Chapter IV (2)

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.

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.

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.

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: