Chapter V: The Alcoholic Ferments (1)

§ I.—On the Origin of Ferment.

Amongst the productions that appear spontaneously, or, we should rather say, without direct impregnation, in organic liquids exposed to contact with the air, there is one that more particularly claims our study. It is that one which, by reason of its active energy as an agent of decomposition, has been distinguished and utilized from the earliest times, and is considered as the type of ferments in general; we mean the ferment of wine, beer, and more generally, of all fermented beverages.

Yeast is that viscous sort of deposit which takes place in the vats or barrels of must or wort that is undergoing fermentation. This kind of ferment presents for consideration a physical fact of the most extraordinary character. Take a morsel of the substance and put it in sweetened water, in must, or in dough, which always contains a little sugar; after a time, the length of which varies, a few minutes often sufficing, we see these liquids or the dough rise, so to speak. This inflation of the mass, which is due to a liberation of carbonic acid gas, may cause it to overflow the vessels containing it, if their capacity is not considerably greater than the volume of the matters fermenting. It is equally remarkable that these phenomena are natural and spontaneous; that is to say that the must, the wort, and the dough are able to rise, as we have termed it, when left to themselves, without the least addition of foreign substances. The only difference that may occur in these phenomena is a certain amount of retardation, in cases where the yeast does not reach the saccharine matters in a perfectly natural form, inasmuch as it then requires a certain time to get itself together before it can begin to act.

It is necessary, indeed, that sugar should be present; for if we abstracted by some means or other from the must or dough all the sugar contained in it, without touching the other constituents, the addition of yeast would produce no gas. Everything would remain quiet until the moment when signs of a more or less advanced putrefaction showed themselves. Yeast is one of the most putrescible of substances, and it is worthy of notice that its alteration is also the consequence of the formation of one or more ferments, very different, however, from that of which we are speaking. As for the nature of yeast, the microscope has taught us what it is. That marvellous instrument, although still in its infancy, enabled Leuwenhoeck, towards the close of the 17th century, to discover that yeast is composed of a mass of cells. In 1835 Cagnard-Latour and Schwann took up Leuwenhoeck’s observations, and by employing a more perfect microscope, discovered that these same cells vegetate and multiply by a process of gemmation. Since then the physical and chemical phenomena already mentioned, such as the raising of the mass, the liberation of carbonic acid gas, and the formation of alcohol, have been announced as acts probably connected with the living processes of a little cellular plant, and subsequent researches have confirmed these views.

In introducing a quantity of yeast into a saccharine wort, it must be borne in mind that we are sowing a multitude of minute living cells, representing so many centres of life, capable of vegetating with extraordinary rapidity in a medium adapted to their nutrition. This phenomenon can occur at any temperature between zero and 55° C. (131° F.), although a temperature between 15° C. and 30° C. (59° F. and 86° F.) is the most favourable to its occurrence.

As regards the rapidity of the budding, the following observations will give some idea of what it is in the case of one of the ferments of natural must. The temperature was between 12° C. and 13° C. (55° F.).

“On October 12, 1861, at ten o’clock in the morning, we crushed some grapes, without filtering the juice that ran from them; afterwards, at different times during the day, we examined the juice under the microscope, until at last, although not before seven o’clock in the evening, we detected a couple of cells, as represented in Fig. 25, _a_.

From that time we kept these contiguous cells constantly in view. At 7.10 we saw them separate and remove to some little distance from each other (Fig. 25, _b_). Between 7 and 7.30 we saw, on each of these cells, a very minute bud originate and grow little by little. These buds developed very near the point of contact, where the disjunction had just taken place. By 7.45 the buds had increased greatly in size (Fig. 25, _c_). By 8 they had attained the size of the mother-cells. By 9 each cell of each couple had put forth a new bud (Fig. 25, _d_). We did not follow the multiplication of the cells any farther, having seen that in the course of two hours two cellules had furnished eight, including the two mother-cells.”[82]

An increase like this, which would have been more rapid at a temperature between 15° and 25° (59° and 77° F.), and still more so between 25° and 30° (77° and 86° F.), may indeed seem surprising. It is really, however, nothing to what sometimes occurs. In choosing proper conditions of temperature and medium, of state and nature of yeast, it has sometimes happened that the bottom of a vessel has become covered with a white deposit of yeast cells, in the course of not more than five or six hours after we had sown a quantity of yeast so small as to effect no change at all in the transparency of the liquid contained in the vessel after it had been shaken up. Such a rapidity of vegetation reminded us of those exotic plants which are said to grow several feet in height in the course of twenty-four hours.

Budding commences in the form of a simple protuberance on the cell—a kind of little boss, as represented in Fig. 26, No. 1. This protuberance goes on increasing, and assumes a spherical or oval form. At the same time, there is a tendency in the points of attachment in the young cell to meet—a kind of strangulation occurs (Fig. 26, No. 2). The junction takes place a little sooner or later, according to the species (Fig. 26, No. 3); the two individuals then separate (Fig. 26, No. 4). In certain cases a single cell may give rise to several protuberances, and, consequently, to several daughter-cells. Where there is only one protuberance or bud we generally see it originate at the thick end and a little on one side of the apex of the oval outline, which, in a greater or less degree, characterizes the cells of the majority of ferments.

Certain authors have maintained that the method of budding which we have just described, and which we think was first promulgated by Mitscherlich, is merely an illusion, and that the cells of yeast break up and scatter their granular contents, and that these scattered granules eventually attach themselves to the cells, growing there, and so giving the appearance of buds or daughter-cells. This error has been revived quite recently.[83] Nothing can be less admissible. We could count the number of yeast cells which we have seen undergo this process of rupture in the course of some ten years of observation, every day of which, we may say, thousands of these cells have passed under our eyes. This breaking up of the cells is really of the most rare occurrence, and may always be explained by some abnormal circumstance affecting the yeast; being indeed a mechanical accident, not a physiological fact. We may easily convince ourselves of this by growing some yeast in a saccharine wort, filtered perfectly clear, and, consequently, deprived of all granular amorphous deposit that might deceive the observer. The cells will be observed to bud and multiply without exhibiting the most minute appearance of granulation, or disruption; moreover, there will always be cells of all sizes, ranging from the smallest visible up to the largest. This very simple piece of observation may be made in all the alcoholic ferments, and with any wort capable of fermenting, and in its presence the hypothesis, which we have been repudiating, cannot hold its own.

In Plate VII. (left side) there is represented a field of yeast, magnified 400 times. We see a mass of disjointed cells, such as appear after fermentations without sufficient aliment; of the kind represented some are nearly spherical, others oval or cylindrical, more or less elongated. If we mix a little of this yeast, of about the size of a pin’s head, with wort, and put the wort into a small, shallow, flat-bottomed basin, having a surface of about 1 square decimetre (10 sq. ins.) exposing it to the surrounding temperature, we shall find next day the bottom of the basin covered with a fine white deposit, of the forms of which we give a sketch in the right half of the plate. In this it will at once be observed that the cells sown have lost their interior granulations, having become more transparent and filled with a gelatinous protoplasm. The principal difference between the two halves of the plate consists in this, that whereas the cells in the left half are isolated and granular, those in the right half are more inflated, more transparent, and provided with buds, which may be seen in every stage of development, from their first appearance till they become as large as the parent cells. They continue to grow until they detach themselves; then they bud in their turn, so that the same figure may furnish examples of cells of the first, second, and third generations. In the right half, the protoplasm contained in the cells exhibits circular spots or vacuoles, which may be made to appear lighter or darker than the rest of the cell by slight movements of the object-glass of the microscope. These spots are due to a migration of the protoplasm towards the sides; they commonly occur in yeast cells the vitality of which, from deficient nourishment, has become suppressed—the shrivelled appearance which they then assume being due to their being forced to live upon themselves, so to speak. However, by introducing such cells into a nutritive and aerated liquid the vacuoles quickly disappear.

In the ordinary yeast, as met with in breweries, the majority of cells show one or more of these vacuoles; if, however, we place a little of this yeast in an aerated wort, and watch under the microscope the changes that occur in the cells, we shall witness, often in the course of a few seconds, a kind of turgescence, a greater tension of the cell-walls, which seem to grow thinner, and a complete disappearance of the vacuoles. At the same time the interior gelatinous matter will become filled with fine granulations that are scarcely visible, but which at a certain distance appear brilliant. At the same time protuberances begin to show themselves, and next day the budding will have already become very active. The newly-formed cells will have such a delicacy of aspect and contour as to be scarcely discernible in the field of the microscope. There will also be a tendency to ramification in the budding, which appearance will be more or less marked according to the kind of alcoholic ferments present, as we shall see presently, attaining its maximum in each case when the cells have been revived after exhaustion by rest and want of food. In the latter case, the process of rejuvenescence may be protracted; but this is not the case with cells of commercial yeast, which is always used within a few days of its formation. And thus, as I said a little ago, speaking of these cells, they often manifest the first signs of their budding in a few seconds.

In our preceding remarks we have expressly assumed that there are many kinds of alcoholic ferment. This is, beyond doubt, the case, as we have given incontestable proofs, first in 1862, in the _Bulletin de la Société chimique_ of Paris, and later on, in 1864 and 1866, in a Note in the _Comptes rendus_, on the diseases of wines, as well as in our “Studies on Wine.” Moreover, we know that brewers have long recognized two distinct methods of fermentation—“high” fermentation and “low” fermentation—and two corresponding yeasts. It is true that the differences presented by these fermentations were believed to be caused by the different conditions under which they took place, and that it was supposed that we might change “high” yeast into “low” yeast, or inversely, by subjecting the first to a low temperature, or the second to a high one. In our observations of 1862, which we have just mentioned, we discovered that must gives rise to several yeasts; that the ferment of “high” beer cannot develop except with great difficulty in must, whilst one of the ferments of the grape grows rapidly and luxuriantly in wort; that it is easy to isolate the smallest of the ferments of the grape from its congeners, by subjecting filtered must to fermentation; and finally, that the secondary fermentations of wines which remain sweet furnish a remarkable ferment, very different in aspect to the ferment of beer.

We have not given specific names to these different ferments, any more than we have to the other microscopic organisms which we have had occasion to study. This was not from any disregard for names, but from a constant fear that, since the physiological functions of these minute forms was the exclusive object of our study, we might be led to attach too much importance to exterior characters. We have often found that forms, having nothing apparently in common, belong to one and the same species, whilst similarity of form may associate species far apart. We shall give some fresh examples of this fact in the present paragraph. A German naturalist, Dr. Rees, who has discovered new proofs of the diversity of alcoholic ferments, putting aside, perhaps rightly, such scruples, has attached specific names to the different kinds of ferments, in his _brochure_ published in 1870, which we have already cited (p. 71). Indeed, we have often ourselves, for brevity’s sake, made use of the names proposed by Dr. Rees.[84]

In a Note inserted in the _Bulletin de la Société chimique de Paris_, in 1862, we figured a ferment of small dimensions, which develops spontaneously in must, filtered or unfiltered, and which is very different from the ordinary ferment of wine. It is the first to make its appearance in the fermentation of the grape, and may even appear alone if the must has been previously well filtered, doubtless because its germs, being smaller than those of other ferments, pass through the filter more easily and in greater number. Fig. 27, extracted from our Note of 1862, represents this ferment, together with some spherical cells of _high_ yeast, with the object of giving a more exact idea of the relative dimensions of these two ferments and their dissimilarities. Dr. Rees has named it _saccharomyces apiculatus_.

The same savant has given the name of _saccharomyces pastorianus_ to the yeast of the secondary fermentations of sweet liquids, such as wine that has remained sweet after its principal fermentation. We have described this yeast in a Note published in 1864, on the diseases of wine, from which we give the following extract:—[85]

“Fig. 6 (Fig. 28 in this work) represents a very interesting variety of alcoholic ferment. It happens pretty often, especially in the Jura, where the vintage takes place about October 15th, when the season is already cold and little favourable to fermentation, that the wine is still sweet at the moment when it is put into casks. This is especially the case in good years, when the sugar is abundant and the proportion of alcohol high, a circumstance which prevents the completion of fermentation when effected at a low temperature. The wine remains sweet in cask sometimes for several years, undergoing a continuous but feeble alcoholic fermentation. In such wines we have always observed the presence of this peculiar ferment. In form it consists of a principal stem, forming nodes at various points, from which short branches arise, ending in spherical or ovoid cells. These cells readily detach themselves, and act as spores of the plant. It is rarely, however, that we see so perfect a vegetation as we have represented, because the different parts fall to pieces, as we have shown in the left half of the figure.”

What is the origin of cellular plants of this remarkable type? Where and how are the ferments of the grape generated?

In Chapter III. § 3 we were on our way to a solution of this question. It has been shown that fermentation cannot take place in the juice of crushed grapes if the must has not come into contact and been mixed with particles of dust on the surface of the grapes, or of the woody part of the bunch. It would, however, be sufficient that a vintage vat, of any capacity whatsoever, should receive the particles of dust existing on a single bunch in some cases, on even a single grape, for the whole mass to enter into fermentation.

What, then, we must ask ourselves, is the nature of these particles of dust? On September 27th, 1872, we picked from a vine, in the neighbourhood of Arbois, a bunch of grapes, of the variety called _le noirin_. The bunch selected, without any injury to a single grape, was brought to our laboratory in a sheet of paper that had been previously scorched in the flame of a spirit lamp, and the grapes were cut off with a pair of fine scissors, which had also been passed through the flame. By means of a badger-hair brush, thoroughly purified in water, each grape to which a portion of its peduncle remained attached, was washed in a little pure water. The successive washing of a dozen grapes in 3 c.c. of water was sufficient to make the water turbid; we then examined it under the microscope. Each field contained many little organized bodies, accidentally associated, now and again, with some very scarce crystalline spicules. As a rule, the organisms consisted of simple, transparent, colourless cells; some, indeed, of larger size had a yellowish brown colour, and were detached or united in irregular masses; and, lastly, there were club-shaped or bottle-shaped vessels, full of spores ready to germinate. We repeated this experiment with bunches of other varieties of grape, and also submitted to examination water in which the outer surfaces of gooseberries, plums, and pears had been washed; the result was in each case the same, that is, we found a great number of the same colourless cells, and the same irregular masses of darker cells, which latter, however, we must not confound with the masses of dead cells sometimes found covering parts of the epidermis of certain fruits.

As we had purposely left each fruit attached to part of its peduncle, we wished to ascertain if these corpuscles proceeded from the grapes or from the wood of the peduncle. For this purpose we washed separately the surface of the grapes and the woody part of the bunch. The water in which the latter was washed was visibly more charged with the minute organisms than that in which the grapes was washed, although the latter was by no means free from them.

Plate VIII. represents these corpuscles as they exist on the surface of fruits, magnified 500 times. The groups, _b_, _b_, _b_, ..., _c_, _c_, ... are of a brown colour, more or less dark, or of a reddish yellow; the cells _a_, _a_, ... are transparent. Amongst them are some spores of ordinary fungoid growths, and several cells which are probably the issue of a germination that had commenced in certain groups which have a hard, yellowish appearance, and which are provided with what seems to be a double case—_b_, _b_, _b_, ..., _c_, _c_, ..., a result of the moisture of the woody part of the bunch, or of rain that fell just before the commencement of our observations.

It is an easy matter to trace the germination of these different varieties of cells with the microscope. We put a drop of the water in which the woody part of a bunch of grapes has been washed into a small quantity of wort, previously boiled and filtered bright. Plate IX. presents a series of developments observed in the case of simple or grouped cells, A, D, G, and J. The process is as follows: The yellowish-brown cells soften and grow larger in the nutritive medium, and gradually become almost transparent and colourless. At the same time we see some very young buds appear on their margins; these rapidly increase in size, and detaching themselves to make room for others, move off as young cells that after a time bud in their turn. The rapidity with which these cells bud and multiply is often extraordinary. The group A and the cell D produced the groups C and F within twenty-four hours, passing through the intermediate stages represented in groups B, E. The cells A and D did not give rise to any filamentous growths, at least whilst under our observation. Some groups of cells, however, put forth, from the first, long filaments, having cross-partitions and resembling the mycelium in ordinary fungoid growths. Together with these, and along their whole length, was an abundance of cells, often in clusters, as represented by Fig. G, the whole of which growth took place in less than twenty-four hours.[86] But apart from contact with the air, there was a complete absence of life.

The figures H, I, J, K, represent other aspects of developing cells and filaments. The cells H are spherical; the cells I have numerous buds, as also have those marked K. These different forms were all produced in the course of twenty-four hours by the cell which may be observed in the centre of the group J. In connection with this same group, J, we may remark that on September 30, 1872, at 10 A.M., we witnessed the detachment of three oval cells at the points _a_, _b_, _c_; by 10.45 other buds of the size represented in our engraving had formed in their place; by about five o’clock that same afternoon these buds, _a_, _b_, _c_, having become transformed into cells, fell off in their turn.[87]

It may be asked, what proof have we that amongst the filamentous and cellular growths which spring from the small, dark bodies existing in the particles of dust adhering to the surface of fruits, and which we here see bud and multiply with such marvellous rapidity, the ferment or ferments of vintage do actually exist? A very simple experiment will prove conclusively that this is the case. When in the course of twenty-four or forty-eight hours, by contact with saccharine must, and in presence of excess of air, the revival and development of the cells has taken place on the bottom of the little troughs employed in our observations; if then we fill up the trough with the same must, so that there remains no free air under the cover-glass, within a very short time—an hour, half-an-hour, or often less—we shall see bubbles of gas rise from the bottom, accompanied by an increase in the deposit of cells. This will be the must fermenting after the submersion of the cellular plants. It follows that the cells, or groups of cells, of a dark colour which cover the grapes, or the woody part of the clusters, are actual germs of the cells of yeast; more correctly speaking, that germs of yeast-cells exist amongst these groups, for it would not be consistent with truth to say that the various germinating forms present in the dust on the surface of grapes must all of them give rise to actual corresponding ferments. Thus the flask-shaped spores _c_, _c_, ... in Plate VIII., are reproductive organs of _alternaria tenuis_, which have probably nothing in common with alcoholic ferment or ferments, properly so called, except their outward form. We may repeat, however, and it is a point of great importance to bear in mind, that the cells of yeast originate from some or other of the little, brownish, organized bodies, which the microscope reveals in such numbers amongst the particles of dust existing on the surface of fruits.

The impossibility, which we have already demonstrated (Chapter III. § 3), of making grape juice ferment apart from the action of external particles of dust, and the knowledge which we have just acquired, that the particles of dust on the surface of the grapes and woody peduncles, at the moment when the grapes have attained maturity, contain certain reproductive cells which give rise to certain ferments, naturally lead us to the investigation of another point, which concerns the period at which these germs make their appearance on the different parts of the vine plant. The two following experiments tend to prove that the ferment can only appear about the time when the grapes attain maturity, and that it disappears during the winter, not to reappear before the end of the following summer.

I. In the month of October, 1873, we procured from a vineyard in the canton of Arbois some of the woody parts of very ripe clusters of grapes, taking the precaution to cut off all the grapes, one by one, with a very clean pair of scissors, whilst still on the vine; we then wrapped up the woody parts of the clusters, thus deprived of their grapes, in thin paper, to convey them to Paris. Our only object at that time was to secure for use in our subsequent studies the ferment-bearing dust found in October on the woody part of the vine, and, more particularly, on the clusters themselves, as already stated. After our return to Paris, and during the course of our experiments in October and November, it sufficed to wash a few scraps of the bunches in a little pure water, in order to obtain the grape-ferment in abundance; but later on in the winter we were astonished to find that the same procedure yielded no ferment, only some moulds. The bunches which, when put into boiled and filtered must, in October, very readily caused that must to ferment, at the end of winter could no longer produce the same effect, however favourable might be the temperature to which we raised the must. The particles of dust on the bunches had, therefore, become sterile, as sources of alcoholic ferments.

II. On February 17th, 1875, we purchased of Chevet, a dealer in provisions, two bunches of white grapes, which were perfectly sound, presenting not the slightest trace of injury or bruise. We took an iron pot full of mercury, which had been heated to 200° C. (392° F.), and then covered over its surface with a sheet of paper that had also been subjected to flame. When the mercury had cooled down we placed several of Chevet’s grapes, singly and in small bunches, on the surface of the metal, and, after having enclosed them in a glass cylinder that had been previously heated with and by means of the mercury, we crushed them in this vessel, in contact with air, by means of a strong, crooked iron wire that had been passed through the flame of a spirit lamp. The object of all these precautions was to prevent any cause of error, such as might have resulted from the accession of particles of dust associated with the mercury, or floating about our laboratory. We then placed our cylindrical jar in an oven, at a temperature of 25° C. (77° F.); but though the experiment was continued for several days following, no fermentation manifested itself. At last, to assure ourselves that the pulp and liquid were, notwithstanding this, well adapted to fermentation, we introduced into the test-flask an almost imperceptible quantity of yeast. This readily developed, and promptly produced fermentation.[91]

It seems possible, therefore, that the germs of ferment may not exist on bunches of sound grapes during winter, and that the well-known experiment of Gay-Lussac on the influence of air on the fermentation of the must of crushed grapes cannot succeed at all times.

The following observations will afford more than sufficient proof of this statement, being, after all, but an easy method of carrying out Gay-Lussac’s experiment, without having recourse to the use of mercury.

It may already be inferred from the preceding facts that there must be, in the course of the year, between the end of winter and autumn, a period when the vegetation of the cellules from which yeast proceeds undergoes a revival. When does this period occur? In other words, how long after winter does sterility of the plant continue, until it is again capable of yielding ferment? To ascertain this, we conducted numerous experiments during the summer and autumn of 1875 and the winter of 1876. Having to conduct them in a vine-growing country—in the vineyards of Arbois, Franche-Comté—at a distance from our laboratory, we were compelled to adopt a simple form of apparatus for our experiments, which, besides being very convenient, was at the same time sufficiently exact for the object we had in view.

Into common test-tubes we poured some preserved must; we then boiled it, with the object of destroying all the germs that it might contain, and then, having passed the flame of a spirit lamp over the upper sides of the tubes, we closed them with corks which had been held in the flame until they began to carbonize (Fig. 32). Having provided ourselves with a series of tubes prepared in this manner, we carried them to a vine, and there dropped into some of them grapes, into others bunches, from which we had taken all the grapes, by cutting their peduncles; into others, fragments of leaves or the wood of the branches. The corks were again passed through the flame and replaced successively in each tube. Some of the grapes we dropped in whole, some we crushed at the bottom of the tubes with an iron rod that had previously been passed through the flame; others, again, at the same moment that we introduced them into the tubes, were cut open with scissors, likewise passed previously through the flame, so that a portion of their interior juice might mix with the must in the tube.

Our experiments gave the following results:—As long as the grapes were green, about the end of July and during the first fortnight of August, we obtained no fermentation in our must. Between the 20th and 25th of August a few tubes underwent fermentation, by the action of the little apiculated ferment; and in the course of September the number of tubes that fermented increased progressively. In each series of tubes, however, we always found a few in which there was a complete absence of fermentation.

Here are a few actual examples. In the beginning of September we placed grapes in thirteen tubes, into some whole, into others crushed ones, taken from bunches of the variety known as the _ploussard_, the fruit being already sufficiently ripe to be very pleasant to the taste. All the tubes of this series failing to give us any trace of fermentation, or anything besides ordinary moulds—which indeed appeared in all our experiments, whether there was or was not fermentation—we began a new series of experiments, under similar conditions, on September 28th, as follows:—

Nos. 1, 2, 3 and 4 tubes containing one uncrushed grape.

No. 5 tube containing two uncrushed grapes.

No. 6 tube containing two crushed grapes.

No. 7 tube containing two crushed grapes, in 2 c.c. of water previously boiled.

No. 8 tube with a fragment of a bunch from which grapes had been cut, and occupying the entire depth of liquid.

No. 9 tube with a fragment of wood from a branch.

Nos. 10, 11, and 12 tubes with a fragment of leaf.

On September 29th and 30th there was no appearance of fermentation in any of the flasks, but all contained flakes of fungoid mycelium. On the 1st of October fermentation more or less marked and active occurred in 2, 3, 4, and 5, in which uncrushed grapes were, accompanied by a general turbidity of the liquid, and a suspension of the development of the fungoid growths. It was still absent in 1, 6, and 7, of which the first contained an entire, the latter crushed grapes. No. 8, containing the woody part of the bunch, was in active fermentation. Nos. 9, 10, 11, and 12, with fragments of branch or leaves, showed no signs of fermentation. The following day No. 1 was fermenting; but from October 5th onwards there was no alteration in the number of fermenting tubes.

In this series we determined the presence of the small apiculated form of yeast (_S. apiculatus_) in the tubes that fermented, only once finding it associated with _saccharomyces pastorianus_.

We need hardly say that the grapes which we employed were perfectly ripe, the vintage having already commenced in some of the Jura cantons.

This experiment shows that, even when the grapes are perfectly matured, it by no means follows that each individual grape must carry germs of ferment, and that some grapes may be crushed, in some instances several together may be crushed, without being able to set up a fermentation. In the presence of these novel facts, those who support the hypothesis of the transformation of the albuminous matter contained in the juice of grapes into yeast will no doubt admit the untenability of their opinions, since their hypothesis requires that every grape or number of grapes, when crushed, should ferment, in contact with air.

On the same day we prepared another series of tubes, using grapes of a variety called the _trousseau_.

Nos. 1, 2, 3, and 4 tubes containing one whole grape.

Nos. 5 and 6 tubes containing some of the wood of a branch.

No. 7 tube containing some of the wood of a branch from which the grapes had been detached.

In the course of the following days fermentation took place in 4, 5, and 7.

In this case three out of four of the uncrushed grapes did not cause the must in which they were placed to ferment; whilst the same must fermented in one of the two tubes containing wood of the branch, and in the other remained unchanged; and, lastly, the tube containing the woody peduncles of the bunch fermented.

We have already remarked that it was more particularly the wood of the bunch that was charged with germs of ferment. The truth of this assertion was proved by the following series of experiments.

On October 2nd, 1875, we charged at the vineyard twenty-four tubes, all of which were about a third filled with pure must that had been previously boiled.

Nos. 1, 2, 3, 4, 5, and 6 tubes containing one crushed grape.

No. 7 tube containing two crushed grapes.

No. 8 tube containing one crushed grape.

Nos. 9, 10, 11, and 12 tubes containing some wood of a branch of the vine.

Nos. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 tubes containing a fragment of the wood of a bunch from which the grapes had all been detached and removed.

In the course of the following days some of these tubes began to ferment; but in others only fungoid mycelia were visible.

On October 7th the following tubes were fermenting: the second of the first eight containing whole or crushed grapes; not one of the four that contained wood of the branches; whilst, on the other hand, of the tubes containing wood of the bunches, 15, 17, 20, 21, 22, 23, and 24 were all in full fermentation. In short, fermentation, and therefore germs of yeast, were present in one single tube out of eight containing grapes; in none of the four tubes containing wood of the vine branch; but in seven out of the dozen containing wood of the bunches. There was no subsequent change in the number of tubes that fermented.

The same day that we arranged this series of tubes we prepared twenty-four other similar ones, using the _wood of bunches preserved from the vintage of the preceding year_. Not one of these twenty-four tubes showed the least sign of fermentation, although they contained grape juice in presence of the wood of the bunches; this was a further confirmation of the sterility of the germs of ferment in the case of bunches of grapes preserved for a sufficient time.

The next question to be considered was, what length of time after the vintage do the germs on the surface of the woody part of bunches of grapes preserve the faculty of producing yeast? The following experiments were undertaken to determine this point.

We have just seen that on October 2 fragments of the woody peduncles introduced with must, on the spot, caused that must to ferment in seven cases out of twelve. In order that we might test the wood of these same bunches during winter we took care to wrap some fragments up in paper previously passed through the flame. We afterwards took occasion to test portions of these fragments as follows:—

On December 21st, 1875, we conducted an experiment with twelve. On the following days all began to show flakes of mycelium, or numerous multiplying cells of _mycoderms_, _torulæ_, and _dematium_; and only four subsequently produced yeast and alcoholic fermentation. From this we may conclude that, three months after the vintage, a large number of the germs of yeast spread over the woody part of the bunches lose their vitality, through desiccation by the surrounding air, since two-thirds of the examples taken had become sterile by that time.

On January 21st we conducted a similar experiment with twelve other tubes. At a temperature between 20° and 25° C. (68° to 77° F.) fermentation occurred in only two of them. On March 2nd we undertook another experiment, again using twelve tubes, and again fermentation occurred in two tubes.

By the beginning of April the sterility was absolute. At that period of the year (April and May) we made numerous experiments of this kind, using the woody parts of fresh bunches of grapes and white grapes preserved from the last vintage, plenty of which were still to be had in a state of freshness at the provision store. We also operated on some wood obtained from a vineyard at Meudon. In a great number of cases no fermentation occurred; it even happened that a whole bunch of fresh black grapes, very ripe, which were bought at Chevet’s on April 16th, and which had been grown in a hot-house, after having been crushed, did not ferment at all.

Up to March not one of the tubes containing the wood of dry bunches brought from the Jura, which had fermented, showed any signs of _apiculatus_ or of _pastorianus_, or anything besides the ordinary low yeast of wine, _saccharomyces ellipsoideus_.[93]

It would be a study of much interest to determine if yeast exists on other species of plants besides the vine. During the winter we could discover it on no others. Once during the winter, experimenting on box, we obtained fermentation in one of our tubes which contained must. In a great number of other experiments we obtained nothing besides moulds and growths of _dematium_, _alternaria_, and _torulaceæ_.

Our observations in Chap. III § 6, taken in connection with those which we have just made, prove that the yeasts of fermentation, after being dried, preserve the faculty of germination longer than the germ-cells which are scattered over the dead wood of the vine.

As might be expected, a microscopical examination of the particles of dust scattered over the surface of the fruit and woody peduncles of grapes, reveals great differences in the number of these fertile particles at different periods of the vegetation of the grapes. As long as the grapes are green and the vine in full activity we find scarcely any, or, at all events, very few spores which seem to belong to ordinary fungoid growths. Towards autumn, however, when the grape is ripening and the leaves becoming yellow, fungoid growths and numerous productions of great fertility accumulate on the vine, on the leaves, the branches, and the bunches. At this period we find the water in which the grapes and the woody parts of the bunches are washed swarming with different kinds of organized corpuscles; it is at this period, too, that the ferment-yielding moulds attain that phase of their vegetation in which, when mixed with the juice of grapes, they produce fermentation.

In the Jura district a peculiar kind of wine, called straw wine (_vin de paille_), is manufactured, which seems to contradict what we have said as to the advent of sterility towards the end of winter in the yeast-germs formed on the surface of preserved bunches of grapes. This straw wine is made of grapes preserved for long after the vintage on straw. From what we have said it might be supposed that fermentation could not occur under these circumstances. We have, in fact, no doubt that it is often really produced by quite different yeast-germs from those which cause fermentation in the vintage gathered in autumn. Fermentation as effected in the manufacture of straw wine is probably due to yeast-dust spread over the utensils of the vine-grower, and derived from the preceding vintage. We have seen (Chap. III. § 6) that yeast may be dried and reduced to powder, and yet preserve its faculty of germination for several months. It would be useful, however, to submit this surmise to the test of experiment, and it would be easy to do so provided we took care to crush the grapes so preserved in very clean vessels, previously heated to a temperature of 100° C. (212° F.), having first rejected every bunch containing injured grapes, which might have fermented or given occasion to the development of yeast. Fermentation, we believe, would not then take place.

Another consequence results from the various facts that we have brought out in relation to the origin of the wine-ferments, which is, that it would be easy to cultivate one or more vine-stocks so that the grapes gathered from them, and crushed to extract their juice, would be unable to ferment spontaneously _even in autumn_. For this purpose it would be sufficient to keep the bunches out of contact with particles of dust during the vegetation of the bunches and the ripening of the grapes, and then to effect the crushing in vessels thoroughly freed from germs of alcoholic ferments. Moreover, every fruit and every vegetable might be submitted to important investigations of this kind, the results of which, in our opinion, could hardly be doubtful.

The following observations, which relate to the polymorphism of _saccharomyces pastorianus_, seem to me to have an important bearing on the history of alcoholic ferments, as presenting a close analogy between the species of ferment and fungoid growths of a higher order, for example, such fungi as _dematium_, which are generally found on dead wood; and we would say that between the vine and other shrubs there is only this difference, that amongst the _dematium_ forms of the vine there occur one or more which are anaërobian, at a certain period of the year, whilst, on the other hand, the _dematia_, _alternaria_, &c., of other shrubs are more generally aërobian. There would be nothing surprising in this result, considering that amongst the mucors, for instance, we find both aërobian and anaërobian forms, and that there are likewise torulæ-ferments or anaërobian forms, as well as torulæ-forms exclusively aërobian.

When _saccharomyces pastorianus_ begins to develop from its natural germs, such as are scattered over the surface of acid fruits, it takes the form of elongated jointed filaments, branching, often pear-shaped, and more or less voluminous. In proportion as the oxygen held in solution in the liquid disappears and the buddings are repeated, the length and diameter of the filaments and cells diminish, and such is the transformation that we might, at last, suppose that we were dealing with a different ferment of smaller dimensions.

Plate X. represents this ferment, at the commencement of fermentation in cherry juice. In the course of a short time there is nothing to be seen but cells of comparatively small size, disjointed and round or oval, and filaments comparatively short and slender. This appearance is indicated in our drawing by the cells _a_, _a_, _a_. As these latter forms multiply with great rapidity, we soon have to search widely over the microscopic field before we find any of the long forms from which they spring. Instead of the forms given in Plate X., we have only those represented in Plate XI. In other words, the aspect of these ferments changes daily, from the very commencement of fermentation. Thus the yeast would appear to grow smaller, coincidently with the progress of fermentation passing from a condition in which it consists of large cells and long ramified filaments, to a condition in which the cells are small and the filaments short. These changes are principally due to an alteration in the method of budding and in the life-processes of the yeast, which speedily exhibits itself when the air supply is reduced, and not through any intermixture of foreign ferments. So, at least, all our observations up to the present time lead us to believe. As soon as the oxygen has been absorbed the cells which form are oval or globular, and the filaments do not lengthen or become so plump.

This is, however, not the only cause of these changes in form and aspect, although the presence of air, in greater or less quantity, has a marked influence on the earlier developments of yeast; there is another circumstance to be taken into account, difficult indeed to state shortly, but which is demonstrated clearly by the microscope, and is connected with the actual state of the germ cells. As a general rule the budding of a cell is not an identical process when the cell is quite young, and when it has become exhausted from want of nourishment. Between these two conditions there is a difference which may be compared with that which exists, for example, between a newly-formed grain which would not germinate, and the same grain matured by rest, if we may use the expression, that is, which has been kept long enough for its germination to be possible. In other words, and as far as our subject is concerned, we are not to expect that, by reviving our old yeast cells and putting them to grow with abundance of air, in a saccharine, nutritive medium, we shall obtain the appearance of the earlier developments of the germ-cells on the surface of sweet and acid fruits. We see this clearly in Plate VII., the right-hand half of which represents the recruited budding of cells, such as those represented in the left-hand half, in a medium peculiarly adapted to their vitality, _and in the presence of much air_. As regards the length and size of filaments and cells, there is little appreciable difference between the two sides. The principal difference consists in the relative freshness and the budding going on in individuals in the right-hand half.

There is a simple means of transforming the small, disjointed forms of the yeast as it occurs in a deposit, at the end of a fermentation, back into the long, tubular, pear-shaped forms peculiar to the germination of the germ-cells, which exist amongst the particles of dust spread over the surface of fruits. Plate X. illustrates the result of the process. For this purpose we must effect as complete an exhaustion as possible of the ferment _saccharomyces pastorianus_, by leaving it to itself for a very long time, without aliment, in contact with pure air, in a damp state; or, better still, in presence of sweetened water. We cultivate some yeast in wort, in one of our two-necked flasks, and then carefully decanting the fermented liquid through the right-hand neck, leave the deposit of yeast on the sides of the flask. The glass stopper which closes the india-rubber tube must be replaced, and the moist yeast be left thus, in contact with pure air. The cells will steadily continue their activity, and so gradually age, without meanwhile losing their vitality. We use the word _age_, as we have already observed, because the period of rejuvenescence in the case of such a yeast is so much the slower the longer the plant has remained in that state.

Under these conditions the yeast rarely dies. It becomes attenuated and shrivelled but still preserves its vitality, that is, the power of reproducing itself after a lapse of several months or even several years. In the end, however, it dies, a fact which is proved by the cells, when sown in a nutritive medium, remaining inert.

To exhaust yeast, without destroying it, sweetened water is preferable. Having decanted the beer, we substitute in its place water sweetened with 10 per cent. of pure sugar. By effecting the substitution in the following manner, we escape the risk of introducing germs from floating particles of dust, which would nullify all experiments of this kind. We prepare, then, a flask containing sweetened water, free from all foreign germs, which we attach to the other flask [_i.e._, two of M. Pasteur’s flasks with two necks, one straight and wide, the other bent and narrow (Fig. 8)]. This is done by taking the india-rubber tube off the flask containing yeast, and removing the glass stopper from the other india-rubber tube attached to the flask containing the sweetened water; then, introducing the right-hand neck of the yeast flask into the india-rubber tube connected with the other, we raise the latter flask so as to pour the sweetened water on to the yeast. At the same time an assistant passes the flame of a spirit-lamp over the bent part of the curved tube attached to the water flask, with the object of destroying the vitality of the germs in the floating particles of dust, which enter the flask in proportion as it is emptied into the other.

The sweetened water, which is thus brought into contact with yeast of greater or less freshness, soon begins to ferment. Fermentation accomplished, the vinous liquid is decanted and replaced by fresh sweetened water, which ferments in its turn, although even at this stage with greater difficulty than the first; this second dose is again decanted, and again replaced by fresh sweetened water, and this process is repeated three or four times. The yeast becomes weaker and weaker, and eventually is unable to cause any fermentation in sweetened water poured on it.

This exhaustion of yeast in sweetened water may be produced more quickly by the following means:—It is sufficient to sow a mere trace of pure yeast in a large quantity of sweetened water, say 100 c.c. (nearly four fluid ounces), that is, instead of pouring the contents of a bottle of sweetened water upon the whole deposit of yeast in the flask which contained the fermented wort, we simply take a little yeast, by means of a fine tube, from the deposit at the bottom of the flask, and introduce it into the flask of sweetened water. This large proportion of liquid is itself sufficient to exhaust the small quantity of yeast, quickly checking the feeble fermentation which it had induced, so feeble indeed as frequently not to be detected by the eye, from the fact that the amount of liquid present is more than sufficient to dissolve any bubbles of carbonic acid gas that might otherwise have been liberated.

It is a remarkable fact that the yeast, which during its protracted stay in the sweetened water becomes enfeebled to such a degree that it can no longer excite the least fermentation in that water, but will remain in its presence for an indefinite time in a state of inert dust, does not die. In some of my experiments the yeast has remained alive in the sweetened water for more than two years.[94] It is almost unnecessary to point out that these results are altogether out of keeping with the various properties that are usually attributed to yeast.[95]

In these experiments we may use yeast-water[96] instead of water sweetened with sugar. Into some flasks of pure yeast-water we put a little yeast, taking all precautions to prevent the introduction of foreign germs. No fermentation results, there being no sugar present; the yeast, however, begins to bud, and this budding is more or less marked according to the quantity of carbohydrate food which we introduce along with the specimen. An interior chemical action also goes on, causing a gradual change in the aspect of the yeast. The plasma of the cells collects about the centres, assuming a yellowish-brown colour, becoming granular, and forming within the cells masses more or less irregular in shape, very rarely spherical.

We may observe here that these conditions seem to be peculiarly adapted to show the character of the interior sporulation of the cells discovered by Dr. Rees. Notwithstanding this we have never succeeded in finding it distinctly, under these circumstances.