The new process of brewing, which it is the principal object of this work to explain, and which will follow as an immediate and inevitable deduction from the novel facts herein demonstrated, cannot be fully understood without a knowledge of all the principles upon which it is founded. One of the most essential of these has reference to the purity of our fermentation. We should gain but little from the use of a yeast uncontaminated by any foreign germs if natural organic substances had the power of organizing themselves by means of spontaneous generation, or by some transformation which took place amongst them, or even by a conversion of certain microscopic beings into certain others. Theories of this kind are still warmly advocated, but, to our thinking, rather from sentimental considerations or prejudice than from any basis of serious experimental proofs.
Be this as it may, we must free our minds from all suppositions which might qualify the exactness of the principles upon which our new process is founded, or cause any doubt in the minds of our readers as to the possibility of its application and the benefits to be derived from its adoption.
§ I.—On the Conditions which cause Variations in the Nature of the Organized Products existing in Infusions.
We have proved by experiment, in the preceding chapter, that a few minutes’ boiling renders liquids, and more especially wort, absolutely free from liability to change when in contact with pure air, that is, air which contains none of the germs of organisms that are continually floating about in the atmosphere.
What is true in the case of wort is equally so in the case of all organic liquids; there is not a single one that could not be rendered inaccessible to any subsequent change if it were brought, first of all, to a suitable temperature, which would vary with the nature of the liquids. Amongst them there are some which, like vinegar, lose their tendency to change after having been rapidly raised to a temperature of not more than 50° C. (122° F.); others, like wine, require a greater heat. Wort, to which no hops have been added, should be subjected to a temperature of not more than 90° C. (194° F.); milk to about 110° C. (230° F.).[21]
It is easy to show that these differences in temperature, which are required to secure organic liquids from ultimate change, depend exclusively upon the state of the liquids, their nature, and, above all, on the conditions that affect their neutrality, whether towards acidity or alkalinity; for it is not difficult to observe that the least changes in these respects lead to considerable variations in the temperatures which we must employ.[22] We could adduce many examples of this. The only difference between the nature of must and that of wine is caused by fermentation. We may say the same thing of wort and beer, and, better still, of new milk and sour milk. Must, to be secured from change, requires a much higher temperature than wine does; similarly, wort requires a much higher temperature than beer. Milk must be heated to about 110° C. (230° F.), as we have just stated, but sour milk would require 20° C. or 30° C. (36° F. or 54° F.) less. Wine, when fresh, ceases to be liable to change after it has been brought to a temperature below 100° C. (212° F.); it requires a temperature of more than 100° C. in the presence of carbonate of lime.
As regards the explanation of the influence which acidity or alkalinity exerts in diminishing or increasing the temperature required to protect infusions and organic matters from ultimate change, although this is a subject which claims special study, we are inclined to believe that acidity permits, and alkalinity prevents, the penetration of moisture into the interior of the cells of the germs belonging to infusions, so that in heating the outer cases of these cells in an alkaline medium we heat the germs in a dry state; and in heating the outer cases in an acid medium we heat the germs when they are moist. We all know that these conditions make a great difference in the resistance which bodies offer to the action of heat. A particle of mould which, in a moist state, cannot survive a temperature of from 60° C. to 100° C. (140° to 212° F.), will preserve its fecundity even if heated to 120° C. (248° F.), if it has been previously well dried.[23]
The nature of the spontaneous productions which we see appear in organic liquids is affected to a remarkable extent by the smallest change in the compositions of those liquids. Generally speaking, as we have often proved in our former works, a feeble acidity is unfavourable to the development of bacteria and infusoria, and, on the other hand, favourable to the growth of mould. A liquid which is neutral or of feeble alkalinity behaves in an exactly inverse manner.
Those who support the theory of spontaneous generation seek to find in the natural differences between the organic productions of various liquids which are simultaneously exposed to the same atmosphere, an argument in favour of their doctrine. These differences, however, are only an effect of the greater or less fitness of a peculiar liquid for a certain kind of growth. When an acid organic liquid, such as the must of grapes for example, is exposed freely to the air, it is besieged simultaneously by spores of _mucedines_, as well as germs of _bacteria_, _leptothrix_, _vibrios_, _&c._, but the latter germs are checked in their development, if, indeed, they are not actually destroyed, by the acidity of the liquid; and we should never find them there in their adult state.
Most moulds, on the other hand, thrive in acid liquids, and therefore they are generally found alone. If we began by saturating must with carbonate of lime, previously dried or otherwise, we should observe opposite phenomena; bacteria, lactic ferments and butyric vibrios would invade our fields long before the spores of mould had time to grow, since the germination of these proceeds very languidly in neutral or alkaline liquids, and an infusion once occupied by a living organism has much trouble in nourishing others; the early developments consume the alimentary substances, especially the oxygen.
Differences as marked as these in the adaptability of certain liquids to certain growths give rise to innumerable illusions, and are one of the chief sources of error in the study of this subject. If we sow in an acid liquid, such as must, an alcoholic ferment, the development of which is not arrested by the acid character of our medium, it will multiply there, and we shall have no difficulty in growing it over and over again in the same acid medium. This being the case, let us suppose that our alcoholic ferment is impure—let us say, mixed with filaments of turned wine, which are due to a ferment checked in its development by the peculiar qualities of the must. In repeating the growth of this alcoholic ferment in the must the filaments which were, by supposition, present in our first sowing, and which cannot reproduce themselves in the must, or do so with great difficulty, will become very scarce in the fields of our microscope; they will not, however, cease to exist, for the repetition of our attempts to grow them only serves to spread the original germs over a larger surface; and, although the eye fails to detect them, they will only have become more difficult to discover. At this point the experimentalist is in danger of falling into error, for when he no longer perceives the foreign ferment he will be inclined to believe that it has vanished, and that he may regard the alcoholic ferment as being free from all impurity, without testing for the purity by a direct experiment.
An example of this mistake is to be found in a recent work by M. Jules Duval. This writer has published a theory according to which yeast becomes transformed into lactic ferment, and likewise into other ferments—that of urea, for example—the only condition of change, according to M. Jules Duval, being that we should cultivate it in suitable mediums. The proofs by which he supports his conclusion are altogether inadmissible, and a simple glance at his experiments enables us to detect innumerable causes of error. M. Duval believes that yeast becomes transformed into lactic ferment from the fact that he obtained a fermentation which furnished lactate of lime and lactic ferment from some sour milk to which he had added some glucose, chalk, and phosphate of ammonia before impregnating it with yeast; but he took no steps to secure himself against introducing into his medium—which was, as a matter of fact, well adapted to lactic fermentation, inasmuch as it was a little alkaline—an alcoholic ferment containing impurities. This was the crucial point in his experiments. M. Duval recognizes this, but he deceives himself when he says, without proof:—“My alcoholic ferment is pure, for I have grown it over and over again in must, preserved in flasks prepared after the manner of those which M. Pasteur uses in his experiments.” Here we have merely a simple assertion; a direct experimental test might have proved that the yeast was impure.
Yeast cannot transform itself into lactic ferment. No matter what the medium may be in which it is sown, _if it is actually pure_ it will never present the least trace of lactic ferment or of any other ferments. By certain changes in the nature of the medium, the temperature, and other conditions, the cells of the ferment may become oval, elongated, spherical, and larger or smaller in size, but they will never produce the most minute quantity of lactic ferment or lactic acid. The whole theory of the transmutation of ferments, which M. Duval has published, is imaginary.[24]
We have asserted that the source from which we obtain our supply of the substances that we expose to contact with the air may likewise furnish a reason for the development of vegetable or animal organisms which make their appearance in our infusions. This may be easily understood. If we expose the infusions to temperatures more or less high, with the object of destroying the vitality of the germs which they may contain, we completely suppress all germs originating from two different sources—those which the infusions may have acquired directly from the atmospheric air, or from the dust upon our utensils, and those which may have been introduced by the materials used in manufacturing our infusions or decoctions, which materials must have been brought from some distance. An infusion, after having been heated to a sufficient degree, can harbour and nourish only such germs as are conveyed to it by the air after the heating. These floating germs are far from being as varied in their nature as it pleases those to believe who are tied down by their arbitrary and faulty interpretation of the knowledge that we have acquired, and the discussions in which we have taken part, during the last fifteen years. Air, unless in violent agitation, can hold only the most minute particles in suspension. The observations which have been recorded concerning organisms of spontaneous growth found in infusions, have always been made in sheltered places—in rooms or laboratories, the atmosphere of which is, relatively speaking, very still. For this reason, in liquids that have been subjected to heat, the _flora_ and _fauna_—if we may use such an expression—are very poor, and all the more so because, as we have recently had occasion to remark, a great number of the organisms which would spring into existence of their own accord, if they were allowed sufficient time for germination, are kept back by others of a more rapid development. The truth of this is evident from the fact that we may observe greater variations in the nature and number of species of living organisms, if we divide one infusion amongst several vessels which are immediately closed up again, than if we leave our infusion in contact with an unlimited volume of surrounding air. By this means we expose each portion of the liquid to no other germs than those existing in a state of suspension in the volume of air introduced into the vessels; and it so frequently happens that we obtain a variety of germs which, coming into contact with a liquid adapted to their nutrition, without having mixed with others, finally multiply there, in consequence of no other organisms of greater activity occurring to impede their slow and laborious propagation.
The nature of the products resulting from raw infusions—that is, those obtained, without heating, from the maceration of organic substances, such as leaves, fruits, grains, or the organs of plants or animals—is much more varied. The reason of this is that such substances generally carry with them not only the particles of dust existing in air that is in motion, but also microscopic parasites, which find a congenial resting-place on their surface or its vicinity. We may cite a few accurate observations on this point, for the subject is one of great interest.
If we boil an infusion of hay, and then expose it to contact with the air in a room, all its productions will be derived from such germs as a comparatively still air can carry about; thus, we shall very rarely find any _colpoda_ in our infusion, for the germs of these infusoria, consisting of rather large cells, can scarcely exist in a state of suspension in motionless air, in spite of their extraordinary diffusion in nature. On the other hand, we almost invariably find _colpoda_ in macerations of raw hay. This difference is easily accounted for; the particles of dust adhering to the surface of hay, especially that which comes from marshy districts, contain the germ-cells of _colpoda_ in abundance. The reason is obvious; rain falls on a meadow and forms little pools of water about the roots of the herbs that grow there; this water remains for some time, and very soon swarms with a multitude of infusoria, especially _colpoda_. Dryness follows; the _colpoda_ becomes encysted, and forms a dust that is wafted by the winds on to the blades of grass, so that the mower will carry away not only his hay, but also myriads of _colpoda_, as well as spores of _mucedines_ and other organisms.[25]
The maceration of pepper will give us some infusoria hardly ever found in other infusions, the reason of this being that such infusoria exist where the pepper grows—in other words, their germs are exotic. That the infusion of a special plant should give us special infusoria, is scarcely more surprising than the discovery of a particular parasite or insect existing on a particular plant, and not on others of a different species that grow near it. Thus it happens that the ferment-germs of must exist on the surface of grapes, whether detached or in clusters. It is only natural that we should find the organ or the plant that is destined later on to become the food of a parasite serving as a habitation for the germs of that parasite.
§ II.—Experiments on Blood and Urine taken in their Normal State, and Exposed to Contact with Air that has been Deprived of the Particles of Dust which it Generally holds in Suspension.
Recourse to the application of heat, in the first place, is an excellent means, as we have just seen, of procuring organic liquids free from all disturbing germs; but there is a still more remarkable and instructive, we may even say more unlooked for, method of securing this result, which may be described as in some measure borrowed from the nature of things. It consists in seeking purity in the natural liquids of animals and plants. It is difficult to understand how the liquids circulating in the organs of animal bodies, such as blood, urine, milk, amniotic fluid, and so on, can possibly secrete the germs of microscopic organisms. There would be excellent opportunities for these germs to propagate themselves if they actually did exist in the liquids appertaining to the animal economy. Life in all probability would become impossible in the presence of such guests. A proof of this is to be found in the multitude of diseases which many of the greatest minds of modern times attribute to parasitic developments of this nature. Medical men of high authority agree in thinking that the questions of contagion and infection will find solutions from the obscurity in which they are now involved in a careful study of ferments, and that hygienists and physicians should labour to secure by every possible means the destruction of the germs of ferments, and should strive to check _their_ development, and prevent the evils which _they_ cause when developed. Great progress has already been made in this direction, and we deem it a signal honour that our researches on the subject have been considered, even by those by whom this progress has been accomplished, as the source from which they derived their first inspirations. We shall, doubtless, be excused by our readers if we recall this fact in relating certain historical details which are especially necessary in order that they may comprehend the principles that we are endeavouring to explain in these “studies.”
It is a matter of regret to us, however, that the facts which we have established should have been accredited with any importance beyond that which is their due. The exaggeration of novel ideas invariably leads to a reaction, which, again, overshooting the mark, brings into disrepute even those points in which such ideas are perfectly just, or, at all events, worthy of serious consideration. There are certain symptoms of such a reaction in the case of our theories: they are evident in the tendency of unreflecting minds to give a total denial to the fact that certain diseases may be derived from certain ferments—organized and living ferments—of the nature of those which have been discovered in the course of the last twenty years. We should be guided by facts, whichever side of the question we espouse, and by facts alone we should test the truth of doubtful discoveries. We are but on the threshold of the exploration of our subject, and we should strive to discover new facts in connection with it, and should deduce from these, whatever they may be, only such conclusions as they may strictly warrant. Unfortunately, there is amongst physicians a tendency to generalize by anticipation. Many of them are men of rare natural or acquired talent, endowed with keen powers of intellect, and the art of expressing themselves fluently and persuasively; but the more eminent they are, the more they are occupied by the duties of their profession, and the less leisure they have for the work of investigation. Urged on by that thirst for knowledge which belongs to superior minds, and perhaps, in some measure, through associating with the upper classes of society, which are becoming more and more interested in science, they eagerly seize upon easy and plausible theories, readily adapted for statement which is general and vague just in proportion to the unsoundness of the facts on which they are based. When we see beer and wine undergo radical changes, in consequence of the harbour which those liquids afford to microscopic organisms that introduce themselves invisibly and unsought into it, and swarm subsequently therein, how can we help imagining that similar changes may and do take place in the case of man and animals? Should we, however, be disposed to think that such a thing must hold true, because it seems both probable and possible, we must, before asserting our belief, recall to mind the epigraph of this work: _the greatest aberration of the mind is to believe a thing to be, because we desire it_.
One of the most distinguished members of the Academy of Medicine, M. Davainne, who was the first to give his attention to rigorous experiments on the influence that organic ferments exercise on the production and propagation of infectious diseases, declares that the idea of his researches on _splenic fever_ and _malignant pustule_ was suggested to him by his perusal of our work on butyric fermentation, published in 1861. In 1850 this gentleman and M. Rayer discovered in the blood of animals attacked by these diseases minute filiform bodies, to which they paid little attention, and which M. Davainne recollected, when he came across our Memoir. He had the foresight to conjecture—a conjecture that was soon confirmed most decisively by his researches—that the former disease, known under the name of _sang de rate_, might be the production of a fermentation analogous to the butyric, in which the minute filiform bodies observed by Rayer and himself, in 1850, played the part which vibrios fill in butyric fermentation. Within two years of this the first works of Messrs. Coze and Feltz appeared. These clever and courageous experimentalists avowed that their beautiful researches had been suggested to them by the perusal of my work on putrefaction, published in 1863. We might also quote the striking and admirably conceived experiments of Dr. Chauveau, on castration.[26] We cannot, however, refrain from reproducing here a letter addressed to us in 1874 by the celebrated Edinburgh surgeon, Mr. Lister:—
“Edinburgh, Feb. 10, 1874.
“DEAR SIR,—Will you permit me to beg your acceptance of a pamphlet which I forward to you by this post, and which describes certain inquiries into a subject upon which you have thrown so much light—the theory of germs and fermentation? It gives me pleasure to think that you will peruse with some interest what I have written about an organism that you were the first to study in your Memoir on lactic fermentation. I do not know if you ever see the records of British surgery. If you have perused them you may have observed from time to time notices of the antiseptic system which I have been endeavouring for the last nine years to bring to perfection. Permit me to take this opportunity of offering you my most hearty thanks for having demonstrated by your brilliant researches the truth of the theory of putrefactive germs, and for having afforded me in this manner the sole means of perfecting the antiseptic system.
“Should you ever come to Edinburgh I am sure that you will be truly gratified to see in our hospital the extent to which the human race has profited by your work. I need hardly add, that I should have great satisfaction in showing you how greatly surgery is indebted to you.
“Excuse the freedom which I have taken in addressing you, on the grounds of our common love for science, and believe in the profound esteem of yours, very sincerely, JOSEPH LISTER.”
The wad dressing of Dr. Alphonse Guérin, surgeon at the Hôtel-Dieu, Paris, which has already rendered great assistance to surgery, and has been the subject of a very favourable report at the Academy of Sciences in Paris, was invented by its author in consequence of certain reflections suggested to him by the perusal of our researches. The commission which framed the report made, through M. Gosselin, some wise reservations in the case of certain theoretical ideas which M. Guérin had not sufficiently proved by experiment. We have no doubt, however, that when the matter is thoroughly examined, facts will confirm the truth of the wide views entertained by the surgeon of the Hôtel-Dieu.
Dr. Déclat has founded quite a new system for the treatment of infectious diseases, which is based upon the use of one of the best known antiseptics, phenic acid. His theory, which he affirms was suggested to him by our studies on fermentations, is, that the diseases which transmit themselves are, each of them, the product of a special ferment, and that both medical and surgical professors of therapeutics should make it their study to prevent exterior ferments from penetrating into the liquids of our economy, or in the event of these ferments having found their way into the system, to discover _antiferments_ for their destruction, without effecting any change in the vitality of the histological elements of the liquid or tissues.
There is no doubt that extreme caution must be exercised in dealing with questions of this kind, as M. Sédillot has authoritatively remarked; but at the same time it cannot be denied that the more such questions are discussed with exactness, the more those celebrated practitioners who originated them are confirmed in the ideas which first guided them. We may give another example of this.
In 1874, in consequence of a communication addressed to the Academy of Sciences by Messrs Gosselin and A. Robin, on the subject of ammoniacal urine, we made the observation that we should endeavour to ascertain if, in all such cases, the urinary fluids were not rendered ammoniacal by the presence of the little ferment of the urea, which we have previously noticed,[27] and which has since then been discussed with remarkable intelligence by M. Van Tieghem, in the thesis which he maintained for his doctor’s degree. The suggestion and the considerations that justified it led to a discussion before the Academy of Medicine, in which contrary opinions were maintained. We lost no time in submitting these to the test of facts. We could not find a single person suffering from ammoniacal urine, in whose case the little ferment which we mentioned was not to be detected. Our predictions were thus completely justified.
As early as 1864 the _Gazette Hebdomadaire de Médecine et de Chirurgie_ published an account of urine made ammoniacal in the bladder, the author of which, Dr. Traube, makes the following observations:—“It was believed that, in consequence of the retention of the liquid, and the resulting distension of the bladder, that organ became irritated and produced a larger quantity of mucus, and that this mucus was the ferment which caused the decomposition of the urea, by virtue of a chemical action peculiar to itself. This belief can no longer be held in presence of M. Pasteur’s discoveries. This observer has demonstrated, in the most conclusive manner, that ammoniacal fermentation, like alcoholic and acetic, is produced by living beings, whose pre-existence in the fermentable liquid is a necessary condition of the process. The preceding fact offers a remarkable proof of M. Pasteur’s theory. In spite of the long duration of the retention, alkaline fermentation was not produced by an excessive secretion of the vesical mucus or of pus: it only began from the moment when the germs of vibrios passed from the outside into the bladder.”[28]
Finally, we may conclude with perfect truth that the liquids of our economy, blood and urine, for instance, may afford a harbour to different ferments, even in the inmost parts of the organs, when external causes enable such ferments to find their way into those liquids, and that diseases of greater or less gravity result from this cause. On the other hand, it must be admitted that the bodies of animals in a state of health afford no means of entrance to these external germs. At the same time, direct experiment alone can convince the mind as to the truth of this latter assertion. Let us take some of the substances that are to be found inside living animals in perfect health, and expose them, in the same condition in which life has formed them, to contact with pure air.
[Illustration: Fig. 5.]
[Illustration: Fig. 6.]
For this purpose we must provide ourselves with a glass flask, joined to a copper tap by means of an india-rubber tube, as shown in Fig. 5. The two branches of the tap should be about twelve centimetres long (about ½-in.), the one which is free tapering off like the end of a pipe. In order to destroy all germs which may exist in the flask, we must join the free end of the copper tube to a platinum tube kept at a very high temperature, after having carefully introduced into the flask a small quantity of water, and expelled all the air by converting the water into steam. Then as we allow the flask to cool, the air which re-enters it will necessarily pass through the hot tube (Fig. 6).
[Illustration: Fig. 7.]
We may cause the water in the flask to boil at a temperature of more than 100° C. (212° F.) by fitting to the free end of the platinum tube a glass tube bent at right angles, which we plunge to any depth in a deep vessel filled with mercury (Fig. 7). Whilst the water is boiling under pressure, we must separate the tube which is plunged in the mercury; the water in the flask will continue to boil at the ordinary pressure. We must then leave the flask to cool. It will gradually become filled with air that has been heated to a high temperature, more than sufficient to burn up all the organic particles of dust which that air could have contained. When the flask is cold we must close the tap and detach it, and proceed to prepare other similar flasks. It will be advisable to close the tap when the temperature of the flask is still a few degrees above that of the surrounding atmosphere: this precaution will cause the air in the cooled flask to have a lower pressure than that of the external atmosphere.
During the interval which must elapse between the preparation and the use of a flask, it is a good thing to keep the free branch of the tap inclined towards the ground, to secure the inside of its tube from the deposit of external particles of dust. Whether this precaution be adopted or not, we must take care to heat this branch in the flame of a spirit lamp just before we bring our flask into requisition.
If we have to study blood, we must take it from a living animal—a dog, for example. We must expose a vein or an artery of the animal, and make an incision into which the end of the free tap-branch, which has previously been heated and allowed to cool, must be introduced and fixed by a ligature in the vein or artery. On opening the tap, the blood will rush into the flask; it must then be closed, and the flask placed in an oven at a certain temperature. We have successfully accomplished these manipulations, thanks to the kind help of our illustrious colleague and friend, M. Claude Bernard.
The operation is nearly the same in the case of urine. The end of the free branch of the tap is introduced into the passage of the urethra; the tap is turned at the moment when the urine is emitted, which is then allowed to pass into the flask, until it is a third or half filled.
The following were the results of our experiments:—Blood underwent no putrefactive change even at the highest temperatures of the atmosphere, but retained the odour of fresh blood, or acquired the smell of lye. Contrary to what we might have expected, the direct oxidation of the constituents of blood by slow combustion was rather sluggish. After subjecting our flasks to a temperature of 25° C. or 30° C. (77° F. to 80° F.) in an oven for several weeks, we observed an absorption of not more than 2 or 3 per cent. of oxygen, which was replaced by a volume of carbonic acid gas of about an equal bulk.[29]
Nearly the same results were obtained in the case of urine; it underwent no radical change; its colour merely assumed a reddish brown tint; it formed some small deposits of crystals, but without becoming at all turbid or putrefying in any way. The direct oxidation of the urinary substances was likewise very sluggish. An analysis of the air in one of the flasks, made forty days after the commencement of the experiment, gave the following results:—
Oxygen 19·2 Carbonic acid 0·8 Nitrogen 80·0 ——- 100·0
These experiments on blood and urine which we have just mentioned date from 1863.[30] Ten years afterwards, in 1873, they were confirmed in an important and striking manner by the results of a very able series of experiments, which were carried out in our laboratory, by M. Gayon, who was formerly a pupil in the _École Normale Supérieure_. M. Gayon proved that what held good in the case of blood and urine, also held good in the case of the substances contained in eggs. He found that the whites of eggs might be exposed for any length of time to contact with air, as also might the yolks, or the white and yolks mixed, without any putrefactive change or fermentation resulting, and without the smallest microscopic germ showing itself, the sole condition being that the air must be freed from all organic particles of dust, germs of mould, _bacteria_, _vibrios_, and other organisms which it holds in suspension. This was only a part of the important facts brought to light by M. Gayon. Amongst other things, he proved that spontaneous putrefaction in eggs is invariably caused by the development of organized ferments, thereby correcting the opposite statements announced by M. Donné and M. Béchamp, who were led by their observations to believe that the change in eggs took place quite independently of the action of _vibrios_ and _mucedines_.[31]
It is almost superfluous to remark how greatly the results of these experiments on blood, urine, and the components of the egg are opposed to the doctrine of spontaneous generation, as also to most modern theories on the generation of ferments. As long as experiments relating to the question of so-called _spontaneous_ generation were made on heated substances, the advocates of _heterogenesis_ had some grounds for asserting that such materials could not satisfy the conditions of spontaneous life, and that we should obtain different results by using natural organic liquids, which, if exposed to contact of pure air would doubtless serve for the production of new beings which did not issue from parents which resembled them. This novel enunciation of the hypothesis of spontaneous generation, the only one, we think, that could be defended after the publication of our Memoir, in 1862, is condemned by the preceding facts.
The same facts completely upset the hypothesis recently maintained by Messrs. Fremy and Trécul on the subject of the causes of fermentation.
“Side by side with the immediate, definite principles which may be formed by synthesis,” says M. Fremy, “such as glucose, oxalic acid, and urea, other substances of greatly inferior stability exist, the constitution of which is considerably more complicated, containing all the elements of living organs, such as carbon, hydrogen, oxygen, nitrogen, and even phosphorus and sulphur; and often salts of lime and of the alkalies besides. These bodies are albumen, fibrin, casein, the congeners of vitellin and others. Chemical synthesis cannot reproduce them. It is impossible, in my opinion, to regard them as immediate, definite principles. I designate them by the general name of _semi-organized_ bodies, because they hold an intermediate place between the immediate principle and the organized tissue.
“These semi-organized bodies, which contain all the elements of organs, have the power, like a dry seed-grain, of existing in a state of organic immobility, and of becoming active under circumstances which favour organic development. By reason of the vital energy that they possess they undergo a succession of decompositions, giving origin to new derivatives, and to the advent of ferments, not by any process of _spontaneous generation_, but by a _vital energy_, which pre-exists in the semi-organized bodies, and is simply carried on, when this energy manifests itself, in these most varied organic changes.”
After having expressed these hypothetical and confused opinions, M. Fremy continues:—“I do not consider, then, that these semi-organized bodies serve merely as nourishment for certain animal and vegetable organisms, which may be the sole agents of fermentations, but I give them a direct _rôle_ and admit that, under the influences which I have already cited[32] they may assume a real and complete organization, and produce ferments which are not derived, as we have seen, from a germ or an ovum but from a semi-organized body, the vital energy of which has become active.[33] It will be seen that these opinions are quite different from those which M. Pasteur has maintained in his works, since they attribute the origin of alcoholic and lactic ferments to an albuminous substance. Taking the case of alcoholic fermentation alone, I assert that, in the production of wine, it is the juice of the grape itself which, in contact with air, produces grains of yeast, by the transformation of the albuminous substances. M. Pasteur, on the contrary, maintains that the grains of yeast are produced by certain germs.”[34]
We have combated these propositions, so extraordinary and unsupported by any rigorous experiments, before the Academy of Sciences, where they were first enunciated. On that occasion we related the facts in connection with blood and urine, which we have just discussed. Could there be any more forcible argument against the theory of our honourable colleague than those facts? Here we had natural albuminous substances, forming part of matter eminently liable to putrefactive change and fermentation, which produced no ferments of any sort whatsoever when brought into contact with air deprived of its organic particles of dust.
Under no known circumstances is albuminous matter transformed into grains of yeast or any other organized ferment, and, to our thinking, nothing can be more chimerical than the gratuitous hypothesis of _hemi-organism_.
We shall proceed to new proofs of this, dealing this time with a liquid formed by the life of a vegetable.
§ III.—Experiments on the Juice contained in Grapes.
In the course of the discussion which took place, at the Academy, on the subject of the generation of ferments, properly so called, much was said about the oldest known fermentation, that of wine. We at once resolved to demolish M. Fremy’s theory, by a decisive experiment on the juice of grapes.
[Illustration: Fig. 8.]
We prepared forty flasks, capable of holding from 250 c.c. to 300 c.c. (from 9 to 11 fl. oz.) and shaped as represented in Fig. 8. These we filled with filtered must, which was perfectly bright, and which, like all acid liquids, would remain sound, after having been boiled for a few seconds, although the ends of the long curved necks of the flasks containing the must might remain constantly open for months or years.
We washed, in a few cubic centimetres of water, part of a bunch of grapes, washing the grapes separately, or the grapes and the wood together, or even the wood of the bunch alone. This washing was easily accomplished by means of a perfectly clean badger’s-hair brush, the water receiving all the particles of dust adhering to the surface of the grapes and the wood of the bunch. By means of a microscope we easily proved that this water held in suspension an infinite number of organized corpuscles, some of them bearing a very close resemblance to the spores of fungoid growths, others to alcoholic ferment, others to _mycoderma vini_, and so on.[35]
[Illustration: Fig. 9.]
We next proceeded to put on one side ten of our forty flasks, to serve for subsequent corroboration; in ten others, by means of the tube which is represented on the right hand side of the flask (Fig. 8), we put a few drops of the water in which the bunch of grapes had been washed; in a third series of ten flasks we put a few drops of the same liquid, after having previously boiled it. Lastly, we introduced into the ten remaining flasks a drop of grape-juice, taken from the inside of an uninjured grape. To do this we had to bend the right-hand tube of each of our last ten flasks, drawing it out to a fine point and closing it in the flame, as represented in Fig. 9 A. This fine closed point was filed round near its extremity and then thrust, as represented in Fig. 9 B, into a grape placed on a hard substance; when the point _b_ was felt to touch the substance supporting the grape it was broken off by a slight pressure sideways at the point _a_, where the file marks had been made. We had taken care to secure a slight vacuum in the flask beforehand; this now caused a drop of the juice to be drawn into the flask. We then drew out the fine point, and closed it immediately in the flame of a spirit lamp. The vacuum was produced by heating the flasks in our hands, or over the flame of a lamp, thus causing a little air to be forced out through the end of the bent tube, which we then closed up with the lamp. When the flask was cool, the slight difference of pressure sufficed to force into it some of the juice contained in the grape, as we have just described. The drop of juice that is sucked into the flask generally remains in the curved part of the fine tube; to mix it with the must we must incline the flask so as to bring the must in contact with the drop; after that we may replace the flask in its natural position.
The following are the results presented by our four series of comparative experiments in the different cases. The first ten flasks—our standard flasks, containing must boiled in contact with pure air—showed no signs of organized products; the must might have remained in them for any number of years without change. Our second series of flasks, which contained the water in which the grapes, separately and in bunch, had been washed, had undergone alcoholic fermentation in every instance; this had manifested itself in all the flasks in the course of about forty-eight hours, the temperature being at about summer heat. At the same time that the yeast made its appearance in the form of little white lines, which gradually joining together formed a deposit on the sides of the flask, we perceived minute flakes of _mycelium_ forming; sometimes as a single fungoid growth, sometimes combined with another, or with many together—these growths being quite independent otherwise of the yeast or alcoholic ferment. In several cases, too, _mycoderma vini_ showed itself on the surface of the liquid in the course of a few days. _Vibrios_ and lactic ferments, properly so called, could not make their appearance, on account of the nature of the liquid.
The flasks of our third series, containing the water in which the bunch of grapes had been washed, and which we boiled before our experiment, remained as free from change as the flasks of our first series had done.
Lastly, our fourth series of flasks, containing the drops of juice taken from inside the grapes, remained equally free from change, although we could not be certain of having removed, in every case, without exception, all causes of error which must inevitably occur sometimes in so delicate an experiment.
These experiments cannot leave the least doubt on our minds:
That must, if boiled, will never ferment when in contact with air that has been freed from the germs which exist in it in a state of suspension.
That must may be fermented, after boiling, by introducing into it a very small quantity of water, in which a bunch of grapes has been washed.
That must will not ferment if we introduce into it some of this same water which has been boiled and afterwards cooled.
That must will not ferment if we introduce into it a small quantity of the juice contained in a grape.[36]
It follows, then, that the ferment which causes grapes to ferment in the vintage tub must come from the exterior, and not the interior of the grapes. Thus, the hypothesis of MM. Trécul and Fremy, according to which albuminous substances transform themselves into grains of yeast by the action of a peculiar vital force, is annihilated; _à fortiori_, there can no longer be a question concerning Liebig’s theory, on the transformation of albuminous substances into ferments, by a process of oxidation.
Our readers may be curious to know what M. Fremy has been able to oppose to such crucial experiments; they could scarcely have imagined the following:—
“In my experiments, which I have varied in every possible manner,” says that gentleman, “I have found that it is almost impossible to discover alcoholic fermentation, appreciable by its results, in a single drop of grape juice, and I may add that this fermentation must be still more difficult to discover when this drop has been drowned in a large quantity of juice that has been previously boiled.”[37]
It will be admitted that we were justified in saying, at the commencement of this paragraph, that we should demolish the theory which was opposed to ours, and which its advocates have been constrained to defend by hypotheses manifestly false.
At the meeting following the one in which M. Fremy declared _that minute quantities do not ferment_, we had the malicious satisfaction of showing a great many very small closed flasks, into each of which we had caused a single drop of the must of crushed grapes to be introduced by suction. We broke the thin points of many of them, in the presence of the Academy, and every one of them showed by a sharp hissing, which was audible at a distance, that fermentation was proceeding in the drop of liquid they contained. M. Fremy was there, but he made no remark.
We may cite some very curious facts on the subject of the period at which the germs that develop yeast are in a condition to be able to cause fermentation.
On July 25th, 1875, in the neighbourhood of Arbois (Jura), the grapes were still green and of the size of peas. We went to a vine that was far from paths and roads, and there, with a pair of small scissors, cut some grapes from off a bunch and let them fall with their short stalks into tubes half filled with gooseberry must, previously rendered unalterable by boiling. These tubes we closed again with all possible precautions, using corks which had been passed through the flame of a spirit-lamp and carried them to our laboratory, where we left them to themselves. Some days afterwards we saw diverse fungoid growths appear in most of the tubes, but not one of them then, or subsequently, presented the least appearance of fermentation. The germs of yeast at that period of the year did not exist either upon the woody part of the bunch, or upon the grapes. In Chapter V. we shall return to observations on this subject.
§ IV.—Wort and Must Exposed to Common Air.
If the principles which we have laid down possess all the value that we attribute to them, if the cause of change in natural or artificial organic liquids does not exist in those liquids themselves, if change considered in itself depends upon the nature and number of the particles of dust in various places, if it is, besides, radically affected by the composition of the liquids, it must necessarily follow that wort or must, whilst, under certain circumstances of exposure to air, it remains absolutely free from life and its results, will, under other circumstances, give rise to a variety of organisms and their corresponding fermentations. This is, in fact, the lesson which direct proofs will teach us. Before entering upon these new observations in detail, we must call the reader’s attention to the difficulty, as experience has shown, of interpreting correctly the facts connected with the spontaneous impregnations of organic liquids.
Gay-Lussac crushed some grapes under a bell-jar filled with mercury, after having washed them in hydrogen, to expel the air adhering to the grapes and the sides of the jar. Having waited for several weeks without detecting any signs of fermentation, he introduced some bubbles of oxygen, and fermentation showed itself the following day. Gay-Lussac concluded that the fermentation of must could not commence without the help of oxygen.[38]
Under the conditions of his experiment nothing could be truer, and we must admire the diffidence with which this great natural philosopher interpreted the fact that he had observed. Another French natural philosopher, however, M. Cagniard-Latour, observed that the ferment of alcoholic fermentation was a little cellular plant. What was its origin in Gay-Lussac’s experiment?
The advocates of the doctrine of spontaneous generation were ready with their explanation, and we have seen how MM. Trécul and Fremy, following many others, did not hesitate to maintain that the little plant with all its particles was produced by the action of oxygen on the albuminous substances contained in the juice of the grapes. The experiments, which we have given in the preceding paragraph, show us positively that germs of the ferment of must exist on the surface of the grape, and that, consequently, Gay-Lussac’s experiment has a more simple and natural explanation. The germs of the ferment existing on the surface of the grape become mixed with the juice of the grape when the latter is crushed; these germs remain inactive in the presence of hydrogen; they vegetate as soon as oxygen is introduced to them.
Moreover, the results of our labours in connection with _spontaneous_ generation, in 1862, teach us that in Gay-Lussac’s experiment the germ of the ferment might also have had its origin either in certain particles of dust adhering to the sides of the glass bell, or upon the mercury; and, in a laboratory where alcoholic fermentation is studied, dust invariably contains dry cells of ferment. The necessity of oxygen for the success of the experiment is surprising, when we reflect that alcoholic fermentation often takes place in liquids that are not exposed to contact with air; but we shall prove by experiment that, notwithstanding what may happen during fermentation, oxygen has the greatest influence on the readiness with which ferment develops itself, and that this gas is indispensable to the revival of withered cells, and still more so to the germination of special cells, which we may consider to be the true germs of the little plant.
The advocates of the doctrine of _spontaneous_ generation have based most of the objections which they vainly urge against their opponents upon erroneous interpretations of certain facts relating to the spontaneous impregnation of organic infusions. Taking a very wrong view of the essential conditions of the phenomena, they require that the assertors of the diffusion of the germs of microscopic organisms should be compelled to place at any one point of space, so to say, all the germs of the products of infusions; a demand which really borders on absurdity. They believe, or feign to believe, that we are bound to admit the existence of germs of must in all places and at all times, on the banks of rivers and on the loftiest mountains, and so on. “Fermentations,” said one of these gentlemen one day, before the Academy, “cannot depend upon chance particles of atmospheric dust. How is it possible that germs of yeast can be present everywhere throughout the universe, ever ready to fall upon must?” It is an established fact that grapes crushed in any part of the globe whatsoever, even on a glacier or at the highest elevations, can set up a fermentation. The explanation of this pretended impossibility is most simple, for we know, from the facts related in the preceding paragraph, that grapes carry on their skins the germs of their own ferments.
In experiments relating to the kind of organisms which we are discussing we must never fail to take into account the action of the particles of dust spread over the articles that are used. Very often an effect that should be attributed to germs adhering to the vessels and utensils used in experiments, the origin of which may be altogether special, is erroneously imputed to the dust-forming germs—that is to say, to those germs which exist in a state of suspension in the air.
In our Memoir of 1862, which we have quoted several times, we have explained that it is almost impossible to draw any serious conclusions from experiments made in a basin of mercury, because of the organic particles of dust which always exist in that metal, and which, without the knowledge of the operator, pass into the interior of the vessel, where they produce certain changes which one is tempted to impute to _heterogenesis_.
In all classical works an experiment of Appert’s, reproduced by Gay-Lussac, is given. This, through a faulty interpretation, led to the hypothesis of the continuity of the causes of fermentation, if we may use such a term, in the atmospheric air.[39]
When we decant bottles of must, which has been preserved by Appert’s method, into other bottles, all the latter soon set up a fermentation: this constitutes the experiment. If it were proved that the must, whilst being decanted, came in contact with atmospheric air alone, as Gay-Lussac believed, we should be compelled to admit, according to the theory of germs, that the must had come in contact with some particles of ferment in the air during decanting. And again, if it were shown that the experiment could succeed in any place whatsoever, we must come to the conclusion that germs of ferment exist everywhere in a state of suspension in the air.
“I have taken,” writes Gay-Lussac, “a bottle of must that had been preserved for a year and was perfectly transparent, and have decanted the must into another bottle, which I then carefully corked and exposed to a temperature of 15° C. to 30° C. (59° F. to 86° F.). Eight days afterwards the must has lost its transparency; fermentation has taken place in it, and soon my must has become transformed into a vinous liquor, sparkling like the best champagne. A second bottle of must that had been preserved for a year, like the preceding one, but which had not been brought into contact with the air, has presented no signs of fermentation, although placed under conditions most favourable to its development.”
The result of this experiment, when roughly made, is correctly described by Gay-Lussac; in other words, it may be proved that if, at the time of vintage, we prepare some bottles of must, after Appert’s process, and, in the course of time, open them and decant their contents into other bottles, we shall soon see the must ferment and deposit yeast. It is, nevertheless, equally certain that the inferences which have been drawn from this celebrated experiment have been founded on error, and that the germs of yeast are very rarely derived from the particles of dust floating in the air with which the must comes in contact. The germs in question are, in our opinion, generally derived, not from the air, but from the sides of the bottles, from corks, from the string employed in corking, from corkscrews, and from a variety of other things. The reason for this is that any room, vault, cellar, or laboratory where the grapes, or must, or vintage, are handled—unless special precautions, of which Appert and Gay-Lussac certainly never thought, are taken—all the utensils, as well as all articles of clothing, and all the sides of the bottles which the hands have touched, are contaminated by cells of ferment derived from must that has fermented, or by germs from the surface of grapes and clusters. Thus, at the moment of decanting the must, a thousand accidental circumstances may lead to the introduction of those germs, the origin of which, as we have seen, may be actually traced to the very grapes which served for the manufacture of the must. In other words, we believe the inference that the germs of yeast which cause the experiment to succeed, are derived from particles of dust floating in the air of the place where we decant the contents of our bottles, to be altogether an erroneous one.
Since the preceding remarks were written, we have endeavoured to repeat this experiment of Gay-Lussac’s in such a manner as we believed would confirm our views, by varying the conditions in such a way as would cause it to succeed or fail, according to the circumstances of the manipulation employed.
On December 7th, 1874, we took two bottles of must which we had preserved, after Appert’s process, in our laboratory from the beginning of October, 1873. Both of these were covered with dust—the dust that floated about in our laboratory. We decanted them as follows:—One bottle, which we handled without special precautions, we uncorked by means of an ordinary corkscrew, and decanted into another bottle that had been well washed, as bottles are washed when they are to be used subsequently. This bottle was taken from a number that had been standing upside down on a drainer for a fortnight. We took no precaution to remove the dust which covered the exterior of the bottle of must, or to purify the washed bottle. The second bottle of must, on the other hand, was decanted after we had removed the dust that covered it; its cork was cut off close to the string, and the flame of a spirit lamp was passed over the string and the surface of the cork; and, as a final precaution, the corkscrew was passed through the flame. As for the bottle into which we subsequently decanted the must, we first plunged it in a hot-water bath kept at 100° C. (212° F.), then took it into a garden to cool upside down in the open air. After these precautions we removed it, and immediately decanted into it the must from the second bottle.
The first bottle showed signs of growths, both on the surface and at the bottom of the must, the day after the operation, and manifested the first symptoms of alcoholic fermentation on December 16th. The contents of the second bottle remained perfectly unchanged after being exposed to the warmth of a stove for several months.
Can anything be more conclusive than these facts? They are in perfect keeping with the views that we have recently expressed, and with the principles that we have maintained for nearly twenty years, on the subject of the causes of change in organic liquids.
It is by no means our intention to assert that in the atmospheric air there exist no germs of ferment in a state of suspension, as fine dust. Beyond all doubt they do so exist in that state; but, as a rule, in comparatively small number, their abundance or scarcity being dependent upon circumstances which control their multiplication, favouring or restricting it, as we are about to prove.
On May 2nd, 1873, we uncorked two ordinary bottles filled with wort, prepared in December, 1872, after Appert’s process. To avoid the causes of error which we have mentioned, we uncorked the bottles in the following manner:—The cork was cut off to the level of the neck; the cork and string were next passed through flame, regardless of burning and charring them; we then gently extracted the cork by means of a corkscrew which also had been passed through the flame.
The bottles thus prepared were placed on the table of an underground room, in which we were continually making experiments on alcoholic fermentation.
_1st bottle._—On May 7th we observed little particles of fungoid growth on the surface of the liquid, and at the bottom were large flakes of _mycelium_.
On May 11th a veil of _mycoderma vini_ had formed: there were no signs of fermentation.
On May 13th vigorous fermentation commenced; it lasted until May 23rd. The microscope revealed yeast in globules of two sizes, the larger of which were considerably less numerous than the others. There were no signs of lactic or butyric ferment.
_2nd bottle._—May 7th, particles of fungoid growth on the surface of the liquid, and also a veil of _mycoderma vini_. On May 11th, 13th, and up to the 23rd, no signs of fermentation were visible. On May 30th fermentation was active. The microscope showed us yeast mixed with butyric _vibrios_.
In this case, alcoholic ferments had come into existence, and from the precautions taken at the moment when the liquid was brought into contact with the external air, it is certain that the advent of the germs of those ferments, as also those of the other organisms which made their appearance—the fungoid growths, _mycoderma vini_ and _vibrios_—could only be accounted for by the fall of particles of dust floating about in the room. It follows, then, that under certain circumstances germs of alcoholic ferment may be found floating in the air; but we can readily show that the peculiar conditions of the place had a large share in bringing about the results obtained by the foregoing experiment.
The same day, May 2nd, 1873, we uncorked, with the precautions that we have already described, four other bottles of the same must. These were placed in a room which was used less frequently than the preceding one, and in which experiments relating to fermentation were seldom conducted.
_1st bottle._—On May 8th we observed on the surface of the liquid large, frothy pieces of mycelium (_mucor mucedo_ or _mucor racemosus_). The liquid was perfectly bright.[40]
May 30th.—No signs of actual fermentation yet visible.
_2nd bottle._—On May 8th we noticed a thin, greasy-looking scum on our liquid, which had become turbid and acquired a sour smell. The microscope showed that this scum was formed of _mycoderma aceti_. On May 30th the scum had assumed a whitish appearance, and seemed to be dead; there was a green spot of _penicillium glaucum_ upon it. No signs of fermentation.
_3rd bottle._—May 8th, patches of fungoid growth on the surface of the liquid. May 30th, thick and abundant fungoid growth, but no fermentation.
_4th bottle._—May 8th, little patches of fungoid growth, and a scum of _mycoderma vini_. May 30th, still no fermentation.
Up to the month of August, 1873, not one of these bottles gave the least sign of alcoholic or other fermentation.
On December 16th, 1872, we uncorked four bottles of wort, which also had been preserved by Appert’s process; these we placed on an oven, where there were always vessels fermenting, at about 25° C. (77° F.), but where none of the manipulations required for the starting or final study of fermentations were practised. The next day a fungoid growth, but unaccompanied by any signs of fermentation, made its appearance, and this state of things lasted for five months, after which we ceased to keep these bottles under observation.
On May 26th, 1873, we uncorked, with all the necessary precautions, ten bottles of wort, which had been preserved from April 9th, and then left them undisturbed in a room where we were constantly engaged in the study of fermentation.
On the following day, some patches of fungoid growth appeared on the surface of the liquids.
May 30th.—Fermentation commenced in one of the bottles.
May 31st.—A second bottle likewise began to ferment.
June 9th.—Four bottles, including the two preceding ones, were now in a state of fermentation. The six bottles that had not fermented were thereupon covered with caps of paper, taken from the centre of a ream of paper and passed through a flame. After this, and up to August 1, when our observations were discontinued, these six bottles underwent no fermentation.
From these examples, which are confirmed by many others that we shall have occasion to mention in the course of this work, it will be seen that the germs of alcoholic ferment are not present in every little point of space, constantly ready to fall upon any object, not even in those places where one is perpetually dealing with that kind of growth.[41] If we conduct our experiments with exactness, we very soon learn that all that has been written on the facility with which saccharine musts may be made to ferment, by being rapidly brought into contact with the surrounding air, is greatly exaggerated.
The germs of ferments, especially of alcoholic ferments—the yeast of beer and the yeast of wine—are not nearly as abundant in atmospheric air, or in the particles of dust spread over the surface of things, as are the spores of fungoid growths. It is easy to understand this, for spores are generally borne by aerial organs in a state of dryness, so that the least breath of wind catches them up and carries them away, whilst ferments are composed of moist cellules that do not readily become dry.
The vacuous flasks, partly filled with organic liquids, which are opened and closed again immediately, frequently give us fungoid growths, but very rarely alcoholic fermentation, although in the latter respect they may not be absolutely sterile. We may cite some proofs of this.
On June 19th, 1872, we prepared seven flasks of saccharine liquid, impregnated with yeast—our flasks were of 300 c.c. capacity (about 10 fl. oz.)—and contained 100 c.c. of the liquid; we then drew out their necks to a small opening, which was sealed during boiling, after the steam had expelled all the air.
On June 29th we opened them in the principal room of our laboratory.
On July 9th, two of the seven flasks gave no sign of organized products; the others were swarming with _mycelia_, submerged or fruiting on the surface of the liquid, and either with or without _bacteria_ entangled in their flakes. In two of the flasks there were visible at the bottom of the liquid some white streaks, which is an indication sometimes of the presence of alcoholic ferment, but much more frequently of a little cellular plant resembling it in appearance, but purely aerial in its growth, that is to say, taking no part in fermentation. Some days afterward, we saw bubbles of gas rising from the bottom of one of these flasks, and then fermentation proceeded so rapidly that we were obliged to open the neck to avoid an explosion. We append a sketch of its ferment (Fig. 10).
[Illustration: Fig. 10.]
The other flask with the white streaks showed no signs of any fermentation.
In this kind of observation we rarely succeed in obtaining active ferments, the reason being that we deal with volumes of air that are too limited for the few germs of ferment that exist in a state of suspension in it.
We are more sure of success if we expose a tolerably large surface of saccharine liquid to the open air, because, under such circumstances, even if the exposure is of short duration, a considerable volume of air will pass over the surface of the liquid.
On May 29th, 1873, at five o’clock in the afternoon, we placed in the underground room previously mentioned, at a height of about two feet, ten porcelain dishes having surfaces of from thirty-five to forty square inches. We had just taken them from boiling water, and after allowing them to cool we placed in each quantities of wort to about one-third of an inch deep, which we poured from bottles uncorked with every precaution against the chance of the wort coming into contact with anything besides the floating particles of dust. On May 30th, at five o’clock, that is after twenty-four hours of exposure to the air of the room, we emptied the contents of the basins separately into glass flasks with long necks, which had been treated with boiling water and then cooled, necks downwards. The beak of the basin, by means of which the liquid was poured, and the funnels—for we used a separate funnel for each flask—had been passed through the flame. The whole ten flasks were then placed in an oven at a temperature of 25° C. (77° F.).[42]
On June 1st six of the flasks gave signs of fermentation, and next day all the flasks were fermenting.
The following are some of the numerous microscopic observations which we made on the liquids and their deposits.
On June 1st the liquid in one of the six flasks which had begun to ferment was covered with a continuous scum of _mycoderma vini_, below which appeared a filamentous network belonging to a _mycelium_, or other fungoid growth. But neither in the liquid itself nor in the deposit could we perceive any cells of the ferment of beer; the field, however, was swarming with active butyric _vibrios_, rather thick and short, their length being about twice their diameter. This fermentation was exclusively butyric.
On June 2nd another of the flasks showed no _vibrios_, but alcoholic ferment in small quantity, much lactic ferment, consisting of little particles contracted at the middle and non-mobile, and, finally, some slender filaments, resembling those represented in Plate I., Nos. 1 and 2.
On June 3rd we examined the liquid in the flask which showed the most marked fermentation. In addition to the flakes of fungoid growths, checked in their development through want of air, we found at least five distinct productions, which are represented in the accompanying sketch (Fig. 11).
_aaa._—Thick cells of alcoholic ferment, the size of which is given in our sketch: thus, 13/450 indicates that the corresponding figure is 13/450 millimetre in length (rather more than 1/1000 inch).
_bbb._—Small alcoholic ferment, such as we generally see in the must of acid and sugared fruits—especially in filtered grape must. Its dimensions varied from 1 to 1-½, or 2, 450ths of a millimetre.[43]
[Illustration: Fig. 11.]
_ccc._—“Low” yeast, of a type resembling that existing in other preparations fermenting in the room.
_ddd._—Enlarged, distended spores of _mucor racemosus_. These are scarce, and have an old appearance. We shall come to them again in a subsequent chapter, where we shall explain their real significance.
_eee._—Short _vibrios_, occurring either contracted or not near the middle. Some were motionless, others vibrating to and fro and executing other movements. These forms belong to the butyric and lactic ferments shown on Plate I.
The preceding series of experiments shows us that, in the case of wort exposed to the air, germs of divers organisms, amongst which various ferments—butyric, lactic, and alcoholic—are to be found, fall simultaneously from the particles of dust floating in the air. We must observe, however, that we were dealing with the air of a laboratory in which we were constantly studying analogous fermentations, and that a different atmosphere would, most likely, give us different results. We shall see a proof of this in the following paragraph, where we shall also find some new facts tending to prove that the germs of alcoholic ferment do not exist amongst the particles of dust floating in the air, in anything like the quantity usually supposed.
§ V.—New Comparative Studies on the Germs held in Suspension by the Air of Different Places which are near each other, but Subjected to Different Conditions affecting the Production and Diffusion of the Particles of Dust found in them.
We may compare the character and the greater or less abundance of similar germs existing in neighbouring localities, by studying the changes which take place in similar liquids exposed simultaneously to the action of the air in those localities. To do this, we must prepare a large number of flasks of the same size, free from air, and containing about equal quantities of a particular liquid—the same being used for all. We must open the same number of these flasks in each of the localities we have selected, and permit the air with all its particles of dust to rush into them; then we must close our flasks again, and observe, day by day, the appearances they present. The results obtained by these means will not furnish us with conclusions applicable to every kind of germ that the air contains at any given moment, but only with conclusions which apply to those germs which can develop in the particular liquid employed. Thus, for example, we could draw no inference as to the nature and relative number of _bacteria_ or _vibrios_, in the case where we employ an acid liquid; for organisms of that kind we must have recourse to neutral or slightly alkaline infusions. On the other hand, liquids having a feeble acid reaction would favour the growth of _mucedines_, _mycoderms_, and certain ferments, as for example, the alcoholic.
On November 26th, 1872, we opened and reclosed thirty flasks containing must kept from the last vintage.
Ten flasks were opened at the bottom of the garden of the _École Normale_.
Ten on the landing of the second floor.
Ten in the principal room of our laboratory, which had been swept out shortly before, by which operation the dust of the floor had been raised and put in motion.
Different objects made their appearance in a certain number of the thirty flasks on the following days; but from December 17th, things remained stationary. The following observations were made at that date:—
Of the ten flasks from the bottom of the garden, only one had undergone any change.
Of the ten flasks from the interior of the building, four had undergone change.
Of the ten flasks from our laboratory, all had undergone change.
The difference in the number of germs held in suspension in the three different places whence we had taken our air was, therefore, considerable.
The difference in the nature of the germs was equally marked. Those flasks of our first two series which had undergone change presented no trace of _torulæ_, or anything besides fungoid growths, whilst three of the last ten contained _torulæ_ associated with fungoid growths.[44]
[Illustration: Plate 3. Torulæ in Process of Development.]
On May 29th, 1873, eighteen flasks, free from air and with necks drawn out to a fine point, containing must, were taken into one of our rooms at the _École Normale_. A jet of gas from an ordinary burner was passed over the surface of the glass down to the surface of the liquid, with the object of burning any particles of dust that might have been deposited from the atmosphere of the laboratory; the points of the flasks were then broken with a pair of ordinary scissors that had been passed through the flame of a spirit lamp; and, lastly, the tops of the flasks were taken off, just above the surface of the liquid, and the eighteen flasks thus became transformed into eighteen basins, each containing about 100 c.c. (about 3-½ fluid ounces) of grape must. These eighteen basins were placed on a table in the room, and left there for five days, precautions being taken to prevent any one from entering the room.
The basins were examined on June 2nd: they all contained little flakes of floating _mycelium_, but none of them had any white streaks on their sides—a proof that they were destitute of _torulæ_—the liquid had remained very bright. With the contents of nine of the basins we filled two long-necked flasks that we had prepared for our purpose—that is to say, had heated to a certain point, just before using them, with the object of removing from their sides any foreign germs which they might have picked up in the laboratory. Up to July 10th, when we deemed it useless to carry our observations further, these flasks presented no signs of fermentation whatever. With the contents of the remaining nine basins, we filled two other long-necked flasks, but before doing so, we kept the basins for twenty-four hours (June 2nd to 3rd) in the basement of our laboratory. These two flasks soon set up an active fermentation, and deposited an abundance of yeast—an additional proof of the great difference in character of the germs floating about the room and those floating about the laboratory.
On June 3rd we exposed, simultaneously, in the aforementioned room and also in our laboratory, seven basins prepared as just described. On June 8th all the basins in the room showed signs of fungoid growths, without any trace of _mycoderma vini_, or lines indicative of the presence of _torulæ_, whilst six of the seven in our laboratory had their sides covered with a white precipitate, and on the surface of the liquids a layer of isolated patches of fungoid growth. The liquid in these latter basins was poured into a long-necked flask, which it nearly filled, and in the course of forty-eight hours it began to show signs of alcoholic fermentation.[45] This is another striking proof of the difference between the number of germs of ferment and _torulaceæ_ in the air of our laboratory and that of an ordinary room.
[Illustration: Fig. 12.]
We append drawings of the _torulæ_ found in the six laboratory basins (see Sketches I., II., III., IV., V., VI. of Fig. 12). The abundance of the germs of these organisms in our laboratory is very striking, and is doubtless due to the nature of the work carried on there, as well as to the power of endurance peculiar to the germs, or the minute vegetative cells of these microscopic plants—a tenacity of life which prevents them from losing their reproductive powers, even after being dried up into dust. But varied as are the formations represented in Fig. 12—and it will be observed that in IV. and VI. we have shown four distinct forms, marked _a_, _a_; _b_, _b_; _m_, _m_; _n_, _n_ respectively—it must not be supposed that they correspond necessarily to distinct species. From the ends of a compound organism like those in No. III, a little spherical cell may detach itself and then, by a process of budding, give rise to a series of other minute spherical cells reproducing the form shown in Nos. I. and II.
The forms figured in No. III. represent one of the types of _mycoderma vini_, which is often found in this branching arborescent state; but it frequently also occurs in short forms, and it is in this shape that it is generally met with on the surface of wines.
It is true that the nature of the substratum has a great influence on the changes of aspect in the organisms which we are describing, but this is not the sole cause of their morphological modifications. We are strongly inclined to believe that each of the cells, or vegetating forms so represented, differing so greatly as they do in aspect, and begotten, all of them, spontaneously in certain appropriate liquids, in a laboratory where researches on fermentation are pursued, is capable of furnishing a distinct variety. In fact, there is not a single one of the cells in the six varieties in Fig. 12, which, taken alone, has not its own peculiar characteristics, which, by hereditary transmission, it can impart, in a greater or less degree, to all the individuals of the generations that succeed it.
We may remark, on the other hand, that nothing can be more favourable to the isolation of different varieties of _torulæ_ or _mycoderma vini_ than the spontaneous impregnations to which we submit our liquids. For when suitable liquids contained in flasks exhausted of air are impregnated with the particles of atmospheric dust, by opening the flasks for a moment and then immediately re-sealing them, it must generally happen that we admit only one species of reproductive organism, so that we shall have a vegetation exclusively of one kind, as being derived from the same mother-cell. If we could take from a crowd composed of men and women separate couples, and forthwith transport each couple to a separate isolated and unpeopled island, they would, in the course of time, beyond doubt form so many distinct tribes.
It is very remarkable that some of the _torulæ_ in Fig. 12 are not ferments; they do not cause sugar to decompose into alcohol and carbonic acid, any more than the _mycoderma vini_ does; but, nevertheless, there may be an absolute similarity in aspect, development, shape, and size between the alcoholic ferment, properly so called, and these _torulaceæ_.
We must here cite a case in proof. On May 28th, 1872, in one of the rooms of our laboratory, we broke the fine points of a series of flasks, similar to those used in our previous experiments, containing must of grapes and deprived of air. We then closed up the ends immediately after the sudden entrance of the exterior air. One of these flasks developed only one kind of organism, which belonged to the _torulaceæ_. On June 7th this was sufficiently abundant to cover all the sides with a white deposit, and the surface of the liquid appeared quite free of any _mycoderma vini_. To assure ourselves that we were actually dealing with one kind of _torulæ_, unattended by fungoid growths, we waited until June 14th, but the aspect of things remained unchanged. On that day we opened the flask; there was no escape of gas to indicate that the interior pressure was greater than the exterior. We then subjected the plant to microscopic examination. It was quite homogeneous, and formed of a mass of cells, absolutely identical in aspect and size with old cells of ordinary yeast (Fig. 13).
[Illustration: Fig. 13.]
We distilled all the liquid, of which there was about 100 c.c., (3-½ fl. oz.) without obtaining any trace of alcohol in our distillate; we collected 33 c.c. (about an ounce) of the distillate, which we distilled again, and we submitted the distillate a third time to distillation; but even then there were no more signs of the presence of alcohol than there had been in our first distillate.[46]
We may safely conclude that our _torula_, in the course of its development in must, with a weight that would have been very appreciable, did not produce, by its action of multiplication 1/10,000 of 1 c.c. of alcohol.
Under the following conditions we obtained a slightly different result, which, nevertheless, confirmed the preceding one.
On July 5th, 1872, we opened and closed twelve flasks similar to those we had used before, the sole difference being that they contained yeast water[47] sweetened with ten per cent. of sugar. One of these flasks furnished us similarly with one kind of _torula_, which bore the greatest resemblance to the ferment of beer. When this _torula_ was beginning to spread all over the bottom of our flask, we shook up the liquid, and turned the flask upside down, with the object of submerging the torula and depriving it of air, at least at the bottom of the neck. For some days, and even months, there were no signs of the liberation of gas. On July 22nd, 1873, after the interval of a year, we opened the flask (which gave no indication of the existence of an interior pressure) and endeavoured to discover the presence of alcohol, by means of successive distillations, as just described. In the two first distillates there seemed to be no alcohol, but in the third we detected its presence in very small quantity. We shall see, later on, that the formation of such a small quantity of alcohol may be attributed to the fact of the plant having been submerged when in full growth, and to its having continued to live for some time after its submersion quite independently of the oxygen contained in the air of the flask.
From the previous facts it is obvious that there exist certain productions of various aspects, the germs of which are particularly abundant in the dust of a laboratory where the phenomena of fermentation are studied, productions essentially aerial, and incapable of giving rise to fermentation, although it may be impossible for the microscope to distinguish their forms from those of true alcoholic ferments.
The idea of some physiological bond between these plants and the ferments which resemble them in so remarkable a manner, is one that impresses itself forcibly and, so to say, instinctively upon the mind. This remark holds good also in the case of _mycoderma vini_, properly so called, when compared with alcoholic ferments. There appears to be no other difference between the _mycoderma vini_ and the _torulæ_ of which we are speaking, than that afforded by peculiarities of physical structure and a certain greasiness in the cells of the former which permits it to exist, in the form of a scum, upon the surface of liquids, that is to say unsubmerged.[48]
We have frequently, but without success, endeavoured to bring about the conversion of these unsubmerged _torulæ_ and _mycoderma vini_ into alcoholic ferments; in other words, we have never succeeded in imparting to these _torulæ_ or to _mycoderma vini_, which bear so striking a resemblance to alcoholic ferments, the permanent fermentative character peculiar to the latter. At one period of our researches, in 1862, and more recently, in 1872, we thought that we had discovered the conditions under which such conversion might be possible, but, as we shall explain in a subsequent chapter, our experiments were affected by certain errors that had escaped our notice.
§ VI.—Yeast may become Dry and be Reduced to Dust without losing its Faculty of Reproduction.
In the preceding paragraphs we have given examples of liquids becoming impregnated with self-sown alcoholic ferments. We shall proceed to show that this little cellular plant may actually exist in the form of dust, floating in the air, after the manner of spores of fungoid growth and the encysted forms of certain infusoria, without losing its powers of reproduction.
On December 16th, 1872, we collected and pressed all the yeast resulting from a brewing of about one hectolitre (about 22 gallons). From the centre of the cake we took a few grammes (50 or 60 grains) of yeast, which we mixed in a porcelain mortar with five times its weight of plaster—both mortar and plaster having been heated, just before, in an oil bath, to a temperature of about 200° C. (392° F.), and then cooled rapidly. The powder thus prepared was immediately done up in a twist of paper, which had been passed through the flame of a spirit lamp, and the twist and its contents were then placed in an oven at a temperature of from 20° C. to 25° C. (about 75° F.). The object of these several precautions was to free the powder composed of the yeast and plaster, if not from the germs contained in floating particles of dust, at all events from those contained in the dust existing on the surface of the articles we used—the mortar, plaster, and paper.
[Illustration: Fig. 14.]
On December 18th, we took up with a platinum spatula, previously passed through the flame, a pinch of the yeast-and-plaster powder, and sowed it in a two-necked flask (Fig. 14) containing some pure wort. We then placed the flask in an oven, at 20° C.
On December 21st, three days after we had sown the powder, fermentation began to manifest itself by the appearance of patches of froth on the surface of the wort. On December 19th and 20th the yeast was sensibly developing, although there was no liberation of gas to denote the presence of actual fermentation. The yeast, examined under the microscope, appeared very pure.
On March 5th, 1873, we took another pinch of the yeast-and-plaster powder from the twist of paper, and placed it in a flask of pure wort, as in the foregoing experiment.
On March 9th, that is, after having been subjected to a heat of 20° C. (68° F.) in the oven for four days, fermentation began to manifest itself by the appearance of patches of froth on the surface of the wort. From this it was evident that the yeast had not been destroyed, but only retarded in its revival.
On July 25th, 1873—that is, after a lapse of seven months and a half—we resumed our experiments, and sowed some more of the yeast-and-plaster powder in another flask of wort. On August 2nd, eight days from the time of our sowing, the little islets of froth appeared on the surface of the liquid. Observed under the microscope, the yeast still seemed pure, and resembled the original yeast; we append a sketch, which will give an idea of its shape (Fig. 15).
[Illustration: Fig. 15.]
On November 7th, 1873, we once more sowed some of the powder. This time the yeast was dead; we observed the flask which contained it, day by day, until February 1st, 1874, without detecting the slightest sign of fermentation or development of the yeast that we had sown. On February 1st, we made a microscopical examination of the yeast, and found it mixed with the plaster and absolutely inert at the bottom of the saccharine liquid; its cells were isolated, very old-looking and granulated, without any appearance that might denote the possibility of their ever budding again.
Thus we determined that alcoholic ferment may be dried at the ordinary temperature of the atmosphere, and preserved, in the form of dust, for a period of seven months or longer, without losing its faculty of reproduction. This faculty evidently diminishes in the course of time, for our dried yeast, after having been kept for seven months and a half—all the other conditions of the two experiments having been precisely the same—required about eight days to develop sufficiently to reveal fermentation, whilst, immediately after the drying, it only required three or four days to accomplish the same thing.
[Illustration: Fig. 16.]
Side by side with these experiments on alcoholic ferment, we carried on exactly similar ones with yeast obtained from “high fermentation” breweries. On December 16th, 1872, we prepared a powder of this yeast and plaster as before. Our last sowing took place on July 25th, 1873, in a flask of pure wort, which showed signs of fermentation on July 27th. We append a sketch (Fig. 16) which gives the general aspect of this “high ferment,” when revived after such a lapse of time; it had preserved the distinctive features of the cells of “high ferment.”
These facts can leave no doubt whatever as to the possibility of cells of yeast existing, in a state of suspension in the air, in the form of fine dust, particularly in a laboratory where researches on alcoholic fermentation are pursued.
Footnote 21:
We have heard of liquids even less sensitive than these, which required a temperature of 120° C. (248° F.) or more, but we have had no opportunity of studying them.
Footnote 22:
See Pasteur, _Mémoire sur les Générations dites Spontanées_ (_Annales de Chimie et de Physique_, t. lxiv. 3^e série, année 1862).
Footnote 23:
See my _Mémoire sur les Générations dites Spontanées_, already cited.
Footnote 24:
Jules Duval (of Versailles), _Nouveaux faits concernant la mutabilité des germes microscopiques. Rôle passif des êtres classés sous le nom de ferments._ (See the _Journal d’Anatomie et de Physiologie_, edited by C. Robin, Sept. and Oct. 1874, and _Compte-rendus de l’Académie des Sciences_, Nov. 1874). M. Béchamps had previously fallen into similar errors.
Footnote 25:
On this subject see the observations of M. Coste (_Compte-rendus de l’Académie_, t. lix. pp. 149 and 358, 1864).
Footnote 26:
Chauveau’s experiments were directed to show that the operation _bistournage_, employed by veterinary surgeons for castrating animals by twisting and subcutaneous rupture of the spermatic cord, an operation which, though leading to the mortification and subsequent absorption of the testicles, is commonly attended with no other mischief to the animal, does, nevertheless, lead to septic effects of a serious character, provided that septic germs—decomposing serum containing _vibrios_, for example—be introduced into the blood current. From the fact that the operation is ordinarily harmless, M. Chauveau concludes that septic organisms are not produced by the action of the constituent gases of the atmosphere—always present in the blood—upon albuminous matter when outside vital influences; whilst, from the success of the direct experiment of introducing septic germs, he concludes that the phenomena always arise from the actual presence of such germs.—D. C. R.
Footnote 27:
See Pasteur, _Mémoire sur les Générations dites Spontanées_, pp. 51 and 52, 1862.
Footnote 28:
DAVAINNE, _Compte-rendus de l’Académie des Sciences_, t. lvii. p. 220, 1863.
COZE and FELTZ, _Recherches cliniques et expérimentales sur les maladies infectieuses_, Paris, J. B. Baillière, 1872. Summary of all their works published before 1865.
Dr. LISTER, Medical and surgical journals, particularly the _Lancet_, 1865-67.
Dr. GUÉRIN, _Compte-rendus de l’Académie des Sciences_, March 23, 1874, and May 28, 1874, also the Report of M. Gosselin, December, 1854.
Dr. SÉDILLOT, _Compte-rendus de l’Académie des Sciences_, November, 1874, t. lxxix. p. 1108.
PASTEUR, _Mémoire sur la fermentation appelée lactique_. (_Annales de Chimie et de Physique_, t. lii. 3^e _série_, 1875.)—_Animalcules infusoires, vivant sans gaz oxygène libre et déterminant des fermentations._ (_Compte-rendus de l’Académie des Sciences_, t. lii. 1861.)—_Recherches sur la putréfaction._ (_Compte-rendus de l’Académie des Sciences_, t. lvi. 1863.)
GOSSELIN, ROBIN, and PASTEUR, _Compte-rendus de l’Académie des Sciences_, January 5, 1874. _Urines ammoniacales._
TRAUBE, _Gazette hebdomadaire de médecine et de chirurgie. Sur la fermentation alcaline de l’urine_, April 8, 1864.
CHAUVEAU, _Putréfaction dans l’animal vivant_. (_Compte-rendus de l’Académie des Sciences_, April 28th, 1873.)
Footnote 29:
We must mention one curious result, which relates to what have been called the _crystals of the blood_. We could hardly have recourse to a better method of preparing these crystals, at least in the case of dog’s blood, which seems to yield them with the greatest facility in any quantity we might desire to procure. Under the circumstances just recounted, in which dog’s blood exposed to contact with pure air underwent no putrefactive change whatever, the crystals of that blood formed with a remarkable rapidity. From the first day that it was placed in the oven and exposed to an ordinary temperature, the serum began gradually to assume a dark brown hue. In proportion as this effect was produced, the globules of blood disappeared, and the serum and the coagulum became filled with very distinct crystals, of a brown or red colour. In the course of a few weeks, not a single globule of blood remained, either in the serum or coagulum; every drop of serum contained thousands of these crystals, and the smallest particle of coagulum, when bruised under a piece of glass, presented to view colourless and very elastic fibrine, associated with masses of crystals, without the slightest trace of blood-globules. Where our observations were protracted, it sometimes happened that all the fibrine collected into one hyaline mass, which gradually expelled every crystal from its interior.
Footnote 30:
PASTEUR, _Comptes rendus de l’Académie des Sciences_, t. lvi. p. 738, 1863.
Footnote 31:
GAYON, _Comptes rendus de l’Académie des Sciences_, and _Annales Scientifiques de l’École Normale Supérieure_, 1874-75.
Footnote 32:
Amongst these influences one of the most important, according to M. Fremy, is “_organic impulse_,”—another gratuitous assumption.
Footnote 33:
FREMY, _Comptes rendus de l’Académie des Sciences_, t. lviii. p. 1167, 1864.
Footnote 34:
FREMY, _Comptes rendus de l’Académie des Sciences_, t. lxxiii. p. 1425, 1871. M. Trécul shares M. Fremy’s opinions, and extends them to the development of different fungoid growths.
Footnote 35:
This observation had already been made by Anthon and H. Hoffmann. “If we scrape the surface of a gooseberry with a blunt knife,” says H. Hoffmann, “and put under the microscope the scrapings, which are of a whitish colour, we shall recognize amongst many varieties of shapeless dirt, earthy particles and other things, the same fungoid spores that we find in the expressed juice, but we shall see them there in infinitely larger quantities. Some of them will be of a dusky colour (_Stemphylium_, _Cladosporium_), and others will be colourless; the shape of these latter will be round or oval, and cylindrical. Most of them will bear resemblance to beads of the _chaplets_ of _Oidium_, _Monilia_, _Torula_ (that is to say, to spores of certain _Hyphomycetes_), which have been detached and carried off by the wind, and have attached themselves to the fruit. Some of these spores will be already provided with short germinating filaments.” (_Annales des Sciences Naturelles, Botanique_, t. xiii. p. 21, 1860).
Footnote 36:
The experiments that we have described give rise to a useful remark. All the organic liquids, boiled or not, in the course of time must take up oxygen from the air. At the same time, and certainly under this influence, they assume an amber or brownish colour, but this effect is only produced when the liquids are placed under conditions of unalterability. Should fermentation or the development of fungoid growths be possible, scarcely any change of colour will take place. Doubtless this non-coloration may be attributed to the fact that these organisms consume the oxygen necessary for coloration. In these experiments on must, all the unchanged flasks assumed a pale yellowish brown colour; those which fermented or contained fungoid growths remained colourless, or nearly so.
Footnote 37:
_Comptes rendus de l’Académie, séance du 28 Octobre, 1872._
Footnote 38:
GAY-LUSSAC, _Annales de Chimie_, t. lxxvi. p. 245; read at the Institute, December 3rd, 1810. Long before Gay-Lussac, it had been remarked that atmospheric air had a great influence on fermentation. See M. Chevreul’s articles on the history of chemistry in the _Journal des Savants_.
Footnote 39:
GAY-LUSSAC, _Annales de Chimie_, t. lxxvi. p. 247, _Mémoire cité_, 1810.
Footnote 40:
It is well to notice that under the influence of fungoid growths, properly so called, the wort of beer speedily becomes bright. We may say that fungoid growths, by their rapid development, clarify the must, which serves to nourish them.
Footnote 41:
It has already been observed in our Memoir on spontaneous generation, that alcoholic fermentation is not always to be obtained by sowing wads of cotton or asbestos, charged with the particles of dust which float through the air, in saccharine musts that are in contact with much air. The air which furnished the particles of dust, in the experiments to which we are alluding, was taken outside the laboratory, in a neighbouring street.
Footnote 42:
The decanting into the flasks is necessary, because of the possibility of the fermentation in the basins being masked. See further on the note on p. 75.
Footnote 43:
This small ferment is very curious, although it scarcely affects industrial fermentation. It was first described in 1862. (PASTEUR, _Bulletin de la Société chimique_, 1862, page 67, and following: _Quelques faits nouveaux au sujet des levûres alcooliques_.) It has since been described by Dr. Rees under the name _Saccharomyces apiculatus_. (Dr. Rees, Leipzig, 1870: _Sur les champignons de fermentation alcooliques_. See also Dr. Engel, _Thèse pour le Doctorat_, Paris, 1872.) If we carefully filter some grape must at the time of vintage, we may be sure that we shall see it appear in the clear liquid at the bottom of our vessel, without intermixture with any other ferment.
Should we not filter the must this ferment will appear all the same, but it will soon become associated with another, thicker in appearance and more elongated, which also is one of the ferments peculiar to the fermentation of grape must.
Footnote 44:
We may remind the reader that in 1862, in our _Mémoire sur les Générations dites spontanées_, we applied the expression of _torula_ to all the little cellular plants of spontaneous growth, excepting _mycelium_, propagated by budding, after the manner of the ferment of beer. At the same time, stress was laid upon the frequent occurrence of their germs, especially in our laboratory, where studies on fermentation were, even then, carried on. Plate III. represents two of these ferments.
Footnote 45:
It is to be remarked that in this case, as in the case recorded § IV. p. 70, in order to detect with certainty any alcoholic ferment, the contents of the basins were transferred to a long-necked flask; since where, as in the basins, a liquid has a large surface exposed to spontaneous impregnation, the strictly alcoholic fermentation may escape observation. The reason of this is that, when a liquid of large surface but small depth is exposed to the air it affords a suitable medium for the active development of moulds, which, by absorbing the oxygen which would dissolve in the liquid, checks the growth of the ferment, or even prevents its germination altogether. As a matter of fact we shall see that for their growth and multiplication ferment _cells_, and still more ferment _germs_ (the difference between the two will appear in Chap. V.), require a larger supply of oxygen just in proportion to their age, state of desiccation, and distance from the budding condition. Now, if the spores of moulds be present and effect a settlement in the liquid, the increase of the ferment, or even its actual germination, is prevented. But by collecting the liquid in a deep and narrow vessel, such as a long-necked flask, after it has been exposed to spontaneous impregnation, we deprive the moulds almost completely of oxygen, and so allow the ferment to exert its peculiar energies. The mere act of transferring enables the liquid to take up a sufficiency of oxygen, and a liberation of gas very speedily shows that fermentative action is going on. We must add further that sometimes in a liquid of large surface and shallow depth, in which but little ferment is formed, the evolution of carbonic acid gas may fail to be detected, by reason of its diffusing itself into the air slowly as it is formed.
Footnote 46:
Here we had to seek for a most minute quantity of alcohol, that no alcoholometer could have indicated. A certain sign of the presence of alcohol is contained in the first few drops distilled; these always assume the form of little drops or _striæ_ or, better still, oily tears, when alcohol is present in the distillate. The distillation should be effected with a small long-necked retort and a Liebig’s condenser. We must carefully watch the neck of the retort at the moment of boiling; should the liquid contain 1/1000 part of its volume of alcohol, we shall observe the indications given above for a short, but appreciable time. 1/10,000 of alcohol is difficult to judge, but with care and practice we may do it without failing. Collecting a third of each distillate, and supposing the limit of appreciation to stop at thousandths, in three distillations we may easily detect the presence of 1/10,000 of 1 c.c. of alcohol in a total volume of 100 c.c.
Footnote 47:
Yeast when macerated in water imparts to it certain soluble nitrogenous materials. The solution so obtained, filtered from yeast globules, is known as _yeast water_.—D. C. R.
Footnote 48:
It is possible that this greasiness in the cells of the common _mycoderma vini_ arises simply from the composition of the liquid in which it vegetates. It is in saccharine liquids that the submerged _torulæ_ are found; fermented liquids more readily give birth to the forms of _torulæ_ and _mycoderma vini_ which exist as a scum. In all probability, however, there is no radical difference between these two kinds of little cellular plants of aerial growth, the floating _torulæ_ and _mycoderma vini_.
CHAPTER IV. The Growth of Different Organisms in a State of Purity: Their _Autonomy_.[49]
Our observations in the preceding chapter will have shown that organic liquids, natural or artificial—the wort of beer amongst others—if exposed to contact with the air, rapidly develop various forms of life. This is a natural consequence of the mode of impregnation. The fertility of the liquid depends on the various microscopic germs which are deposited in it by the common air, and these, again, as regards their nature and number, are dependent upon the situation of the vessel containing them, its height above the ground, the time of year, the disturbance of the atmosphere, and other causes.
The fortuitous association of other forms, in growths which we believe to be uniform and independent, constitutes one of the principal difficulties that occur in the study of the lower organisms, particularly that of fungoid growths. The fact that the germs of many of these little beings exist in the atmosphere in the form of dust, invisible to the naked eye, or, as such, spread over the surface of the different materials and objects used in experiments, exposes the student to constant risk of wrongly interpreting the results which come under his notice. He has sown a plant, and is observing the course of its development. Without his knowledge, spores of another plant have got mixed with his growth, and germinated. In his ignorance, he will attribute all that he sees, all the changes which he describes and which he sketches, and all the conclusions which he draws, to the one plant which engages his attention. If he is dealing with _bacteria_, _vibrios_, and, generally speaking, the infinite variety of mobile microscopic organisms, his embarrassment will be greater still. Again, inasmuch as the medium which serves as substratum for growths has a considerable influence on the fertility of the germs in contact with it, as well as on their ulterior development, it often happens that germs deposited fortuitously by the particles of dust which fall from the atmosphere or collect on objects are fertile and multiply with rapidity, whilst those which have been directly sown, no matter in what number, remain sterile, or multiply very slowly. If we place in a young wine some _mycoderma aceti_, we shall obtain _mycoderma vini_; by placing some _mycoderma vini_ in an old wine, especially if it is a little acid, we shall obtain _mycoderma aceti_.[50] It is from facts of this kind, wrongly interpreted, that many errors have crept into our knowledge of the lower organisms, and that we are constantly seeing old discussions crop up, both on the subject of so-called _spontaneous_ generation and on that of the theory of fermentation. At every step in the course of this work we shall see the trace of these complications, as well as the influence they have had on the progress of our knowledge.
In opposition to these results, we will study the case of wort sown directly with germs distinctly of one kind, unmixed with any other.
§ I.—Growth of Penicillium Glaucum and Aspergillus Glaucus in a State of Purity—Proofs that these Fungoid Growths do not become Transformed into the Alcoholic Ferments of Beer or Wine.—Preliminary Enquiry into the Cause of Fermentation.
Let us again take one of the flasks furnished with two necks such as we have already described, and let it be supposed that this flask contains a quantity of saccharine wort, brewed some considerable time ago, which has undergone no change whatever, except in colour, the slow process of oxidation having gradually darkened the original colour of the liquid. What we have to do is to drop into this unchanged and fertile liquid some grains or spores of _penicillium_ which are free from the slightest trace of the spores or germs of other microscopic organisms.
One means of effecting this consists in taking up with a pair of metallic forceps, previously heated, a piece of platinum wire, one or two centimetres (about 3/4 in.) in length, which we also pass through the flame of a spirit lamp, and with which, as soon as it is cold, we touch a mass of sporanges of a growth of _penicillium_. No matter how few spores may be taken up on the end of the platinum wire, we shall have far more than we require for the impregnation of the liquid. At the moment of charging the point of the wire we withdraw the glass stopper which closes the india-rubber tube on the right-hand neck of the flask (Fig. 14) and drop the wire through that tube; we then replace the glass stopper, after having, by way of additional precaution, passed it rapidly through the flame of the lamp. There is no doubt that we expose ourselves to error in consequence of having to convey the wire through the surrounding air, and also, in consequence of having previously to open the flask; but, as we have already remarked, this double cause of error has never, we may say, interfered with the exactness of our experiments, the volume of air with which we are concerned being exceedingly limited. Moreover, our flask being in free communication with the exterior air, by means of the opening in the curved, slender tube, there is no inrush of air when we withdraw the stopper. The chance of encountering a spore or fecund germ and introducing it into the flask on the wire that is charged with the others, is so remote that we have considered it unnecessary to adopt a more perfect apparatus, which might easily have been devised had we felt that it was necessary.
A more serious cause of error may occur in the preceding method; resulting from the possible impurity of the spores taken from a field of _penicillium_ which has developed in contact with common air. This field receives, every instant, and has received throughout its growth, particles of dust which have fallen from the atmosphere; thus, it may not be, and, as a matter of fact, is not, free from the germs of other fungoid growths.[51]
The operator, without knowing it, may frequently sow, besides the _penicillium_, which is all he can see, spores of _mucor mucedo_ and _mycoderma vini_, in short, of all the most common fungoid growths.
[Illustration: Fig. 17.]
This process of impregnation, therefore, does not afford us sufficient safeguards, but by means of the following device we shall render it more satisfactory. Let us take a series of flasks shaped as in Fig. 17, containing an organic liquid suitable to the development of fungoid growths, that is to say, slightly acid—yeast-water, plain or sugared, the wort of beer, or Raulin’s fluid[52] will answer our purpose; let us boil the liquid, and having previously drawn out the necks, let us close the ends in the flame of a lamp whilst the steam is escaping, as soon as we judge that the air has been nearly all expelled. Having prepared ten or twenty of these flasks in this manner, when they are cold we may break their points in any place we may be in. The air will rush into the flasks, and we must then seal them up again in the flame of the lamp, and put them aside for future observations. In a certain number of these flasks, as we have already explained in our experiments carried on after this fashion, we shall see some fungoid growths appear, first in the shape of flakes of _mycelium_ floating in the liquid, and afterwards coming to the surface to fructify. Now, it often happens that _penicillium glaucum_ appears alone, so numerous are the spores of this fungus floating in the air. Under such conditions, we shall evidently obtain a field of sporanges quite free from the presence of other organisms. If we now take off the neck of one of the flasks containing the pure _penicillium_, and take out some of the germs with our platinum wire,[53] we shall thus obtain with most certainty spores of _penicillium_ free from impurities.
Our readers will excuse the length of these details and the minutiæ of our precautions, but we shall again and again see that to neglect them, or any part of them, is to expose ourselves to hazard in drawing sure conclusions from facts which come under our observation.
[Illustration: Fig. 18.]
On June 17th, 1872, we placed some pure spores of _penicillium_ in a series of three flasks containing wort (Fig. 18), observing all the precautions that we have indicated. We shall designate these flasks by the letters A, B, C. On the following day the spores germinated, and the liquid became full of flakes of mycelium, some of which came to the surface to fructify. The temperature varied between 25° C. and 30° C. (77° to 86° F.).
On June 22nd, small patches, with whitish borders and green centres, developed on the liquids. We then shook up the flask A, in order to submerge the plant and the spores.[54] We also shook the flask B, after observing the precaution of sealing up the slender bent tube.[55] The flask C was attached on one side to an aspirator, on the other to a tube filled with cotton, and every day we renewed the air in it.
During the weeks and months over which our observations extended, there was not the least formation of yeast in these flasks; moreover, we have frequently repeated this and other experiments of a similar kind, without ever detecting the appearance of either ordinary yeast or any other true alcoholic ferment. The experiments may be made with saccharine juices that are highly favourable to the development of _bacteria_ and lactic ferment. These latter appear equally incapable of transformation into yeast, which has never been seen to develop in experiments where they were used, if proper precautions have been taken to secure a pure growth. Should we neglect any of the precautions that are necessary to secure the purity of our spores, we may of course obtain different results. If, for instance, we sow spores of _penicillium_ grown in free contact with the atmosphere, and consequently exposed to the particles of dust floating therein, we shall frequently observe, mixed with the fruiting hyphæ of the fungoid growth, yeast and _mycoderma vini_ and _torulæ_, or even _bacteria_ and lactic ferment. Thus we shall be led to believe, in all good faith, that we have under our eyes examples of the changes of spores of fungoid growth into cells of ferment, or proofs of the conversion of _bacteria_ or lactic ferment into the same cells.
Causes of error of this nature have induced some German naturalists to believe that they have succeeded in proving, beyond the possibility of doubt, that a number of fungoid growths may produce alcoholic ferment, and that they have clearly demonstrated that spores of these fungi may become transformed into yeast. In 1856 M. Bail, and, about the same time, Berkeley, and, later on, H. Hoffmann and Hallier, have successively entertained these views, which were introduced into science by M. Turpin. We have combated them since the year 1861.[56] Since that period they have lost rather than gained ground abroad, in spite of the growing favour bestowed on the Darwinian system. One of the mycologists who enjoy the most legitimate authority beyond the Rhine, M. de Bary, has arrived, as we have, at absolutely negative results.
A simple perusal of what has been written in favour of the transformations which we are discussing causes us to entertain the gravest doubts as to the correctness of results which are quoted as decisive. We need only give one example, which we extract from a paper by M. H. Hoffmann.
“In some cases,” this author writes, “and under favourable circumstances, I have been able to see the ferment produce filaments, both small specimens that could be examined immediately under the microscope, and also large specimens, and I have recognized, amongst other varieties, _penicillium glaucum_, _ascophora mucedo_, _ascophora elegans_, and _periconia hyalina_, sometimes isolated, sometimes intermixed. This result is most easily to be obtained by the following method:—Pour a small quantity of water into a test-tube, which should then be placed slantingly: introduce some fresh yeast into the middle part of the tube, and close the tube with a wad of cotton, to prevent the entrance of exterior particles of dust. In this vapour-filled receptacle we shall sometimes see flakes develop. It seems that Messrs. Berkeley and G. H. Hoffmann have also obtained _penicillium_ from ferment, by a similar process.”[57]
Why, however, should it not be admitted that _penicillium_ found under such conditions may be derived from spores of that growth, adhering to the sides of the tube before it is closed with the wad, or mixed with the yeast that is put into the tube?
The facts alleged during the last few years by M. Bail, who persists in his views, in spite of their apparently greater exactness, are also far from being satisfactory.[58]
M. Trécul, who is almost the only one in France, besides Messrs. Ch. Robin and Fremy, to participate in these errors, does not confine himself to affirming the change of the spores of _penicillium_ into yeast, and _vice versâ_; his system is a far more extensive one. “According to my observations,” he says, “there would be the following series of changes; albuminous matter changed into _bacteria_, or directly into alcoholic ferment, or into _mycoderma_; _bacteria_ into lactic ferment, there becoming immoveable; lactic ferment into alcoholic ferment; alcoholic ferment into _mycoderma cerevisiæ_; finally, _mycoderma cerevisiæ_ into _penicillium_.[59]”
M. Trécul does not stop here. He goes on to explain the principal of these changes, as though his testimony were quite beyond refutation:
“If we use a perfectly filtered wort, containing no granulations, and prepared at a temperature between 60° C. and 70° C. (140° F. and 160° F.), there will first of all appear a multitude of fine granules that will develop into active bacteria, which, losing the faculty of motion, will constitute lactic ferment, as I have repeatedly pointed out. A few days after the appearance of the first granules, we shall perceive others rather larger in size and isolated. These will increase in size, and, in the course of time, assume the form of little globuloid, or elliptic cells; they will not commence to bud before they have attained a comparatively large size, approaching that of ordinary yeast, consequently, there will be a considerable interval of time, during which the young cells will present no buds, especially if we work at a low temperature, as from 20° C. to 35° C. (68° F. to 95° F.)
“As for the transformation of the spores of _penicillium_ into alcoholic ferment, the possibility of which M. Pasteur also denies, I have very often obtained it by using liquids, such as boiled wort and sugared barley water, which had stood for a month or six weeks without setting up an alcoholic fermentation. These liquids, sown with spores of different forms of _penicillium_, chosen when young and in full growth, fermented after a varying number of days, even at a temperature of 12° C. (54° F.), the condition of fermentation being that the flasks were closed with very elastic corks, which had been boiled for a quarter or half an hour; these corks, as I have already pointed out, it is best to keep for a month after the boiling, to make sure, by drying them thoroughly again, of destroying any _mycelia_ adhering to them. It is necessary to keep the flasks stoppered that the corks may be always moist, and it is also advisable to shake the flasks once or twice a day, to secure the submersion of the spores. If these conditions are carried out, we shall soon see the spores increase in size, gradually lose their green colour and then bud, and often a very active fermentation will manifest itself. All the spores will be transformed, if the flasks are perfectly air-tight.”[60]
Such is the manner in which M. Trécul regards these changes. His is entirely a system of spontaneous generation, worked out into minutest detail, from the transformation of the albuminous substances to the formation of cells of the higher organisms, passing from the disintegration of the original substances to the formation of very fine granules, from these to the creation of active _bacteria_, which last, in their turn, become lactic ferment through the simple cessation of their faculty of moving and so on. We regard all this as purely imaginary. As a matter of fact, M. Trécul’s argument is based on the successive phenomena which manifest themselves in filtered wort “containing no granulations.” As M. Trécul reasons, this condition is a necessity, for he starts with the assertion that the albuminous substances in the wort become changed into granulations “that will develop into active bacteria.” This is another of M. Trécul’s illusions. No doubt we may filter hopped wort to almost perfect clearness, but we can only do this when it is cold. If we filter it warm, it will be bright as long as it remains warm, but as soon as cold it will appear turbid, in consequence of the great number of minute granules floating in it. Again, cold wort, however little it may be or has been in contact with air, undergoes a process of oxidation, and this oxidation, which acts principally on the colouring or resinous matter, causes a deposit of fine granules, the number of which is constantly increasing as oxidation goes on. These granules form an absolutely inert precipitate which, under no possible circumstances, can become transformed into active bacteria. Nothing can be easier than to prove this fact, by taking some of our two-necked flasks (Fig. 4) and preparing pure wort in them, by boiling, and then leaving it to cool and undergo the process of oxidation. In wort thus exposed to the air—the air being pure and free from germs—there will be formed a granular deposit, which will never become active or transform itself into any kind of organism whatever.
We may also remark that whilst M. Trécul used wort in his experiments, he does not tell us if it was hopped or not. Had M. Trécul informed us that the facts which he described applied to unhopped wort, we should reply that a temperature of 60° C. or 70° C. (140° F. or 160° F.) is quite insufficient to kill the germs of bacteria existing in such a wort. Hopped wort should be heated to 70° C. or 75° C. (160° F. to 170° F.), that it may remain inert after having cooled down in contact with pure air; unhopped wort must be heated to about 90° C. (194° F.).
In short, whatever may be the case, it must be evident to our readers that the active bacteria observed by M. Trécul existed in the form of germs in the wort that he used, and that what he observed was nothing more than the development of these germs when brought into contact with the air held in solution in the liquid.
As for the success of M. Trécul’s experiments on the _penicillium_, we have no doubt that that gentleman has sown germs of yeast or _torulæ_, which bear so striking a resemblance to yeast, at the same time that he sowed spores, since he took his spores from sporangia of _penicillium_ that had been exposed to contact with ordinary air. The conditions under which M. Trécul conducted his experiment rendered it difficult for the spores—although, relatively, much more numerous than the contaminating germs of ferment with which they happened to be associated—to make way against these latter, since the spores were unable to continue their development in a medium that was deprived of oxygen. On the other hand, the cells of ferment might easily have multiplied to such an extent as to make the discovery of the spores that had been sown a matter of some difficulty, since those spores would have been lost amongst the great number of cells of ferment. This was, probably, one of the causes which led to the mistaken notion that such spores underwent conversion into cells of ferment. Now although botanists describe several varieties of _penicillium glaucum_, we do not suppose that the cause of the difference between our results and those obtained by M. Trécul can be attributed to our having operated upon a different variety of _penicillium_ from that which he used. Supposing that there had been a great difference between our two varieties, still M. Trécul declares that he has realized the phenomenon with numerous varieties of this fungus.
M. Trécul expresses himself as follows: “I have used spores of _penicillium_ of several varieties in these experiments: Firstly, thick, green, elliptic spores of a variety of _penicillium_ that grows on lemons; secondly, elliptic spores of a bluish colour and smaller than the preceding, of another variety of _penicillium_ found on lemons; thirdly, spherical spores of the variety termed _penicillium crustaceum_; fourthly, spores of the _penicillium_ that develops on the yeast of beer.[61]”
This criticism of M. Trécul’s opinions was written on the occasion of a discussion at the Academy, after we had been induced to read over again the remarks which he had published on the subject. We were so impressed by the positive manner in which his conclusions were stated, that we asked ourselves, once more, which of us could be mistaken, and once more, also, we applied ourselves to fresh experiments, which we conducted with every possible precaution, following, as far as we could without falling into the errors of which we accuse the learned botanist, the mode of procedure that he adopted. As his descriptions struck us as being at times insufficient, we resolved to ask him for certain explanations _vivâ voce_ (November 3rd, 1873), which he gave us with the greatest willingness.
“Every variety of _penicillium_,” said M. Trécul, “especially when young and vigorous, is amenable to transformation into ferment. This is the way in which I operated; I had some little flasks of 30 c.c. to 40 c.c. (about 1-½ fluid ounces) in capacity, filled quite full with wort, or, at least, containing very little air, closed perfectly air-tight with corks which I had kept for a quarter of an hour in boiling water. These flasks when corked were heated to 60° C. or 70° C. (140° to 158° F.). After they had cooled I uncorked them, and introduced into them the spores which had been prepared as follows: I placed on a piece of glass some spores of the variety of _penicillium_ that I wished to study, taken with a pair of forceps from a mouldy lemon, and I mixed these spores with a drop of wort and observed them under the microscope to assure myself that they contained nothing of a foreign nature; then I poured my drop of wort from the piece of glass into one of the flasks, which I recorked and laid down. The transformation into ferment took place next day.”
Provided with these new data we set to work again and prepared a series of little flasks which were filled quite full with hopped wort, or contained but very little air, as M. Trécul had recommended. These we heated in a hot water bath to 70° C. (158° F.) at least; we then impregnated them, observing the necessary precautions, which we described at the commencement of this paragraph—not working in the evidently defective manner in which M. Trécul had done. Taking his spore-seeds from a field of sporanges exposed to the air, and afterwards manipulating them, in contact with the air, in water on a piece of glass, before he made his microscopical examination, his experiments were conducted under circumstances in every way conducive to the introduction of causes of error. One of the most serious of those causes is that which results from the substratum of the spores as taken from a mouldy lemon. If M. Trécul will examine under the microscope the water in which any lemon has been washed—even a sound lemon, unattacked by any fungoid growth—he will immediately see the cause of error to which his method of working exposes him. Germs of microscopic organisms exist abundantly on the surface of all fruits.
Having impregnated the liquids in our flasks with spores from a quantity of pure sporangia grown in a closed vessel, gathered on the point of a platinum wire, which had first been heated and then allowed to cool, we found that in each case, without exception, germination took place, then a _mycelium_ was developed, which soon, however, ceased to grow from want of a proper supply of air; but not in any single case was there the faintest trace of fermentation, formation of yeast, or appearance of _bacteria_ or lactic ferment.
We repeated these experiments, using unhopped wort instead and obtained similar negative results. We had previously determined that it was necessary to heat flasks of hopped wort to 70° C. (158° F.), at least, and those of unhopped wort to 90° C. (194° F.) to secure them from further change.
In short, contrary to the assertions of M. Trécul, M. H. Hoffmann, and other naturalists, it is not true that the spores of _penicillium_ can change into alcoholic ferment.
Regarded from another point of view, growths of pure _penicillium_ will give us some remarkable results, the interpretation of which seems to us to be intimately connected with the physiological theory of fermentation that we shall discuss in a subsequent chapter. It is a question as to the production of alcohol whilst the life of the plant is carried on under certain conditions of growth.
If we distil saccharine liquids on the surface or in the body of which we have grown _penicillium_, and repeat the distillation in the manner that we have already described for the detection of the minutest quantities of alcohol, we shall readily find that those liquids frequently do contain a little ordinary alcohol. Moreover, if we regard the quantities of alcohol produced, which are always very minute, seldom exceeding 1 or 1·5 thousandth of the total volume of the liquids, we shall find that there is no fixed proportion between this alcohol and the weights of the plants formed. It is possible, for instance, that we may obtain more alcohol from one plant than from another weighing a hundred times as much. Often, however, when the vegetation is abundant we cannot make out the occurrence of alcohol in spite of the sensitiveness of the process described (p. 78).
What can be the cause of these varying results relating to the production or non-production of alcohol in the vegetation of the little plant? The numerous experiments that we have made seem to demonstrate positively that they are dependent upon variations in the amount of air or oxygen that is supplied to the fungoid growths, whether, that is, the vegetating mycelium alone be submerged, or the whole plant with its organs of fructification. When the plant has at its disposal an excess of oxygen, as much as its vitality can dispose of, there is no alcohol, or very little, formed. If, on the other hand, the plant vegetates with difficulty, in presence of an insufficiency of oxygen, the proportion of alcohol increases; in other words, the plant shows a certain tendency to behave after the manner of ferments.
Some time ago, wishing to assure ourselves that the spores of _penicillium_ could not become transformed into ferment, we sowed some pure spores in small flasks, holding from 50 c.c. to 100 c.c. (from 2 to 4 fl. oz.), which contained very little air, and which were sealed hermetically after the sowing. Under these conditions, the germination and growth of the spores proceeded with great difficulty, and soon ceased through want of air. The total weight of the little plant was too small to be determined. In cases of this kind, if we distil the whole of the liquid we shall often see the alcohol appear in the second distillation, even though the weight of the plant may have been scarcely appreciable. If, on the other hand, side by side with experiments of this kind, we grow pure _penicillium_ in flasks containing air and having quantities of saccharine liquids equal to the quantities in the small flasks of which we have been speaking, the plant, in consequence of the large volume of air at its disposal, will develop vigorously, and in the course of even a few days will have become perceptibly heavier. In distilling the subjacent liquid, however, we shall generally find that it contains no alcohol at all, even though the weight of the plant be half a gramme, or more (6 or 7 grains).
These results apply to all the fungoid growths that we have studied, but they vary considerably with the nature of the organisms. _Aspergillus glaucus_ is, in this respect, one of the most curious.
On June 15th, 1873, we impregnated three flasks of wort, A, B, C, with pure spores of _aspergillus glaucus_. The development was rapid and the fructification abundant. On June 20th, we shook up the liquid and the supernatant fungoid growths in the three flasks; the flasks A and B were then treated as follows:—
We distilled the Liquid in A to discover the presence of alcohol; but could find none.
[Illustration: Fig. 19.]
The flask B was connected with a test flask (Fig. 19), into which the liquid, together with its fungoid growth, was transferred from B. The next day, June 21st, the mycelium which was on the surface of the liquid, in the neck of the flask, was studded with bubbles of gas; these we dispersed by shaking. On June 22nd, many others had formed again, and a large flake of mycelium that had risen from the bottom of the test flask had been stopped at the bottom of the neck, quite distended by gas bubbles. We liberated the gas by shaking, but the bubbles formed again by the next day, and this effect continued for several days; nevertheless the liberation of gas was not continuous, as is the case in an ordinary fermentation.
On July 20th we drew off the liquid and distilled it; it was still very sweet, but though it contained a sensible quantity of alcohol, the microscope failed to detect a single cell of ordinary alcoholic ferment.
These results show that the _aspergillus_ when in full growth, with plenty of air at its disposal, does not yield alcohol, and that if we submerge it, so as to prevent the oxygen of the air from readily coming into contact with its various parts, it decomposes sugar, after the manner of yeast, forming carbonic acid gas and alcohol.
These effects were still more marked in the case of the flask C, the liquid in which, after having been shaken up, was not decanted to any great depth in our test flask, as had been the case with B. From June 21st, there was _mycelium_ on the surface of the liquid, studded with large bubbles of gas, which formed again after having been liberated by shaking. This last flask was examined on November 1st, 1873. Its aspect was unchanged; the liquid was covered with _mycelium_ loaded with sporanges and borne up by large old bubbles that had not disappeared. The following was the analysis of the liquid:—
Alcohol 1·2 Glucose 84·0 Dextrine (?) 32·0
The liquid was very bright, and contained an amorphous granular deposit, formed by the wort after it had been boiled, at the time when we prepared our flasks. We crushed a small quantity of mycelium that had risen to the surface of the liquid, and obtained a field such as is represented in Fig. 20. Amongst the ordinary filaments of mycelium belonging to the plant, which are not represented in our engraving, and which were not more than 1/300 of a millimetre (nearly 1/7500 in.) in diameter, we perceived much larger ones, swollen and contorted in the most singular manner, and measuring as much as 1/50 of a millimetre across their broadest parts. There was also a multitude of the ordinary spores of _aspergillus_ mixed with others of larger size, and big, inflated cells, with irregular or spherical protuberances, full of granular matter. As there are all the stages between the normal spores of the plant and the big cells, and between these latter and the filaments, it must be admitted that the whole of this strange vegetation results from spores which change their structure under the influence of special conditions to which they are exposed.[62] Beyond all doubt these cells and irregularly shaped segments, in vegetating with difficulty, gave rise to the fermentation, which, although insignificant, was sufficiently marked to produce more than a gramme (15 grains) of alcohol. The oxygen of the air failing, or existing in insufficient quantity for the regular development of the filaments of mycelium belonging to the plant, and for the germination of its submerged spores, filaments and spores vegetated as the yeast of beer might have done if deprived of oxygen.
[Illustration: Fig. 20.]
If we study the vegetation of _aspergillus glaucus_ with this preconceived idea, we shall soon recognize the fact that these spherical forms of mycelium are the result of a greater or less deprivation of air. The filaments of this mycelium which develop freely in the aerated liquid are young and transparent, small in diameter, and exhibit the ordinary ramifications. Those which are situated about the centre, in the denser or more complicated parts, to which the oxygen cannot penetrate in consequence of its absorption by the surrounding parts, are more granular in appearance as well as larger, and inclined to develop swellings. We can observe no _conidia_[63] on these filaments, but we may say that they are on the point of appearing, for the spherical segments often tend to assume an appearance of close jointing, as when they take the form of those rows of swelling, or cells, which has given rise to the idea of the _chaplets of the conidia-cellules_. This is represented in the accompanying sketches (Fig. 21), which we have purposely contrasted with two similar ones which relate to the _mucor_, of which we shall soon speak. The _conidia_ of these latter are very remarkable, and their fermentative character becomes apparent as soon as their growths are deprived of air.
[Illustration: Fig. 21 A. Growth with abundant air-supply at the edge of the mycelium crust.]
[Illustration: Fig. 21 B. Growth with reduced air-supply in the central and deeper parts of the mycelium.]
[Illustration: Fig. 21 C. Growth with abundant air-supply at the edge of the mycelium tuft.]
[Illustration: Fig. 21 D. Growth with reduced air-supply in the central and deeper parts.]
It is scarcely necessary to add that in these vegetations of aspergillus, which were accompanied by a corresponding alcoholic fermentation, it was impossible to find cells of yeast; and that, notwithstanding this, the liquid was so adapted to ordinary alcoholic fermentation, that, when we added a small quantity of yeast to it, in the course of a few hours, a most active alcoholic fermentation declared itself.
We may give some other facts relating to a crop of _aspergillus glaucus_ which was also grown in ordinary hopped wort, and which was left to itself for a year.
A two-necked flask, holding 300 c.c. (rather more than 10 fl. oz.) was prepared and impregnated on December 21st, 1873, and was then placed in an oven at a temperature of 25° C. (77° F.). The fungoid growth developed in isolated tufts, which subsequently united, but without entirely covering the surface of the wort. A few tufts also vegetated at the bottom of the liquid; those on the surface soon became surrounded by large bubbles of gas.
On December 12th, 1874, we examined the liquid and the plant, which for a long time had appeared dead. Its mycelium was formed of aged, granulated filaments, with few swellings. The weight of the dry fungoid growth was 0·50 gramme (about 8 grains) for a total volume of liquid of 122 c.c. (4-1/4 fluid ounces). We obtained 4·4 c.c. of alcohol of 15°, which was about seven times the weight of the plant. Finally, we determined the acidity of the liquid, and found 2·8 grammes, in equivalents of sulphuric acid, a quantity greatly in excess of the total acidity of an equal volume of wort, a fact which shows us that fermentation caused by _aspergillus glaucus_ is accompanied by the formation of an organic acid, the nature of which it would be interesting to determine. M. Gayon has commenced the study of this subject in our laboratory.
In concluding our observations on the _aspergillus glaucus_, we may give the comparative results of two growths that were obtained under precisely similar conditions, in flasks of exactly the same size, but differing in this respect—that one of them was constantly subjected to a current of pure air that played on the liquid. In the course of a few days, when the fungoid growth in the flask that had been aerated had attained a considerable size, in comparison with the other, we broke the flasks in order that we might take out the two growths and compare their weights. After drying them at 100° C. (212° F.) we found:—
Growth in the aerated flask 0·92 Growth in the closed flask 0·16 Ratio of weights, 92/16 = 5·75.
Again, although we had taken the precaution of condensing in a U tube, over which cold water played, the vapours carried away by the current of air, the liquid in the aerated flask gave no evidence of alcohol. That in the other flask contained a very appreciable quantity, although the weight of fungoid growth in that flask was scarcely a sixth part of what it was in the other.
The preceding facts taken altogether, seem to us to demonstrate once more, in the most conclusive manner:—
Firstly, That neither _penicillium_ nor _aspergillus glaucus_ can change into yeast, even under conditions that are most favourable to the life of that ferment.
Secondly, That a fungoid growth which vegetates by using the oxygen of the air, and which derives from the oxidating action of that gas, the heat that it requires to enable it to perform the acts necessary to its nutrition, may continue to live, although with difficulty, in the absence of oxygen; that, in such a case, the forms of its mycelian or sporic vegetation undergo a change, the plant, at the same time, evincing a great tendency to act as alcoholic ferment, that is to say, decomposing sugar and forming carbonic acid gas, alcohol, and other substances which we have not determined, and which probably vary with different growths.
Such, at least, is one interpretation of the facts that we have reviewed. The observations in the following paragraphs and chapters may the more incline our readers to accept it as the true one.
§ II.—Growth of Mycoderma Vini in a state of Purity—Confirmation of our original Conjectures as to the cause of Fermentation—Mycoderma Vini does not Change into Yeast, although it may give rise to Fermentation.
The efflorescence of wine, cider, and beer is pretty generally known.[64] Fermented liquors cannot be exposed to the air without soon becoming covered with a white film, which grows thick and becomes wrinkled in a marked manner in proportion as it is deprived of room wherein to spread horizontally, in accord with the extraordinary multiplication of the cellules that compose it. The rapidity of this multiplication is sometimes astounding. During the heat of summer, when the medium is well adapted to the life of the plant, we may count the number of cells which grow in the course of a few hours by millions. The absorption of the oxygen necessary to the activity of this growth, and the heat developed in the film, as well as the liberation of carbonic acid gas, that result from it are considerable. A piece of glass covering the mycoderma, at some distance above it, becomes wet with moisture, that soon accumulates to form large drops of water. The quantity of oxygen absorbed is so great that we never see any other fungoid growth on the surface of this film, although the air is constantly depositing on it, as dust, spores of an entirely different character; for, notwithstanding that the warm and moist surface is in contact with an atmosphere that is being continually renewed, yet the _mycoderma_ appropriates to itself all the oxygen contained in the air. When, however, the vegetation begins to languish, we often find, on the other hand, that the plant becomes associated with other species of mycoderma, notably _mycoderma aceti_, as well as other fungi, amongst which _penicillium glaucum_ generally appears. This is one of the facts which, wrongly interpreted, have led to the belief that _mycoderma vini_ or _cerevisiæ_ may possibly, or even readily, become transformed into _penicillium_, and _vice versâ_.[65] As the study of the growth of _mycoderma vini_ on the surface of saccharine liquids and in their depths, unaccompanied by any other species, has the most important bearing on the theory of alcoholic fermentation, we may pursue it through a few examples with all the detail that it allows of.
[Illustration: Fig. 22.]
On June 21st, 1872, we sowed some _mycoderma vini_ in three flasks, with double necks, A, B, C (Fig. 22), containing some wort. The spores employed for the purpose were obtained from plants growing on sweetened yeast-water in an ordinary closed flask. This had been impregnated with spores from plants grown on wort, which in turn had sprung from spores taken directly from _mycoderma vini_ growing on wine.
The several impregnations were effected by means of a platinum wire, held by forceps, both having been first cleaned by passing through flame, and then smeared with the fungoid films.
By this series of growings in closed vessels, which were but momentarily open at the time when we dropped the spores into them, we secured the separation of the mycoderma from all foreign organisms; and more particularly from germs of _mycoderma aceti_, which is generally found along with it, but which propagates with difficulty in neutral saccharine liquids.
On the following days films of _mycoderma vini_ had spread over the surface of the liquid in the three flasks. To all appearance they were very pure; and the microscope showed the complete absence of any mixture of _mycoderma aceti_, lactic ferment, or other foreign growths.[66]
On June 26th we decanted and distilled the liquid in A without finding any trace of alcohol. We shook up the liquids in B and C, with all due precautions, so as to submerge their films as much as possible, and then we raised the temperature of the flasks to 26° C. or 28° C. (82° F.). For some days afterwards we saw a constant succession of minute bubbles of carbonic acid gas rising through the liquid, which remained bright under the part of the film that had not fallen in. It had all the appearance of a slow but continuous fermentation.
On June 29th we decanted and distilled the liquid in B, and found in it an appreciable quantity of alcohol, which showed itself in the first distillation. The flask C, which was shaken afresh, continued to give signs of fermentation, but, some days later, the evolution of the bubbles ceased.
On July 15th, 1873, we examined the flask with its film and its deposit of _mycoderma vini_, without finding a trace of any foreign growths, either in the shape of _penicillium glaucum_, or _mucor mucedo_, or _rhyzopus nigrans_, or _mycoderma aceti_, or, in short, any of the organisms which could not have failed to appear on the surface of a substratum so peculiarly adapted to their development, had it been in the nature of _mycoderma vini_ to transform itself into one or other of those common fungoid growths. The liquid, moreover, still remained sweet, and did not contain any cells of actual yeast. We may conclude then that when one or more of these fungi occur, after an interval of some days, in a growth of _mycoderma vini_ conducted in contact with common air, it does so in consequence of that air having, without the knowledge of the observer, impregnated the liquid spontaneously with germs of these foreign organisms.
There might perhaps be room for some fear that the conditions of growth in our flasks were not favourable to the simultaneous appearance of these common fungoid growths along with the _mycoderma vini_. On June 24th, 1872, we sowed, in three flasks of sugared yeast-water, prepared as before—in the first, _mycoderma vini_, together with _penicillium glaucum_; in the second, _mycoderma vini_, together with _mucor mucedo_; in the third, _mycoderma vini_ alone.
We effected this by plunging the platinum wire, which we used for impregnating the liquids, into the pure film of _mycoderma vini_, and then touching with the wire the sporanges of the other fungus. On June 29th, we saw on the surface of our first flask some green patches of _penicillium_, along with some spots of _mycoderma vini_; in the second flask a voluminous mycelium of _mucor mucedo_, distended by large bubbles, had risen to the surface of the liquid, and was entirely covered by a film of _mycoderma vini_. As for the liquid in the third flask, there were only a few spots of very pure _mycoderma vini_. This last flask, after being kept in an oven at 25° C. (77° F.) for several months, still contained nothing but _mycoderma vini_, unmixed with any other fungoid growth whatever.
We may therefore be sure that _mycoderma vini_, vegetating on the surface of liquids adapted to its nutrition, in contact with air deprived of its germinating dust, will not present the least sign of a transformation into any of these other common fungi, or into yeast, however long may be the duration of its exposure to contact with that pure air.
We may now return to that feeble and limited production of carbonic acid gas and alcohol, the formation of which we have shown experimentally to take place at a high temperature, after submerging the film of _mycoderma vini_.[67] There can be no doubt that we have here a phenomenon similar in every point to that presented by _penicillium_ and _aspergillus_, which we studied in the preceding paragraph. When the germs or jointed filaments of _mycoderma vini_, growing on a saccharine substratum in contact with the air, are in the full activity of life, this activity is carried on at the expense of the sugar and other materials in the liquid, in the same way that animals consume the oxygen of the air and evolve carbonic acid.
The consumption of the different materials is attended with a proportionate formation of new materials, development of structure, and reproduction of organisms.
Under these conditions, not only does the _mycoderma vini_ not form a sufficiency of alcohol for analytical determination, but, if any alcohol exists in the subjacent liquid, the _mycoderma_ consumes it, converting it into water and carbonic acid gas, by fixation of the oxygen of the air.[68] If, however, we suddenly submerge the _mycoderma_, we shall obtain a different result. If, on the one hand, the conditions of life of this fungus are incompatible with the altered circumstances in which it is placed, the plant must perish, just as an animal does when deprived of oxygen. But if, in spite of these changed conditions of nutrition, it can still continue in life, we should expect to see marked changes in its organic structure, or chemical metamorphoses. The result of our observations points to the continuance of life, in a distinct though sluggish and fugacious activity, accompanied by the phenomena of alcoholic fermentation, that is, the evolution of carbonic acid gas, and the production of alcohol.
If we take a drop of liquid charged with disjointed cells of _mycoderma_, a day or two immediately after the submersion of the film, we shall observe changes, small but appreciable, in the aspect of a great number of these cells; they will show increase in size, their protoplasm will be in process of modification, and many of them will have put forth little buds. It will be quite evident, however, that these acts of interior nutrition and the changes of tissue resulting from them, proceed with difficulty; the buds when they form will soon wither, and there will be no multiplication of new cells. These changes will, nevertheless, be accompanied by the decomposition of sugar into alcohol and carbonic acid.
In comparing these facts with those which we have pointed out in connection with the cultivation of _penicillium_ and _aspergillus_, we are compelled to admit that the production of alcohol and carbonic acid gas from sugar—in one word, alcoholic fermentation—is a chemical action, connected with the vegetable life of cells which may differ greatly in their nature, and that it takes place at the moment when these cells, ceasing to have the power of freely consuming the materials of their nutrition by respiratory processes—that is, by the absorption of free oxygen—continue to live by utilizing oxygenated matters which, like sugar or such unstable substances, produce heat by their decomposition. The character of ferment thus presents itself to us, not as being peculiar to any particular being or to any particular organ, but as a general property of the living cell. This character is always ready to manifest itself, and, in reality, does manifest itself as soon as life ceases to perform its functions under the influence of free oxygen, or without a quantity of that gas sufficient for all the acts of nutrition. Thus we should see it appear and disappear concomitantly with that mode of life; feeble and fugacious in its action when the conditions of this vitality are of a similarly restricted character; intense, on the other hand, and of long duration and productive of large quantities of carbonic acid gas and alcohol, when the conditions are such that the plant or cell can multiply with facility in this novel manner. To this we may attribute all possible degrees of activity in fermentation, as well as the existence of ferments of every variety of form and of very different species. It may readily be imagined that sugar may undergo decomposition in a quite different manner from that of which we have spoken, that instead of alcohol, carbonic acid gas, glycerine, and similar substances, it may yield lactic, butyric, acetic, and other acids. It would be only one definite class of cellular organisms, the members of which resembled each other more or less, that decomposed sugar into alcohol and carbonic acid; others, specifically different, would act in a different manner. In short, we may say that the number of these living organisms is a measure of the number of different ferments.
Plate IV. represents in its two halves the condition of the _mycoderma vini_ at two different and unequal periods after its submersion. In the left-hand semi-circle, it is evident that many of the figures are swollen, that modification of their protoplasm has taken place, and incipient budding is going on in several of them. A budding of this kind would not wither; the buds would grow and, detaching themselves, would form new cells capable of budding in their turn. We should have under our eyes all the characteristics of a yeast, which, beyond doubt, would give rise to a very active fermentation, inasmuch as it would belong to the order of phenomena of nutrition and vital energy of which we are speaking. Instead, however, of insisting upon the acceptance of our interpretations, based on a few facts merely, let us go on to accumulate facts, varying the conditions as much as possible. Our examples, taken singly, may seem insufficient to establish the theory that it will be our endeavour to substantiate, but taken together we trust that they will secure our readers’ confidence.
We may now, perhaps with advantage, introduce two new expressions to embody the preceding facts, by the help of which we may often shorten our subsequent explanations. Since life can continue, under certain conditions, away from contact with the oxygen of the air, and since the altered nutrition is accompanied by a phenomenon which is of great scientific as well as industrial importance, we may divide living beings into two classes, _aërobian_, that is those which cannot live without air, and _anaërobian_, which, strictly speaking, and for a time, can do without it; these latter would be ferments, properly so called. Again, since we can conceive, in an entire organism, some organ or even a cell capable of existing, at least momentarily, apart from the influence of the air, and endowed at a given moment with the character of a ferment, we may, in like manner, make use of the expression _anaërobian_ cell, in opposition to a cell that is _aërobian_.
As long ago as 1863, in our work on putrefaction, we proposed to adopt the preceding expressions, and since then we have had the satisfaction of seeing them used by different authors in France and other countries.
One of the principal assertions in this paragraph relates to the non-transformation of _mycoderma vini_ into other moulds or into yeast.[69]
[Illustration: Plate 4. Mycoderma Vini functioning as an Alcoholic Ferment.—Right Half, showing Appearance of Spores just Sown; Left Half, their Appearance after an Interval of Submerged Life.]
For a long time, like Turpin and many other observers, although we had no belief in the transformation of _mycoderma vini_ into any one of the common moulds, yet we did believe in its transformation into alcoholic ferment. In the course of more elaborate researches, however, we at last discovered that our previous experiments had been vitiated from the same source of error which we have so often had occasion to point out as affecting the observations of our opponents, namely, the fortuitous and spontaneous introduction, unknown to the experimentalist, of germs of the very plant for whose appearance by way of transformation he is seeking.
When we consider that every fermented vinous liquor, when put on draught, is liable to efflorescence, it is difficult to avoid the supposition that this efflorescence is primarily due to cells of the yeast that has caused the liquid to ferment, from which cells the liquid could not be completely freed, no matter how bright it might have been, and which come to the surface of the liquid to live after the manner of fungoid growths. We wished to test this supposition by means of experiments. So great, however, was the resemblance between the forms possible to yeast and mycoderma, of which latter efflorescence is really composed, that we quite despaired of being able to solve the question by microscopical examination, that is, by observing the actual conversion of a cell of yeast into a cell of mycoderma. In order, then, to overcome that difficulty, we endeavoured to produce an inverse transformation—that of mycoderma into yeast. We imagined that we should doubtless obtain this result by submerging some of the efflorescence of wine or beer in a saccharine liquid well adapted to alcoholic fermentation. By submerging the mycoderma we would do away with the ordinary conditions of life in this kind of fungoid growth; for we would thus prevent the supply of oxygen from the air, since that oxygen would always be excluded, in the most effectual manner possible, by the portion of mycoderma that would remain on the surface of the liquid, even after the submersion process; and on the other hand, we would be subjecting our growth to the ordinary conditions of ferment life, which acts at the bottom or in the bulk of liquids fermenting.
Our experiments were conducted in the following manner:—In some flat porcelain basins, we grew some pure _mycoderma vini_[70] on fermented liquids, such as wine or beer, or on artificially vinous liquids, such as alcoholized yeast-water, taking care to boil these liquids previously to kill any germs of yeast or other organism that they might contain. The basins themselves, as well as the plates of glass with which they were to be covered, were plunged into boiling water just before they were wanted for use. As soon as the film of mycoderma had become well developed and thick, and even wrinkled—a process requiring not more than two or three days during summer heat—we decanted the subjacent liquid, by means of a siphon, so as to leave the film on the bottom of the basin. We then diffused the whole mass of efflorescence in a saccharine liquid that had been boiled and afterwards cooled down in a closed vessel; generally, we used wort or must preserved by Appert’s process. After that, we emptied the mixture of saccharine liquid and efflorescence into long-necked flasks that had likewise been previously heated, as also had the funnels used in the process of transference.
It seemed to us that experiments conducted with all these precautions must be free from causes of error. It was true that we were working more or less in contact with atmospheric air, but all that we had to fear for the soundness of the conclusions which we might draw was the presence of germs of alcoholic ferment, and we considered how few of these there are amongst floating particles of dust. Consequently, if we succeeded in observing the advent of yeast in each of the long-necked flasks, accompanied by an active alcoholic fermentation, we thought that we might, without danger of error, admit as a fact the transformation of cells of mycoderma into cells of yeast. Again, we thought that we should probably find in the forms of the cells of yeast which were directly derived from the cells of mycoderma, a more or less elongated structure, which would be a convincing proof of the transformation that we were seeking, if, indeed, such transformation were possible.
Strange to say, everything happened in a manner that seemed to realize our expectations. The saccharine worts in the flasks in which we had mixed and submerged the mycoderma, fermented in the course of a few days; the yeast first appeared in elongated shapes; lastly, we could see under the microscope that many of the cells or jointed filaments of mycoderma were inflated and presented the appearance of undoubted gradations between their natural state and that of the cells of yeast which soon formed part of the deposit in the vessels. In spite of all this, however, we were the victims of an illusion.
In experiments conducted as we have just described, the yeast which appears, and which soon sets up an active alcoholic fermentation, is introduced in the first place by atmospheric air, from which germs are constantly falling either upon the film of mycoderma or upon the objects that are employed in the successive manipulations. Two peculiarities in these experiments first opened our eyes to the existence of this cause of error. We sometimes found at the bottom of the flasks in which we had submerged the efflorescence, along with the cells of mycoderma, large, spherical cells of _mucor mucedo_ or _racemosus_, ferment-cells that we shall soon learn to recognize in studying this curious fungoid growth. The existence of _mucor mucedo_ or _racemosus_, where we had only sown _mycoderma vini_, was to us a proof that one or more spores of that mucor had been introduced by the surrounding air. If then, we reasoned, the air can introduce spores of mucor into our field of operations, why should it not introduce cells of yeast, especially in our laboratory? Again, it sometimes happened that a negative result was obtained. Harassed by doubts about the reality of this transformation, which accorded so well with, the physiological theory of fermentation we had been led to adopt, we repeated the experiments many times, and in some cases we failed to detect any appearance whatever of a transformation of mycoderma into yeast cells, although the conditions under which each of the experiments was conducted had been as similar as could be.
We were at a loss to account for this inactivity in the cells of the mycoderma. Even in the most favourable cases of the supposed fermentation, it was evident that a host of cells of _mycoderma vini_ did not become cells of yeast; but how could it possibly be admitted that amongst the millions of submerged cells, none were adapted for transformation, if that transformation were at all possible?
Thereupon, to find a way out of the difficulty, we resolved to modify completely the conditions of our experiments, and to apply to the research that we had in view a mode of cultivation that might completely, or nearly so, obviate the sole cause of error that we suspected, namely, the possible fall of cells or germs of yeast during the manipulations. We secured this by the use of flasks with two tubes, the right hand one of which was closed by means of a piece of india-rubber tubing with a glass stopper, the other one being drawn out in the shape of a swan’s neck. The use of these flasks, which was then new to us, permitted us to grow mycoderma and to study it under the microscope without fear of disturbance from exterior particles of dust. This time we obtained the results given in the first part of this paragraph. We no longer observed yeast or alcoholic fermentation following the submersion of the efflorescence, either in the flasks themselves, or in the test-flasks attached to them, as represented in Fig. 19. We observed, however, that kind of alcoholic fermentation of which we have already spoken and which is due to the mycoderma itself, a fermentative action that is still more instructive than the one which we thought we had determined, and certainly not less calculated to support the theory of fermentation which we have already briefly sketched.
In an age when ideas involving transformation of species are so readily accepted, perhaps in consequence of their requiring no rigorous experimental work, it is not without interest to consider that, in the course of our researches upon the growths of microscopic plants in a state of purity, we once were inclined to believe in the transformation of one organism into another—the transformation of _mycoderma vini_ or _cerevisiæ_ into yeast, and that, on that occasion, we were altogether wrong, through having ourselves fallen a victim to the identical source of error which confidence in our theory of germs had led us so frequently to detect as affecting the observations of others.
§ III.—Growth of Mycoderma Aceti in a State of Purity.
The study of _mycoderma aceti_ has not escaped the numerous causes of error which are apt to attend all observations made on microscopic organisms. This little fungus is still believed by many authors to be one of those polymorphous species capable of great modifications, according to the conditions of their cultivation—it could be, in turns, bacterium, vibrio, yeast, &c. Respecting it, we have seen resuscitated under a modern name, in the course of the last few years, the old hypothesis of Buffon concerning _organic molecules_, that of Turpin concerning the _punctiform globulines_ of barley, milk, and albumen, and the theory maintained by Dr. Pineau, of Nancy, and by Pouchet concerning _proliferous pellicles_.[71]
M. Béchamp, Professor in the Faculty of Medicine at Montpellier, disdaining to adopt the expressions which we have just used, has substituted for them that of _microzyma_, whilst adhering to the opinions and errors represented by the other expressions. This savant designates under the name of _microzyma_ all those punctiform globulines that are met with in most organic liquids when submitted to the microscope; and attributes to them, with Turpin, the faculty of playing the part of ferments, as well as of transforming themselves into yeast and various other organisms. They are contained in milk, blood, eggs, the infusion of barley, and such like; nay, we may even find them in chalk, and so we have the fine discovery of _Microzyma cretae_ as a distinct species!
Those who, like ourselves, cannot see in these granulations of organic liquids ought besides things whose nature is still undetermined, term them _molecular granules_, or, in reference to their Brownian movements, _mobile granules_. Indefinite expression is the best exponent of imperfect knowledge; when a precise terminology is invented, without any basis of precise ideas derived from a rigid observation of facts, sooner or later the hypothetical facts disappear, but the terminology prematurely created to explain them, hangs about the Science, and, bearing an erroneous interpretation, retards rather than promotes real progress.
We may here introduce a summary of Turpin’s system, as given by himself. It forms a complete biogenesis, which leaves far behind it M. Béchamp’s theory of _microzymata_, M. Fremy’s descriptions of _hemi-organism_, and M. Trécul’s account of the genesis of bacteria and lactic ferment:—
“When a mucous substance presents nothing visible through the microscope, as, for example, gelatinous matter, dissolved gum, the white of eggs, or plant-sap, simply thickened on its way to _cambium_, we call it _organic matter_ or _organizable matter_. We attribute to it the fecundating power of organic life in the simplest degree; we consider it as material still isolated from organization. We suppose that the invisible molecules, of which this organizable matter is composed, come together, combine and serve through this association in the construction of the different elementary forms of future tissues.
“May we not with greater truth believe that organizable matter is of varied origin, formed of innumerable globulines, too minute and transparent as yet to be observed by our present microscopical means, and that these globulines which are always endowed with motion and a special vital centre, are all capable, although many of them _do_ abort, of separate development either into a formative element of tissue or into a mucedinous plant?
“Organizable matter may, according to its successive states of development or age, and according to the different forms it takes in the tissues, be distinguished by special names:—
“1.—We may term matter _organizable_ as long as the globulines composing it are not yet visible to microscopes of existing power.
“2.—We may speak of _amorphous_ or _globuline tissue_ when even the globulines, previously invisible, have increased so as to be seen under the microscope, the term amorphous, or shapeless, being here applied to the association of globulines, and not to the globulines themselves.
“3.—Then we have _vesicular tissue_, when the globulines, continuing to increase, have developed in such a manner as to present a mass of continuous vesicles, still empty or already containing a new generation of globulines.
“4.—Lastly we have _filamentous_ or _tubular tissue_, when the globulines, instead of vesiculating, form threads or tubes.”[72]
Such are the purely hypothetical and exploded ideas which MM. Fremy, Trécul, Béchamp, H. Hoffmann, Hallier, and others would revive in our own day, in opposition to a theory so clear and so well supported by facts as that of germs floating in the air, or spread over the surface of objects, as fruits, dry or green wood, and so on.
M. Béchamp believes that he has discovered that _mother of vinegar_, introduced into various saccharine liquids, in the presence of carbonate of lime, generates bacteria, which, with the sugar or dregs, produce butyric, lactic, and acetic acids, and that this same mother of vinegar, without the addition of the carbonate of lime, “generates, on the other hand, the fine cells, which produce the normal alcoholic fermentation of cane sugar.” Further, M. Béchamp advances the hypothesis that mother of vinegar is a conglomeration of _microzymata_, and, as he fails to see in the experiments on which he bases the conclusions which we have just given, that bacteria and ferment cells are the result of spontaneous impregnation, having no connection with the presence of mother of vinegar, on which he experimented, he arrives at this conclusion: “In the experiments which I have just described, things happened as though the microzyma, under some peculiarly favourable conditions, had been the parent both of the bacteria and the cells.”[73] ...
The object of the following experiments was the study of these assumed transformations of the _mycoderma aceti_ in saccharine liquids, in the presence and in the absence of carbonate of lime.
We prepared some two-necked flasks, containing as a growing medium a liquid composed of one-third of Orleans vinegar, and two-thirds of a white wine used by vinegar-makers in Orleans. This liquid is peculiarly adapted to the development of _mycoderma aceti_.
On December 13th, 1872, we sowed the little plant in a state of purity, by means of a piece of platinum wire, in the manner already explained in connection with propagation of other fungoid growths. On December 19th a young and thin film of _mycoderma aceti_ covered the surface of the liquid. We then poured out the liquid through the right-hand tube, at the same time heating the end of the bent tube, to purify the air that passed into the flask. The whole film of _mycoderma aceti_ remained adhering to the interior sides of the flask during this decanting. The question then was how to convey this film of the little plant into a saccharine liquid of a particular kind. We effected this easily by the following means: After having emptied the flask, as just described, instead of re-closing the india-rubber nozzle on the end of the right-hand tube, we attached it to a test-flask containing the saccharine liquid on which we wished to operate. This had been previously boiled in the test-flask, and when we attached the neck of the test-flask, previously slightly drawn out and curved, to the india-rubber tube, the liquid was still very warm. We permitted the liquid in the test-flask to cool down, and, then, taking up the test-flask, we decanted its contents into the other flask, in which, as we have already said, the film of _mycoderma aceti_ had been left. In this way the film became partly submerged, partly spread over the surface of the new liquid. Experiments were made with two saccharine liquids, must and wort. In the case of the latter, from December 22nd the whole surface of the liquid was covered by a film of _mycoderma aceti_, which even spread up the moist sides of the flask above the level of the liquid. In the case of the must, on the other hand, the plant for some time did not seem to be developing; on December 24th, however, it was visibly spreading over the surface of the must. The following days we frequently shook up the films to separate them, and spread them over the subjacent liquid. There were no signs of alcoholic fermentation.
On December 30th we introduced several grammes (50 or 60 grains) of carbonate of lime into each of the flasks, an operation of little difficulty, which we effected in a manner similar to that just described. We substituted for the test-flask another flask—or, better still, a simple glass tube—containing carbonate of lime that had been subjected to great heat in the flask or tube, and there left to cool down. When cold, we poured the powdered carbonate of lime into the liquid in the flask, in this way avoiding the possibility of any error from the introduction with the carbonate of lime of any foreign germ.
In neither case did we obtain alcoholic fermentation, nor was there any appearance of lactic fermentation, or bacteria, or _vibrios_, properly so called. The flasks remained in the oven, at a temperature of about 25° C. (77° F.), until the end of January, 1873, when we made a microscopical examination of their deposits, exercising greater care and precaution than we had adopted in the case of those examinations which we had made from time to time in the course of the experiment to assure ourselves of the nature of the organisms present.[74] The result was that we never found anything besides the _mycoderma aceti_, which had developed, although with great difficulty, on the surface of the liquids neutralized with carbonate of lime. The beaded filaments had, under these circumstances, only become a little larger than they had been in the unsweetened acid liquids.
_Mycoderma aceti_, then, grown on sweetened acid or neutral liquids, grown in the absence or in the presence of carbonate of lime, undergoes no transformation into bacteria or vibrios or yeast, if only we operate with pure germs, free from the dust floating in the air, and from that which, unknown to the operator, may be introduced by means of the vessels and materials employed. It may be asked, do we, therefore, absolutely, reject the theory of the polymorphism of _mycoderma aceti_? On the contrary, we have endeavoured to prove the existence of this polymorphism again and again in a variety of ways. We have been mostly concerned with physiological polymorphism; that is, our efforts have been directed to ascertain if _mycoderma aceti_ might be, for example, the _aërobian_ form of a ferment from which it differed physiologically, as, for instance, lactic ferment, which, in shape, sometimes bears a striking resemblance to _mycoderma aceti_. We have not succeeded in discovering anything of the kind up to the present time.
What, in view of the positive proofs to the contrary, we do absolutely reject in the matter of this mycoderma, is the theory of polymorphisms, advocated by M. Béchamp and other authors, which, in our judgment, can only be founded on incomplete and erroneous observations.
§ IV.—Growth of Mucor Racemosus in a state of Purity—Example of Life more active and lasting when removed from the influence of Air.
Side by side with the facts explained in the last paragraph, the study of varieties of the genus _mucor_, grown in natural or artificial saccharine liquids, is of great importance to the establishment of the physiological theory of fermentation, which we shall explain later on. There is a very remarkable work on the subject of this mucedinous fungus by a German botanist, M. Bail, who, in 1857, declared that _mucor mucedo_ caused alcoholic fermentation, and could change into ordinary yeast. The first assertion, relating to the alcoholic fermentation that this fungoid growth which is everywhere so abundant may cause, is quite correct; the second which relates to its faculty of changing into yeast is erroneous.[75]
On June 13th, 1872, we sowed by the help of a platinum wire in some wort, contained in two-necked flasks, A, B, and C, several of the minute sporange-bearing filaments of _mucor_ along with the heads containing the spores.
On June 14th, there was no mycelium visible to the naked eye in the liquids.
On June 15th mycelium was very abundant, and was borne up by bubbles of gas. In addition to this there were a few scattered patches of bubbles on the surface of the liquid, showing that fermentation had commenced.
On June 16th fermentation continued to show itself by the frothy state of the crusts of mycelium buoyed up by the bubbles of gas.
On June 17th we attached B and C separately, as indicated in Fig. 19 (p. 101) to test-flasks, into which we transferred nearly all their contents. Some clusters of entangled filaments of mycelium remained on the surface of the liquids in the test-flasks.
On June 18th a very slow fermentation commenced in the test-flasks; it continued for some days without becoming more active. A little bubble would slowly rise from the bottom of the vessel, succeeded after a short interval by another, and so on. The temperature of the oven was 24° C. (75° F.). On June 22nd we raised it to 28° C. (82° F.). The fermentation became more rapid, a constant succession of bubbles rose quickly from the bottom of the test-flasks; still there was none of the vivacity of an alcoholic fermentation produced by yeast.
On June 25th the fermentation was in much the same condition, if anything rather less active.
On June 28th temperature 25° C. (77° F); fermentation had stopped.
On June 29th we raised the temperature to 27° C. (81° F.) again, and some slight revival of fermentation manifested itself.
The increase in temperature, therefore, as might have been expected, exercises a considerable influence on this kind of fermentation.
The vessels were then left to themselves, and during the course of three months they did not show the least sign of fermentation; moreover, we did not observe, either on the interior walls of the empty flasks, or on the surface or throughout the body of the liquid in the test-glasses, any fungoid production or organism whatever different from _mucor_ itself.
The same observations apply to the vessel A; in this case the liquid that remained in the flask was covered with a gelatinous and frothy mycelium.
On October 20th, 1872, after a lapse of three months and a half, we poured the liquid from the test-flask attached to flask C back again to that flask. The test-flask connected with flask B we left untouched alongside the other flasks to serve as a means of comparison.
On October 21st, 22nd, 23rd, we observed nothing; on succeeding days, however, some patches of bubbles appeared on the surface of the liquid in flask C, and clusters of mycelium buoyed up by the bubbles of gas which they imprisoned. Life had resumed its course, and with life fermentation had recommenced. What had been the cause of this change in the condition of the liquid, after an absolute quiescence of three months? There can be but one answer to this question: for in the other vessels there was no corresponding movement, or sign of life to be detected. In this vessel, however, an aeration of the plant had evidently taken place, consequent on the decantation and contact with the atmosphere of the flask, which communicated with the exterior air through the curved tube. This aeration had been absent or ineffective before decantation, in consequence of the great depth of liquid in the test-flask, the surface of which, too, was covered by a mass of mycelium filaments, itself effectually opposing any aeration of the liquid. Moreover, the surface of the liquid in the narrow neck of the test-flask had necessarily been covered by a layer of carbonic acid gas. We may investigate more thoroughly the influence of aeration, and its relation to the resumption of life in the mycelium of _mucor_, by restoring the liquid to its previous condition of depth and so cutting off again contact with the air.
For this purpose, on October 31st we decanted once more the liquid and its deposit from the flask into the test-glass. The same evening a slight but continuous fermentation, with formation of froth, appeared on the surface of the liquid in the neck of the test-glass. Fermentation although never vigorous, continued the following days, and until December 20th.
Between December 20th and 23rd, it ceased altogether to manifest itself by liberation of gas. As for the flask B, during all this time it had remained quite inactive and in the same state in which it had existed since June 29th, although the oven had on several days been heated to 28° C. (82° F.).
On December 23rd, 1872, wishing to assure ourselves of the state of the plant in flask B, we subjected it to the same operation to which the flask C had been subjected on October 20th: that is to say, we poured the contents of the test-glass back into the connected flask, with the object of supplying the plant with oxygen.
On December 24th, 25th, 26th, 27th, there was no apparent change.
On December 28th bubbles of gas began to be evolved carrying up clusters of mycelium to the surface of the liquid. It was evident, therefore, that the quiescence in the test-glass attached to flask B, was solely due to deprivation of air, as had happened in the case of the test-glass attached to flask C, up to the date of October 31st.
On this day, December 28th, we re-decanted the contents of the flask into the test-glass, and the following day a continuous but feeble fermentation proceeded. This lasted until January 22nd, although very sluggish in character; it is evident that these effects were exactly the same as those which took place in flask C.[76]
We should observe before we proceed further, that we took specimens from the flasks A, B, C, at different times between June and January, and that the microscope never revealed the least trace of yeast in them. We may note besides that, during this interval, we impregnated fresh flasks of wort with specimens taken from the deposits in the flasks A, B, C, and that we always obtained reproduction of the _mucor_ and its peculiar fermentation without the least appearance of ordinary ferment.
The inferences from the results that we have just detailed follow readily, and are besides of great interest. In the first place, it is evident that even if the _mucor mucedo_ may be able to produce alcoholic fermentation, it is totally incapable of changing into yeast. The two plants are necessarily and radically distinct, and, if different authors have succeeded in obtaining them mixed one with another in growths of _mucor_, this intermixture was doubtless the result of a spontaneous sowing of the yeast, the germs of which abound, particularly in the particles of dust existing in the atmosphere of any laboratory in which studies relating to fermentation are pursued.
This, however, is not the most striking inference from the facts which the cultivation of these organisms revealed. The _mucor_ is evidently a plant, at the same time _aërobian_ and _anaërobian_. If we had sown the spore-bearing filaments of _mucor_ on slices of pear, lemon, or similar fruit, we should have seen the spores germinate, tubes of mycelium ramifying on the surface of the substratum, and reproducing sporiferous aerial _hyphae_. In this case the plant would have effected all its phenomena of nutrition by absorbing oxygen and emitting carbonic acid, after the manner of animals, as, in our essay on the organic corpuscles which exist in a state of suspension in the atmosphere, we have shown to be the case generally with fungoid growths. Under these circumstances, the only sugar decomposed would have been a quantity equivalent to that assimilated in forming the cellulose of the young tissues of the fungus, or in entering into combination, either with the elements of ammonia or with the sulphur of the sulphates, or the phosphorus of the phosphates, to form the albuminous substances of the interior of the cells.[77] In this case the sugar used up would furnish no alcohol, or at least, if alcohol were formed, it would be decomposed immediately. All _aerial_ growths take place in the same manner; and such is the nature of nutrition and life in all the larger forms.
In our flasks, on the other hand, the life of the little plant functions quite differently. Deprived of oxygen, or having at its disposal but an insufficient quantity of that gas, after a life of activity in contact with air, it can, nevertheless, live apart from the direct action of that element, and the combinations to which it gives rise. On the other hand, we see all the signs of alcoholic fermentation appear; that is, a notable proportion of sugar, in comparison with the weight of solid matter assimilated and fixed by the plant, is decomposed into alcohol and carbonic acid gas; and this decomposition continues as long as life itself continues in the cells, and they remain submerged, this last condition being effected by the decantation of the liquid and its deposit into the test-glass. Along with the disappearance of the phenomena of vital activity in the cells, the fermentation ceases absolutely, or at least is no longer visible externally, by reason of its extreme feebleness. The cells then assume an old, shrivelled, worn-out appearance, with irregular outlines and granular markings. Their life is merely suspended, however, not extinct; for if they be supplied once more with oxygen, and suffered to exist under the influence of that gas, they will vegetate again, and become capable of producing fermentation afresh, even after having been excluded from the air for a considerable time.
Oxygen then presents itself to us as being endowed with a certain determining stimulus in the matter of nutritive action enabling this action to be prolonged beyond the point where the direct influence of oxygen ceases. In time the energy that has been imparted to the cells will die away, and then also fermentation will cease, to be resumed, however, when the plant is once more submitted to the revivifying action of the gas. It seems as though the vital energy derived from the influence of gaseous oxygen were capable of effecting an assimilation of oxygen, not in the gaseous state, but existing in some state of combination, and hence its power of causing the decomposition of sugar. Looking at the matter in this light, it seems to us that we may discover in it a fact of general occurrence, that this peculiar action of the oxygen and the cells is to be seen in all living beings. For indeed is there any cell which, if suddenly and completely deprived of air, would perish forthwith, and absolutely? Probably there is not a single one that would do so. With certain modifications of greater or less amount the assimilative and excretive acts which have taken place during life must be carried on after the suppression of oxygen, resulting in fermentations ordinarily obscure and feeble, but in the case of the cells of ferments, properly so called, manifesting an activity both greater in amount and more enduring.
Let us now proceed to compare the weight of alcohol formed by the _mucor_ during fermentation with the weight of the plant itself.
_First experiment._—One of the double-necked flasks contained at starting 120 c.c. (about 4 fl. oz.) of wort.
On January 2nd, 1873, we attached this flask to a test-glass, containing a deposit of _mucor_ ferment (Fig. 19, p. 101), a few drops of which we poured into the wort in the flask, to impregnate it. On January 3rd we decanted the wort from the flask into the test-glass; under these conditions we have seen that the wort must ferment.
On January 18th the fermentation in the test-glass ceased. On July 31st, 1873, we transferred the liquid from the test-glass back to the flask. On August 4th, 1873, we again decanted this same liquid from the flask into the test-glass. On December 25th, 1873, we once more removed the liquid from the test-glass to the flask, and allowed it to remain so until December 23rd, 1874, on which day we submitted it to examination. It was found to contain per 100 c.c. (3-½ fl. oz.)
Grains. Grammes.[78]
Total weight of the fungus 5·7 0·37
Absolute alcohol 50·9 3·3
Acidity, estimated in its equivalent of 1·7 0·11 sulphuric acid
Sugar, determined by cupric solution 82·2 5·2
Dextrine (?) 24·6 1·6
The total weight of fungoid growth being 0·37 gramme, and the total weight of absolute alcohol for the 120 c.c. of fermented liquid being 4 grammes, we had, consequently, from ten to eleven times by weight more alcohol than fungus.
_Second experiment._—On June 13th, 1872, we sowed two or three sporiferous heads of _mucor_ in some wort contained in one of the double-necked flasks. The temperature of our oven varied between 23° C. and 25° C. (73° F. to 77° F.) The total volume of liquid was 120 c.c., as before.
June 15th, mycelium had developed, buoyed up on bubbles of gas.
June 16th, patches of bubbles, due to fermentation, covered the surface of the liquid.
June 17th, we transferred the liquid to the test-glass.
June 28th, fermentation in the test-flask had ceased.
June 28th, fermentation recommenced, the temperature of the oven being raised to 27° C. (80° F.).
October 20th, the liquid was transferred back from the test-glass to the flask.
October 24th, mycelium had developed, supported by big bubbles on the surface of the liquid in the flask.
October 31st, we retransferred the liquid to the test-glass.
November 1st, a feeble, but continuous fermentation commenced. This was kept up until January 2nd, 1873, on which day we transferred the liquid, with its deposit from the test-glass to the flask, when it now seemed to be quite inert. We left it in this flask until December 24th, 1874, without its manifesting during this long interval any sign of fermentation; nor did the fungus appear to grow at all.
We then submitted the liquid to analysis, and found in it, per 100 c.c.—
Grammes.[79]
Total weight of fungoid growth 0·25
Absolute alcohol 3·4
Acidity, estimated in its equivalent of sulphuric 0·12 acid
Sugar, determined by copper solution 6·2
Sugar, determined after treatment by boiling with 1·0 sulphuric acid, and deduction of amount of sugar already obtained (dextrine)?
The total weight of absolute alcohol for the 120 c.c. of fermented liquid was 4·1 grammes—that is, the weight of the alcohol was sixteen or seventeen times that of the plant.
[Illustration: Plate 5. Mucor, Vegetating Submerged, in Deficit of Air.]
The structure of the plant differs considerably when it lives surrounded by air, and when it is more or less completely deprived of that fluid. If it has an abundance of air at its disposal, if it vegetates on the surface of a moist substance or in a liquid in which the air held in solution may be renewed without being incessantly displaced by carbonic acid gas, we shall see it develop as an ordinary fungoid growth, with a mycelium consisting of filaments more or less slender, branching, and entangled, sending up from the surface of the liquid aerial organs of fructification. This is the well-known form of vegetation of the common _mucor_. On the other hand, if we compel the _mucor_ to live in a saccharine liquid with insufficiency of air, at least for some of its parts, the mode of vegetation will change completely, as we have seen in the case of _penicillium_, _aspergillus_, and _mycoderma vini_ when submerged, but with this difference, that in the case of the _mucor_ the changes in question, and the activity of nutrition under these new conditions, are much more marked than in the case of those other organisms. The spores grow larger and the filaments of mycelium which do develop are much stronger than those in the normal plant. These filaments put forth, here and there, other filaments which detach themselves and vegetate at the side of the others, being terminated or interrupted by chains of large cells, species of spores which can live by budding and reproducing cells similar to themselves or by elongating into filaments.
Plate V. represents the living plant submerged at a little depth, and having, consequently, still at its disposal a certain quantity of air, insufficient, however, to supply the oxygen needed for all the acts of nutrition. In this case the _mucor_ appears very different, morphologically, from what it is when in free contact with air. Here it forms short filaments, having a diameter double or triple that of the filaments of the ordinary mycelium with branches and buds all over, and what is especially characteristic, forming a network of chains of cells, sometimes spherical, sometimes oval or pear-shaped, which are the actual spores. These, as soon as they are detached, bud in their turn, and reproduce either cells or branching tubes; these cells or the chaplets which they form being known under the name _mycelian spores_ or _conidia_. Our plate gives these different aspects very correctly, and affords us a good idea of the luxuriant state of this remarkable vegetation.
Plate VI. represents the plant living at a greater depth with less air, expending, by means of sugar as source of heat, the energy which it acquired in vegetating under the influence of the oxygen of the air. The filaments are fewer and older in aspect, and the number of cellular forms is proportionately larger than in the former case, the budding giving rise by preference to spherical or oval cells. On a single cell we often see two, three, four, five, six, and even more buds.
When the buds of the oval or spherical cells detach themselves whilst young, they often resemble in form and size cells of ordinary yeast, nor can even considerable experience in this kind of observation always enable us to distinguish them. Hence we may easily understand how many have come to believe, with so skilful a botanist as Dr. Bail, in the transformation of _mucor_ into yeast.
With the forms represented in Plates V. and VI., the plant is more of a ferment than of a fungoid growth. In such cases the weight of sugar decomposed in comparison with the weight of new cell-globules formed is very considerable, an effect which is more marked the less air the plant has at its disposal. Under the latter conditions, however, vegetation is slow and laborious, and the ferment very soon assumes an aspect of age, and we must constantly rejuvenate the cells by bringing them into contact with oxygen, and subjecting them to the action of limited quantities of that gas, and so promote their vegetation and prolong their fermentative activity. This effect we brought about when we retransferred the liquid and its deposit of _mucor_ from the test-glass to the flask, thus bringing them into contact with fresh air. We saw cells that appeared old, dark, and highly granulated, become inflated, grow more transparent, and fill with a gelatinous protoplasm, the few granulations which they still exhibited assuming a brilliant appearance when we succeeded in distinguishing them; and finally, a very active budding was set up. Under this reviving influence life could continue once more away from the air, although with difficulty, so that fermentation would be most intense if the large filaments and their conidia were constantly being removed from and to the action of air.
The preceding plates show several instances of this rejuvenescence of the old cells of _mucor_ ferment.
[Illustration: Plate 6. Ferment of Mucor.]
We have omitted to represent amongst the old cells some cells which have their granulations collected about the centre, with an empty space between the granulations and the exterior borders.[80] In this state, cells are generally dead and incapable of any revival. It is impossible to avoid being impressed by the striking analogies which exist between all these facts and those presented by cells of yeast.
In concluding our study of the vegetation of _mucor_ as a mould and _mucor_ as a ferment, we may again remark that the most striking analogies also exist between the preceding observations and those we have seen in the case of _penicillium_, _aspergillus_, and _mycoderma vini_. These latter plants do not furnish alcohol or carbonic acid gas by direct fermentation of the sugar, as long as we let them vegetate with plenty of air at their disposal. Once submerged, however, their vital aspect changes; on the one hand the cells or filaments of the mycelia evince a tendency to become larger; on the other hand there is a tendency to greater closeness in these latter, and, consequently, a transition to the state of _conidia_. Lastly, there is a correlative budding of cells, accompanied by a formation of alcohol and liberation of carbonic acid gas; in short, all the ordinary signs of alcoholic fermentation.
The principal difference in the case of _mucor_ consists in this, that the vegetation of this latter, under the conditions of insufficient aeration or none at all, is more decided, both as to extent and duration.
It may be thought that all the varieties of _mucor_ are capable of yielding the kind of ferment that we have just mentioned. But this is not the case; and here we have another striking proof of the great physiological differences presented by forms of vegetation so intimately connected with each other that, in botanical classifications, they must be put as closely as possible together. Of this fact we have the most striking example in _mycoderma vini_ and the alcoholic ferments, properly so called, which so closely resemble each other in form and development that they might be supposed to be identical, at least, according to our present knowledge, but which differ so widely in their physiological aspects.
On November 17th, 1873, we found a very beautiful specimen of _mucor mucedo_ on a pear, under a glass bell jar. It was a mass of perfectly straight filaments, simple and isolated, very large in comparison with those ordinarily met with, each terminating in a sporange, identical to that of _mucor mucedo_, and proportionately well developed. We are able to distinguish _mucor racemosus_ from _mucor mucedo_ only by the circumstance of its having on its sporange-bearing hyphæ lateral branches which also terminate in sporanges.
We sowed only one of the terminal heads of the large erect hyphae in some wort, in which it soon produced an abundant mycelium, but without the least appearance of gas. For a very long time, up to January 7th, 1875, we studied the developments of this organism, which remained all the time perfectly pure, in consequence of our having cultivated it in one of our two-necked flasks on pure wort.
The total volume of liquid, which was 130 c.c., (4·57 fl. oz.) contained 2·3 grammes (35·3 grains) of alcohol. In spite of this rather large proportion of alcohol, a clear sign of undoubted fermentation, the plant had yielded no _conidia_ at all, nor any cell-globules of ferment. Some of the filaments, however, were larger than the rest, and exhibited irregularly-shaped swellings, which in some cases were of enormous size. Whilst the natural mycelial filaments, by which we mean the vegetating part of mucor, which were supplied with abundance of air, only measured 3/450 of a millimetre[81] in diameter, the filaments that had grown probably with an insufficiency of oxygen, and performed the functions of ferment, measured 8/450, and the swellings as much as 30/450 of a millimetre in diameter, as represented in Fig. 24.
[Illustration: Fig. 24.]
In concluding this paragraph, we may mention a very able research on fermentation which we have lately studied, the author of which, Dr. Fitz, communicated it to the Chemical Society of Berlin in 1873. In section II., page 48, this author explains his observations in a manner conformable to our own views, as may be seen from the following passage of the memoir:—
“In the presence of oxygen, the ferment of _mucor_ develops into a mycelium and consumes the sugar; in the absence of oxygen, on the other hand, the spores develop into ferment of _mucor_, that buds and decomposes the sugar into the products of fermentation.
“The properties of _mucor mucedo_ in a fermentable liquid, in the presence or in the absence of oxygen, accord perfectly with the theory of fermentation established by Pasteur in 1861 (_Comptes rendus de l’Académie des Sciences_, t. lii., p. 1260). According to this theory a fermentative fungus needs oxygen for its development; if it finds any free oxygen it utilizes the whole of it, assimilating one part of the sugar and burning the other; whilst in the absence of free oxygen, the fungus appropriates what it requires from the sugar.”
Footnote 49:
In the course of this work we shall combat, by means of experimental proofs which appear to us irrefragable, the opinions which many writers entertain on the subject of certain transformations of organisms—that of _penicillium glaucum_ into ferment, or _mycoderma_; of _bacteria_ into lactic ferment; of ferment into _vibrios_; of _mycoderma aceti_ into ferment, and so on. Nevertheless, we shall pronounce no _a priori_ opinion on the question whether the inferior organisms, which will be the subject of this chapter, and which include yeast and the ferments properly so called, are perfect beings in their habitual form, or whether they are susceptible of polymorphism. It is with this reservation that we employ the word _autonomy_. If we claim polymorphism for any species, we shall not do so without furnishing proofs. Some organs detached from higher organisms, and some beings in a certain phase of their existence, may reproduce themselves under a special form, with special properties, when brought into media and under conditions that are unfit for the production of the plant or animal under its other shape or ordinary mode of reproduction. Modern Science affords many examples of this, and certain alcoholic ferments present us with analogous facts; but to wish to stretch these facts beyond their due significance, and to admit a polymorphism that cannot be proved, in consequence of a belief that it is possible, or on the faith of confused observations, is to indulge in gratuitous assertion from a mere spirit of system.
Footnote 50:
See, on this subject, the author’s _Études sur le Vinaigre_, Paris, 1868, p. 76, note; and especially _Études sur le Vin_, 2nd Edition, 1873, p. 19.
Footnote 51:
Some observations in the preceding chapter enable us to account for the vast number of germs which are constantly falling on the surface of everything. We may here allude to the use we have made of flasks, shaped as in Fig. 17, and holding from 250 c.c. to 300 c.c., which are a third part filled with an organic liquid, and are closed up when boiling. They contain no air when cool, and are opened in series of 10, 20, &c., out of doors, and closed up again immediately. The air rushes violently into the vacuum, and thus we introduce about 200 c.c. of air, with all the particles of dust contained in that air, into each flask. It has been proved that a certain number of these flasks undergo change in the course of time, the number of those changing and the nature of their changes being in close proportion to the probable number and nature of the floating germs able to develop in the particular nutritive liquid used. If we work at great elevations, far from houses and the dirt of towns and inhabited plains, as we did at Montanvert, near the _Mer de Glace_, change will seldom occur. The opposite will be the case if we work in a place like the living-room of the little, dirty, ill-kept inn at Montanvert. In a laboratory where fermentation is studied we obtain certain kinds of germs which often differ from those found in the air of the open country. If we desire to have organisms in all our flasks, we have only to stir up the dust on the ground or on surrounding objects at the moment when we open the flasks. This simple and easy experiment clearly shows us that it is impossible for a field of sporanges of fungoid growth, existing in an uncovered vessel or on the surface of a fruit, to escape becoming mixed with germs that are foreign to the little plant; in other words, the student who sows spores of _penicillium_, which he has collected from one place or another on a brush, exposes himself to serious causes of error.
Footnote 52:
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 ordinary fungoid growths; he has given a formula for the composition of such a medium. It is this that we call here “Raulin’s fluid” for abbreviation.
Water 1,500 Sugar Candy 70 Tartaric Acid 4 Nitrate of Ammonia 4 Phosphate of Ammonia 0·6 Carbonate of Potassium 0·6 Carbonate of Magnesia 0·4 Sulphate of Ammonia 0·25 Sulphate of Zinc 0·07 Sulphate of Iron 0·07 Silicate of Potassium 0·07
J. RAULIN. Paris, Victor Masson, 1870. _Thèse pour le doctorat._
Footnote 53:
If we do not wish to take the chance of procuring the pure _penicillium_ by means of these spontaneous sowings, effected by opening and then closing in the flame a certain number of flasks with drawn-out points, we may utilize one of the flasks, which, having been opened and closed again, has notwithstanding developed no organized forms, as follows:—We impregnate the contained liquid directly, by dropping into it from a metallic wire spores taken from any growth of _penicillium_ exposed to the common air; and then from the new field of sporanges formed by this sowing in the flask that has been re-closed, we must, later on, take the pure spores that we require. This method is quicker and almost as safe.
We should add that, if we wish to use for our purpose spores of _penicillium_ from a closed flask, in which the plant has fructified, we must be careful not to leave the plant too long closed up. A few days after the sowing the growth of the fungus is arrested, in consequence of all the oxygen being absorbed, and its place being supplied by a mixture of carbonic acid and nitrogen; and the spores, if kept too long in this atmosphere, will all perish.
Footnote 54:
To shake the liquid without danger of introducing exterior particles of dust, we apply the flame of the spirit lamp to the drawn-out neck of the flask, and close up the open end; we may then shake our flask without risk. We must afterwards reopen the end of the drawn-out neck for the purpose of re-establishing communication with the exterior air.
Footnote 55:
The flask B was closed with the lamp in consequence of one of the objects of these experiments being to test M. Trécul’s experiments on the transformation of _penicillium_ into ferment. Strangely enough, according to M. Trécul, as we shall see later on, the spores of _penicillium_ refuse to change into ferment, if the vessels in which they are sown are not “perfectly air-tight.”
Footnote 56:
_Bulletin de la Société Philomathique._
Footnote 57:
HERMANN HOFFMANN, _Études Mycologiques sur la Fermentation_. _Botanische Zeitung_ and _Annales des Sciences Naturelles_, 4^e série, t. xiii. p. 24, 1860.
Footnote 58:
_Communication sur l’Origine et le Développement de quelques Champignons._ Dantzig, 1867.
Footnote 59:
TRÉCUL, _Comptes rendus de l’Académie_, t. lxxiii. p. 1454; December 28, 1871.
Footnote 60:
TRÉCUL, _Comptes rendus de l’Académie_, t. lxxv. p. 1169, November 11, 1872. A proof of M. Trécul’s carelessness in experiments of this kind is the fact that in studying the fertility of an impregnated wort, he often obtains different productions. Our experiments give opposite results. If we sow nothing, we obtain nothing. If we sow a plant, we obtain a similar plant; or, should there be any difference, the change may be traced, beyond question, to its origin in the plant sown, and is the consequence of some alteration in the conditions of our experiment.
Footnote 61:
_Comptes rendus des Séances de l’Académie des Sciences_, t. lxxv. p. 1220; Nov. 18, 1872.
Footnote 62:
Since writing the above we have experienced some doubt as to whether the forms of development represented in Fig. 20 are actually those of the _aspergillus glaucus_, which we supposed our fungoid growth to be. In some of the later sketches of our observations we find similar forms, which belong to a bluish kind of _penicillium_, with rather large spores. Fortunately, this doubt affects our argument in no essential particular. It matters very little what variety of fungoid growth it is that gives rise to alcoholic fermentation attended by peculiarities of shape that only occur in the development of its spores when air fails it.
Footnote 63:
By the term _conidia_ is meant certain chains of cells, which are in reality mycelial spores.
Footnote 64:
See PASTEUR, _Études sur le Vin_, 1st Edition, pp. 20 and following.
Footnote 65:
Since writing this paragraph, we have found in M. Ch. Robin’s _Journal d’Anatomie et de Physiologie_, an article signed by that gentleman, and entitled _Sur la Nature des Fermentations_, &c. (July-August, 1875), in which the learned microscopist says:—“The _torula cerevisiæ_ is derived from the _mycoderma cerevisiæ_. My observations leave no doubt on my mind that _penicillium glaucum_ is one of the forms evolved from spores or ferments that have preceded it, as M. Trécul showed a long time ago, and that, moreover, the spores of _penicillium_, germinating in suitable media, give us the sporical form termed _mycoderma_.”
We take the liberty to observe that these assertions of M. Robin’s are purely gratuitous. Up to the present time it has been impossible to discover a suitable medium for the proof of these different transformations or polymorphisms. From the time of Turpin, who firmly believed that he had observed these changes, to our own, none of the microscopists who have affirmed these transformations have succeeded in adducing any convincing proof of them, and M. Trécul’s latest observations, especially as regards _penicillium_ and its transformation into ferment or into the _mycoderma_ of beer, have been positively disproved by ours, supported, as they are, by proofs that we consider irrefutable.
Footnote 66:
It is a very easy matter to study the liquids and growths in our flasks during the course of a single experiment. We take out the glass stopper that closes the india-rubber tube on the straight-neck, and, by means of a long rod or a glass tube previously passed through the flame, take up a quantity, which we draw out immediately for microscopical examination. We then replace the glass stopper, taking care to pass it through the flame before doing so, to burn up any organic particles of dust that it may have picked up from the table on which we laid it.
Footnote 67:
We may prove the occurrence of alcoholic fermentation by the cells of submerged _mycoderma vini_ in a different manner. To do this, after having made all our preparations as before and shaken up the film of _mycoderma vini_ in its liquid, we must attach our flask to a test flask (Fig. 19), and pass the turbid liquid into the latter. On succeeding days we shall detect a very protracted fermentation in the test flask; there will be a succession of minute bubbles rising from the bottom, but in small number at a time. The fermentation is very evident whilst it lasts, but is rather sluggish, and, although of very long duration, ceases long before the sugar is exhausted.
This experiment proves better than any other the non-transformation of _mycoderma vini_ into other ordinary fungoid growths. For after decanting the liquid into the test flasks, the sides of the experimental flask remain covered with streaks of _mycoderma vini_ along with some of the liquid. Moreover, the flask is refilled with air, and this air is being constantly renewed, in part, by variations of the temperature of the oven, so that the _mycoderma_ remaining on the sides is thus placed under the most favourable conditions for transformation into other fungoid growths, if that were possible. It is still more easy to detach the experimental from the test flask, and to pass pure air into it, once or twice a day, or constantly. In any case, we shall never see anything besides the _mycoderma vini_ spring up within it.
Footnote 68:
See PASTEUR, _Comptes rendus des Séances de l’Académie des Sciences_, t. liv., 1862, and t. lv., 1862. _Études sur les Mycodermes, &c._
Footnote 69:
In a subsequent chapter we shall prove that yeast is likewise incapable of transformation into _mycoderma vini_.
Footnote 70:
We secured the purity of our mycoderma by the same means that we have already described for the procuring of spores of _penicillium_ or other fungoid growths in a state of purity.
Footnote 71:
BUFFON, _Histoire de l’Homme_, t. viii., edition 12mo, 1778; TURPIN, _Mémoires de l’Académie des Sciences_, t. xvii.; Dr. PINEAU, _Annales des Sciences Naturelles_, t. iii., 1845; POUCHET, _Traité de la Génération dite Spontanée_, p. 335, 1859. See also our _Mémoire sur les Générations dites Spontanées_, 1862, pp. 100 and following, in which we give a _resumé_ of some of these theories.
Footnote 72:
The following is Turpin’s application of his theory to the formation of the ferments of fruits (_Mémoires de l’Académie_, t. xvii., 1840, p. 155), where also, on p. 171 the above quotation will be found:—_Ferments Produced by the Filtered Juice of the Pulp of Different Fruits_—“By the word pulp we mean the soft and juicy cellular tissue of the fleshy part, mesocarp or middle layer of the pericarp of certain ripe fruits. This cellular tissue, which is very abundant in the peach and all stone-fruit, in the apple and pear, in the orange and grape, and similar fruits, is the same as that which forms the body of a leaf. Being in every case composed of a simple agglomeration of contiguous mother-vesicles, which are always filled with globulines that are more or less developed, more or less coloured, and individually endowed with a special vital centre, it is not surprising that its globulines when free and detached from the compound organisms to which they belong, and from association with its vegetable life, should, when placed in a suitable medium, themselves vegetate and become transformed, under these new influences, into a mucedine, with filaments and articulations. Such are the very fine, and, consequently, very transparent globulines, which, when left to themselves in sweetened water, grow and become vesicular, producing other globulines in their interior, then bud, vegetate into mucedinous filaments, decompose sugar, and produce all the effects that constitute what we term _alcoholic fermentation_.”
Footnote 73:
BÉCHAMP, _Recherches sur la Nature et l’Origine des Ferments (Annales de Chimie et de Physique, 4^e série_, t. xxiii., and _Comptes rendus de l’Académie des Sciences_, Oct. 23, 1871).
Footnote 74:
We need scarcely here observe, having done so on previous occasions, that whenever we opened our flasks to obtain specimens, we made use of a fine tube, previously passed through the flame of a spirit lamp, and that we also passed this flame over the surface of the india-rubber, glass stopper, &c., to consume the organic particles of dust which floating about might introduce themselves at the moment when we opened the right-hand tube of the flask.
Footnote 75:
Ever since the year 1861 (see p. 92), this question of the possible transformation of the ordinary fungi, especially _penicillium_ and _mucor mucedo_, into yeast has engaged our attention. The results attained have been entirely negative; but hitherto only the conclusions of our work have been published, some account of which was given at the meeting of the _Société Philomathique_ of March 30th, 1861. The following extract is from the _Bulletin_ of the society:—“Meeting of March 30th, 1861. At this meeting a paper was read by M. Pasteur ‘On the supposed changes in the form and vegetation of yeast-cells, depending on the external condition of their development.’ It is well-known that Leuwenhoeck was the first to describe the globules of yeast, and that M. Cagnard-Latour discovered their faculty of multiplying by budding. This interesting vegetable organism has been the subject of a host of researches by chemists and botanists. The latter, from the days of Turpin and Kutzing, have almost unanimously regarded yeast as a form of development of various inferior vegetable types, especially _penicillium_. The studies of this subject which seem to have won most favour during the last few years are those of MM. Wagner, Bail, Berkeley, and H. Hoffmann. The researches of these botanists seem to strengthen and confirm the original observations of Turpin and Kutzing. M. Pouchet has, quite recently, expressed the same ideas, and has determined certain points in connection with them with much precision of detail. M. Pasteur has long studied this important question, which is so intimately connected with the essential nature of yeast and with those phenomena of the polymorphism of the inferior types of vegetable life, to which most of the remarkable works of M. Tulasne relate; he has, however, arrived at results that are altogether negative, and he declares that he was unable to detect the transformation of yeast into any of the _mucedines_ whatsoever, and, inversely, that he could never succeed in producing the smallest quantity of yeast from ordinary _mucedines_.” These same results we communicated to the _Société Chimique_ of Paris, at a meeting held April 12th, 1861. Throughout the investigation of which we have just indicated the conclusions, we insisted on the necessity of cultivating the separate organisms in a state of purity in all researches relating to these inferior forms of life, if we desire to attain to sure inferences about them; and the method of working, which we recommended, did not differ essentially from that adopted in the present work. Since then the study of these growths has been conducted with the utmost precautions; and other apparatus, perhaps as safe as those which we employ and better adapted than ours for the study of polymorphism of species, have been invented by botanists of great skill—M. de Bary, in Germany, and M. Van Tieghem, in France.
Footnote 76:
We found, after the lapse of another year, in December, 1873, that the ferment of the _mucor_ in the test glass might still be easily revived; that it was able to propagate, both in the mycelium and in the cellular form, in wort, and that it might produce a fermentation, more or less active, according to the condition of aeration; in short, that it was capable of producing all the characteristic phenomena described. By means of the method of cultivation that we employ, our study, which was continued for years, was pursued without the least fear of any foreign fungoid growths being introduced into the vessels, although they remained constantly open, and the air in them was being perpetually renewed by the action of diffusion and variations of temperature. In 1875 nothing remained alive in our flask, and further revival became impossible.
Footnote 77:
We do not here take into account certain phenomena of oxidation of which the fungoid growths are the seat, and which remind us of those that are presented in so remarkable a degree by _mycoderma vini_ and _mycoderma aceti_.
Footnote 78:
[There are 15·43 grains in the gramme.]
Footnote 79:
[For English equivalent see Experiment 1, p. 135.]
Footnote 80:
The figure given below supplies this omission. The cells that are isolated or are in chains, _b.b.b._, show this state of the old cells. The cells _a.a.a._ are younger, and may be more easily revived. We may see by the dimensions of some of these how greatly, in certain cases, the cells of _mucor_ resemble cells of yeast; nevertheless, in the state of the contents and the aspect of the outlines, there are always some differences sufficiently appreciable to strike the practised observer.
[Illustration: Fig. 23.]
The figures adjoining the cells indicate fractions of a millimetre. (A millimetre may be taken as ½5-in.)
Footnote 81:
[0·000089 in., 0·00026 in. and O·00089 in. respectively.]
