Chapter IV (1)

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.

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.

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.

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.

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.

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.

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]

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.