Chapter IV

.; such, too, is the case with the fruits employed in the experiments of Messrs. Lechartier and Bellamy, and in our own experiments, the results of which not only confirm those obtained by these gentlemen, but even extend them, in so far as we have shown that fruits, when surrounded with carbonic acid gas, immediately produce alcohol. When surrounded with air, they live in their aërobian state, and we have no ferment-action; immersed immediately afterwards in carbonic acid gas, they now assume their anaërobian state, and at once begin to act upon the sugar in the manner of ferments, and emit heat. As for seeing in these facts anything like a confirmation of the theory of hemi-organism, imagined by M. Fremy, the idea of such a thing is absurd. The following, for instance, is the theory of the fermentation of the vintage, according to M. Fremy.[129]

“To speak here of alcoholic fermentation alone,”[130] our author says, “I hold that in the production of wine it is the juice of the fruit itself that, in contact with air, produces grains of ferment, by the transformation of the albuminous matter; M. Pasteur, on the other hand, maintains that the fermentation is produced by germs existing outside the skin of the grapes.”

Now what bearing on this purely imaginary theory can the fact have, that a whole fruit, immersed in carbonic acid gas, immediately produces alcohol and carbonic acid? In the preceding passage, which we have borrowed from M. Fremy, an indispensable condition of the transformation of the albuminous matter is the contact with air and the crushing of the grapes. Here, however, we are dealing with _uninjured fruits in contact with carbonic acid gas_. Our theory, on the other hand, which, we may repeat, we have advocated since 1861, maintains that all cells become fermentative when their vital action is protracted in the absence of air, which are precisely the conditions that hold in the experiment on fruits immersed in carbonic acid gas. The vital energy is not immediately suspended in their cells, and the latter are deprived of air. Consequently, fermentation must result. Moreover, we may add, if we destroy the fruit, or crush it before immersing it in the gas, it no longer produces alcohol or fermentation of any kind, a circumstance that may be attributed to the fact of the destruction of vital action in the crushed fruit. On the other hand, in what way ought this crushing to affect the hypothesis of hemi-organism? The crushed fruit ought to act quite as well, or even better than that which is uncrushed. In short, nothing can be more directly opposed to the theory of the mode of manifestation of that hidden force to which the name of hemi-organism has been given, than the discovery of the production of these phenomena of fermentation in fruits surrounded with carbonic acid gas; whilst the theory, which sees in fermentation a consequence of vital energy in absence of air, finds in these facts the strictest confirmation of an express prediction, which from the first formed an integral part of its statement.

We should not be justified in devoting further time to opinions which are not supported by any serious experiment. Abroad, as well as in France, the theory of the transformation of albuminous substances into organized ferments had been advocated long before it was taken up by M. Fremy. It no longer commands the slightest credit, nor do any observers of note any longer give it the least attention; it might even be said that it has become a subject of ridicule.

An attempt has also been made to prove that we have contradicted ourselves, inasmuch as in 1860 we published our opinion that alcoholic fermentation can never occur without a simultaneous occurrence of organization, development, and multiplication of globules; or continued life, carried on from globules already formed.[131] Nothing, however, can be truer than that opinion, and at the present moment, after fifteen years of study devoted to the subject, since the publication to which we have referred, we need no longer say “we think,” but instead, “we affirm” that it is correct. It is, as a matter of fact, to alcoholic fermentation, properly so called, that the charge to which we have referred relates—to that fermentation which yields, besides alcohol, carbonic acid, succinic acid, glycerine, volatile acids, and other products. This fermentation undoubtedly requires the presence of yeast-cells, under the conditions that we have named. Those who have contradicted us have fallen into the error of supposing that the fermentation of fruits is an ordinary alcoholic fermentation, identical with that produced by beer-yeast, and that, consequently, the cells of that yeast must, according to our own theory, be always present. There is not the least authority for such a supposition. When we come to exact quantitative estimations—and these are to be found in the figures supplied by Messrs. Lechartier and Bellamy—it will be seen that the proportions of alcohol and carbonic acid gas produced in the fermentation of fruits differ widely from those that we find in alcoholic fermentations, properly so called, as must necessarily be the case, since, in the former, the ferment-action is effected by the cells of a fruit, but in the latter by cells of ordinary alcoholic ferment. Indeed we have a strong conviction that each fruit would be found to give rise to a special action, the chemical equation of which would be different from that in the case of other fruits. As for the circumstance that the cells of these fruits cause fermentation, without multiplying, this comes under the kind of activity, which we have already distinguished by the expression _continuous life in cells already formed_.

We will conclude this paragraph with a few remarks on the subject of the equations of fermentations, which have been suggested to us principally in attempts to explain the results derived from the fermentation of fruits immersed in carbonic acid gas.

Originally, when fermentations were put amongst the class of decompositions by contact-action, it seemed probable, and, in fact, was believed, that every fermentation had its own well-defined equation, which never varied. In the present day, on the contrary, it must be borne in mind that the equation of a fermentation varies essentially with the conditions under which that fermentation is accomplished, and that a statement of this equation is a problem no less complicated than that in the case of the nutrition of a living being. To every fermentation may be assigned an equation in a general sort of way, an equation, however, which, in numerous points of detail, is liable to the thousand variations connected with the phenomena of life. Moreover, there will be as many distinct fermentations brought about by one ferment as there are fermentable substances capable of supplying the carbon element of the food of that same ferment, in the same way that the equation of the nutrition of an animal will vary with the nature of the food which it consumes. As regards fermentation producing alcohol, which may be effected by several different ferments, there will be, in the case of a given sugar, as many general equations as there are ferments, whether they be ferment-cells, properly so called, or cells of the organs of living beings functioning as ferments. In the same way the equation of nutrition varies in the case of different animals nourished on the same food. And it is from the same reason that ordinary wort produces such a variety of beers when treated with the numerous alcoholic ferments which we have described. These remarks are applicable to all ferments alike; for instance, butyric ferment is capable of producing a host of distinct fermentations, in consequence of its ability to derive the carbonaceous part of its food from very different substances, from sugar, or lactic acid, or glycerine, or mannite, and many others.

When we say that every fermentation has its own peculiar ferment, it must be understood that we are speaking of the fermentation considered as a whole, including all the accessory products. We do not mean to imply that the ferment in question is not capable of acting on some other fermentable substance and giving rise to fermentation of a very different kind. Moreover, it is quite erroneous to suppose that the presence of a single one of the products of a fermentation implies the co-existence of a particular ferment. If, for example, we find alcohol among the products of a fermentation, or even alcohol and carbonic acid gas together, this does not prove that the ferment must be an alcoholic ferment, belonging to alcoholic fermentations, in the strict sense of the term. Nor, again, does the mere presence of lactic acid necessarily imply the presence of lactic ferment. As a matter of fact, different fermentations may give rise to one or even several identical products. We could not say with certainty, from a purely chemical point of view, that we were dealing, for example, with an alcoholic fermentation, properly so called, and that the yeast of beer must be present in it, if we had not first determined the presence of all the numerous products of that particular fermentation, and that they were present in those proportions, characteristic of that fermentation under conditions similar to those under which the fermentation in question had occurred. In works on fermentation, the reader will often find those confusions against which we are now attempting to guard him. It is precisely in consequence of not having had their attention drawn to such observations that some have imagined that the fermentation in fruits, immersed in carbonic acid gas, is in contradiction to the assertion which we originally made in our Memoir on alcoholic fermentation, published in 1860, the exact words of which we may here repeat:—“The chemical phenomena of fermentation are related essentially to a vital activity, beginning and ending with the latter; we believe that alcoholic fermentation never occurs”—we were discussing the question of ordinary alcoholic fermentation produced by the yeast of beer—“without the simultaneous occurrence of organization, development, and multiplication of globules, or continued life, carried on by means of globules already formed. The general results of the present Memoir seem to us to be in direct opposition to the opinions of MM. Liebig and Berzelius.” These conclusions, we repeat, are as true now as they ever were, and are as applicable to the fermentation of fruits, of which nothing was known in 1860, as they are to the fermentation produced by means of yeast. Only, in the case of fruits, it is the cells of the parenchyma that function as ferment, _by a continuation of their vital activity in carbonic acid gas_, whilst in the other case the ferment consists of the cells of yeast.

There should be nothing very surprising in the fact that fermentation can originate in fruits and form alcohol, without the presence of yeast, if the fermentation of fruits were not confounded completely with ordinary alcoholic fermentation, yielding the same products and in the same proportions. It is through the misuse of words that the fermentation of fruits has been termed _alcoholic_, in a way which has misled many persons.[132] In this fermentation, neither alcohol nor carbonic acid gas exists in those proportions in which they are found in fermentations produced by yeast; and although we may determine in it the presence of succinic acid, glycerine, and a small quantity of volatile acids,[133] the relative proportions of these substances will be different from what they are in the case of alcoholic fermentation.

§ III.—Reply to certain Critical Observations of the German Naturalists, Oscar Brefeld and Moritz Traube.

The essential point of the theory of fermentation, which we have been concerned in proving in preceding paragraphs, may be briefly put in the statement that ferments, properly so called, constitute a class of beings possessing the faculty of living out of contact with free oxygen; or, more concisely still, we may say, fermentation is a result of life without air.

If our affirmation were inexact, if ferment-cells did require for their growth or for their increase in number or weight, as all other vegetable cells do, the presence of oxygen, whether gaseous or held in solution in liquids, this new theory would lose all value, its very _raison d’être_ would be gone, at least as far as the most important part of fermentations is concerned. This is precisely what M. Oscar Brefeld has endeavoured to prove, in a Memoir read to the Physico-Medical Society of Wurzburg, on July 26th, 1873, in which, although we have ample evidence of the great experimental skill of its author, he has, nevertheless, in our opinion, arrived at conclusions entirely opposed to fact.

“From the experiments which I have just described,” he says, “it follows, in the most indisputable manner, that _a ferment cannot increase without free oxygen_. Pasteur’s supposition that a ferment, unlike all other living organisms, can live and increase at the expense of oxygen held in combination, is, consequently, altogether wanting in any solid basis of experimental proof. Moreover, since, according to the theory of Pasteur, it is precisely this faculty of living and increasing at the expense of the oxygen held in combination that constitutes the phenomenon of fermentation, it follows that the whole theory, commanding though it does such general assent, is shown to be untenable; it is simply inaccurate.”

The experiments to which Dr. Brefeld alludes, consisted in keeping under continued study with the microscope, in a room specially prepared for the purpose, one or more cells of ferment in wort, in an atmosphere of carbonic acid gas, free from the least traces of free oxygen. We have, however, recognized the fact that the increase of a ferment out of contact with air is only possible in the case of a very young specimen; but our author employed brewer’s yeast taken after fermentation, and to this fact we may attribute the non-success of his growths. Dr. Brefeld, without knowing it, operated on yeast in one of the states in which it requires gaseous oxygen to enable it to germinate again. A perusal of what we have previously written on the subject of the revival of yeast, according to its age, will show how widely the time required for such revival may vary in different cases. What may be perfectly true of the state of a yeast to-day may not be so to-morrow, since yeast is continually undergoing modifications. We have already shown the energy and activity with which a ferment can vegetate in the presence of free oxygen, and we have pointed out the great extent to which a very small quantity of oxygen held in solution in fermenting liquids can operate at the beginning of fermentation. It is this oxygen that produces revival in the cells of the ferment and enables them to resume the faculty of germinating and continuing their life, and of multiplying when deprived of air.

In our opinion, a simple reflection should have guarded Dr. Brefeld against the interpretation which he has attached to his observations. If a cell of ferment cannot bud or increase without absorbing oxygen, either free or held in solution in the liquid, the ratio between the weight of ferment formed during fermentation and that of oxygen used up must be constant. We had, however, clearly established, as far back as 1861, the fact that this ratio is extremely variable, a fact, moreover, which is placed beyond doubt by the experiments described in the preceding paragraph. Though but small quantities of oxygen are absorbed, a considerable weight of ferment may be generated; whilst if the ferment has abundance of oxygen at its disposal, it will absorb much, and the weight of yeast formed will be still greater. The ratio between the weight of ferment formed and that of sugar decomposed may pass through all stages between certain very wide limits, the variations depending on the greater or less absorption of free oxygen. And in this fact, we believe, lies one of the most essential supports of the theory which we advocate. In denouncing the impossibility, as he considered it, of a ferment living without air or oxygen, and so acting in defiance of that law which governs all living beings, animal or vegetable, Dr. Brefeld ought also to have borne in mind the fact which we have pointed out, that alcoholic yeast is not the only organized ferment which lives in an anaërobian state. It is really a small matter that one more ferment should be placed in a list of exceptions to the generality of living beings, for whom there is a rigid law in their vital economy which requires for continued life a continuous respiration, a continuous supply of free oxygen. Why, for instance, has Dr. Brefeld omitted the facts bearing on the life of the vibrios of butyric fermentation? Doubtless he thought we were equally mistaken in these: a few actual experiments would have put him right.

These remarks on the criticisms of Dr. Brefeld are also applicable to certain observations of M. Moritz Traube’s, although, as regards the principal object of Dr. Brefeld’s attack, we are indebted to M. Traube for our defence. This gentleman maintained the exactness of our results before the Chemical Society of Berlin, proving by fresh experiments that yeast is able to live and multiply without the intervention of oxygen. “My researches,” he said, “confirm in an indisputable manner M. Pasteur’s assertion that the multiplication of yeast can take place in media which contain no trace of free oxygen.... M. Brefeld’s assertion to the contrary is erroneous.” But, immediately afterwards, M. Traube adds: “Have we here a confirmation of Pasteur’s theory? By no means. The results of my experiments demonstrate, on the contrary, that this theory has no sure foundation.” What were these results? Whilst proving that yeast could live without air, M. Traube, as we ourselves did, found that it had great difficulty in living under these conditions; indeed he never succeeded in obtaining more than the first stages of true fermentation. This was doubtless for the two following reasons—first, in consequence of the accidental production of secondary and diseased fermentations, which frequently prevent the propagation of alcoholic ferment; and, secondly, in consequence of the original exhausted condition of the yeast employed. As long ago as 1861 we pointed out the slowness and difficulty of the vital action of yeast when deprived of air, and a little way back, in the preceding paragraph, we have called attention to certain fermentations that cannot be completed under such conditions without going into the causes of these peculiarities. M. Traube expresses himself thus: “Pasteur’s conclusion, that yeast in the absence of air is able to derive the oxygen necessary for its development from sugar, is erroneous; its increase is arrested, even when the greater part of the sugar still remains undecomposed. _It is in a mixture of albuminous substances that yeast, when deprived of air, finds the materials for its development._” This last assertion of M. Traube’s is entirely disproved by those fermentation experiments in which, after suppressing the presence of albuminous substances, the

## action, nevertheless, went on in a purely inorganic medium, out of

contact with air, a fact of which we shall give irrefutable proofs.[134]

§ IV.—Fermentation of Dextro-Tartrate of Lime.[135]

Tartrate of lime, in spite of its insolubility in water, is capable of complete fermentation in a mineral medium.

If we put some pure tartrate of lime, in the form of a granulated, crystalline powder, into pure water, together with some sulphate of ammonia and phosphates of potassium and magnesium, in very small proportions, a spontaneous fermentation will take place in the deposit in the course of a few days, although no germs of ferment have been added. A living, organized ferment, of the vibrionic type, filiform, with tortuous motions, and often of immense length, forms spontaneously by the development of some germs derived in some way from the inevitable

## particles of dust floating in the air or resting on the surface of the

vessels or materials which we employ. The germs of the vibrios concerned in putrefaction are diffused around us on every side, and, in all probability, it is one or more of these germs that develop in the medium in question. In this way they effect the decomposition of the tartrate, from which they must necessarily obtain the carbon of their food, without which they cannot exist, while the nitrogen is furnished by the ammonia of the ammoniacal salt, the mineral principles by the phosphate of potassium and magnesium, and the sulphur by the sulphate of ammonia. How strange to see organization, life, and motion originating under such conditions! Stranger still to think that this organization, life, and motion are effected without the participation of free oxygen. Once the germ gets a primary impulse on its living career by access of oxygen, it goes on reproducing indefinitely, absolutely without atmospheric air. Here then we have a fact which it is important to establish beyond the possibility of doubt, that we may prove that yeast is not the only organized ferment able to live and multiply when out of the influence of free oxygen.

Into a flask, like that represented in Fig. 67, of 2·5 litres (about four pints) in capacity, we put:—

Pure, crystallized, neutral tartrate of 100 grammes. lime

Phosphate of ammonia 1 gramme.

Phosphate of magnesium 1 gramme.

Phosphate of potassium 0·5 gramme.

Sulphate of ammonia 0·5 gramme.

(1 gramme=15·43 grains.)

To this we added pure distilled water, so as to entirely fill the flask.

In order to expel all the air dissolved in the water and adhering to the solid substances, we first placed our flask in a bath of chloride of calcium, in a large cylindrical white iron pot, set over a flame. The exit-tube of the flask was plunged in a test-tube of Bohemian glass three-quarters full of distilled water, and also heated by a flame. We boiled the liquids in the flask and test-tube for a sufficient time to expel all the air contained in them. We then withdrew the heat from under the test-tube, and immediately afterwards covered the water which it contained with a layer of oil, and then permitted the whole apparatus to cool down.

[Illustration: Fig. 67.]

Next day we applied a finger to the open extremity of the exit-tube, which we then plunged in a vessel of mercury. In this particular experiment which we are describing, we permitted the flask to remain in this state for a fortnight. It might have remained for a century without ever manifesting the least sign of fermentation, the fermentation of the tartrate being a consequence of life, and life after the boiling no longer existed in the flask. When it was evident that the contents of the flask were perfectly inert, we impregnated them rapidly, as follows:—All the liquid contained in the exit-tube was removed by means of a fine caoutchouc tube, and replaced by about 1 c.c. (about 17 minims) of liquid and deposit from another flask, similar to the one we have described, but which had been fermenting spontaneously for twelve days; we lost no time in refilling completely the exit-tube with water which had been first boiled and then cooled down in carbonic acid gas. This operation lasted only a few minutes. The exit-tube was again plunged under mercury. Subsequently the tube was not moved from under the mercury, and as it formed part of the flask, and there was neither cork nor india-rubber, any introduction of air was consequently impossible. The small quantity of air introduced during the impregnation was insignificant, and it might even be shown that it injured rather than assisted the growth of the organisms, inasmuch as these consisted of adult individuals which had lived without air and might be liable to be damaged or even destroyed by it. Be this as it may, in a subsequent experiment we shall find the possibility removed of any aeration taking place in this way, however infinitesimal, so that no doubt may linger on this subject.

The following days the organisms multiplied, the deposit of tartrate gradually disappeared, and a sensible ferment action was manifest on the surface, and throughout the bulk of the liquid. The deposit seemed lifted up in places, and was covered with a layer of a dark-grey colour, puffed up, and having an organic and gelatinous appearance. For several days, in spite of this action in the deposit, we detected no disengagement of gas, except when the flask was slightly shaken, in which case rather large bubbles adhering to the deposit rose, carrying with them some solid particles, which quickly fell back again, whilst the bubbles diminished in size as they rose, from being partially taken into solution, in consequence of the liquid not being saturated. The smallest bubbles had even time to dissolve completely before they could reach the surface of the liquid. In course of time the liquid was saturated, and the tartrate was gradually displaced by mammillated crusts, or clear, transparent crystals of carbonate of lime at the bottom and on the sides of the vessel.

The impregnation took place on February 10th, and on March 15th the liquid was nearly saturated. The bubbles then began to lodge in the bent part of the exit-tube, at the top of the flask. A glass measuring-tube containing mercury was now placed with its open end over the point of the exit-tube under the mercury in the trough, so that no bubble might escape. A steady evolution of gas went on from the 17th to the 18th, 17·4 c.c. (1·06 cubic inches) having been collected. This was proved to be nearly absolutely pure carbonic acid, as indeed might have been suspected from the fact that the evolution did not begin before a distinct saturation of the liquid was observed.[136]

The liquid, which was turbid on the day after its impregnation, had, in spite of the liberation of gas, again become so transparent that we could read our handwriting through the body of the flask. Notwithstanding this, there was still a very active operation going on in the deposit, but it was confined to that spot. Indeed, the swarming vibrios were bound to remain there, the tartrate of lime being still more insoluble in water saturated with carbonate of lime than it is in pure water. A supply of carbonaceous food, at all events, was absolutely wanting in the bulk of the liquid. Every day we continued to collect and analyze the total amount of gas disengaged. To the very last, it was composed of pure carbonic acid gas. Only during the first few days did the absorption by the concentrated potash leave a very minute residue. By April 26th all liberation of gas had ceased, the last bubbles having risen in the course of April 23rd. The flask had been all the time in the oven, at a temperature between 25° C. and 28° C. (77° F. and 83° F.). The total volume of gas collected was 2·135 litres (130·2 cubic inches). To obtain the whole volume of gas formed we had to add to this what was held in the liquid in the state of acid carbonate of lime. To determine this we poured a portion of the liquid from the flask into another flask of similar shape, but smaller, up to a gauge-mark on the neck.[137] This smaller flask had been previously filled with carbonic acid. The carbonic acid of the fermented liquid was then expelled by means of heat, and collected over mercury. In this way we found a volume of 8·322 litres (508 cubic inches) of gas in solution, which, added to 2·135 litres, gave a total of 10·457 litres (638·2 cubic inches) at 20° and 760, which calculated to 0° C. and 760 mm. atmospheric pressure (32° F. and 30 inches) gave a weight of 19·70 grammes (302·2 grains) of carbonic acid.

Exactly half of the lime of the tartrate employed got used up in the soluble salts formed during fermentation; the other half was partly precipitated in the form of carbonate of lime, partly dissolved in the liquid by the carbonic acid. The soluble salts seemed to us to be a mixture or combination of 1 equivalent of metacetate of lime, with 2 equivalents of the acetate, for every 10 equivalents of carbonic acid produced, the whole corresponding to the fermentation of 3 equivalents of neutral tartrate of lime.[138] This point, however, is worthy of being studied with greater care: the present statement of the nature of the products formed is given with all reserve. For our point, indeed, the matter is of little importance, since the equation of the fermentation does not concern us.

[Illustration: Fig. 68.]

After the completion of fermentation there was not a trace of tartrate of lime remaining at the bottom of the vessel: it had disappeared gradually as it got broken up into the different products of fermentation, and its place was taken by some crystallized carbonate of lime—the excess, namely, which had been unable to dissolve by the action of the carbonic acid. Associated, moreover, with this carbonate of lime there was a quantity of some kind of animal matter, which, under the microscope, appeared to be composed of masses of granules mixed with very fine filaments of varying lengths, studded with minute dots, and presenting all the characteristics of a nitrogenous organic substance.[139] That this was really the ferment is evident enough from all that we have already said. To convince ourselves more thoroughly of the fact, and at the same time to enable us to observe the mode of

## activity of the organism, we instituted the following supplementary

observation. Side by side with the experiment just described, we conducted a similar one, which we intermitted after the fermentation was somewhat advanced, and about half of the tartrate dissolved. Breaking off, with a file, the exit tube at the point where the neck began to narrow off, we took some of the deposit from the bottom by means of a long, straight piece of tubing, in order to bring it under microscopical examination. We found it to consist of a host of long filaments of extreme tenuity, their diameter being about 1/1000th of a millimetre (0·000039 in.); their length varied, in some cases being as much as 1/20th of a millimetre (0·0019 in.). A crowd of these long vibrios were to be seen creeping slowly along, with a sinuous movement, showing three, four, or even five flexures. The filaments that were at rest had the same aspect as these last, with the exception that they appeared punctate, as though composed of a series of granules arranged in irregular order. No doubt these were vibrios in which vital action had ceased, exhausted specimens which we may compare with the old granular ferment of beer, whilst those in motion may be compared with young and vigorous yeast. The absence of movement in the former seems to prove that this view is correct. Both kinds showed a tendency to form clusters, the compactness of which impeded the movements of those which were in motion. Moreover, it was noticeable that the masses of these latter rested on tartrate not yet dissolved, whilst the granular clusters of the others rested directly on the glass, at the bottom of the flask, as if, having decomposed the tartrate, the only carbonaceous food at their disposal, they had then died at the spot where we captured them from inability to escape, precisely in consequence of that state of entanglement which they combined to form, during the period of their

## active development. Besides these we observed vibrios of the same

diameter, but of much smaller length, whirling round with great rapidity, and darting backwards and forwards; these were probably identical with the longer ones, and possessed greater freedom of movement, no doubt in consequence of their greater shortness. Not one of these vibrios could be found throughout the mass of the liquid.

We may remark that as there was a somewhat putrid odour from the deposit in which the vibrios swarmed, the action must have been one of reduction, and no doubt to this fact was due the greyish coloration of the deposit. We suppose that the substances employed, however pure, always contain some trace of iron, which becomes converted into the sulphide, the black colour of which would modify the originally white deposit of insoluble tartrate and phosphate.

But what is the nature of these vibrios? We have already said that we believe that they are nothing but the ordinary vibrios of putrefaction, reduced to a state of extreme tenuity by the special conditions of nutrition involved in the fermentable medium used; in a word, we think that the fermentation in question might be called putrefaction of tartrate of lime. It would be easy enough to determine this point by growing the vibrios of such a fermentation in media adapted to the production of the ordinary forms of vibrio; but this is an experiment which we have not ourselves tried.

One word more on the subject of these curious beings. In a great many of them there appears to be something like a clear spot, a kind of bead, at one of their extremities. This is an illusion arising from the fact that the extremity of these vibrios is curved, hanging downwards, thus causing a greater refraction at that particular point, and leading us to think that the diameter is greater at that extremity. We may easily undeceive ourselves if we watch the movements of the vibrio, when we will readily recognize the bend, especially as it is brought into the vertical plane passing over the rest of the filament. In this way we will see the bright spot, the head disappear, and then reappear.

The chief inference that it concerns us to draw from the preceding facts is one which cannot admit of doubt, and which we need not insist on any further—namely, that vibrios, as met with in the fermentation of neutral tartrate of lime, are able to live and multiply when entirely deprived of air.

§ V.—Another Example of Life Without Air—Fermentation of Lactate of Lime.

As another example of life without air, accompanied by fermentation properly so called, we may lastly cite the fermentation of lactate of lime in a mineral medium.

In the experiment described in the last paragraph, it will be remembered that the ferment-liquid and the germs employed in its impregnation came in contact with air, although only for a very brief time. Now, notwithstanding that we possess exact observations which prove that the diffusion of oxygen and nitrogen in a liquid absolutely deprived of air, so far from taking place rapidly, is, on the contrary, a very slow process indeed; yet we were anxious to guard the experiment that we are about to describe from the slightest possible trace of oxygen at the moment of impregnation.

We employed a liquid prepared as follows: Into from 9 to 10 litres (somewhat over 2 gallons) of pure water the following salts[140] were introduced successively, viz:—

Pure lactate of lime 225 grammes Phosphate of ammonia 0·75 grammes Phosphate of potassium 0·4 grammes Sulphate of magnesium 0·4 grammes Sulphate of ammonia 0·2 grammes [1 gramme=15·43 grains.]

[Illustration: Fig. 69.]

On March 23rd, 1875, we filled a 6 litre (about 11 pints) flask, of the shape represented in Fig. 69, and placed it over a heater. Another flame was placed below a vessel containing the same liquid, into which the curved tube of the flask was plunged. The liquids in the flask and in the basin were raised to boiling together, and kept in this condition for more than half-an-hour, so as to expel all the air held in solution. The liquid was several times forced out of the flask by the steam, and sucked back again; but the portion which re-entered the flask was always boiling. On the following day, when the flask had cooled, we transferred the end of the delivery tube to a vessel full of mercury and placed the whole apparatus in an oven at a temperature varying between 25° C. and 30° C. (77° F. and 86° F.); then, after having refilled the small cylindrical tap-funnel with carbonic acid, we passed into it with all necessary precautions 10 c.c. (0·35 fl. oz.) of a liquid similar to that described, which had been already in active fermentation for several days out of contact with air and now swarmed with vibrios. We then turned the tap of the funnel, until only a small quantity of liquid was left, just enough to prevent the access of air. In this way the impregnation was accomplished without either the ferment-liquid or the ferment-germs having been brought in contact, even for the shortest space, with the external air. The fermentation, the occurrence of which at an earlier or later period depends for the most part on the condition of the impregnating germs, and the number introduced in the act, in this case began to manifest itself by the appearance of minute bubbles from March 29th. But not till April 9th did we observe bubbles of larger size rise to the surface. From that date onward they continued to come in increasing number, from certain points at the bottom of the flask, where a deposit of earthy phosphates existed; and at the same time the liquid, which for the first few days remained perfectly clear, began to grow turbid in consequence of the development of vibrios. It was on the same day that we first observed a deposit on the sides of carbonate of lime in crystals.

It is a matter of some interest to notice here that, in the mode of procedure adopted, everything combined to prevent the interference of air. A portion of the liquid expelled at the beginning of the experiment, partly because of the increased temperature in the oven and

## partly also by the force of the gas, as it began to be evolved from the

fermentative action, reached the surface of the mercury, where, being the most suitable medium we know for the growth of bacteria, it speedily swarmed with these organisms.[141] In this way any passage of air, if such a thing were possible, between the mercury and the sides of the delivery-tube was altogether prevented, since the bacteria would consume every trace of oxygen which might be dissolved in the liquid lying on the surface of the mercury. Hence it is impossible to imagine that the slightest trace of oxygen could have got into the liquid in the flask.

Before passing on we may remark that in this ready absorption of oxygen by bacteria we have a means of depriving fermentable liquids of every trace of that gas with a facility and success equal or even greater than by the method of preliminary boiling. Such a solution as we have described, if kept at summer heat, without any previous boiling, becomes turbid in the course of twenty-four hours from a _spontaneous_ development of bacteria; and it is easy to prove that they absorb all the oxygen held in solution.[142] If we completely fill a flask of a few litres capacity (about a gallon) (Fig. 67) with the liquid described, taking care to have the delivery-tube also filled, and its opening plunged under mercury, and, forty-eight hours afterwards, by means of a chloride of calcium bath, expel from the liquid on the surface of the mercury all the gas which it holds in solution, this gas, when analyzed, will be found to be composed of a mixture of nitrogen and carbonic acid gas, _without the least trace of oxygen_. Here, then, we have an excellent means of depriving the fermentable liquid of air; we have simply to completely fill a flask with the liquid, and place it in the oven, merely avoiding any addition of butyric vibrios before the lapse of two or three days. We may wait even longer; and then, if the liquid does not become impregnated spontaneously with vibrio germs, the liquid, which at first was turbid from the presence of bacteria, will become bright again, since the bacteria when deprived of life, or, at least, of the power of moving, after they have exhausted all the oxygen in solution, will fall inert to the bottom of the vessel. On several occasions, we have determined this interesting fact, which tends to prove that the butyric vibrios cannot be regarded as another form of bacteria, inasmuch as, on the hypothesis of an original relation between the two productions, butyric fermentation ought in every case to follow the growth of bacteria.

We may also call attention to another striking experiment, well suited to show the effect of differences in the composition of the medium upon the propagation of microscopic beings. The fermentation which we last described commenced on March 27th and continued until May 10th; that to which we are now to refer, however, was completed in four days, the liquid employed being similar in composition and quantity to that employed in the former experiment. On April 23rd, 1875, we filled a flask of the same shape as that represented in Fig. 69, and of similar capacity, viz., 6 litres, with a liquid composed as described at page 293. This liquid had been previously left to itself for five days in large open flasks, in consequence of which it had developed an abundant growth of bacteria. On the fifth day a few bubbles, rising from the bottom of the vessels, at long intervals, betokened the commencement of butyric fermentation, a fact, moreover, confirmed by the microscope, in the appearance of the vibrios of this fermentation in specimens of the liquid taken from the bottom of the vessels, the middle of its mass, and even in the layer on the surface that was swarming with bacteria. We transferred the liquid so prepared to the 6-litre flask arranged over the mercury. By evening a tolerably active fermentation had begun to manifest itself. On the 24th this fermentation was proceeding with astonishing rapidity, which continued during the 25th and 26th. During the evening of the 26th it slackened, and on the 27th all signs of fermentation had ceased. This was not, as might be supposed, a sudden stoppage, due to some unknown cause; the fermentation was actually completed, for when we examined the fermented liquid on the 28th we could not find the smallest quantity of lactate of lime. If the needs of industry should ever require the production of large quantities of butyric acid, there would, beyond doubt, be found in the preceding fact valuable information in devising an easy method of preparing that product in abundance.[143]

Before we go any further, let us devote some attention to the vibrios of the preceding fermentations.

On May 27th, 1862, we completely filled a flask, capable of holding 2·780 litres (about five pints), with the solution of lactate and phosphates.[144] We refrained from impregnating it with any germs. The liquid became turbid from a development of bacteria, and then underwent butyric fermentation. By June 9th the fermentation had become sufficiently active to enable us to collect in the course of twenty-four hours, over mercury, as in all our experiments, about 100 cc. (about 6 cubic inches) of gas. By June 11th, judging from the volume of gas liberated in the course of twenty-four hours, the activity of the fermentation had doubled. We examined a drop of the turbid liquid. Here are the notes accompanying the sketch (Fig. 70) as they stand in our note-book:—“A swarm of vibrios, so active in their movements that the eye has great difficulty in following them. They may be seen in pairs throughout the field, apparently making efforts to separate from each other. The connection would seem to be by some invisible, gelatinous thread, which yields so far to their efforts that they succeed in breaking away from actual contact, but yet are, for a while, so far restrained that the movements of one have a visible effect on those of the other. By and by, however, we see a complete separation effected, and each moves on its separate way with an activity still greater than it had before.”

[Illustration: Fig. 70.]

One of the best methods that can be employed for the microscopical examination of these vibrios, quite out of contact with air, is the following:—After butyric fermentation has been going on for several days in a flask, A (Fig. 71), we connect this flask by an india-rubber tube with one of the flattened bulbs previously described, page 156 (Fig. 31), which we then place on the stage of the microscope (Fig. 71). When we wish to make an observation we close, under the mercury, at the point _b_, the end of the drawn-out and bent delivery-tube. The continued evolution of gas soon exerts such a pressure within the flask, that when we open the tap _r_, the liquid is driven into the bulb _l l_, until it becomes quite full and the liquid flows over into the glass V. In this manner we may bring the vibrios under observation without their coming into contact with the least trace of air, and with as much success as if the bulb, which takes the place of an object glass, had been plunged into the very centre of the flask. The movements and fissiparous multiplication of the vibrios may thus be seen in all their beauty, and it is indeed a most interesting sight. The movements do not immediately cease when the temperature is suddenly lowered, even to a considerable extent, 15° C. (59° F.) for example; they are only slackened. Nevertheless, it is better to observe them at the temperatures most favourable to fermentation, even in the oven where the vessels employed in the experiment are kept at a temperature between 25° C. and 30° C. (77° F. and 86° F.).

[Illustration: Fig. 71.]

We may now continue our account of the fermentation which we were studying when we made this last digression. On June 17th that fermentation produced three times as much gas as it did on June 11th, when the residue of hydrogen, after absorption by potash, was 72·6 per cent.; whilst on the 17th it was only 49·2 per cent. Let us again discuss the microscopic aspect of the turbid liquid at this stage. Appended is the sketch we made (Fig. 72) and our notes on it:—“A most beautiful object: vibrios all in motion, advancing or undulating. They have grown considerably in bulk and length since the 11th; many of them are joined together into long sinuous chains, very mobile at the articulations, visibly less active and more wavering in proportion to the number that go to form the chain, or the length of the individuals.” This description is applicable to the majority of the vibrios which occur in cylindrical rods and are homogeneous in aspect. There are others, of rare occurrence in chains, which have a clear corpuscle, that is to say, a portion more refractive than the other parts of the segments, at one of their extremities. Sometimes the foremost segment has the corpuscle at one end, sometimes at the other. The long segments of the commoner kind attain a length of from 10 to 30 and even 45 thousandths of a millimetre. Their diameter is from 1-½ to 2, very rarely 3, thousandths of a millimetre.[145]

[Illustration: Fig. 72.]

[Illustration: Fig. 73.]

On June 28th, fermentation was quite finished; there was no longer any trace of gas, nor any lactate in solution. All the infusoria were lying motionless at the bottom of the flask. The liquid clarified by degrees, and in the course of a few days became quite bright. Here we may inquire, were these motionless infusoria, which from complete exhaustion of the lactate, the source of the carbonaceous part of their food, were now lying inert at the bottom of the fermenting vessel—were they dead beyond power of revival?[146] The following experiment leads us to believe that they were not perfectly lifeless, and that they behave in the same manner as the yeast of beer, which, after it has decomposed all the sugar in a fermentable liquid, is ready to revive and multiply in a fresh saccharine medium. On April 22nd, 1875, we left in the oven, at a temperature of 25° C. (77° F.), a fermentation of lactate of lime that had been completed. The delivery tube of the flask, A, (Fig. 73) in which it had taken place had never been withdrawn from under the mercury. We kept the liquid under observation daily, and saw it gradually become brighter; this went on for fifteen days. We then filled a similar flask, B, with the solution of lactate, which we boiled, not only to kill the germs of vibrios which the liquid might contain, but also to expel the air that it held in solution. When the flask, B, had cooled, we connected the two flasks, avoiding the introduction of air[147], after having slightly shaken the flask, A, to stir up the deposit at the bottom. There was then a pressure, due to carbonic acid at the end of the delivery tube of this latter flask, at the point _a_, so that on opening the taps _r_ and _s_, the deposit at the bottom of flask A was driven over into flask B, which in consequence was impregnated with the deposit of a fermentation that had been completed fifteen days before. Two days after impregnation, the flask B began to show signs of fermentation. It follows, that the deposit of vibrios of a completed butyric fermentation may be kept, at least for a certain time, without losing the power of causing fermentation. It furnishes a butyric ferment, capable of revival and action in a suitable, fresh, fermentable medium.

The reader who has attentively studied the facts which we have placed before him cannot, in our opinion, entertain the least doubt on the subject of the possible multiplication of the vibrios of a fermentation of lactate of lime out of contact with atmospheric oxygen. If fresh proofs of this important proposition were necessary, they might be found in the following observations, from which it may be inferred that atmospheric oxygen is capable of suddenly checking a fermentation produced by butyric vibrios, and rendering them absolutely motionless, so that it cannot be necessary to enable them to live. On May 7th, 1862, we placed in the oven a flask holding 2·580 litres (4-½ pints), and filled with the solution of lactate of lime and phosphates, which we had impregnated on the 9th with two drops of a liquid in butyric fermentation. In the course of a few days fermentation declared itself: on the 16th it was in progress, but feebly; on the 18th it was active; on the 30th it was very active. On June 1st it yielded hourly 35 c.c. (2·3 cubic inches) of gas, containing ten per cent. of hydrogen. On the 2nd we began the study of the action of air on the vibrios of this fermentation. To do this we cut off the delivery-tube on a level with its point of junction to the flask, then with a 50 c.c. pipette we took out that quantity (1-3/4 fl. oz.) of liquid which was, of course, replaced at once by air. We then reversed the flask with the opening under the mercury, and shook it every ten minutes for more than an hour. Wishing to make sure, to begin with, that the oxygen had been absorbed, we connected under the mercury the beak of the flask by means of a thin india-rubber tube filled with water, with a small flask, the neck of which had been drawn out, and was filled with water; we then raised the large flask with the smaller kept above it. A Mohr’s clip, which closed the india-rubber tube, and which we then opened, permitted the water contained in the small flask to pass into the large one, whilst the gas, on the contrary, passed upwards from the large flask into the small one. We analyzed the gas immediately, and found that, allowing for carbonic acid and hydrogen, it did not contain more than 14·2 per cent. of oxygen, which corresponds to an absorption of 6·6 c.c., or of 3·3 c.c. (0·2 cubic inch) of oxygen for the 50 c.c. (3·05 cubic inches) of air employed. Lastly, we again established connection by an india-rubber tube between the flasks, after having seen by microscopical examination that the movements of the vibrios were very languid. Fermentation had become less vigorous without having actually ceased, no doubt because some portions of the liquid had not been brought into contact with the atmospheric oxygen, in spite of the prolonged shaking that the flask had undergone after the introduction of the air. Whatever the cause might have been, the significance of the phenomenon is not doubtful. To assure ourselves further of the effect of air on the vibrios, we half filled two test tubes with the fermenting liquid taken from another fermentation which had also attained its maximum of intensity, into one of which we passed a current of air, into the other carbonic acid gas. In the course of half an hour, all the vibrios in the aerated tube were dead, or at least motionless, and fermentation had ceased. In the other tube, after three hours’ exposure to the effects of the carbonic acid gas, the vibrios were still very active, and fermentation was going on.

There is a most simple method of observing the deadly effect of atmospheric air upon vibrios. We have seen in the microscopical examination made by means of the apparatus represented in Fig. 71, how remarkable were the movements of the vibrios when absolutely deprived of air, and how easy it was to discern them. We will repeat this observation, and at the same time make a comparative study of the same liquid, under the microscope, in the ordinary way, that is to say, by placing a drop of the liquid on an object-glass, and covering it with a thin glass slip, a method which must necessarily bring the drop into contact with air, if only for a moment. It is surprising what a remarkable difference is observed immediately between the movements of the vibrios in the bulb and of those under the glass. In the case of the latter we generally see all movement at once cease near the edges of the glass, where the drop of liquid is in direct contact with the air; the movements continue for a longer or shorter time about the centre, in proportion as the air is more or less intercepted by the vibrios at the circumference of the liquid. It does not require much skill in experiments of this kind to enable one to see plainly that immediately after the glass has been placed on the drop, which has been affected all over by atmospheric air, the whole of the vibrios seem to languish and to manifest symptoms of illness—we can think of no better expression to explain what we see taking place—and that they gradually recover their

## activity about the centre, in proportion as they find themselves in a

part of the medium that is less affected by the presence of oxygen.

Some most curious facts are to be found in connection with an observation, the correlative and inverse of the foregoing, on the ordinary aërobian bacteria. If we examine below the microscope a drop of liquid full of these organisms under a coverslip, we very soon observe a cessation of motion in all the bacteria which lie in the central portion of the liquid, where the oxygen rapidly disappears to supply the necessities of the bacteria existing there; whilst, on the other hand, near the edges of the cover-glass the movements are very active, in consequence of the constant supply of air. In spite of the speedy death of the bacteria beneath the centre of the glass, we see life prolonged there if by chance a bubble of air has been enclosed. All round this bubble a vast number of bacteria collect in a thick, moving circle, but as soon as all the oxygen of the bubble has been absorbed they fall apparently lifeless, and are scattered by the movement of the liquid.[148]

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

We may pause a moment to consider an interesting question in reference to the two characters under which vibrios appear in butyric fermentations. What is the reason that some vibrios exhibit refractive corpuscles, generally of a lenticular form, such as we see in Fig. 72? We are strongly inclined to believe that these corpuscles have to do with a special mode of reproduction in the vibrios, common alike to the anaërobian forms which we are studying, and the ordinary aërobian forms in which also the corpuscles of which we are speaking may occur. The explanation of the phenomenon, from our point of view, would be that, after a certain number of fissiparous generations, and under the influence of variations in the composition of the medium, which is constantly changing through fermentation as well as through the active life of the vibrios themselves, cysts, which are simply the refractive corpuscles, form along them at different points. From these gemmules we have ultimately produced vibrios, ready to reproduce others by the process of transverse division for a certain time, to be themselves encysted later on. Various observations incline us to believe that, in their ordinary form of minute, soft, exuberant rods, the vibrios perish when submitted to desiccation, but when they occur in the corpuscular or encysted form they possess unusual powers of resistance, and may be brought to the state of dry dust and be wafted about by winds. None of the matter which surrounds the corpuscle or cyst seems to take part in the preservation of the germ, when the cyst is formed, for it is all re-absorbed, gradually leaving the cyst bare. The cysts appear as masses of corpuscles, in which the most practised eye cannot detect anything of an organic nature, or anything to remind one of the vibrios which produced them; nevertheless, these minute bodies are endowed with a latent vital action, and only await favourable conditions to develop long rods of vibrios. We are not, it is true, in a position to adduce any very forcible proofs in support of these opinions. They have been suggested to us by experiments, none of which, however, have been absolutely decisive in their favour. We may cite one of our observations on this subject.

In a fermentation of glycerine in a mineral medium—the glycerine was fermenting under the influence of butyric vibrios—after we had determined the, we may say, exclusive presence of lenticular vibrios, with refractive corpuscles, we observed the fermentation, which, for some unknown reason, had been very languid, suddenly become extremely

## active, but now through the influence of ordinary vibrios. The gemmules

with brilliant corpuscles had almost disappeared; we could see but very few, and those now consisted of the refractive bodies alone, the bulk of the vibrios accompanying them having undergone some process of re-absorption.

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

It is a question whether differences in the aspect and nature of vibrios, which depend upon their more or less advanced age, or are occasioned by the influence of certain conditions of the medium in which they propagate, do not bring about corresponding changes in the course of the fermentation and the nature of its products. Judging at least from the variations in the proportions of hydrogen and carbonic acid gas produced in butyric fermentations, we are inclined to think that this must be the case; nay, more, we find that hydrogen is not even a constant product in these fermentations. We have met with butyric fermentations of lactate of lime which did not yield the minutest trace of hydrogen, or anything besides carbonic acid. Fig. 74 represents the vibrios which we observed in a fermentation of this kind. They present no special features. Butyl alcohol is, according to our observations, an ordinary product, although it varies and is by no means a necessary concomitant of these fermentations. It might be supposed, since butylic alcohol may be produced, and hydrogen be in deficit, that the proportion of the former of these products would attain its maximum when the latter assumed a minimum. This, however, is by no means the case; even in those few fermentations that we have met with in which hydrogen was absent, there was no formation of butylic alcohol.

[Illustration: Fig. 74.]

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

We are compelled here to admit that vibrios frequently abound in liquids exposed to the air, and that they appropriate the atmospheric oxygen, and could not withstand a sudden removal from its influence. Must we, then, believe that such vibrios are absolutely different from those of butyric fermentations? It would, perhaps, be more natural to admit that in the one case there is an adaptation to life with air, and in the other case an adaptation to life without air; each of these varieties perishing when suddenly transferred from its habitual condition to that of the other, whilst by a series of progressive changes one might be modified into the other.[149] We know that in the case of alcoholic ferments, although these can actually live without air, propagation is wonderfully assisted by the presence of minute quantities of air; and certain experiments, which we have not yet published, lead us to believe that, after having lived without air, they cannot be suddenly exposed with impunity to the influence of large quantities of oxygen.

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

The question has been raised whether vibrios, especially those which we have shown to be the ferment of butyric and many other fermentations, are, in their nature, animal or vegetable. M. Ch. Robin attaches great importance to the solution of this question, of which he speaks as follows[150]:—“The determination of the nature, whether animal or vegetable, of organisms, either as a whole or in respect to their anatomical parts, assimilative or reproductive, is a problem which has been capable of solution for a quarter of a century. The method has been brought to a state of remarkable precision, experimentally, as well as in its theoretical aspects, since those who devote their attention to the organic sciences consider it indispensable in every observation and experiment to determine accurately, before anything else, whether the object of their study is animal or vegetable in its nature, whether adult or otherwise. To neglect this is as serious an omission for such students, as for chemists would be the neglecting to determine whether it is nitrogen or hydrogen, urea or stearine that has been extracted from a tissue, or which it is whose combinations they are studying in this or that chemical operation. Now, scarcely any one of those who study fermentations, properly so called, and putrefactions, ever pay attention to the preceding data.... Among the observers to whom I allude even M. Pasteur is to be found, who, even in his most recent communications, omits to state definitely what is the nature of many of the ferments which he has studied, with the exception, however, of those which belong to the cryptogamic group called _torulaceæ_. Various passages in his works seem to show that he considers the cryptogamic organisms called _bacteria_, as well as those known as _vibrios_, as belonging to the animal kingdom (see _Bulletin de l’Académie de Médecine_, Paris, 1875, pp. 249, 251, especially 256, 266, 267, 289, and 290). These would be very different, at least physiologically, the former being _aërobian_, whilst the vibrios are _anaërobian_, that is to say, requiring no air to enable them to live, and being killed by oxygen, should it be dissolved in the liquid to any considerable extent.”

We are unable to see the matter in the same light as our learned colleague does; to our thinking, we should be labouring under a great delusion were we to suppose “that it is quite as serious an omission not to determine the animal or vegetable nature of a ferment as it would be to confound nitrogen with hydrogen, or urea with stearine.” The importance of the solutions of disputed questions often depends upon the point of view from which these are regarded. As far as the result of our labours is concerned, we devoted our attention to these two questions exclusively:—1. Is the ferment, in every fermentation properly so called, an organized being? 2. Can this organized being live without air? Now, what bearing can the question of the animal or vegetable nature of the ferment, of the organized being, have upon the investigation of these two problems? In studying butyric fermentation, for example, we endeavoured to establish these two fundamental points:—1. _The butyric ferment is a vibrio._ 2. _This vibrio may dispense with air in its life, and, as a matter of fact, does dispense with it in the act of producing butyric fermentation._ We did not consider it at all necessary to pronounce any opinion as to the animal or vegetable nature of this organism, and, even up to the present moment, the idea that vibrio is an animal and not a plant is, in our minds, a matter of sentiment rather than of conviction.

M. Robin, however, would have no difficulty in determining the limits of the two kingdoms. According to him, “every variety of cellulose is, we may say, insoluble in ammonia, as also are the reproductive elements of plants, whether male or female. Whatever phase of evolution the elements which reproduce a new individual may have reached, treatment with this reagent, either cold or raised to boiling, leaves them absolutely intact under the eyes of the observer, except that their contents, from being

## partially dissolved, become more transparent. Every vegetable, whether

microscopic or not, every mycelium, and every spore thus preserves in its entirety its special characteristics of form, volume, and structural arrangements; whilst in the case of microscopic animals, or the ova and microscopic embryos of different members of the animal kingdom, the very opposite is the case.”

We should be glad to learn that the employment of a drop of ammonia would enable us to pronounce an opinion, with this degree of confidence, on the nature of the lowest microscopic beings; but is M. Robin absolutely correct in his assumptions? That gentleman himself remarks that spermatozoa, which belong to animal organisms, are insoluble in ammonia, the effect of which is merely to make them paler. If a difference of action in certain reagents, in ammonia, for example, were sufficient to determine the limits of the animal and vegetable kingdoms, might we not argue that there must be a very great and natural difference between moulds and bacteria, inasmuch as the presence of a small quantity of acid in the nutritive medium facilitates the growth and propagation of the former, whilst it is able to prevent the life of bacteria and vibrios? Although, as is well known, movement is not an exclusive characteristic of animals, yet we have always been inclined to regard vibrios as animals, on account of the peculiar character of their movements. How greatly they differ in this respect from the diatomacæ, for example! When the vibrio encounters an obstacle it turns, or after having assured itself by some visual effort or other that it cannot overcome it, it retraces its steps. The colpoda—undoubted infusoria—behave in an exactly similar manner. It is true one may argue that the zoospores of certain cryptogamia exhibit similar movements; but do not these zoospores possess as much of an animal nature as do the spermatozoa? As far as bacteria are concerned, when, as already remarked, we see them crowd round a bubble of air in a liquid to prolong their life, oxygen having failed them everywhere else, how can we avoid believing that they are animated by an instinct for life, of the same kind as that which we find in animals. M. Robin seems to us to be wrong in supposing that it is possible to draw any absolute line of separation between the animal and vegetable kingdoms. The settlement of this line, however, we repeat again, no matter what it may be, has no serious bearing upon the questions that have been the subject of our researches.

In like manner the difficulty which M. Robin has raised in objecting to the employment of the word _germ_, when we cannot specify whether the nature of that germ is animal or vegetable, is in many respects an unnecessary one. In all the questions which we have discussed, whether we were speaking of fermentation or spontaneous generation, the word _germ_ has been used in the sense of _origin of living organism_. If Liebig, for example, said of an albuminous substance that it gave birth to ferment, could we contradict him more plainly than by replying: “No; ferment is an organized being, the germ of which is always present, and the albuminous substance merely serves by its occurrence to nourish the germ and its successive generations.”

In our Memoir of 1862, on so-called _spontaneous_ generations, would it not have been an entire mistake to have attempted to assign specific names to the microscopic organisms which we met with in the course of our observations? Not only would we have met with extreme difficulty in the attempt, arising from the state of extreme confusion which even in the present day exists in the classification and nomenclature of these microscopic organisms, but we should have been forced to sacrifice clearness in our work besides; at all events, we should have wandered from our principal object, which was the determination of the presence or absence of life in general, and had nothing to do with the manifestation of a particular kind of life in this or that species, animal or vegetable. Thus we have systematically employed the vaguest nomenclature, such as _mucors_, _torulæ_, _bacteria_, and _vibrios_. There was nothing arbitrary in our doing this, whereas there is much that is arbitrary in adopting a definite system of nomenclature, and applying it to organisms but imperfectly known, the differences or resemblances between which are only recognizable through certain characteristics, the true signification of which is obscure. Take, for example, the extensive array of widely different systems that have been invented during the last few years for the species of the genera bacterium and vibrio in the works of Cohn, H. Hoffmann, Hallier, and Billroth. The confusion which prevails here is very great, although we do not of course by any means place these different works on the same footing as regards their respective merits.

M. Robin is, however, right in recognizing the impossibility of maintaining in the present day, as he formerly did, “that fermentation is an exterior phenomenon, going on outside cryptogamic cells, a phenomenon of contact. It is probably,” he adds, “an interior and molecular action at work in the inmost recesses of the substance of each cell.” From the day when we first proved that it is possible for all organized ferments, properly so called, to spring up and multiply from their respective germs, sown, whether consciously or by accident, in a mineral medium free from organic and nitrogenous matters other than ammonia, in which medium the fermentable matter alone is adapted to provide the ferment with whatever carbon enters into its composition, from that time forward the theories of Liebig, as well as that of Berzelius, which M. Robin formerly defended, have had to give place to others more in harmony with facts. We trust that the day will come when M. Robin will likewise acknowledge that he has been in error on the subject of the doctrine of spontaneous generation, which he continues to affirm, without adducing any direct proofs in support of it, at the end of the article to which we have been here replying.

We have devoted the greater part of this chapter to the establishing with all possible exactness the extremely important physiological fact of life without air, and its correlation to the phenomena of fermentations properly so called—that is to say, of those which are due to the presence of microscopic cellular organisms. This is the chief basis of the new theory that we propose for the explanation of these phenomena. The details into which we have entered were indispensable on account of the novelty of the subject no less than on account of the necessity we were under of combating the criticisms of the two German naturalists, Drs. Oscar Brefeld and Traube, whose works had cast some doubts on the correctness of the facts upon which we had based the preceding propositions. We have much pleasure in adding that at the very moment when we were revising the proofs of this chapter, we received from M. Brefeld an essay, dated from Berlin, January, 1876, in which, after describing his later experimental researches, he owns with praiseworthy frankness that Dr. Traube and he were both of them mistaken. Life without air is now a proposition which he accepts as perfectly demonstrated. He has witnessed it in the case of _mucor racemosus_, and has also verified it in the case of yeast. “If,” he says, “after the results of my previous researches, which I conducted with all possible exactness, I was inclined to consider Pasteur’s assertions as inaccurate, and to attack them, I have no hesitation now in recognizing them as true, and in proclaiming the service which Pasteur has rendered to science in being the first to indicate the exact relation of things in the phenomenon of fermentation.” In his later researches. Dr. Brefeld has adopted the method which we have long employed for demonstrating the life and multiplication of butyric vibrios in the entire absence of air, as well as the method of conducting growths in mineral media associated with the fermentable substance. We need not pause to consider certain other secondary criticisms of Dr. Brefeld. A perusal of the present work will, we trust, convince him that they are based on no surer foundation than were his former criticisms.

To bring one’s self to believe in a truth that has just dawned upon one is the first step towards progress; to persuade others is the second. There is a third step, less useful perhaps, but highly gratifying nevertheless, which is, to convince one’s opponents.

We, therefore, have experienced great satisfaction in learning that we have won over to our ideas an observer of singular ability, on a subject which is of the utmost importance to the physiology of cells.

§ VI.—Reply to the Critical Observations of Liebig, Published in 1870.[151]

In the Memoir which we published, in 1860, on alcoholic fermentation, and in several subsequent works, we were led to a different conclusion on the causes of this very remarkable phenomenon from that which Liebig had adopted. The opinions of Mitscherlich and Berzelius had ceased to be tenable in the presence of the new facts which we had brought to light. From that time we felt sure that the celebrated chemist of Munich had adopted our conclusions, from the fact that he remained silent on this question for a long time, although it had been until then the constant subject of his study, as is shown by all his works. Suddenly there appeared in the _Annales de Chimie et de Physique_ a long essay, reproduced from a lecture delivered by him before the Academy of Bavaria in 1868 and 1869. In this Liebig again maintained, not, however, without certain modifications, the views which he had expressed in his former publications, and disputed the correctness of the principal facts enunciated in our Memoir of 1860, on which were based the arguments against his theory.

“I had admitted,” he says, “that the resolution of fermentable matter into compounds of a simpler kind must be traced to some process of decomposition taking place in the ferment, and that the action of this same ferment on the fermentable matter must continue or cease according to the prolongation or cessation of the alteration produced in the ferment. The molecular change in the sugar would, consequently, be brought about by the destruction or modification of one or more of the component parts of the ferment, and could only take place through the contact of the two substances. M. Pasteur regards fermentation in the following light:—The chemical action of fermentation is essentially a phenomenon correlative with a vital action, beginning and ending with it. He believes that alcoholic fermentation can never occur without the simultaneous occurrence of organization, development, and multiplication of globules, or continuous life, carried on from globules already formed. But the idea that the decomposition of sugar during fermentation is due to the development of the cellules of the ferment, is in contradiction with the fact that the ferment is able to bring about the fermentation of a pure solution of sugar. The greater part of the ferment is composed of a substance that is rich in nitrogen and contains sulphur. It contains, moreover, an appreciable quantity of phosphates, hence it is difficult to conceive how, in the absence of these elements in a pure solution of sugar undergoing fermentation, the number of cells is capable of any increase.”

Notwithstanding Liebig’s belief to the contrary, the idea that the decomposition of sugar during fermentation is intimately connected with a development of the cellules of the ferment, or a prolongation of the life of cellules already formed, is in no way opposed to the fact that the ferment is capable of bringing about the fermentation of a pure solution of sugar. It is manifest to any one who has studied such fermentation with the microscope, even in those cases where the sweetened water has been absolutely pure, that ferment-cells do multiply, the reason being that the cells carry with them all the food-supplies necessary for the life of the ferment. They may be observed budding, at least many of them, and there can be no doubt that those which do not bud still continue to live; life has other ways of manifesting itself besides development and cell-proliferation.

If we refer to the figures on page 81 of our Memoir of 1860, Experiments D, E, F, G, H, I, we shall see that the weight of yeast, in the case of the fermentation of a pure solution of sugar, undergoes a considerable increase, even without taking into account the fact that the sugared water gains from the yeast certain soluble parts, since, in the experiments just mentioned, the weights of solid yeast, washed and dried at 100° C. (212° F.), are much greater than those of the raw yeast employed, dried at the same temperature.

In these experiments we employed the following weights of yeast, expressed in grammes (1 gramme = 15·43 grains)—

2·313 2·626 1·198 0·699 0·326 0·476

which became after fermentation, we repeat, without taking into account the matters which the sugared water gained from the yeast—

grammes. grains.

2·486 [Increase 0·173 = 2·65 2·963 “ 0·337 = 5·16 1·700 ” 0·502 = 7·7 0·712 “ 0·013 = 0·2 0·335 ” 0·009 = 0·14 0·590 “ 0·114 = 1·75

Have we not in this marked increase in weight a proof of life, or, to adopt an expression which may be preferred, a proof of a profound chemical work of nutrition and assimilation?

We may cite on this subject one of our earlier experiments, which is to be found in the _Comptes rendus de l’Académie_ for the year 1857, and which clearly shows the great influence exerted on fermentation by the soluble portion that the sugared water takes up from the globules of ferment:—

“We take two equal quantities of fresh yeast that have been washed very freely. One of these we cause to ferment in water containing nothing but sugar, and, after removing from the other all its soluble particles—by boiling it in an excess of water and then filtering it to separate the globules—we add to the filtered liquid as much sugar as was used in the first case along with a mere trace of fresh yeast, insufficient, as far as its weight is concerned, to affect the results of our experiment. The globules which we have sown bud, the liquid becomes turbid, a deposit of yeast gradually forms, and, side by side with these appearances, the decomposition of the sugar is effected, and in the course of a few hours manifests itself clearly. These results are such as we might have anticipated. The following fact, however, is of importance. In effecting by these means the organization into globules of the soluble part of the yeast that we used in the second case, we find that a considerable quantity of sugar is decomposed. The following are the results of our experiment: 5 grammes of yeast caused the fermentation of 12·9 grammes of sugar in six days, at the end of which time it was exhausted. The soluble portion of a like quantity of 5 grammes of the same yeast caused the fermentation of 10 grammes of sugar in nine days, after which the yeast developed by the sowing was likewise exhausted.”

How is it possible to maintain that, in the fermentation of water containing nothing but sugar, the soluble portion of the yeast does not act, either in the production of new globules or the perfection of old ones, when we see, in the preceding experiment, that after this nitrogenous and mineral portion has been removed by boiling, it immediately serves for the production of new globules, which, under the influence of the sowing of a mere trace of globules, causes the fermentation of much sugar?[152]

In short, Liebig is not justified in saying that the solution of pure sugar, caused to ferment by means of yeast, contains none of the elements needed for the growth of yeast, neither nitrogen, sulphur, nor phosphorus, and that, consequently, it should not be possible, by our theory, for the sugar to ferment. On the contrary, the solution does contain all these elements, as a consequence of the introduction and presence of the yeast.

Let us proceed with our examination of Liebig’s criticisms:—

“To this,” he goes on to say, “must be added the decomposing action which yeast exercises on a great number of substances, and which resembles that which sugar undergoes. I have shown that malate of lime ferments readily enough through the action of yeast, and that it splits up into three other calcareous salts, namely the acetate, the carbonate, and the succinate. If the action of yeast consists in its increase and multiplication, it is difficult to conceive this action in the case of malate of lime and other calcareous salts of vegetable acids.”

This statement, with all due deference to the opinion of our illustrious critic, is by no means correct. Yeast has no action on malate of lime, or on other calcareous salts formed by vegetable acids. Liebig had previously, much to his own satisfaction, brought forward urea as being capable of transformation into carbonate of ammonia during alcoholic fermentation in contact with yeast. This has been proved by us to be erroneous. It is an error of the same kind that Liebig again brings forward here. In the fermentation of which he speaks (that of malate of lime), certain spontaneous ferments are produced, the germs of which are associated with the yeast, and develop in the mixture of yeast and malate. The yeast merely serves as a source of food for these new ferments without taking any direct part in the fermentations of which we are speaking. Our researches leave no doubt on this point, as is evident from the observations on the fermentations of tartrate of lime previously given.

It is true that there are circumstances under which yeast brings about modifications in different substances. Doebereiner and Mitscherlich, more especially, have shown that yeast imparts to water a soluble material, which liquefies cane-sugar and produces inversion in it by causing it to take up the elements of water, just as diastase behaves to starch or emulsin to amygdalin.

M. Berthelot also has shown that this substance may be isolated by precipitating it with alcohol, in the same way as diastase is precipitated from its solutions.[153] These are remarkable facts, which are, however, at present but vaguely connected with the alcoholic fermentation of sugar by means of yeast. The researches in which we have proved the existence of special forms of living ferments in many fermentations, which one might have supposed to have been produced by simple contact action, had established beyond doubt the existence of profound differences between those fermentations, which we have distinguished as fermentations proper, and the phenomena connected with soluble substances. The more we advance, the more clearly we are able to detect these differences. M. Dumas has insisted on the fact that the ferments of fermentation proper multiply and reproduce themselves in the process, whilst the others are destroyed.[154] Still more recently M. Müntz has shown that chloroform prevents fermentations proper, but does not interfere with the action of diastase (_Comptes rendus_, 1875.) M. Bouchardat had already established the fact that “hydrocyanic acid, salts of mercury, ether, alcohol, creosote, and the oils of turpentine, lemon, cloves, and mustard destroy or check alcoholic fermentation, whilst in no way interfering with the glucoside fermentations” (_Annales de Chimie et de Physique_, 3rd series, t. xiv., 1845.) We may add, in praise of M. Bouchardat’s sagacity, that that skilful observer has always considered these results as a proof that alcoholic fermentation is dependent on the life of the yeast-cell, and that a distinction should be made between the two orders of fermentation.

M. Paul Bert, in his remarkable studies on the influence of barometric pressure on the phenomena of life, has recognized the fact that compressed oxygen is fatal to certain ferments, whilst under similar conditions it does not interfere with the action of those substances classed under the name of _soluble ferments_, such as diastase (the ferment which inverts cane sugar), emulsin, and others. During their stay in compressed air, ferments proper ceased their activity, nor did they resume it, even after exposure to air at ordinary pressures, provided the access of germs was prevented.

We now come to Liebig’s principal objection, with which he concludes his ingenious argument, and to which no less than eight or nine pages of the _Annales_ are devoted.

Our author takes up the question of the possibility of causing yeast to grow in sweetened water, to which a salt of ammonia and some yeast-ash have been added—a fact which is evidently incompatible with his theory that a ferment is always an albuminous substance on its way to decomposition. In this case the albuminous substance does not exist; we have only the mineral substances which will serve to produce it. We know that Liebig regarded yeast, and, generally speaking, any ferment whatever, as being a nitrogenous, albuminous substance which, in the same way as emulsin, for example, possesses the power of bringing about certain chemical decompositions. He connected fermentation with the easy decomposition of that albuminous substance, and imagined that the phenomenon occurred in the following manner:—“The albuminous substance on its way to decomposition possesses the power of communicating to certain other bodies that same state of mobility by which its own atoms are already affected; and through its contact with other bodies it imparts to them the power of decomposing or of entering into other combinations.” Here Liebig failed to perceive that the ferment, in its capacity of a living organism, had anything to do with the fermentation.

This theory dates back as far as 1843. In 1846 Messrs. Boutron and Fremy, in a Memoir on lactic fermentation, published in the _Annales de Chimie et de Physique_, strained the conclusions deducible from it to a most unjustifiable extent. They asserted that one and the same nitrogenous substance might undergo various modifications in contact with air, so as to become successively alcoholic, lactic, butyric, and other ferments. There is nothing more convenient than purely hypothetical theories, theories which are not the necessary consequences of facts; when fresh facts which cannot be reconciled with the original hypothesis are discovered, new hypotheses can be tacked on to the old ones. This is exactly what Liebig and Fremy have done, each in his turn, under the pressure of our studies, commenced in 1857. In 1864 Fremy devised the theory of _hemi-organism_, which meant nothing more than that he gave up Liebig’s theory of 1843, together with the additions which Boutron and he had made to it in 1846; in other words, he abandoned the idea of albuminous substances being ferments, to take up another idea, that albuminous substances, in contact with air, are peculiarly adapted to undergo organization into new beings—that is, the living ferments which we had discovered—and that the ferments of beer and of the grape have a common origin.

This theory of hemi-organism was word for word the antiquated opinion of Turpin, as may be readily seen by referring to