We employed a liquid prepared as follows: Into from 9 to 10 litres (somewhat over 2 gallons) of pure water the following salts [Footnote: Should the solution of lactate of lime be turbid, it may be clarified by filtration, after previously adding a small quantity of phosphate of ammonia, which throws down phosphate of lime. It is only after this process of clarification and filtration that the phosphates of the formula are added. The solution soon becomes turbid if left in contact with air, in consequence of the spontaneous formation of bacteria.] were introduced successively, viz:
Pure lactate of lime. … . … . … . … . .. 225 grammes
Phosphate of ammonia. … . … . … . … . .. 0.75 grammes
Phosphate of potassium. … . … . … . … .. 0.4 grammes
Sulphate of magnesium. … . … . … . … … 0.4 grammes
Sulphate of ammonia. … . … . … . … . … 0.2 grammes
(1 gramme = 15.43 grains.)
[Illustration with caption: Fig. 11]
On March 23rd, 1875, we filled a 6 litre (about 11 pints) flask, of the shape represented in FIG. 11, and placed it over a heater. Another flame was placed below a vessel containing the same liquid, into which the curved tube of the flask plunged. The liquids in the flask and in the basin were raised to boiling together, and kept in this condition for more than half-an-hour, so as to expel all the air held in solution. The liquid was several times forced out of the flask by the steam, and sucked back again; but the portion which re-entered the flask was always boiling. On the following day when the flask had cooled, we transferred the end of the delivery tube to a vessel full of mercury and placed the whole apparatus in an oven at a temperature varying between 25 degrees C. and 30 degrees C. (77 degrees F. and 86 degrees F.) then, after having refilled the small cylindrical tap-funnel with carbonic acid, we passed into it with all necessary precautions 10 cc. (0.35 fl. oz) of a liquid similar to that described, which had been already in active fermentation for several days out of contact with air and now swarmed with vibrios. We then turned the tap of the funnel, until only a small quantity of liquid was left, just enough to prevent the access of air. In this way the impregnation was accomplished without either the ferment-liquid or the ferment- germs having been brought in contact, even for the shortest space, with the external air. The fermentation, the occurrence of which at an earlier or later period depends for the most part on the condition of the impregnating germs, and the number introduced in the act, in this case began to manifest itself by the appearance of minute bubbles from March 29th. But not until April 9th did we observe bubbles of larger size rise to the surface. From that date onward they continued to come in increasing number, from certain points at the bottom of the flask, where a deposit of earthy phosphates existed; and at the same time the liquid, which for the first few days remained perfectly clear, began to grow turbid in consequence of the development of vibrios. It was on the same day that we first observed a deposit on the sides of carbonate of lime in crystals.
It is a matter of some interest to notice here that, in the mode of procedure adopted, everything combined to prevent the interference of air. A portion of the liquid expelled at the beginning of the experiment, partly because of the increased temperature in the oven and partly also by the force of the gas, as it began to be evolved from the fermentative action, reached the surface of the mercury, where, being the most suitable medium we know for the growth of bacteria, it speedily swarmed with these organisms. [Footnote: The naturalist Cohn, of Breslau, who published an excellent work on bacteria in 1872, described, after Mayer, the composition of a liquid peculiarly adapted to the propagation of these organisms, which it would be well to compare for its utility in studies of this kind with our solution of lactate and phosphates. The following is Cohn's formula:
Distilled water. … . … . … . ..20 cc. (0.7 fl. oz.)
Phosphate of potassium. … . … …0.1 gramme (1.5 grains)
Sulphate of magnesium. … . … . 0.1 gramme (1.5 grains)
Tribasic phosphate of lime. … … 0.01 gramme (0.15
grain)
Tartrate of ammonia. … . … . … 0.2 gramme (3 grains)
This liquid, the author says, has a feeble acid reaction and forms a perfectly clear solution.] In this way any passage of air, if such a thing were possible, between the mercury and the sides of the delivery-tube was altogether prevented, since the bacteria would consume every trace of oxygen which might be dissolved in the liquid lying on the surface of the mercury. Hence it is impossible to imagine that the slightest trace of oxygen could have got into the liquid in the flask.
Before passing on we may remark that in this ready absorption of oxygen by bacteria we have a means of depriving fermentable liquids of every trace of that gas with a facility and success equal or even greater than by the preliminary method of boiling. Such a solution as we have described, if kept at summer heat, without any previous boiling, becomes turbid in the course of twenty-four hours from a SPONTANEOUS development of bacteria; and it is easy to prove that they absorb all the oxygen held in solution. [Footnote: On the rapid absorption of oxygen by bacteria, see also our Memoire of 1872, sur les Generations dites Spontanees, especially the note on page 78.] If we completely fill a flask of a few litres capacity (about a gallon) (Fig. 9) with the liquid described, taking care to have the delivery-tube also filled, and its opening plunged under mercury, and, forty- eight hours afterwards by means of a chloride of calcium bath, expel from the liquid on the surface of the mercury all the gas which it holds in solution, this gas, when analyzed, will be found to be composed of a mixture of nitrogen and carbonic acid gas, WITHOUT THE LEAST TRACE OF OXYGEN. Here, then, we have an excellent means of depriving the fermentable liquid of air; we simply have completely to fill a flask with the liquid, and place it in the oven, merely avoiding any addition of butyric vibrios, before the lapse of two or three days. We may wait even longer; and then, if the liquid does become impregnated spontaneously with vibrio germs, the liquid, which at first was turbid from the presence of bacteria, will become bright again, since the bacteria, when deprived of life, or, at least, of the power of moving, after they have exhausted all the oxygen in solution, will fall inert to the bottom of the vessel. On several occasions we have determined this interesting fact, which tends to prove that the butyric vibrios cannot be regarded as another form of bacteria, inasmuch as, on the hypothesis of an original relation between the two productions, butyric fermentation ought in every case to follow the growth of bacteria.
We may also call attention to another striking experiment, well suited to show the effect of differences in the composition of the medium upon the propagation of microscopic beings. The fermentation which we last described commenced on March 27th and continued until May 10th; that to which we are now to refer, however, was completed in four days, the liquid employed being similar in composition and quantity to that employed in the former experiment. On April 23, 1875, we filled a flask of the same shape as that represented in Fig. 11, and of similar capacity, viz., 6 litres, with a liquid composed as described at page 69. This liquid had been previously left to itself for five days in large open flasks, in consequence of which it had developed an abundant growth of bacteria. On the fifth day a few bubbles, rising from the bottom of the vessels, at long intervals, betokened the commencement of butyric fermentation, a fact, moreover, confirmed by the microscope, in the appearance of the vibrios of this fermentation in specimens of the liquid taken from the bottom of the vessels, the middle of its mass, and even in the layer on the surface that was swarming with bacteria. We transferred the liquid so prepared to the 6 litre flask arranged over the mercury. By evening a tolerably active fermentation had begun to manifest itself. On the 24th this fermentation was proceeding with astonishing rapidity, which continued during the 25th and 26th. During the evening of the 26th it slackened, and on the 27th all signs of fermentation had ceased. This was not, as might be supposed, a sudden stoppage due to some unknown cause; the fermentation was actually completed, for when we examined the fermented liquid on the 28th we could not find the smallest quantity of lactate of lime. If the needs of industry should ever require the production of large quantities of butyric acid, there would, beyond doubt, be found in the preceding fact valuable information in devising an easy method of preparing that product in abundance. [Footnote: In what way are we to account for so great a difference between the two fermentations that we have just described? Probably it was owing to some modification effected in the medium by the previous life of the bacteria, or to the special character of the vibrios used in impregnation. Or, again, it might have been due to the action of the air, which, under the conditions of our second experiment, was not absolutely eliminated, since we took no precaution against its introduction at the moment of filling our flask, and this would tend to facilitate the multiplication of anaerobian vibrios, just as, under similar conditions, would have been the case if we had been dealing with a fermentation by ordinary yeast.]
Before we go any further, let us devote some attention to the vibrios of the preceding fermentations.