When we published, in 1860, the details of the experiment in question, we pointed out at some length the difficulties of conducting it successfully, and the possible causes of failure. We called attention particularly to the fact that saccharine mineral media are much more suited for the nutrition of bacteria, lactic ferment, and other lowly forms, than they are to that of yeast, and in consequence readily become filled with various organisms from the spontaneous growth of germs derived from the particles of dust floating in the atmosphere. The reason why we do not observe the growth of alcoholic ferments, especially at the commencement of the experiments, is because of the unsuitableness of those media for the life of yeast. The latter may, nevertheless, form in them subsequent to this development of other organized forms, by reason of the modification produced in the original mineral medium by the albuminous matters that they introduce into it. It is interesting to peruse, in our Memoir of 1860, certain facts of the same kind relating to fermentation by means of albumens—that of the blood for example, from which, we may mention incidentally, we were led to infer the existence of several distinct albumens in the serum, a conclusion which, since then, has been confirmed by various observers, notably by M. Bechamp. Now, in his experiments on fermentation in sweetened water, with yeast-ash and a salt of ammonia, there is no doubt that Liebig had failed to avoid those difficulties which are entailed by the spontaneous growth of other organisms than yeast. Moreover, it is possible that, to have established the certainty of this result, Liebig should have had recourse to a closer microscopical observation than from certain passages in his Memoir he seems to have adopted. We have little doubt that his pupils could tell us that Liebig did not even employ that instrument without which any exact study of fermentation is not merely difficult but well-nigh impossible. We ourselves, for the reasons, mentioned, did not obtain a simple alcoholic fermentation any more than Liebig did. In that particular experiment, the details of which we gave in our Memoir of 1860, we obtained lactic and alcoholic fermentation together; an appreciable quantity of lactic acid formed and arrested the propagation of the lactic and alcoholic ferments, so that more than half of the sugar remained in the liquid without fermenting. This, however, in no way detracted from the correctness of the conclusion which we deduced from the experiment, and from other similar ones; it might even be said that, from a general and philosophical point of view—which is the only one of interest here—the result was doubly satisfactory, inasmuch as we demonstrated that mineral media were adapted to the simultaneous development of several organized ferments instead of only one. The fortuitous association of different ferments could not invalidate the conclusion that all the nitrogen of the cells of the alcoholic and lactic ferments was derived from the nitrogen in the ammoniacal salts, and that all the carbon of those ferments was taken from the sugar, since, in the medium employed in our experiment, the sugar was the only substance that contained carbon. Liebig carefully abstained from noticing this fact, which would have been fatal to the very groundwork of his criticisms, and thought that he was keeping up the appearance of a grave contradiction by arguing that we had never obtained a simple alcoholic fermentation. It would be unprofitable to dwell longer upon the subject of the difficulties which the propagation of yeast in a saccharine mineral medium formerly presented. As a matter of fact, the progress of our studies has imparted to the question an aspect very different from that which it formerly wore; it was this circumstance which emboldened us to offer, in our reply to Liebig before the Academy of Sciences in 1871, to prepare, in a saccharine mineral medium, in the presence of a commission to be appointed by our opponent, any quantity of ferment that he might require, and to effect the fermentation of any weight of sugar whatsoever.
Our knowledge of the facts detailed in the preceding chapter concerning pure ferments, and their manipulation in the presence of pure air, enables us completely to disregard those causes of embarrassment that result from the fortuitous occurrence of the germs of organisms different in character from the ferments introduced by the air or from the sides of vessels, or even by the ferment itself.
Let us once more take one of our double-necked flasks, which we will suppose is capable of containing three or four litres (six to eight pints).
Let us put into it the following:
Pure distilled water.
Sugar candy. … . … . … . … . … . 200 grammes
Bitartrate of potassium. … . … . 1.0 grammes
Bitartrate of ammonia. … . … … 0.5 grammes
Sulphate of ammonia.,. … . … … 1.5 grammes
Ash of yeast. … . … . … . … . … 1.5 grammes
(1 gramme = 15.43 grains)
Let us boil the mixture, to destroy all germs of organisms that may exist in the air or liquid or on the sides of the flask, and then permit it to cool, after having placed, by way of extra precaution a small quantity of asbestos in the end of the fine curved tube. Let us next introduce a trace of ferment into the liquid, through the other neck, which, as we have described, is terminated by a small piece of india-rubber tube closed with a glass stopper.
Here are the details of such an experiment:—
On December 9th, 1873, we sowed some pure ferment—saccharomyces pastorianus. From December 11, that is, within so short a time as forty-eight hours after impregnation, we saw a multitude of extremely minute bubbles rising almost continuously from the bottom, indication that at this point the fermentation had commenced. On the following days, several patches of froth appeared on the surface of the liquid. We left the flask undisturbed in the oven, at a temperature of 25 degrees C. (77 degrees F.) On April 24, 1874, we tested some of the liquid, obtained by means of the straight tube, to see if it still contained any sugar. We found that it contained less than two grammes, so that 198 grammes (4.2 oz. Troy) had already disappeared. Some time afterwards the fermentation came to an end; we carried on the experiment, nevertheless, until April 18, 1875.
There was no development of any organism absolutely foreign to the ferment, which was itself abundant, a circumstance that, added to the persistent vitality of the ferment, in spite of the unsuitableness of the medium for its nutrition, permitted the perfect completion of fermentation. There was not the minutest quantity of sugar remaining. The total weight of ferment, after washing and drying at 100 degrees C. (212 degrees F.), was 2.563 grammes (39.5 grains).
In experiments of this kind, in which the ferment has to be weighed, it is better not to use any yeast-ash that cannot be dissolved completely, so as to be capable of easy separation from the ferment formed. Raulin's liquid [Footnote: M. Jules Raulin has published a well-known and remarkable work on the discovery of the mineral medium best adapted by its composition to the life of certain fungoid growth; he has given a formula for the composition of such a medium. It is this that we call here "Raulin's liquid" for abbreviation.