Our author takes up the question of the possibility of causing yeast to grow in sweetened water, to which a salt of ammonia and some yeast-ash have been added--a fact which is evidently incompatible with his theory that a ferment is always an alb.u.minous substance on its way to decomposition. In this case the alb.u.minous substance does not exist; we have only the mineral substances which will serve to produce it. We know that Liebig regarded yeast, and, generally speaking, any ferment whatever, as being a nitrogenous, alb.u.minous substance which, in the same way as emulsin, for example, possesses the power of bringing about certain chemical decompositions. He connected fermentation with the easy decomposition of that alb.u.minous substance, and imagined that the phenomenon occurred in the following manner: "The alb.u.minous substance on its way to decomposition possesses the power of communicating to certain other bodies that same state of mobility by which its own atoms are already affected; and through its contact with other bodies it imparts to them the power of decomposing or of entering into other combinations." Here Liebig failed to perceive that the ferment, in its capacity of a living organism, had anything to do with the fermentation.
This theory dates back as far as 1843. In 1846 Messrs. Boutron and Fremy, in a Memoir on lactic fermentation, published in the Annales de Chimie et de Physique, strained the conclusions deducible from it to a most unjustifiable extent. They a.s.serted that one and the same nitrogenous substance might undergo various modifications in contact with air, so as to become successively alcoholic, lactic, butyric, and other ferments. There is nothing more convenient than purely hypothetical theories, theories which are not the necessary consequences of facts; when fresh facts which cannot be reconciled with the original hypothesis are discovered, new hypotheses can be tacked on to the old ones. This is exactly what Liebig and Fremy have done, each in his turn, under the pressure of our studies, commenced in 1857. In 1864 Fremy devised the theory of hemi-organism, which meant nothing more than that he gave up Liebig"s theory of 1843, together with the additions which Boutron and he had made to it in 1846; in other words, he abandoned the idea of alb.u.minous substances being ferments, to take up another idea, that alb.u.minous substances in contact with air are peculiarly adapted to undergo organization into new beings--that is, the living ferments which we had discovered--and that the ferments of beer and of the grape have a common origin.
This theory of hemi-organism was word for word the antiquated opinion of Turpin. * * * The public, especially a certain section of the public did not go very deeply into an examination of the subject. It was the period when the doctrine of spontaneous generation was being discussed with much warmth. The new word hemi-organism, which was the only novelty in M. Fremy"s theory, deceived people. It was thought that M. Fremy had really discovered the solution of the question of the day. It is true that it was rather difficult to understand the process by which an alb.u.minous substance could become all at once a living and budding cell. This difficulty was solved by M. Fremy, who declared that it was the result of some power that was not yet understood, the power of "organic impulse." [Footnote: FREMY, Comptes rendus de l"Academie, vol. lviii., p. 1065, 1864.]
Liebig, who, as well as M. Fremy, was compelled to renounce his original opinions concerning the nature of ferments, devised the following obscure theory (Memoir by Liebig, 1870, already cited):
"There seems to be no doubt as to the part which the vegetable organism plays in the phenomenon of fermentation. It is through it alone that an alb.u.minous substance and sugar are enabled to unite and form this particular combination, this unstable form under which alone, as a component part of the mycoderm, they manifest an action on sugar. Should the mycoderm cease to grow, the bond which unites the const.i.tuent parts of the cellular contents is loosened, and it is through the motion produced therein that the cells of yeast bring about a disarrangement or separation of the elements of the sugar into molecules."
One might easily believe that the translator for the Annales has made some mistake, so great is the obscurity of this pa.s.sage.
Whether we take this new form of the theory or the old one, neither can be reconciled at all with the development of yeast and fermentation in a saccharine mineral medium, for in the latter experiment fermentation is correlative to the life of the ferment and to its nutrition, a constant change going on between the ferment and its food-matters, since all the carbon a.s.similated by the ferment is derived from sugar, its nitrogen from ammonia and phosphorus from the phosphates in solution. And even all said, what purpose can be served by the gratuitous hypothesis of contact-action or communicated motion? The experiment of which we are speaking is thus a fundamental one; indeed, it is its possibility that const.i.tutes the most effective point in the controversy. No doubt Liebig might say, "but it is the motion of life and of nutrition which const.i.tutes your experiment, and this is the communicated motion that my theory requires." Curiously enough, Liebig does endeavour, as a matter of fact, to say this, but he does so timidly and incidentally: "From a chemical point of view, which point of view I would not willingly abandon, a VITAL ACTION is a phenomenon of motion, and, in this double sense of LIFE M. Pasteur"s theory agrees with my own, and is not in contradiction with it (page 6)." This is true.
Elsewhere Liebig says:
"It is possible that the only correlation between the physiological act and the phenomenon of fermentation is the production, in the living cell, of the substance which, by some special property a.n.a.logous to that by which emulsin exerts a decomposing action on salicin and amygdalin, may bring about the decomposition of sugar into other organic molecules; the physiological act, in this view, would be necessary for the production of this substance, but would have nothing else to do with the fermentation (page 10)." To this, again, we have no objection to raise.
Liebig, however, does not dwell upon these considerations, which he merely notices in pa.s.sing, because he is well aware that, as far as the defence of his theory is concerned, they would be mere evasions. If he had insisted on them, or based his opposition solely upon them, our answer would have been simply this: "If you do not admit with us that fermentation is correlated with the life and nutrition of the ferment, we agree upon the princ.i.p.al point. So agreeing, let us examine, if you will, the actual cause of fermentation;--this is a second question, quite distinct from the first. Science is built up of successive solutions given to questions of ever increasing subtlety, approaching nearer and nearer towards the very essence of phenomena. If we proceed to discuss together the question of how living, organized beings act in decomposing fermentable substances, we will be found to fall out once more on your hypothesis of communicated motion, since according to our ideas, the actual cause of fermentation is to be sought, in most cases, in the fact of life without air, which is the characteristic of many ferments."
Let us briefly see what Liebig thinks of the experiment in which fermentation is produced by the impregnation of a saccharine mineral medium, a result so greatly at variance with his mode of viewing the question. [Footnote: See our Memoir of 1860 (Annales de Chimie et de Physique, vol. lviii, p. 61, and following, especially pp. 69 and 70, where the details of the experiment will be found).] After deep consideration he p.r.o.nounces this experiment to be inexact, and the result ill-founded. Liebig, however, was not one to reject a fact without grave reasons for doing so, or with the sole object of evading a troublesome discussion. "I have repeated this experiment," he says, "a great number of times, with the greatest possible care, and have obtained the same results as M. Pasteur, excepting as regards the formation and increase of the ferment." It was, however, the formation and increase of the ferment that const.i.tuted the point of the experiment. Our discussion was, therefore, distinctly limited to this: Liebig denied that the ferment was capable of development in a saccharine mineral medium, whilst we a.s.serted that this development did actually take place, and was comparatively easy to prove. In 1871 we replied to M. Liebig before the Paris Academy of Sciences in a Note, in which we offered to prepare in a mineral medium, in the presence of a commission to be chosen for the purpose, as great a weight of ferment as Liebig could reasonably demand. [Footnote: PASTEUR, Comptes rendus de l"Academie des Sciences, vol. lxxiii., p. 1419.
1871.] We were bolder than we should, perhaps, have been in 1860; the reason was that our knowledge of the subject had been strengthened by ten years of renewed research. Liebig did not accept our proposal, nor did he even reply to our Note. Up to the time of his death, which took place on April 18th, 1873, he wrote nothing more on the subject. [Footnote: In his Memoir of 1870, Liebig made a remarkable admission: "My late friend Pelouze," he says, "had communicated to me nine years ago certain results of M. Pasteur"s researches on fermentation. I told him that just then I was not disposed to alter my opinion on the cause of fermentation, and that if it were possible, by means of ammonia, to produce or multiply the yeast in fermenting liquors, industry would soon avail itself of the fact, and that I would wait to see if it did so; up to the present time, however, there had not been the least change in the manufacture of yeast. "We do not know what M. Pelouze"s reply was; but it is not difficult to conceive so sagacious an observer remarking to his ill.u.s.trious friend that the possibility of deriving pecuniary advantage from the wide application of a new scientific fact had never been regarded as the criterion of the exactness of that fact. We could prove, moreover, by the undoubted testimony of very distinguished practical men, notably by that of M. Pezeyre, director of distilleries, that upon this point also Liebig was mistaken.]
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 alb.u.minous 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 alb.u.mens--that of the blood for example, from which, we may mention incidentally, we were led to infer the existence of several distinct alb.u.mens 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 pa.s.sages 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 quant.i.ty 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 a.s.sociation 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 circ.u.mstance 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 quant.i.ty 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 embarra.s.sment 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 pota.s.sium. ....... . 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 quant.i.ty 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 gla.s.s stopper.
Here are the details of such an experiment:--
On December 9th, 1873, we sowed some pure ferment--saccharomyces pastoria.n.u.s. From December 11, that is, within so short a time as forty-eight hours after impregnation, we saw a mult.i.tude 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 circ.u.mstance 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 quant.i.ty 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.
Water ... ... ... ... ... ... 1,500 Sugar candy ... ... ... ... ... 70 Tartaric acid ... ... ... ... . . 4 Nitrate of ammonia ... ... ... . . 4 Phosphate of ammonia ... ... ... . 0.6 Carbonate of pota.s.sium ... ... ... 0.6 Carbonate of magnesia ... ... ... . 0.4 Sulphate of ammonia ... ... ... . . 0.25 Sulphate of zinc ... ... ... ... 0.07 Sulphate of iron ... ... ... ... 0.07 Silicate of pota.s.sium ... ... ... . 0.07 --J. Raulin, Paris, Victor Ma.s.son, 1870. These pour le doctorat.] may be used in such cases with success.
All the alcoholic ferments are not capable to the same extent of development by means of phosphates, ammoniacal salts, and sugar.
There are some whose development is arrested a longer or shorter time before the transformation of all the sugar. In a series of comparative experiments, 200 grammes of sugar-candy being used in each case, we found that whilst saccharomyces pastoria.n.u.s effected a complete fermentation of the sugar, the caseous ferment did not decompose more than two-thirds, and the ferment we have designated NEW "HIGH" FERMENT not more than one-fifth: and keeping the flasks for a longer time in the oven had no effect in increasing the proportions of sugar fermented in these two last cases.
We conducted a great number of fermentations in mineral media, in consequence of a circ.u.mstance which it may be interesting to mention here. A person who was working in our laboratory a.s.serted that the success of our experiments depended upon the impurity of the sugar-candy which we employed, and that if this sugar had been pure--much purer than was the ordinary, white, commercial sugar-candy, which up to that time we had always used--the ferment could not have multiplied. The persistent objections of our friend, and our desire to convince him, caused us to repeat all our previous experiments on the subject, using sugar of great purity, which had been specially prepared for us, with the utmost care, by a skilful confectioner, Seugnot. The result only confirmed our former conclusions. Even this did not satisfy our obstinate friend, who went to the trouble of preparing some pure sugar for himself, in little crystals, by repeated crystallizations of carefully selected commercial sugar-candy; he then repeated our experiments himself. This time his doubts were overcome. It even happened that the fermentations with the perfectly pure sugar instead of being slow were very active, when compared with those which we had conducted with, the commercial sugar-candy.
We may here add a few words on the non-transformation of yeast into penicillium glauc.u.m.
If at any time during fermentation we pour off the fermenting liquid, the deposit of yeast remaining in the vessel may continue there, in contact with air, without our ever being able to discover the least formation of penicillium glauc.u.m in it. We may keep a current of pure air constantly pa.s.sing through the flask; the experiment will give the same result. Nevertheless, this is a medium peculiarly adapted to the development of this mould, inasmuch as if we were to introduce merely a few spores of penicillium an abundant vegetation of that growth will afterwards appear on the deposit. The descriptions of Messrs. Turpin, Hoffmann, and Trecul have, therefore, been based on one of these illusions which we meet with so frequently in microscopical observations.
When we laid these facts before the Academy, [Footnote: PASTEUR, Comptes rendus de l"Academie, vol. lxxviii., pp. 213-216.] M.
Trecul professed his inability to comprehend them: [Footnote: TRECUL, Comptes rendus de l"Academie, vol. lxxviii., pp. 217, 218.] "According to M. Pasteur," he said, "the yeast of beer is ANAEROBIAN, that is to say, it lives in a liquid deprived of free oxygen; and to become mycoderma or penicillium it is above all things necessary that it should be placed in air, since, without this, as the name signifies, an aerobian being cannot exist. To bring about the transformation of the yeast of beer into mycoderma cerevisiae or into penicillium glauc.u.m we must accept the conditions under which these two forms are obtained. If M.
Pasteur will persist in keeping his yeast in media which are incompatible with the desired modification, it is clear that the results which he obtains must always be negative."
Contrary to this perfectly gratuitous a.s.sertion of M. Trecul"s we do not keep our yeast in media which are calculated to prevent its transformation into penicillium. As we have just seen, the princ.i.p.al aim and object of our experiment was to bring this minute plant into contact with air, and under conditions that would allow the penicillium to develop with perfect freedom. We conducted our experiments exactly as Turpin and Hoffmann conducted theirs, and exactly as they stipulate that such experiments should be conducted--with the one sole difference, indispensable to the correctness of our observations, that we carefully guarded ourselves against those causes of error which they did not take the least trouble to avoid. It is possible to produce a ready entrance and escape of pure air in the case of the double-necked flasks which we have so often employed in the course of this work, without having recourse to the continuous pa.s.sage of a current of air. Having made a file-mark on the thin curved neck at a distance of two or three centimetres (an inch) from the flask, we must cut round the neck at this point with a glazier"s diamond, and then remove it, taking care to cover the opening immediately with a sheef of paper which has been pa.s.sed through the flame, and which we must fasten with a thread round the part of the neck still left. In this manner we may increase or prolong the fructification of fungoid growths, or the life of the aerobian ferments in our flasks.
What we have said of Penicillium glauc.u.m will apply equally to Mycoderma cerevisiae. Notwithstanding that Turpin and Trecul may a.s.sert to the contrary, yeast, in contact with air as it was under the conditions of the experiment just described, will not yield Mycoderma vini or Mycoderma cerevisiae any more than it will Penicillium.
The experiments described in the preceding paragraphs on the increase of organized ferments in mineral media of the composition described, are of the greatest physiological interest. Amongst other results, they show that all the proteic matter of ferments may be produced by the vital activity of the cells, which, apart altogether from the influence of light or free oxygen (unless indeed, we are dealing with aerobian moulds which require free oxygen), have the power of developing a chemical activity between carbohydrates, ammoniacal salts, phosphates, and sulphates of pota.s.sium and magnesium. It may be admitted with truth that a similar effect obtains in the case of the higher plants, so that in the existing state of science we fail to conceive what serious reason can be urged against our considering this effect as general. It would be perfectly logical to extend the results of which we are speaking to all plants, and to believe that the proteic matter of vegetables, and perhaps of animals also, is formed exclusively by the activity of the cells operating upon the ammoniacal and other mineral salts of the sap or plasma of the blood, and the carbo-hydrates, the formation of which, in the case of the higher plants, requires only the concurrence of the chemical impulse of green light.
Viewed in this manner, the formation of the proteic substances, would be independent of the great act of reduction of carbonic acid gas under the influence of light. These substances would not be built up from the elements of water, ammonia, and carbonic acid gas, after the decomposition of this last; they would be formed where they are found in the cells themselves, by some process of union between the carbo-hydrates imported by the sap, and the phosphates of pota.s.sium and magnesium and salts of ammonia. Lastly, in vegetable growth, by means of a carbo-hydrate and a mineral medium, since the carbo-hydrate is capable of many variations, and it would be difficult to understand how it could be split up into its elements before serving to const.i.tute the proteic substances, and even cellulose substances, as these are carbo-hydrates. We have commenced certain studies in this direction.
If solar radiation is indispensable to the decomposition of carbonic acid and the building up of the primary substances in the case of higher vegetable life, it is still possible that certain inferior organisms may do without it and nevertheless yield the most complex substances, fatty or carbo-hydrate, such as cellulose, various organic acids, and proteic matter; not, however, by borrowing their carbon from the carbonic acid which is saturated with oxygen, but from other matters still capable of acquiring oxygen, and so of yielding heat in the process, such as alcohol and acetic acid, for example, to cite merely carbon compounds most removed from organization. As these last compounds, and a host of others equally adapted to serve as the carbonaceous food of mycoderms and the mucedines, may be produced synthetically by means of carbon and the vapour of water, after the methods that science owes to Berthelot, it follows that, in the case of certain inferior beings, life would be possible even if it should be that the solar light was extinguished. [Footnote: See on this subject the verbal observations which we addressed to the Academy of Sciences at its meetings of April 10th and 24th, 1876].
THE GERM THEORY AND ITS APPLICATIONS TO MEDICINE AND SURGERY
[Footnote: Read before the French Academy of Sciences, April 29th, 1878. Published in Comptes Rendus de l" Academie des Sciences, lx.x.xvi., pp. 1037-43.]
The Sciences gain by mutual support. When, as the result of my first communications on the fermentations in 1857-1858, it appeared that the ferments, properly so-called, are living beings, that the germs of microscopic organisms abound in the surface of all objects, in the air and in water; that the theory of spontaneous generation is chimerical; that wines, beer, vinegar, the blood, urine and all the fluids of the body undergo none of their usual changes in pure air, both Medicine and Surgery received fresh stimulation. A French physician, Dr.
Davaine, was fortunate in making the first application of these principles to Medicine, in 1863.
Our researches of last year, left the etiology of the putrid disease, or septicemia, in a much less advanced condition than that of anthrax. We had demonstrated the probability that septicemia depends upon the presence and growth of a microscopic body, but the absolute proof of this important conclusion was not reached. To demonstrate experimentally that a microscopic organism actually is the cause of a disease and the agent of contagion, I know no other way, in the present state of Science, than to subject the microbe (the new and happy term introduced by M. Sedillot) to the method of cultivation out of the body. It may be noted that in twelve successive cultures, each one of only ten cubic centimeters volume, the original drop will be diluted as if placed in a volume of fluid equal to the total volume of the earth. It is just this form of test to which M. Joubert and I subjected the anthrax bacteridium. [Footnote: In making the translation, it seems wiser to adhere to Pasteur"s nomenclature.
Bacillus anthracis would be the term employed to-day.-- Translator.] Having cultivated it a great number of times in a sterile fluid, each culture being started with a minute drop from the preceding, we then demonstrated that the product of the last culture was capable of further development and of acting in the animal tissues by producing anthrax with all its symptoms. Such is--as we believe--the indisputable proof that ANTHRAX IS A BACTERIAL DISEASE.
Our researches concerning the septic vibrio had not so far been convincing, and it was to fill up this gap that we resumed our experiments. To this end, we attempted the cultivation of the septic vibrio from an animal dead of septicemia. It is worth noting that all of our first experiments failed, despite the variety of culture media we employed--urine, beer yeast water, meat water, etc. Our culture media were not sterile, but we found--most commonly--a microscopic organism showing no relationship to the septic vibrio, and presenting the form, common enough elsewhere, of chains of extremely minute spherical granules possessed of no virulence whatever. [Footnote: It is quite possible that Pasteur was here dealing with certain septicemic streptococci that are now know to lose their virulence with extreme rapidity under artificial cultivation.--Translator.]
This was an impurity, introduced, unknown to us, at the same time as the septic vibrio; and the germ undoubtedly pa.s.sed from the intestines--always inflamed and distended in septicemic animals-- into the abdominal fluids from which we took our original cultures of the septic vibrio. If this explanation of the contamination of our cultures was correct, we ought to find a pure culture of the septic vibrio in the heart"s blood of an animal recently dead of septicemia. This was what happened, but a new difficulty presented itself; all our cultures remained sterile. Furthermore this sterility was accompanied by loss in the culture media of (the original) virulence.
It occurred to us that the septic vibrio might be an obligatory anaerobe and that the sterility of our inoculated culture fluids might be due to the destruction of the septic vibrio by the atmospheric oxygen dissolved in the fluids. The Academy may remember that I have previously demonstrated facts of this nature in regard to the vibrio of butyric fermentation, which not only lives without air but is killed by the air.
It was necessary therefore to attempt to cultivate the septic vibrio either in a vacuum or in the presence of inert gases--such as carbonic acid.
Results justified our attempt; the septic vibrio grew easily in a complete vacuum, and no less easily in the presence of pure carbonic acid.
These results have a necessary corollary. If a fluid containing septic vibrios be exposed to pure air, the vibrios should be killed and all virulence should disappear. This is actually the case. If some drops of septic serum be spread horizontally in a tube and in a very thin layer, the fluid will become absolutely harmless in less than half a day, even if at first it was so virulent as to produce death upon the inoculation of the smallest portion of a drop.