seemed

so obscure; we can see how putrescible fluids can be inoculated

by the dust of the air, and how it is that putrid diseases are

permanent in the world.

The Academy will permit me, before leaving these interesting

results, to refer to one of their main theoretical consequences.

At the very beginning of these researches, for they reveal an

entirely new field, what must be insistently demanded? The

absolute proof that there actually exist transmissible,

contagious, infectious diseases of which the cause lies

essentially and solely in the presence of microscopic organisms.

The proof that for at least some diseases, the conception of

spontaneous virulence must be forever abandoned--as well as the

idea of contagion and an infectious element suddenly originating

in the bodies of men or animals and able to originate diseases

which propagate themselves under identical forms: and all of

those opinions fatal to medical progress, which have given rise

to the gratuitous hypotheses of spontaneous generation, of

albuminoid ferments, of hemiorganisms, of archebiosis, and many

other conceptions without the least basis in observation. What is

to be sought for in this instance is the proof that along with

our vibrio there does not exist an independent virulence

belonging to the surrounding fluids or solids, in short that the

vibrio is not merely an epiphenomenon of the disease of which it

is the obligatory accompaniment. What then do we see, in the

results that I have just brought out? A septic fluid, taken at

the moment that the vibrios are not yet changed into germs, loses

its virulence completely upon simple exposure to the air, but

preserves this virulence, although exposed to air on the simple

condition of being in a thick layer for some hours. In the first

case, the virulence once lost by exposure to air, the liquid is

incapable of taking it on again upon cultivation: but, in the

second case, it preserves its virulence and can propagate, even

after exposure to air. It is impossible, then, to assert that

there is a separate virulent substance, either fluid or solid,

existing, apart from the adult vibrio or its germ. Nor can it be

supposed that there is a virus which loses its virulence at the

moment that the adult vibrio dies; for such a substance should

also lose its virulence when the vibrios, changed to germs, are

exposed to the air. Since the virulence persists under these

conditions it can only be due to the germ corpuscles--the only

thing present. There is only one possible hypothesis as to the

existence of a virus in solution, and that is that such a

substance, which was present in our experiment in nonfatal

amounts, should be continuously furnished by the vibrio itself,

during its growth in the body of the living animal. But it is of

little importance since the hypothesis supposes the forming and

necessary existence of the vibrio. [Footnote: The regular limits,

oblige me to omit a portion of my speech.]

I hasten to touch upon another series of observations which are

even more deserving the attention of the surgeon than the

preceding: I desire to speak of the effects of our microbe of pus

when associated with the septic vibrio. There is nothing more

easy to superpose--as it were--two distinct diseases and to

produce what might be called a SEPTICEMIC PURULENT INFECTION, or

a PURULENT SEPTICEMIA. Whilst the microbe-producing pus, when

acting alone, gives rise to a thick pus, white, or sometimes with

a yellow or bluish tint, not putrid, diffused or enclosed by the

so-called pyogenic membrane, not dangerous, especially if

localized in cellular tissue, ready, if the expression may be

used for rapid resorption; on the other hand the smallest abscess

produced by this organism when associated with the septic vibrio

takes on a thick gangrenous appearance, putrid, greenish and

infiltrating the softened tissues. In this case the microbe of

pus carried so to speak by the septic vibrio, accompanies it

throughout the body: the highly-inflamed muscular tissues, full

of serous fluid, showing also globules of pus here and there, are

like a kneading of the two organisms.

By a similar procedure the effects of the anthrax bacteridium and

the microbe of pus may be combined and the two diseases may be

superposed, so as to obtain a purulent anthrax or an anthracoid

purulent infection. Care must be taken not to exaggerate the

predominance of the new microbe over the bacteridum. If the

microbe be associated with the latter in sufficient amount it may

crowd it out completely--prevent it from growing in the body at

all. Anthrax does not appear, and the infection, entirely local,

becomes merely an abscess whose cure is easy. The microbe-

producing pus and the septic vibrio (not) [Footnote: There is

undoubtedly a mistake in the original. Pasteur could not have

meant to say that both bacteria are anaerobes. The word "not" is

introduced to correct the error.--Translator.] being both

anaerobes, as we have demonstrated, it is evident that the latter

will not much disturb its neighbor. Nutrient substances, fluid or

solid, can scarcely be deficient in the tissues from such minute

organisms. But the anthrax bacteridium is exclusively aerobic,

and the proportion of oxygen is far from being equally

distributed throughout the tissues: innumerable conditions can

diminish or exhaust the supply here and there, and since the

microbe-producing pus is also aerobic, it can be understood how,

by using a quantity slightly greater than that of the bacteridium

it might easily deprive the latter of the oxygen necessary for

it. But the explanation of the fact is of little importance: it

is certain that under some conditions the microbe we are speaking

of entirely prevents the development of the bacteridium.