  § 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 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 “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. Fig. 25. Fig. 25. 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.” 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 Fig. 26. Fig. 26. 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. 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, Plate 7. Yeast-cells—Worn out and Dissociated (left), after Revival in a Sweet Wort (right). Plate 7. Yeast-cells—Worn out and Dissociated (left), after Revival in a Sweet Wort (right). 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. 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 Fig. 27. Fig. 27. 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. Fig. 28. Fig. 28. “Fig. 6 (Fig. 28 in this work) represents a very interesting 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. Plate 8. Fertile Mould-cells from the Outer Surface of Grapes. Plate 8. Fertile Mould-cells from the Outer Surface of Grapes. 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 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 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. Plate 9. Various Examples of the Mode of Growth of Mould-cells from the Outer Surface of Grapes. Plate 9. Various Examples of the Mode of Growth of Mould-cells from the Outer Surface of Grapes. 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 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 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 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 Fig. 32. Fig. 32. 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 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. 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. 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 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. 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 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. Plate 10. One of the Ferments of Acid Fruits at the Commencement of Fermentation in its Natural Medium. 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 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 Plate 11. Saccharomyces Pastorianus, in course of Regular Growth. Plate 11. accharomyces Pastorianus, in course of Regular Growth. 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 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 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 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. In these experiments we may use yeast-water 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. The fact which should claim all our attention, we repeat, is, that this exhausted, shrivelled-up, aged-looking yeast preserves its faculty of germination for several years; that, moreover, this faculty may be aroused by placing it in aerated nutritive media, in which case it will exhibit all the peculiarities which, under similar conditions, characterize some of the germ-cells found on the surface of our sweet domestic fruits. In other words, this yeast, instead of multiplying, as it always does in the course of several growths in saccharine musts, in the form of cells which detach themselves readily as soon as they have nearly attained the form and size of the mother-cells, begins to shoot out into such beautiful forms as those of dematium pullulans, producing like that ferment long, well-grown, branching filaments, as well as plump and frequently pyriform cells, as represented in Plate X. The following figures (33 to 37) and descriptions of the observations to which they relate will furnish fresh proofs of our assertions. In these figures we see saccharomyces pastorianus, which has been exhausted in sweetened water or in yeast-water, undergo revival in saccharine musts, give rise to elongated, branching, pear-shaped forms, such as belong to the original ferments of fruits, and afterwards assume the most minute forms that we find in fermentations progressing or completed. Let us examine Fig. 33. The history of this growth is as follows:— Fig. 33. Fig. 33. The next figure (34) represents the earliest forms of germination of another specimen of saccharomyces pastorianus in Fig. 34. Fig. 34. Fig. 35. Fig. 35. Fig. 35 represents saccharomyces pastorianus again, as it appears after having been exhausted by two years preservation Fig. 36 represents the germination of this ferment, which had previously been revived in a flask of wort, at the temperature of the air, in May, 1875. The following are the details of our observation:— We sowed a trace of the exhausted yeast (Fig. 35) in a flask of wort on May 16th. The sketch (Fig. 36) was made on May 19th, but on the 18th there was a sensible revival. It will be seen how much the little ferment had developed in the course of three days from the time when the process commenced. If we had waited a few days longer before taking our sample, we should probably have had difficulty in finding any cells or filaments of the large ferment form, as there would have been so few of them in comparison with the others. Fig. 36. Fig. 36. In the above figure we should remark the chain of large cells and long-jointed processes, a, b, c, d : d is one of the cells that we sowed; it has become transparent, and its contents, which are slightly granular, have lost their brownish tint; c is a large cell which sprang from the preceding one; its outline Fig. 37. 1 Div. = 1/450th of millimetre (1/11250th of in.) Fig. 37. 1 Div. = 1/450th of millimetre (1/11250th of in.). The following is one of the most curious of the forms presented by saccharomyces pastorianus, occurring after exhaustion in a sweet mineral liquid. The ferment, taken from a closed vat, in which it had been used for beer, was sown in the mineral liquid on July 4th, 1873. The following days the ferment developed feebly, but perceptibly, and gradually increased in bulk. The flask was left to itself in an oven at 25° C. (77° F.) until December 3rd, when we ascertained that all the sugar had fermented. We then sowed a trace of the deposit, which had become abundant, in a flask of pure wort. On December 4th there was no perceptible change. On December 5th, however, fermentation was in active progress; a large quantity of froth covered the surface of the liquid, and a considerable deposit of ferment had already taken We may briefly summarize the leading facts demonstrated in the above paragraph. We have seen that there are different alcoholic ferments. In the fermentation of natural saccharine juices, which, especially when acid, so readily undergo a decided alcoholic fermentation, the ferments originate in certain germ-cells, which are spread in the form of minute spherical bodies of a yellow or brown colour, isolated or in groups, over the exterior surface of the epidermis of the plant, and which are gifted with an extraordinary power of budding with ease and rapidity in fermentable liquids. The presence of atmospheric oxygen is indispensable to the germination of these germ-cells, a fact which explains Gay-Lussac’s observation that atmospheric oxygen is necessary for the commencement of spontaneous fermentation in must. There seems every reason to believe that at this period of the year one or more of the varieties of dematium furnish cells of yeast, or even that the ordinary aËrobian varieties of In this manner we arrive at the confirmation of an idea entertained by most authors who have studied yeast closely—namely, that it must be an organ detached from some more complex vegetable form. We may also add that in the case of saccharomyces the chains of filaments, both tubular and fusiform, and septate cells more or less pyriform originating in them, when attentively observed, remind us forcibly of the filamentous chains and spore-balls, or conidia of mucor racemosus when submerged, so that one might suppose that the spore-ferment of our dematium is itself an organ detached from some still more complex vegetable form, in the same way that conidia-ferment of mucor racemosus belongs to that more complex fungoid growth. In the following passage De Bary uses, for the first time, the words dematium pullulans (Hofmeister, vol. ii. p. 182, 1866). The German naturalist begins by citing the opinions of Bail, Berkeley, and H. Hoffmann, the first of whom maintains that mucor mucedo becomes transformed into the yeast of beer, the second that yeast is a peculiar state of penicillium, and the third that it may be generated by fungi of very different nature, and especially by penicillium glaucum and mucor mucedo. He goes on to say: “I have taken great pains to repeat the experiments of Bail, Berkeley, and H. Hoffmann, but I have never been able to confirm the results which they have stated, either in the case of growths in microscopic cells or in experiments performed in test-tubes with the purest possible substances—specially prepared solutions or must of wine and spores of penicillium, mucor mucedo, botrytis cinerea, &c.” On this point M. De Bary arrives at exactly the same results which we communicated to the SociÉtÉ Philomathique and the SociÉtÉ Chimique of Paris, as already given in Chap. IV. § 4, p. 128, note. M. De Bary goes on to say: “In researches of this kind We shall conclude this paragraph with a remark that has doubtless presented itself to the minds of our readers, which is, that it would be impossible to carry out the experiments we have described if we could not make sure of dealing with pure ferments, or, at least, with mixtures the components of which are sufficiently well known for us to assign to each the effect produced by it in the total phenomena observed. It would be extremely difficult to continue growths of yeast-deposit in sweetened water or in a moist atmosphere if the little plant were mixed with spores of other fungoid growths, a variety of ferment-forms, and germs of bacteria, vibrios, or § II.—On “Spontaneous” Ferment.The expression spontaneous ferment may be applied to any ferment that appears in a fermentable liquid without having been purposely sown in it. In this respect the ferments mentioned in the preceding paragraph, those of all saccharine juices of fruit which ferment when left to themselves—the ferments of wine, for example—are spontaneous. The term, however, is not altogether appropriate, because, after all, the process is the same as if an actual sowing had been made, since, In the course of the researches which we undertook in order to ascertain whether mycoderma vini, or vinous efflorescence, became transformed, in the case of beer, into actual alcoholic ferment—researches which were the more protracted and varied in consequence of their leading to the condemnation as erroneous, on the faith of new and more precise experiments, such as those given in Chap. IV. § 2, of that transformation, in which we had for long believed—we had occasion to observe several spontaneous fermentations of this kind in various saccharine liquids. We then proceeded to describe our method of conducting the experiments. Having brought about the development of a film of mycoderma vini or cerevisiÆ on the surface of a liquid, fermented or not, we submerged that film in wort, which we afterwards put into long-necked flasks, in which alcoholic fermentation generally took place in the course of a few days. This fermentation in no way resulted from the transformation of the cells constituting the efflorescence into ferment. The mycodermic film merely acted as a receptacle of true ferment germs, wafted thither with the particles of dust floating in the air of the laboratory, which germs developed in the liquid into actual alcoholic ferments amongst the cells of the submerged mycoderma. By conducting experiments in this manner we Plate 12. Ferment-cells from a Spontaneous Fermentation just starting. Plate 12. Ferment-cells from a Spontaneous Fermentation just starting. Here let me describe one of these experiments. In the beginning of March, 1872, we grew some mycoderma vini, obtained from wine, on some wort contained in a shallow basin. On March 6th we submerged the efflorescence and put it all together, liquid and film, into a long-necked flask. On March 9th we detected incipient fermentation, and on March 12th we took a sketch of the yeast of the deposit, as given in Plate XII. This is the large and long branching, more or less pear-shaped form, which occurs at the beginning of fermentation in the sweet and acid musts of our domestic fruits. On March 16th we made another sketch of the deposit, in which the proportion of cells, in the form of elongated segments and filaments, reminded us, in some measure, of the filamentous mycelium of typical fungoid growths much diminished. In this case, however, the majority of cells were oval, round, and in short segments. On this day, March 16th, we added some fresh wort to that which had fermented, with the object of prolonging the duration of Fig. 38. Fig. 38. Spontaneous ferment, therefore, very often occurs in this large ferment-form, which, by repeated developments in the act of fermentation, becomes reduced by degrees after successive generations to the ferment which, following Dr. Rees, we have named saccharomyces pastorianus, a polymorphous ferment which must be studied closely that it may not be confounded with others, inasmuch as it is so universally diffused that we very seldom fail to find it in any ferment which has been exposed in contact with ordinary air, at least, we may repeat, in a laboratory devoted to researches on fermentation. We have found the same thing occur in a brewery, being there mixed with the ferments used in brewing. There are, no doubt, several varieties of this saccharomyces. We sometimes find amongst the spontaneous ferments which repeated growths have brought to a more or less uniform state, the forms represented in Plate XI., but the cells and segments much smaller. Amongst others, Dr. Rees has distinguished a saccharomyces exiguus. Fig. 38 represents another spontaneous ferment, which appeared in a boiled saccharine wort, which entered into fermentation after being exposed to the air of the laboratory. § III.—On “High” and “Low” Ferments.The ferments mentioned in the preceding paragraphs do not belong, properly speaking, to industrial products; that is to say, in actual practice there are no operations in which the ferments of fruits and spontaneous ferments are employed for the purpose. It is quite true that these ferments are the cause of the fermentations from which wine, cider, gin, rum, gentiana, mead, &c., are derived, but these fermentations are spontaneous, they take place without the intervention of man, and without man’s directing their production, or taking any notice of the agent which starts them. In the manufacture of beer, on the other hand, the practice is quite different. We may say that the wort is never left to ferment spontaneously, the fermentation being invariably produced by the addition of yeast formed on the spot in a preceding operation, or procured from some other working brewery, which, again, had at some time been supplied from a third brewery, which itself had derived it from another, and so on, as far back as the oldest brewery that can be imagined. A brewer never prepares his own yeast. We have already had occasion to remark that the interchange of yeasts amongst breweries is a time-honoured custom, which has been observed in all countries at all periods, as far back as we can trace the history of brewing. The yeasts which in the present day produce beer in the brewery of Tourtel, near Nancy, in that of GrÜber, at Strasburg, that of Dreher, at Vienna, and others, came originally These reflections may seem to favour the supposition that there is a real difference between “high” yeast and “low” yeast, and that both of these differ from spontaneous ferments and the ferments of domestic fruits. These are propositions demanding most careful consideration, for it is generally admitted Fig. 39. Fig. 39. “High” Ferment.—Fig. 39 represents some “high” yeast taken from a deposit after fermentation, and Fig. 40 the same yeast in course of propagation in some aerated wort. In comparing “high” yeast with other alcoholic ferments at the same stage of development, there are three points which are especially striking: the diameter of its cells is relatively large, their general aspect is rounder, and when they are undergoing propagation their mode of budding produces a markedly ramified appearance, so that the cells always occur in clusters and branches. Fig. 40 gives a very exact idea of these characters. To investigate satisfactorily the branching habit of growth peculiar to this ferment we should examine it during the first few hours of its propagation, when, under the influence of the oxygen dissolved in the fermentable liquid, its vital activity is greatest. Later on, often on the day following the sowing, the groups become disconnected, and at the end of the fermentation the cells have quite separated from each other, not more than 2 or 3 per cent. remaining united, and even these in groups of not more than two cells together. This is represented in Fig. 39. To give an idea of the rapidity with which this ferment multiplies, we may state that our sketch (Fig. 40) was made under the following conditions:—On April 28th, 1874, we caused a flask of wort to ferment by means of a trace of “high” yeast. On the morning of the next day, that is Fig. 40. Let us next suffer our yeast to exhaust itself by keeping it in a great excess of sweetened water for a very long time; we shall then be able to observe its process of revival, and With this object in view, on May 6th, 1874, we impregnated two fresh flasks of sweetened water with some of the contents of the before-mentioned flask, which we had refilled with sweetened water on May 2nd. On May 13th we decanted the liquid, which was still very sweet, from one of these two fresh flasks, which could hardly be said to have fermented at all—the quantity of yeast in them being so small—and replaced it with some wort. Strange to say, on the morning of the 14th we found an appreciable growth of yeast, and a froth of carbonic acid gas on the surface of the liquid. The yeast therefore was not dead, although its fermentative powers had been exhausted. There was, however, no remarkable feature in connection with its revival, nor did we find the slightest trace of any of the elongated ferment-form. What we got was simply the ramified groups of “high” yeast again, in round cells, but nothing more. Fearing that our yeast might not have remained for a sufficient time in the sweetened water for exhaustion, we set aside, for a whole year, the other flask which we had prepared on May 6th. On May 16th, 1875, we decanted the sweetened liquid and replaced it with wort. This time, however, there was no revival of the yeast; it had perished. Fortunately, we had also saved the flask of yeast and sweetened water which was prepared on May 2nd, 1874, as already mentioned, and in this case, as will be seen, the vitality of the yeast had not been extinguished, doubtless, in consequence of the formation of what we shall presently designate by the name of aËrobian ferment. On May 16th, 1875, we decanted the liquid from this last flask, and replaced it with wort. On the next day the surface of the wort was covered with a thin froth, indicating the commencement of fermentation. The microscope revealed nothing extraordinary, or indicative of the fermentation of any special ferment. To assure ourselves that our ferment had remained “high,” we sowed some of it in a fresh flask of wort on May 19th, and then, seven hours after impregnation, It would seem, therefore, that “high” yeast cannot, under any circumstances, assume the form and character of the ferment saccharomyces pastorianus, or of other known ferments. We are justified, therefore, in regarding it as a distinct species of ferment, an opinion which is supported by other circumstances. 1. In equal quantities of saccharine wort a considerably greater growth of “high” yeast is obtained than of other yeasts. We need no very rigorous proofs to convince ourselves of this fact: for by simply causing equal volumes of the same wort to ferment, the one being pitched with saccharomyces pastorianus, for example, the other with “high” yeast, we shall obtain a perceptibly greater volume of “high” yeast than of the other, in certain cases even five or six times as much. 2. “High” yeast is of a tougher texture than the others, separating, when the fermented liquor and its deposit is shaken up, into lumps which refuse to disappear; whereas saccharomyces pastorianus diffuses through the whole liquid with the greatest ease. 3. “High” yeast produces a special beer, with a peculiar flavour, well known to consumers, but little esteemed at the present day. Hence the gradual displacement of breweries worked on the old “high” fermentation system by others in which “low” yeast (of which more anon) alone is employed. 4. Lastly, one characteristic of “high” yeast, which it shares in common with some other ferments, although not with all, and which, from a practical point of view, deserves special mention, is that as fermentation proceeds the yeast rises to the surface of the liquid. Whilst the process of the manufacture of beer by this ferment is going on, the yeast is seen to work out of the bung-holes, flowing over in considerable quantity. The ferment named after the author, as well as “low” yeast, does not possess this property: it remains “Low” Ferment.—Whilst high yeast performs its functions in the breweries in which it is used at somewhat high temperatures—namely, between 16° C. and 20° C. (60° F. to 68° F.)—“low” yeast is never employed at a higher temperature than 10° C. (50° F.), and it is even thought preferable that it should not be subjected to more than 6° C., 7° C., or 8° C. (43° F. to 46° F.). At these comparatively low temperatures “high” yeast would have no perceptible action, whereas it is at such temperatures that “low” yeast best performs its functions. In our Memoir on alcoholic fermentation, published in 1860, in the Annales de Chimie et de Physique, the idea of the identity of the two yeasts was accepted; but we had at that time made no special observations of our own on the subject. Upon closer investigation we are inclined to believe that the two yeasts are quite distinct. We might keep our “high” yeast at the lowest temperatures that it can bear, and repeat our growths under these conditions; or, on the other hand, we might subject the “low” yeast to temperatures higher than those at which it ordinarily grows, without ever succeeding in changing the first into the second or the second into the first, supposing, of course, that each of our yeasts was pure to begin with. If they were intermixed the change in the conditions of development would cause one or the Fig. 41. Fig. 41. It is true that brewers generally are of a different opinion. Most of them assert that “low” yeast cultivated at a high temperature becomes “high” yeast; and conversely, that “high” yeast becomes “low” by repeated growths at a low temperature. Many have told us that they have proved this. Nevertheless it is our belief that the success of such transformation has been but apparent, attributable in each case to the fact, as we have just stated, of their having operated on a mixture of the two yeasts. Mitscherlich, and various authors after him, have asserted that “high” yeast propagates by budding, and “low” yeast, on the contrary, by spores, formed by the endogenous division of the protoplasm of the cells, and set free by the rupture of the cell-wall, which then, increasing in size, assume the character of ordinary cells. But we have never been able to confirm this. Fig. 41 represents a field of low yeast, taken from the deposit in a vat after the fermentation of the beer was finished. The granular matter mixed with the cells is altogether amorphous although in many cases perfectly spherical. It is a product in no way related to this yeast (see Plate I., No. 7). Comparing Fig. 41 with Fig. 40 (p. 189), it may be seen that the general aspect of low yeast is distinguished, in its early stages, although in no very decided manner, from that of “high” yeast, by being slightly smaller and less round or spherical in its cells than the latter. As to the case of “high” yeast, the deposits of “low” yeast after fermentation appear as scattered, isolated cells; we do not find more than two or three per cent. of united cells. Nevertheless the two yeasts present, as we shall see, quite marked differences in the character of their budding and multiplication. Fig. 42. Fig. 42. On May 28th, 1875, we put a trace of pure, unicellular, “low” yeast, taken at the end of a fermentation, into a flask of wort. On May 29th, sixteen hours after impregnation, the temperature during the night having been 15° C. (59° F.), we made a sketch of the yeast before its development had become apparent to the naked eye. No perceptible development, that is to say, no visible deposit at the bottom of the liquid and formation of patches of froth on the surface, took place before May 30th. A mere glance at Fig. 42 will be sufficient to enable us to detect a considerable difference between it and Fig. 40, which represents the multiplication of the cells of “high” § IV.—On the Existence and Production of Other Species of Ferment.Our present knowledge of the alcoholic ferments embraces the following, without taking into account the ferment-form of mucor:— The ferment named after the author, which is found associated with the ferments of the grape and other domestic fruits, and with spontaneous ferments in general. The ferment of “high” beer. The ferment of “low” beer. To these must be added the ordinary ferment of wine, and that called apiculatus, although, indeed, these last are of little practical importance, since, in general, they soon become lost amongst others of greater vitality, in the spontaneous fermentation of fruits. These are not the only alcoholic ferments; a study of the germ-cells diffused over the surface of fruits, grains, and stalks of all vegetables in different countries, would doubtless lead to the discovery of many new ones. We are even inclined to believe that one ferment might give rise to a multitude of others. The investigations which we have undertaken in this direction are as yet not far advanced; we may, however, be allowed merely to state the principle which governs them. A ferment is a combination of cells, the individuals of which must differ more or less from each other. Each of these cells Spontaneous ferments, properly so called, of which we have already spoken, are, after all, the result of sowings of this kind. Originating in liquids which have been boiled, and then left to themselves in contact with the air in a place where cells or germs must have existed, these ferments must necessarily often spring from single germs or from a limited few, and this also would probably be a means of developing distinct varieties of ferments. New “High” Ferment.—We met with this ferment accidentally, under the following circumstances:—On February 12th, 1873, we had brewed in the laboratory about 2-½ hectolitres (rather over 50 gallons) of wort, 10 litres (about two gallons) of which were set aside to cool in a white-iron trough, and left during the night exposed to free contact with air in the underground part of the laboratory, where we have a small experimental brewery. Next day we put some of this latter into a bottle; the wort soon began to show evidence of change, various productions made their appearance on the surface of the liquid, and a deposit of yeast settled at the bottom. On May 23rd, perceiving bubbles of gas and a steady fermentation set up in the wort, which remained all the time corked up, and fearing that the bottle might burst by the increasing internal pressure, we drew the cork. A considerable liberation of gas at once took place, accompanied by a voluminous foam which half emptied the bottle. A microscopical examination of the deposit from the disturbed liquid led to the discovery of a very homogeneous yeast, associated with various other organisms; it was clearly a yeast which we had not hitherto met with amongst the spontaneous ferments which we had had occasion to study. Thinking that this might be a new species of ferment which would probably produce a beer that was also unknown, we set to work to purify it by cultivation in flasks of pure wort, during the months of May, June, and July. Our last growths, of August 4th, 1873, were preserved, in order that we might assure ourselves of the purity of the beer, and, consequently, of the ferment. On November 15th its purity was established. On that date we made some beer with this ferment, which had now been left to itself for several months in contact with pure air. The beer which we obtained resembled no known variety; consequently the ferment must Fig. 43. Fig. 43. Fig. 43 represents the rejuvenescence of this ferment. Comparing this figure with Fig. 42, we see that this ferment presents a considerable resemblance to “low” yeast in dimensions, method of budding, and oval shape; but the feature which distinguishes it essentially from “low” yeast is that it rises to the surface, like “high” yeast. Buoyed up by the gas during fermentation, it forms a layer of yeast on the surface of the fermenting liquid, where it remains after the head has fallen. Some of this head of yeast likewise adheres to the sides of the vessel above the level of the liquid. In short, by the greater regularity of its forms and the uniform dimensions of its cells, this ferment is to be easily distinguished from saccharomyces pastorianus; its aspect, which is oval instead of spherical, and the ramified form of its chains of cells, which is less marked than in the case of “high” yeast, also prevent our confounding it with the latter ferment; in its rising character it differs absolutely from “low” yeast; lastly, it may be distinguished from all other ferments by the flavour of the beer that it produces. The ferment which we discovered in this accidental way may be utilized. Indeed, we may ask, is it not to be found already in our beer? We are inclined to believe that it is. After the war of 1870, some Viennese traders established at Maisons-Alfort, near Paris, a manufactory of yeast for bakers. They saccharified by means of malt a mixture of the meals of rye, Caseous Ferment.—We give the title caseous for a reason that will presently appear, to a ferment which we came across also accidentally. We were trying different methods of purifying yeasts, and for this purpose had composed a liquid formed of:
Quantities of this liquid were placed in several of our double-necked flasks, submitted to boiling, then, after cooling, impregnated with different ferments, and kept in a water-bath at 50° C. (122° F.) for one hour. In operating under these conditions with brewers’ “high” yeast, say, for instance, with what is called Dutch yeast, a kind well known in distilleries, fermentation shows itself in the course of a few days, in spite of the increased temperature to which our liquid, which is hopped and slightly acid and alcoholic, has been subjected. The time required for the resumption of fermentation depends both upon the degree of temperature to which the yeast has been exposed and upon the duration of its exposure. These, however, are not the points Fig. 44. Fig. 44. Fig. 45. Fig. 45. Figs. 44 and 45 represent this new ferment magnified to the same extent as the other ferments have generally been, that is 400/1; it will readily be seen how different its form is from that of “high” yeast, how far it is from having the spherical aspect and mode of budding characteristic of that ferment. In Fig. 45 the ferment is represented in a mass; in Fig. 44 we see the ramified groups, the cells and segments of which form, after separation, the yeast of the deposit. It thus appears to be composed of jointed branches of greater or less length, which, at the junctions of the segments, put forth similar cells or segments of a round, oval, pyriform, cylindrical, or other shape; in all its characters recalling the description of dematium. Moreover, the cells and segments exhibit a greater sharpness of outline, as well as a more marked transparency and refractive power than are found in the majority of ferments; but the most curious physical characteristic of this ferment is its plasticity and elasticity, if we may use those terms. It can only be made to diffuse through water with great difficulty; when shaken up in it, it sinks to the bottom quickly When caseous ferment is sown in a saccharine medium charged with mineral salts, its aspect, form, and mode of budding differ completely from what they are when the ferment exists in a natural medium, such as wort or other liquid adapted to the nutrition and life of ferments. Fig. 46. Fig. 46. Fig. 46 represents this ferment in course of development, forty-eight hours after it had been sown in a saline medium (we employed Raulin’s fluid, substituting bi-tartrate for the “High” yeast from a “high” fermentation brewery in the Ardennes, after having been exposed to heat under the conditions given above, likewise produced caseous ferment, without a trace of “high” ferment, just as happened in the case of the Dutch yeast. All the “high” yeasts used in brewing seem to behave in the same manner. What conclusion are we to draw from these facts? Apparently that “high” yeast is modified by heat in an acid and alcoholic medium, giving rise to caseous ferment. On the other hand, it might be conceived that the “high” yeasts on which we experimented were not pure, but contained, in a state of intermixture, some caseous ferment, and that by the application of a temperature of 50° C. (122° F.) to our alcoholic medium, the high ferment was all killed and the caseous ferment alone survived. It is a remarkable fact that this latter hypothesis, improbable as it seems, inasmuch as the microscope revealed no intermixture of ferments, seems, nevertheless, to be a true one. As a matter of fact, if we subject to a temperature of 50° C. for one hour in the medium in which it acts, not the “high” yeast of commerce but “high” yeast that is absolutely pure, this will perish utterly, and the wort after cooling may remain for years in an oven without either undergoing fermentation or developing any growth whatever of “high” ferment or “caseous” ferment. On the other hand, if we impregnate this same alcoholic liquid with some of the caseous ferment and then heat the vessel to 50° C. for one hour, the caseous ferment will go on reproducing itself after the liquid has cooled down. Fig. 47. Fig. 47. This conclusion is supported by the following fact, which also tends to prove that in the case of the “high” English pale ales, caseous ferment plays a most important part. In the medium already described, we sowed the deposit from a bottle of good English pale ale. After having been heated the yeast went on growing, and we obtained the very beautiful specimen of caseous ferment represented in Fig. 47. The two dark globules are dead cells which had been killed. Two minute segments of lactic ferment are also visible in the sketch—the yeast which we sowed was, of course, impure—and their presence Fig. 48. Fig. 48. § V.—On a New Race of Alcoholic Ferments: AËrobian Ferments.Mention has already been made of certain researches which we undertook with the object of ascertaining whether mycoderma vini, or efflorescence of wine, and mycoderma cerevisiÆ, or efflorescence of beer, which grow equally well in all fermented Whilst engaged in these researches, we were pursuing others in relation to the converse of the proposition just discussed, that is to say, respecting the possibility of ferment becoming transformed into mycoderma vini or mycoderma cerevisiae. Our experiments in connection with this subject chiefly consisted in various endeavours by way of exhausting the yeast and subsequent revival of its growth. This exhaustion was effected by growing the yeast in excess of sweetened water, and at other times in unsweetened yeast-water, our efforts being directed to deprive it of all power of fermenting. We afterwards caused it to develop afresh in highly aerated, nutritious liquids, in order that we might see how it reproduced itself, and if its new form were that of a mycoderma. The yeast after having lost its power as a ferment, and being no longer able to act in pure sweetened water, nevertheless reproduced itself when placed in fermentable media, holding in solution materials adapted to its nutrition; yet we never succeeded in obtaining any organism besides the ferment, and, indeed, the identical variety of ferment on which we had operated. In no case was mycoderma vini or cerevisiÆ produced, and we concluded that we were justified in stating that whenever the mycoderma vini appeared on the surface of a fermented or fermentable liquid, its germ must have been introduced by the surrounding air, In a laboratory where alcoholic fermentations are studied, these germs of mycoderma vini exist in great abundance on the surfaces of different objects. This fact admits of easy proof; we have merely to open in such a laboratory some flasks containing yeast-water deprived of air, or yeast-water sweetened, or any natural saccharine medium, or any fermented liquid, which till the moment when our flasks were closed had been kept boiling (Chap. IV.); it would be a very rare thing, indeed, if mycoderma vini did not develop in most of these flasks after the air was readmitted, especially if, shortly before this operation, the dust lying on the surface of the tables or floor of the laboratory had been stirred up by dusting or sweeping. This series of experiments, the salient points of which we have just given, conducted with a view to ascertain whether yeast could be transformed into mycoderma, has led the way to certain results of special interest, results which concern all alcoholic ferments, and which in all probability will be found in the long run to apply to all aËrobian ferments. It being necessary for the conduct of our experiments to preserve our yeast in a state of purity for an indefinite period, often for a great length of time, in contact with pure air, we discovered that yeast was possessed of extraordinary vitality, and that it rarely perished completely throughout, inasmuch as we could almost invariably cause it to revive by bringing it into contact with fresh, fermentable liquid. This revival of the yeast—and it is to this point that we are most anxious to direct the attention of our readers—is effected from two distinct sources:— 1. By those cells of yeast which have not perished. 2. By cells of new formation. We may give an example to explain this more clearly. In one of our two-necked flasks we cause some pure wort to ferment by employing yeast also in a state of purity. Fermentation It may easily be understood how this kind of production has escaped notice up to the present time. The conditions of our experiment were, in many respects, novel; a saccharine liquid had never before been caused to ferment by means of pure yeast, absolutely free from foreign germs; a fermented liquid had not previously been exposed to contact with pure air for an indefinite time. On the other hand, all ordinary fermented liquids, when left to themselves in contact with air, are a ready prey to mycoderma vini or aceti at their surface, and then give rise to true fungoid growths. The appearance of these organisms, which always takes place soon, has thus constantly concealed or prevented the development of the true aËrobian ferments. In repeating the experiment described any alcoholic ferment may be used, and each one will be found to produce its own peculiar fungoid form of ferment. Another point worthy of notice is that these aËrobian ferments, when they put forth buds in the act of fermentation, reproduce the forms of the original ferment, at least apparently so. In this respect they cannot be distinguished, notwithstanding the fact, surprising Notwithstanding these facts, it would be difficult to discover any very appreciable differences between the forms of the cells of any particular yeast and those of its aËrobian ferment in course of development. So true is this, that the aËrobian ferment of saccharomyces pastorianus might even be caused to put on the forms of dematium pullulans, which we have had occasion to observe specially characterize this ferment after the cells have been subjected to a prolonged process of senescence. On August 6th, 1873, we took some of the ferment saccharomyces pastorianus from a flask of wort that had undergone fermentation, and sowed a scarcely perceptible quantity of it in another flask containing a saline medium, composed as follows:—
In the course of the following days the ferment began to develop, although with difficulty, the fermentation revealing itself by collections of bubbles appearing here and there on the surface of the liquid. We left the flask undisturbed till the 25th of November following. On that day we found a very white deposit of ferment covering the yeast-ash that had not been taken into solution, and a ring of aËrobian ferment on a level with the surface of liquid; all the sugar had disappeared; the liquid contained 5·2 per cent. of alcohol, by volume, at a temperature of 15° C. (59° F.); and, lastly, in consequence of the purity of the materials employed, there was no trace of the formation of fungoid growths, whether of mycoderma vini or of mycoderma cerevisiÆ, on the surface of the liquid, or of vibrios or lactic-ferment below the surface. Thus then we see—and several other examples throughout this work confirm the fact—that saccharine liquids holding mineral salts in solution are as capable of complete fermentation as any media of natural composition. It is true that ferment develops slowly and with difficulty in them, and at times takes on rather curious forms, but, nevertheless, it does develop in the media and carry on a fermentation in which not the Fig. 49. Fig. 49. Fig. 49 represents the ferment as it appeared when examined on August 11th, 1873. We can no longer recognize in it any saccharomyces pastorianus. The general appearance is spherical, and there are a number of clusters of budding cells which remind one at first sight of the mode of germination of brewers’ “high” yeast. At a, a, a, we see globules from which irregular abortive filaments have sprung, a proof of difficult germination. No such monstrosities could ever have occurred if we had used beer-wort or must as our nutritive medium. On November 25th we made another examination and sketch of the ferment, the appearance of which did not differ materially from that given above. The general appearance was the same, consisting mostly of globules joined together in clusters of two or three or more. No separation, such as occurs in the case of Fig. 50. Taking some fresh yeast from the bottom of the liquid we examined and made a sketch of it (Fig. 50). The field was filled with round and oval cells, jointed and ramified filaments, budding and multiplying in the most remarkable manner, reminding us of the germination of the cells of yeast exhausted The aËrobian ferment of “high” yeast, in whatever medium we cultivated it, presented no peculiarity, as far as its forms were concerned. It was composed of cells of spherical shape, like ordinary “high” yeast, and germinated in the same way as the latter. Fig. 51. Fig. 51. Fig. 51 represents the revival of this aËrobian ferment. We recognize here the branched mode of budding and spherical contour characteristic of “high” yeast proper. Nor does the aËrobian ferment of “low” yeast present any special Fig. 52. Fig. 52. Fig. 52 represents the aËrobian ferment of yeast used in “low”-fermentation breweries, examined forty-eight hours after pitching. We find that groups resembling that at a are of very rare occurrence. They are to be seen only at the very beginning, generally only for the first few hours of the renewed activity. Very soon, however, they develop cells which are of the size of the oval cells budding at b. Fig. 53. Fig. 53. Fig. 53 represents the aËrobian caseous yeast which forms Fig. 54. Fig. 54. On May 27th, 1875, we sowed, in a flask of wort, a trace of a pellicle of this kind, which had formed on the surface of a flask in which fermentation had been set up by means of caseous yeast in May of the preceding year. On May 30th fermentation began to reveal its presence by a voluminous froth, and the newly-formed yeast had reached the bottom of the flask. A small quantity was taken out by a capillary glass tube, and a sketch of the ferment made; this is given in Fig. 54. Amongst the cells which occupy the field there are groups of some of larger size. These are not distinct forms mixed with the others, but simply another illustration of the fact that old cells in course of revival, especially when they have been exhausted in sweetened water, as we have just observed of the aËrobian ferment of “low” yeast, commence with forms of larger diameter or more elongated than the ordinary forms peculiar to the ferment which at a later stage are developed from them. We have seen how marked and exaggerated this feature was in the case of saccharomyces pastorianus. Let us again call attention to the forms of aËrobian ferment furnished by the yeast which we have already described under the name of new “high” yeast. Fig. 55 represents this Fig. 55. Fig. 55. Fig. 56. Fig. 56. On November 27th, 1873, we sowed a trace of this ferment in a flask of wort. From the 29th, with a continuous temperature of 25° C. (77° F.), a considerable deposit of yeast began to form, and the froth of fermentation covered the whole surface of the liquid. We took a little of this deposit for examination; it is represented in Fig. 56. The field is occupied with oval cells of great uniformity. We recognize the aspect of the original yeast (Fig. 43). Here and there, indeed, we come across some cells of larger size, such as those at a and b, which is another illustration of the remark that we have just made respecting the forms which revived exhausted cells take on at the commencement of a new germination. The aËrobian ferment of “high” yeast appears in the form of small isolated teats on the surface of the fermented liquid. It develops rather sluggishly, and has no great vitality. The aËrobian form of “low” yeast develops as a somewhat fragile layer, the least agitation precipitating it to the bottom of the vessel in a cloud of very small irregular flocks, that do not diffuse through the liquid as they fall. With free access to air it retains life for a long time. The aËrobian ferment of caseous yeast forms a continuous greasy-looking pellicle, gradually thickening, which breaks up into fragments when shaken. With a supply of air it lives very long, and the pellicle gradually increases in thickness. In reviewing these ferments we may naturally ask ourselves the question whether the “high” ferments of which we have spoken—the industrial one concerned in the “high” fermentation of breweries, and the other which we have termed new “high” ferment—are not aËrobian ferments of “low” yeasts. We are inclined to think that the ferment which in the preceding paragraph we termed new “high” ferment, may, perhaps, be the aËrobian form of the “low” yeast employed by Alsatian and German brewers. We have studied this new “high” ferment side by side with the aËrobian ferment of “low” yeast, and the result we have arrived at is, that in appearance and mode of germination, as well as in the flavour and quality of the beers which they produce, they greatly resemble one another. In the last respect, however, we cannot say that the identity is quite absolute, and hence it is with some doubt that we suggest the possible identity of the two In writing these lines an idea suggests itself which might be profitably made the subject of serious experimental study. What would be the peculiar properties of the aËrobious ferment-form of an aËrobian yeast? Certain facts incline us to believe that these forms differ from each other just as a “low” yeast differs from its aËrobian ferment. If this were actually the case it would be very interesting to compare the peculiar properties of an indefinite series of aËrobian ferments, all derived from a common origin. We find recorded in our laboratory notes that a certain aËrobian ferment of the second generation produced a beer different from that produced by the same ferment of the first generation, being possessed of a fragrance so marked that, on entering our laboratory, in which only a few litres of this beer were fermenting, we were at once struck by the powerful odour which it emitted. § VI.—The Purification of Commercial Yeasts.We have already stated that the researches detailed in the preceding chapter require for their successful prosecution that the ferments on which we experiment should be absolutely free from germs of other organisms, and we have shown how impossible it would be, if this condition were not complied with, to follow for weeks or months, sometimes even years, the changes which occur in a yeast maintained in contact with air, either in sweetened water or in a liquid which has fermented under its influence. Equally necessary is it that the In general, the inconveniences resulting from the impurity of a yeast employed do not immediately manifest themselves, in consequence of the enormous preponderance of the true yeast, which, in comparison with the foreign germs that contaminate it, may be so great that microscopical examination fails to reveal even the presence of these latter. Again, it is a well-known fact that the abundance of one growth in a limited medium operates to the prejudice of a less abundant one, inasmuch as the first consumes the nutritive materials at the expense of the second, and more particularly the needful amount of oxygen. It follows, that when a saccharine liquid is impregnated with commercial yeast, nothing but yeast may be detected for a time, and one is led to believe in the purity of the subsequent growth. This, however, supposes that the external conditions, as well as those of the medium of growth, are equally adapted to the life of the yeast and that of those organisms present as impurities; for if these conditions rather favoured the nutrition of the latter, we should be sure to find their proper developments appearing at an early stage. For example, when the growth of yeast becomes sluggish, we have invariably the development of such after-growths. The principal germs, having exhausted the saccharine liquid which has fermented under their influence and is no longer adapted for their growth, cease to develop, and have their place taken by ferments of disease, spores of moulds, mycodermata, &c., the growth of which proceeds more or less rapidly, in proportion as the character of the liquid and the surrounding temperature are more or less suited to their growth. Here, too, we have an explanation of the rapid change that In the process of brewing, as soon as fermentation is finished, or rather, as soon as certain physical effects are produced, for instance, when the beer falls bright, or, as the French say technically, when the yeast breaks up, At the same time, by way of comparison, we impregnated other flasks of wort with pure ferments. None of the beers of this series acquired a bad taste or produced foreign ferments; they only became flat. When we review the operations of the brewer’s art, we are surprised by the comparative perfection to which that art has been brought by the laborious experience of years, and the more so when we consider that, as regards the question of the diseases of beer, the brewer has never been guided by any such rigorous principles as those which we have explained in this work. We have already given proofs of this in our first chapter. The beer is racked and separated from its yeast before fermentation has entirely ceased. The principal reason for this is that it is necessary that the beer, after being run into cask, should work again and undergo a secondary fermentation, in order that it may not be invaded by the parasites, of which we have already spoken, as would not fail to be the case if the beer were suffered to remain in a state of perfect quiescence. Not only is the beer racked before it has attained its limit of attenuation, but in addition to this, and also with the view of checking the development of parasites, it is placed in cellars sensibly cooler than the temperature of fermentation, low as that is in the case of “low” beers: the temperature of the cellars being not higher than 2° or 3° C. (36° F.). Unfortunately, the requirements of trade prevent our complying This vinous flavour develops more especially in English beers when these are kept. It is an easy matter to show that in English beers, after their manufacture, saccharomyces pastorianus and the ferment which we have termed caseous, which also imparts a peculiar flavour, form almost exclusively, notwithstanding the fact that the yeast used in the manufacture of English beer is a ferment essentially distinct from saccharomyces pastorianus. The secondary fermentation which takes place in “high” and “low” beers stored in cask after manufacture, is very often due to this same ferment, which may be recognized by elongated jointed cells, at times more or less ramified, as well as by the influence which it exercises upon the flavour of the beer. We may add that the general result of our researches has convinced us that “high” yeast cannot transform itself, any more than “low” yeast can, into the ferment of which we are speaking, and that whenever a beer produced by means of “high” or “low” yeast develops a foreign ferment, this ferment must have existed in the original yeast in the form of germs, which, from their extreme scarcity, often fail to be detected by means of the microscope. The best proof that we can give of this is the fact that a beer produced by means of “high” or “low” yeast, if left to itself for months In certain cases the intermixture of ferments is to be feared almost as much as the presence of diseased ferments, when these latter have not developed to any great extent. We have often seen our fermentations invaded by ferments differing absolutely from those which we originally employed. The repetition of growths, and more particularly changes in the composition of our fermentable media, purposely made with the view of attaining certain results, often produce complications of this kind. For a long time we were unable to realize the true significance of the results of some of our experiments, in consequence of the facts which we have just explained, as well as those detailed in the preceding paragraph, having escaped our notice; indeed, our ignorance of those facts added greatly to the difficulty and length of our researches. Our labours from the commencement of this work to the date of its publication have extended over not less than five years, and no one can know better than ourselves with what advantage we might devote a still longer time to it; but, as Lavoisier says, one would never give anything to the world if he delayed doing so until he fully attained unto his ideal aims, which always seem more distant the more one increases one’s efforts in the attempt. Our preceding observations show how extremely important it is to employ pure yeasts to obtain, on the one hand, well flavoured beers, whilst adhering to the processes at present existing in breweries, and on the other, beers of good keeping qualities, In the case of intermixture of alcoholic ferments, we may sometimes manage to effect their separation by taking advantage of their unequal vitalities in different media of cultivation. On December 17th, 1872, we made a powder of commercial Dutch yeast and plaster, as described in Chapter III. § 6. The Dutch yeast was a “high” ferment. On July 25th, 1873, we sowed a portion of this dried mixture in a flask of pure wort. From July 27th patches of bubbles from fermentation were visible on the surface. On August 2nd the fermentation was completed. The yeast, examined under the microscope, was apparently pure, formed of spherical cells of a fine “high” ferment. We poured away the fermented liquid, observing every necessary precaution, and left in the flask almost all the deposit of yeast, and not more than one or two cubic centimetres (about half a tea-spoonful) of beer. On November 15th following the yeast, examined afresh, still seemed pure and still exhibited the form of round cells of “high” yeast, only that they had taken on a very aged aspect, showing a double contour, and filled with granulations collected irregularly about the centre. Such are the precise characteristics of dead cells; nevertheless it was still possible that some living cells yet remained. To assure ourselves of this we took some of the yeast and placed it in a flask of pure wort. On the 19th a little froth from fermentative action appeared on the surface. We then examined the yeast and discovered that it was no longer “high” yeast, but a small ferment of rather irregular appearance, in which the jointed cells of saccharomyces pastorianus, as it usually appears after a succession of growths, predominated. It must not be imagined here that what we saw was a transformation of one yeast into another. The Let us take, as another example of purification of the same kind, the case of the different ferments of the vintage. When must begins to ferment the apiculated ferment invariably appears, and becomes afterwards associated, more or less, with the saccharomyces pastorianus, in the presence of which the multiplication of the apiculated ferment soon ceases. Saccharomyces pastorianus, in its turn, is gradually displaced by the ferment which we have termed the ordinary ferment of wine, and which Dr. Rees has named saccharomyces ellipsoÏdeus. On the subject of these changes in the proportion of the ferments of wine, the Note which we published in 1862 in the Bulletin It is evident from what we have just said that the principal part of the deposits of yeast in the sediment of fermented grapes, at the time when the wine is first racked, which in the Jura, is called l’entonnaison, is composed of the ordinary ferment of wine, the saccharomyces ellipsoÏdeus, and that the cells of apiculated ferment are scarcely discoverable with the microscope, being scattered amongst an infinite multitude of other ferments. Fig. 57. Fig. 57. We procured from Arbois, on January 20th, 1875, some wine yeast taken from a large barrel of the preceding vintage, racked on January 18th. The ferment was very irregular. Some of its cells were very old, of a yellowish colour, and full of granulations—amongst these a certain number formed jointed segments, rather elongated, and probably belonging to saccharomyces pastorianus. The other cells were transparent, and apparently still young. This mixture of the two ferments is represented in Fig. 57. No doubt if we had searched carefully we should also have found some cells of saccharomyces apiculatus. On January 21st we sowed a small quantity of this Fig. 58. Fig. 58. Fig. 58 represents the yeast formed, which evidently had sprung from the transparent cells seen in Fig. 57, and doubtless belonging to the ordinary ferment of wine, saccharomyces ellipsoÏdeus. Here we have another example of the natural separation of ferments brought about by the death of one or two of them, or by extreme differences in the time of their revival. The purification of ferments may be accomplished by various methods, according as we have to deal with an intermixture of ferments, or to regard as our principal object the expulsion of ferments of disease, such as vibrio germs, lactic ferment, the filamentous ferment of turned beer, mycoderma aceti or mycoderma vini. One method of easy application consists in sowing the yeast in water sweetened with 10 per cent. of sugar. This liquid should be first boiled, and preserved in the two-necked flasks which we have so often described. Sweetened water is a very exhaustive medium for ferments, and the organisms mixed with them. A great many cells perish in it, and the chances are that the foreign germs, which are always scarce in comparison with the great number of cells of ferment, may be amongst those which die, or those which become so exhausted that when the yeast, after this treatment, is sown in wort, they disappear, and allow those cells which have remained vigorous enough to develop alone. The addition of a little tartaric acid to the saccharine solution—say, from 1/1000 to 2/1000 part by weight—often facilitates the destruction of certain germs of impurity. Mycoderma aceti and mycoderma vini do not find suitable life-conditions in the sweetened water; they soon disappear if cultivated alternately in sweetened water and wort. There is, therefore, this advantage in cultivating yeasts in shallow basins, that the multiplication of the alcoholic ferments is promoted, and that of most of the disease-ferments is checked. There is an exception, indeed, in the case of mycodermata; but of all disease-ferments these are the most easily got rid of, by repeating our growths before they make their appearance. Notwithstanding this, our two-necked flasks, which also contain much air at first, are to be preferred to the shallow basins, inasmuch as they are a perfect safeguard against the germs floating in the surrounding air, as well as those of the ferment saccharomyces pastorianus. Another method is suggested to us by the curious results of which we have already spoken, obtained by sowing yeasts in a wort rendered acid and alcoholic by the addition of bi-tartrate of potash and alcohol. Experience proves that many disease-ferments find great difficulty in withstanding a succession of growths in wort to which 1-½ per cent. of tartaric acid and from 2 to 3 per cent. of alcohol have been added. Such a mixture, however, is equally well adapted to the requirements of saccharomyces pastorianus, and we must always Another method of purification, which is perhaps quicker, although inferior in other respects, consists in the employment of carbolic acid—that is to say, in purifying our yeast by successive growths, we may add to every 100 c.c. (3-½ fluid ounces) of wort that we employ from ten to twelve drops of phenol water, containing 10 per cent. of the acid. The action of the phenol, which at first is invariably combined with that of the oxygen of the air, tends to destroy the vitality of many of the cells sown, involving to some extent also the yeast which we are interested in preserving. But amongst the number of cells that are affected those which are less abundant, that is to say, those which are present as impurities, are paralyzed relatively in much greater proportion. If the acid does not destroy them it greatly checks their development, and the cells of yeast, which multiply continuously in vast numbers (for the fermentation goes on in spite of the phenol, if this is added in small quantity), gradually choke the foreign germs in a succession of growths. By these different means, which are employed separately or combined with one another, we generally manage to obtain the yeast which we wish to purify in a very pure state. We need scarcely add that it is always well, in the case of our purifications, to begin with specimens which are already as pure as it is possible to obtain them. In making our choice the microscope is our best guide, but it is not a sufficient one. We should be strangely deceived if we believed in the purity of a yeast for the sole reason that when examined under the microscope it appeared to contain nothing of a foreign nature. The best means of assuring ourselves of the purity of a yeast consists in making some beer in one of our two-necked flasks, and leaving In the course of a series of practical experiments that we were carrying out in the large brewery of Tourtel, at Tantonville, in 1875, in connection with the new process of brewing, which will be explained in Chap. VII., the following circumstance occurred. We had purified some of the yeast of the brewery, by means of a succession of growths and adding a few drops of phenol, and had obtained a yeast of irreproachable purity. It happened that this yeast, which was repeatedly cultivated in the brewery during the summer of 1875, from six to ten hectolitres (130 to 220 gallons) of wort being used on each occasion, always produced a beer that had a yeast-bitten flavour and defective clarifying powers, notwithstanding that it possessed remarkable keeping properties, which it owed to the pureness of the ferment employed. As a matter of fact, the beer suffered no injury from journeys of more than 300 miles, by slow trains, in ordinary casks, containing from 50 to 100 litres (10 to 20 gallons), during the great heats of June and July, or from being subsequently stored for two months in a cellar, the temperature of which rose during that time from 12° to 18° C. (54° to 65° F.). The temperature of fermentation had been 13° C. (55° F.). Beer from the same brewery, made with the same wort by the ordinary process, did not remain sound for three weeks in this same cellar. |
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