On May 27th, 1862, we completely filled a flask capable of holding 2.780 litres (about five pints) with the solution of lactate and phosphates. [Footnote: In this case the liquid was composed as follows: A saturated solution of lactate of lime, at a temperature of 25 degrees C. (77 degrees F.), was prepared, containing for every 1OO cc. (3 1/2 fl. oz.) 25.65 grammes (394 grains) of the lactate, C6 H5 O5 Ca O (NEW NOTATION, C6 H10 Ca O6) This solution was rendered very clear by the addition of 1 gramme of phosphate of ammonia and subsequent filtration. For a volume of 8 litres (14 pints) of this clear saturated solution we used (1 gramme = 15.43 grains):
Phosphate of ammonia. … . … . … . … 2 grammes
Phosphate of potassium. … . … . … . … 1 gramme
Phosphate of magnesium. … . … . … . … 1 gramme
Sulphate of ammonia. … . … . … . … 0.5 gramme]
We refrained from impregnating it with any germs. The liquid became turbid from a development of bacteria and then underwent butyric fermentation. By June 9th the fermentation had become sufficiently active to enable us to collect in the course of twenty-four hours, over mercury, as in all our experiments, about 100 cc. (about 6 cubic inches) of gas. By June 11th, judging from the volume of gas liberated in the course of twenty-four hours, the activity of the fermentation had doubled. We examined a drop of the turbid liquid. Here are the notes accompanying the sketch (Fig. 12) as they stand in our note-book: "A swarm of vibrios, so active in their movements that the eye has great difficulty in following them. They may be seen in pairs throughout the field, apparently making efforts to separate from each other. The connection would seem to be by some invisible, gelatinous thread, which yields so far to their efforts that they succeed in breaking away from actual contact, but yet are, for a while, so far restrained that the movements of one have a visible effect on those of the other. By and by, however, we see a complete separation effected, and each moves on its separate way with an activity greater than it ever had before."
[Illustration with caption: Fig. 12]
One of the best methods that can be employed for the
microscopical examination of these vibrios, quite out of contact with air, is the following. After butyric fermentation has been going on for several days in a flask, (Fig. 13), we connect this flask by an india-rubber tube with one of the flattened bulbs previously described, which we then place on the stage of the microscope (Fig. 13). When we wish to make an observation we close, under the mercury, at the point B, the end of the drawn- out and bent delivery-tube. The continued evolution of gas soon exerts such a pressure within the flask, that when we open the tap R, the liquid is driven into the bulb LL, until it becomes quite full and the liquid flows over into the glass V. In this manner we may bring the vibrios under observation without their coming into contact with the least trace of air, and with as much success as if the bulb, which takes the place of an object glass, had been plunged into the very centre of the flask. The movements and fissiparous multiplication of the vibrios may thus be seen in all their beauty, and it is indeed a most interesting sight. The movements do not immediately cease when the temperature is suddenly lowered, even to a considerable extent, 15 degrees C. (59 degrees F.) for example; they are only slackened. Nevertheless, it is better to observe them at the temperatures most favourable to fermentation, even in the oven where the vessels employed in the experiment are kept at a temperature between 25 degrees C. and 30 degrees C. (77 degrees F. and 86 degrees F.).
[Illustration: Fig. 13]
We may now continue our account of the fermentation which we were studying when we made this last digression. On June 17th that fermentation produced three times as much gas as it did on June 11th, when the residue of hydrogen, after absorption by potash, was 72.6 per cent.; whilst on the 17th it was only 49.2 per cent. Let us again discuss the microscopic aspect of the turbid liquid at this stage. Appended is the sketch we made (Fig. 14) and our notes on it: "A most beautiful object: vibrios all in motion, advancing or undulating. They have grown considerably in bulk and length since the 11th; many of them are joined together in long sinuous chains, very mobile at the articulations, visibly less active and more wavering in proportion to the number that go to form the chain, of the length of the individuals." This description is applicable to the majority of the vibrios which occur in cylindrical rods and are homogeneous in aspect. There are others, of rare occurrence in chains, which have a clear corpuscle, that is to say, a portion more refractive than other parts of the segments, at one of their extremities. Sometimes the foremost segment has the corpuscle at one end, sometimes the other. The long segments of the commoner kind attain a length of from 10 to 30 and even 45 thousandths of a millimetre. Their diameter is from 1 1/2 to 2, very rarely 3, thousandths of a millimetre. [Footnote: 1 millimetre = 0.039 inch: hence the dimensions indicated will be—length, from 0.00039 to 0.00117, or even 0.00176 in.; diameter, from 0.000058 to 0.000078, rarely 0.000117 in.—D. C. R.]
[Illustration: Figure 14.]
On June 28th, fermentation was quite finished; there was no longer any trace of gas, nor any lactate in solution. All the infusoria were lying motionless at the bottom of the flask. The liquid clarified by degrees, and in the course of a few days became quite bright. Here we may inquire, were these motionless infusoria, which from complete exhaustion of the lactate, the source of the carbonaceous part of their food, were now lying inert at the bottom of the fermenting vessel—were they dead beyond the power of revival? [Footnote: The carbonaceous supply, as we remarked, had failed them, and to this failure the absence of vital action, nutrition, and multiplication was attributable. The liquid, however, contained butyrate of lime, a salt possessing properties similar to those of the lactate. Why could not this salt equally well support the life of the vibrios? The explanation of the difficulty seems to us to lie simply in the fact that lactic acid produces heat by its decomposition, whilst butyric acid does not, and the vibrios seem to require heat during the chemical process of their nutrition.] The following experiment leads us to believe that they were not perfectly lifeless, and that they might behave in the same manner as the yeast of beer, which, after it has decomposed all the sugar in a fermentable liquid, is ready to revive and multiply in a fresh saccharine medium. On April 22nd, 1875, we left in the oven at a temperature of 25 degrees C. (77 degrees F.) a fermentation of lactate of lime that had been completed. The delivery tube of the flask A, (Fig. 15), in which it had taken place, had never been withdrawn from under the mercury. We kept the liquid under observation daily, and saw it gradually become brighter; this went on for fifteen days. We then filled a similar flask, B, with the solution of lactate, which we boiled, not only to kill the germs of vibrios which the liquid might contain, but also to expel the air that it held in solution. When the flask, B, had cooled, we connected the two flasks, avoiding the introduction of air, [Footnote: To do this it is sufficient, first, to fill the curved ends of the stop-cocked tubes of the flasks, as well as the india-rubber tube C C which connects them, with boiling water that contains no air.] after having slightly shaken the flask, A, to stir up the deposit at the bottom. There was then a pressure due to carbonic acid at the end of the delivery tube of this latter flask, at the point A, so that on opening the taps R and S, the deposit at the bottom of flask A was driven over into flask B, which in consequence was impregnated with the deposit of a fermentation that had been completed fifteen days before. Two days after impregnation the flask B began to show signs of fermentation. It follows that the deposit of vibrios of a completed butyric fermentation may be kept, at least for a certain time, without losing the power of causing fementation. It furnishes a butyric ferment, capable of revival and action in a suitable fresh fermentable medium.