[Illustration: Fig. 15]

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

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

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

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

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

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

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

[Illustration: Fig. 16]

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