Chapter XVII (9)

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 cc. (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. [Footnote: Carbonic add being considerably more soluble than other gases possible under the circumstances.--ED.]

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 degrees C. and 28 degrees C. (77 degrees F. and 83 degrees 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 the gaugemark on the neck. [Footnote: We had to avoid filling the small flask completely, for fear of causing some of the liquid to pass on to the surface of the mercury in the measuring tube. The liquid condensed by boiling forms pure water, the solvent affinity of which for carbonic acid, at the temperature we employ, is well known. 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 the 2.135 litres, gave a total of 10.457 litres (638.2 cubic inches) at 20 degrees and 760 mm., which, calculated to 0 degrees, C. and 760 mm. atmospheric pressure (32 degrees F. and 30 inches) gave a weight of 19.700 grammes (302.2 grains) of carbonic acid.

Exactly half of the lime in 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. [Footnote: The following is a curious consequence of these numbers and of the nature of the products of this fermentation. The carbonic acid liberated being quite pure, especially when the liquid has been boiled to expel all air from the flask, and capable of perfect solution, it follows that the volume of liquid being sufficient and the weight of tartrate suitably chosen--we may set aside tartrate of lime in an insoluble, crystalline powder, alone with phosphates at the bottom of a closed vessel full of water, and find soon afterwards in their place carbonate of lime, and in the liquid soluble salts of lime, with a mass of organic matter at the bottom, without any liberation of gas or appearance of fermentation ever taking place, except as far as the vital action and transformation in the tartrate are concerned. It is easy to calculate that a vessel or flask of five litres (rather more than a gallon) would be large enough for the accomplishment of this remarkable and singularly quiet transformation, in the case of 50 grammes (767 grains) of tartrate of lime.]. 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.

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. [Footnote: We treated the whole deposit with dilute hydrochloric acid, which dissolved the carbonate of lime and the insoluble phosphates of calcium and magnesium; afterwards filtering the liquid through a weighed filter paper. Dried at 100 degrees C. (212 degrees F.), the weight of the organic matter thus obtained was 0.54 gramme (8.3 grains), which was rather more than 1/200 of the weight of fermentable matter.] 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 punctuate, 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 on 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 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 fermentation in media adapted to the production of the ordinary forms of vibrio; but this is an experiment which we have not ourselves tried.

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

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

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

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

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

We employed a liquid prepared as follows: Into from 9 to 10 litres (somewhat over 2 gallons) of pure water the following salts [Footnote: Should the solution of lactate of lime be turbid, it may be clarified by filtration, after previously adding a small quantity of phosphate of ammonia, which throws down phosphate of lime. It is only after this process of clarification and filtration that the phosphates of the formula are added. The solution soon becomes turbid if left in contact with air, in consequence of the spontaneous formation of bacteria.] were introduced successively, viz:

Pure lactate of lime. ... . ... . ... . ... . .. 225 grammes
Phosphate of ammonia. ... . ... . ... . ... . .. 0.75 grammes
Phosphate of potassium. ... . ... . ... . ... .. 0.4 grammes
Sulphate of magnesium. ... . ... . ... . ... ... 0.4 grammes
Sulphate of ammonia. ... . ... . ... . ... . ... 0.2 grammes
(1 gramme = 15.43 grains.)

On March 23rd, 1875, we filled a 6 litre (about 11 pints) flask, of the shape represented in FIG. 11, and placed it over a heater. Another flame was placed below a vessel containing the same liquid, into which the curved tube of the flask plunged. The liquids in the flask and in the basin were raised to boiling together, and kept in this condition for more than half-an-hour, so as to expel all the air held in solution. The liquid was several times forced out of the flask by the steam, and sucked back again; but the portion which re-entered the flask was always boiling. On the following day when the flask had cooled, we transferred the end of the delivery tube to a vessel full of mercury and placed the whole apparatus in an oven at a temperature varying between 25 degrees C. and 30 degrees C. (77 degrees F. and 86 degrees F.) then, after having refilled the small cylindrical tap-funnel with carbonic acid, we passed into it with all necessary precautions 10 cc. (0.35 fl. oz) of a liquid similar to that described, which had been already in active fermentation for several days out of contact with air and now swarmed with vibrios. We then turned the tap of the funnel, until only a small quantity of liquid was left, just enough to prevent the access of air. In this way the impregnation was accomplished without either the ferment-liquid or the ferment- germs having been brought in contact, even for the shortest space, with the external air. The fermentation, the occurrence of which at an earlier or later period depends for the most part on the condition of the impregnating germs, and the number introduced in the act, in this case began to manifest itself by the appearance of minute bubbles from March 29th. But not until April 9th did we observe bubbles of larger size rise to the surface. From that date onward they continued to come in increasing number, from certain points at the bottom of the flask, where a deposit of earthy phosphates existed; and at the same time the liquid, which for the first few days remained perfectly clear, began to grow turbid in consequence of the development of vibrios. It was on the same day that we first observed a deposit on the sides of carbonate of lime in crystals.

It is a matter of some interest to notice here that, in the mode of procedure adopted, everything combined to prevent the interference of air. A portion of the liquid expelled at the beginning of the experiment, partly because of the increased temperature in the oven and partly also by the force of the gas, as it began to be evolved from the fermentative action, reached the surface of the mercury, where, being the most suitable medium we know for the growth of bacteria, it speedily swarmed with these organisms. [Footnote: The naturalist Cohn, of Breslau, who published an excellent work on bacteria in 1872, described, after Mayer, the composition of a liquid peculiarly adapted to the propagation of these organisms, which it would be well to compare for its utility in studies of this kind with our solution of lactate and phosphates. The following is Cohn's formula:

Distilled water. ... . ... . ... . ..20 cc. (0.7 fl. oz.)
Phosphate of potassium. ... . ... ...0.1 gramme (1.5 grains)
Sulphate of magnesium. ... . ... . 0.1 gramme (1.5 grains)
Tribasic phosphate of lime. ... ... 0.01 gramme (0.15
grain)
Tartrate of ammonia. ... . ... . ... 0.2 gramme (3 grains)

This liquid, the author says, has a feeble acid reaction and forms a perfectly clear solution.] In this way any passage of air, if such a thing were possible, between the mercury and the sides of the delivery-tube was altogether prevented, since the bacteria would consume every trace of oxygen which might be dissolved in the liquid lying on the surface of the mercury. Hence it is impossible to imagine that the slightest trace of oxygen could have got into the liquid in the flask.

Before passing on we may remark that in this ready absorption of oxygen by bacteria we have a means of depriving fermentable liquids of every trace of that gas with a facility and success equal or even greater than by the preliminary method of boiling. Such a solution as we have described, if kept at summer heat, without any previous boiling, becomes turbid in the course of twenty-four hours from a SPONTANEOUS development of bacteria; and it is easy to prove that they absorb all the oxygen held in solution. [Footnote: On the rapid absorption of oxygen by bacteria, see also our Memoire of 1872, sur les Generations dites Spontanees, especially the note on page 78.] If we completely fill a flask of a few litres capacity (about a gallon) (Fig. 9) with the liquid described, taking care to have the delivery-tube also filled, and its opening plunged under mercury, and, forty- eight hours afterwards by means of a chloride of calcium bath, expel from the liquid on the surface of the mercury all the gas which it holds in solution, this gas, when analyzed, will be found to be composed of a mixture of nitrogen and carbonic acid gas, WITHOUT THE LEAST TRACE OF OXYGEN. Here, then, we have an excellent means of depriving the fermentable liquid of air; we simply have completely to fill a flask with the liquid, and place it in the oven, merely avoiding any addition of butyric vibrios, before the lapse of two or three days. We may wait even longer; and then, if the liquid does become impregnated spontaneously with vibrio germs, the liquid, which at first was turbid from the presence of bacteria, will become bright again, since the bacteria, when deprived of life, or, at least, of the power of moving, after they have exhausted all the oxygen in solution, will fall inert to the bottom of the vessel. On several occasions we have determined this interesting fact, which tends to prove that the butyric vibrios cannot be regarded as another form of bacteria, inasmuch as, on the hypothesis of an original relation between the two productions, butyric fermentation ought in every case to follow the growth of bacteria.

We may also call attention to another striking experiment, well suited to show the effect of differences in the composition of the medium upon the propagation of microscopic beings. The fermentation which we last described commenced on March 27th and continued until May 10th; that to which we are now to refer, however, was completed in four days, the liquid employed being similar in composition and quantity to that employed in the former experiment. On April 23, 1875, we filled a flask of the same shape as that represented in Fig. 11, and of similar capacity, viz., 6 litres, with a liquid composed as described at page 69. This liquid had been previously left to itself for five days in large open flasks, in consequence of which it had developed an abundant growth of bacteria. On the fifth day a few bubbles, rising from the bottom of the vessels, at long intervals, betokened the commencement of butyric fermentation, a fact, moreover, confirmed by the microscope, in the appearance of the vibrios of this fermentation in specimens of the liquid taken from the bottom of the vessels, the middle of its mass, and even in the layer on the surface that was swarming with bacteria. We transferred the liquid so prepared to the 6 litre flask arranged over the mercury. By evening a tolerably active fermentation had begun to manifest itself. On the 24th this fermentation was proceeding with astonishing rapidity, which continued during the 25th and 26th. During the evening of the 26th it slackened, and on the 27th all signs of fermentation had ceased. This was not, as might be supposed, a sudden stoppage due to some unknown cause; the fermentation was actually completed, for when we examined the fermented liquid on the 28th we could not find the smallest quantity of lactate of lime. If the needs of industry should ever require the production of large quantities of butyric acid, there would, beyond doubt, be found in the preceding fact valuable information in devising an easy method of preparing that product in abundance. [Footnote: In what way are we to account for so great a difference between the two fermentations that we have just described? Probably it was owing to some modification effected in the medium by the previous life of the bacteria, or to the special character of the vibrios used in impregnation. Or, again, it might have been due to the action of the air, which, under the conditions of our second experiment, was not absolutely eliminated, since we took no precaution against its introduction at the moment of filling our flask, and this would tend to facilitate the multiplication of anaerobian vibrios, just as, under similar conditions, would have been the case if we had been dealing with a fermentation by ordinary yeast.]

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

On May 27th, 1862, we completely filled a flask capable of holding 2.780 litres (about five pints) with the solution of lactate and phosphates. [Footnote: In this case the liquid was composed as follows: A saturated solution of lactate of lime, at a temperature of 25 degrees C. (77 degrees F.), was prepared, containing for every 1OO cc. (3 1/2 fl. oz.) 25.65 grammes (394 grains) of the lactate, C6 H5 O5 Ca O (NEW NOTATION, C6 H10 Ca O6) This solution was rendered very clear by the addition of 1 gramme of phosphate of ammonia and subsequent filtration. For a volume of 8 litres (14 pints) of this clear saturated solution we used (1 gramme = 15.43 grains):

Phosphate of ammonia. ... . ... . ... . ... 2 grammes
Phosphate of potassium. ... . ... . ... . ... 1 gramme
Phosphate of magnesium. ... . ... . ... . ... 1 gramme
Sulphate of ammonia. ... . ... . ... . ... 0.5 gramme]

We refrained from impregnating it with any germs. The liquid became turbid from a development of bacteria and then underwent butyric fermentation. By June 9th the fermentation had become sufficiently active to enable us to collect in the course of twenty-four hours, over mercury, as in all our experiments, about 100 cc. (about 6 cubic inches) of gas. By June 11th, judging from the volume of gas liberated in the course of twenty-four hours, the activity of the fermentation had doubled. We examined a drop of the turbid liquid. Here are the notes accompanying the sketch (Fig. 12) as they stand in our note-book: "A swarm of vibrios, so active in their movements that the eye has great difficulty in following them. They may be seen in pairs throughout the field, apparently making efforts to separate from each other. The connection would seem to be by some invisible, gelatinous thread, which yields so far to their efforts that they succeed in breaking away from actual contact, but yet are, for a while, so far restrained that the movements of one have a visible effect on those of the other. By and by, however, we see a complete separation effected, and each moves on its separate way with an activity greater than it ever had before."

One of the best methods that can be employed for the

microscopical examination of these vibrios, quite out of contact with air, is the following. After butyric fermentation has been going on for several days in a flask, (Fig. 13), we connect this flask by an india-rubber tube with one of the flattened bulbs previously described, which we then place on the stage of the microscope (Fig. 13). When we wish to make an observation we close, under the mercury, at the point B, the end of the drawn- out and bent delivery-tube. The continued evolution of gas soon exerts such a pressure within the flask, that when we open the tap R, the liquid is driven into the bulb LL, until it becomes quite full and the liquid flows over into the glass V. In this manner we may bring the vibrios under observation without their coming into contact with the least trace of air, and with as much success as if the bulb, which takes the place of an object glass, had been plunged into the very centre of the flask. The movements and fissiparous multiplication of the vibrios may thus be seen in all their beauty, and it is indeed a most interesting sight. The movements do not immediately cease when the temperature is suddenly lowered, even to a considerable extent, 15 degrees C. (59 degrees F.) for example; they are only slackened. Nevertheless, it is better to observe them at the temperatures most favourable to fermentation, even in the oven where the vessels employed in the experiment are kept at a temperature between 25 degrees C. and 30 degrees C. (77 degrees F. and 86 degrees F.).

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

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

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?

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

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

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

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

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

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

M. Robin, however, would have no difficulty in determining the limits of the two kingdoms. According to him, "every variety of cellulose is, we may say, insoluble in ammonia, as also are the reproductive elements of plants, whether male or female. Whatever phase of evolution the elements which reproduce a new individual may have reached, treatment with this reagent, either cold or raised to boiling, leaves them absolutely intact under the eyes of the observer, except that their contents, from being partially dissolved, become more transparent. Every vegetable whether microscopic or not, every mycelium and every spore, thus preserves in its entirety its special characteristics of form, volume and structural arrangements; whilst in the case of microscopic animals, or the ova and microscopic embryos of different members of the animal kingdom, the very opposite is the case."