Chapter III

. § 3), of making grape juice ferment apart from the action of external

## particles of dust, and the knowledge which we have just acquired, that

the particles of dust on the surface of the grapes and woody peduncles, at the moment when the grapes have attained maturity, contain certain reproductive cells which give rise to certain ferments, naturally lead us to the investigation of another point, which concerns the period at which these germs make their appearance on the different parts of the vine plant. The two following experiments tend to prove that the ferment can only appear about the time when the grapes attain maturity, and that it disappears during the winter, not to reappear before the end of the following summer.

I. In the month of October, 1873, we procured from a vineyard in the canton of Arbois some of the woody parts of very ripe clusters of grapes, taking the precaution to cut off all the grapes, one by one, with a very clean pair of scissors, whilst still on the vine; we then wrapped up the woody parts of the clusters, thus deprived of their grapes, in thin paper, to convey them to Paris. Our only object at that time was to secure for use in our subsequent studies the ferment-bearing dust found in October on the woody part of the vine, and, more

## particularly, on the clusters themselves, as already stated. After our

return to Paris, and during the course of our experiments in October and November, it sufficed to wash a few scraps of the bunches in a little pure water, in order to obtain the grape-ferment in abundance; but later on in the winter we were astonished to find that the same procedure yielded no ferment, only some moulds. The bunches which, when put into boiled and filtered must, in October, very readily caused that must to ferment, at the end of winter could no longer produce the same effect, however favourable might be the temperature to which we raised the must. The particles of dust on the bunches had, therefore, become sterile, as sources of alcoholic ferments.

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

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

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

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

[Illustration: 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 ferment; and in the course of September the number of tubes that fermented increased progressively. In each series of tubes, however, we always found a few in which there was a complete absence of fermentation.

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

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

No. 5 tube containing two uncrushed grapes.

No. 6 tube containing two crushed grapes.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

No. 7 tube containing two crushed grapes.

No. 8 tube containing one crushed grape.

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

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

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

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

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

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

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

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

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

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

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

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

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

As might be expected, a microscopical examination of the particles of dust scattered over the surface of the fruit and woody peduncles of grapes, reveals great differences in the number of these fertile

## particles at different periods of the vegetation of the grapes. As long

as the grapes are green and the vine in full activity we find scarcely any, or, at all events, very few spores which seem to belong to ordinary fungoid growths. Towards autumn, however, when the grape is ripening and the leaves becoming yellow, fungoid growths and numerous productions of great fertility accumulate on the vine, on the leaves, the branches, and the bunches. At this period we find the water in which the grapes and the woody parts of the bunches are washed swarming with different kinds of organized corpuscles; it is at this period, too, that the ferment-yielding moulds attain that phase of their vegetation in which, when mixed with the juice of grapes, they produce fermentation.

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

[Illustration: 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 every vegetable might be submitted to important investigations of this kind, the results of which, in our opinion, could hardly be doubtful.

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

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

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

[Illustration: 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 represented in the left-hand half, in a medium peculiarly adapted to their vitality, _and in the presence of much air_. As regards the length and size of filaments and cells, there is little appreciable difference between the two sides. The principal difference consists in the relative freshness and the budding going on in individuals in the right-hand half.

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

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

To exhaust yeast, without destroying it, sweetened water is preferable. Having decanted the beer, we substitute in its place water sweetened with 10 per cent. of pure sugar. By effecting the substitution in the following manner, we escape the risk of introducing germs from floating

## particles of dust, which would nullify all experiments of this kind. We

prepare, then, a flask containing sweetened water, free from all foreign germs, which we attach to the other flask [_i.e._, two of M. Pasteur’s flasks with two necks, one straight and wide, the other bent and narrow (Fig. 8)]. This is done by taking the india-rubber tube off the flask containing yeast, and removing the glass stopper from the other india-rubber tube attached to the flask containing the sweetened water; then, introducing the right-hand neck of the yeast flask into the india-rubber tube connected with the other, we raise the latter flask so as to pour the sweetened water on to the yeast. At the same time an assistant passes the flame of a spirit-lamp over the bent part of the curved tube attached to the water flask, with the object of destroying the vitality of the germs in the floating particles of dust, which enter the flask in proportion as it is emptied into the other.

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

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

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

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

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

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:—

Some spontaneous yeast which, after repeated cultivation, had acquired the aspect represented in Plate XI.—which aspect the _saccharomyces pastorianus_ generally assumes under these circumstances—was exhausted in sweetened water, and subsequently revived in must at 10° C. to 11° C. (51° F.). At this temperature germination was not very marked before the end of eight days; at a temperature of 20° C. (68° F.), it only took three days, under similar conditions. The sketch includes but one of the long branches from which the ferment cells and the budding joints took rise, but there were a great number more. Some of the forms represented in the figure bear a striking resemblance, it appears to us, to some of those of _dematium_, in Plate IX.; and even we may trace out the several peculiarities of form which distinguish the figures in the latter plate.

[Illustration: Fig. 33.]

The next figure (34) represents the earliest forms of germination of another specimen of _saccharomyces pastorianus_ in wort, after it had been exhausted by four successive growths in sweetened water. We here see the large ferment-form which appears at the commencement of fermentation, in acid fruits, such as cherries and gooseberries (Plate IX.), associated with smaller forms, which follow it and emanate from it, in proportion as the process of budding is repeated. The field was covered with this minute form, and we had to search about considerably before we could find any of the large cells and the long, branching, jointed filaments which we have sketched. The reason of this was, that these large, extended filaments only appear at the beginning, when there is still an abundance of air, giving place, after repeated budding, to minute cells or short filaments, the ever-increasing number of which soon hides the others from sight.

[Illustration: Fig. 34.]

[Illustration: Fig. 35.]

Fig. 35 represents _saccharomyces pastorianus_ again, as it appears after having been exhausted by two years preservation in yeast-water, in contact with pure air. Strange to say, it has lost its elongated appearance, and would appear to have originated from a round ferment. The cells are much exhausted, and most of them seem to have a double border; their interior is very granular and of a yellowish colour. One might readily take the specimen to be a dead old ferment, which, however, it by no means is.

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.

[Illustration: 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 is clear, and it is full of fine, yellowish granules, which present a perfect resemblance to the large ferment-cells of fruits, proceeding from the germ-cells on the surface of those fruits, when it begins to appear in sweet juices; _b_ is a long filament, sprung from the preceding cell; and, last of all, _a_ is a joint and its bud, in which the border is not yet very clearly defined; it has scarcely any granules, and is finer than the others, belonging, in short, to the small ferment form represented in Plate XI. Here, then, we see the transition of the large ferment to the small, on the same branch, after two generations from the germination of the germ-cell _d_. This observation corroborates the opinion maintained by us, that in Figs. 33, 34, 36, as in Plate X., we have not a mixture of two ferments, the one consisting of large, elongated filaments, the other of small cells, but one and the same ferment, the differences in the form and size of which depend on particular conditions. The smallest ferment-form very soon becomes the only one visible, and it preserves its peculiar appearance in successive growths from inability to return to the full, elongated, filamentous forms before undergoing a prolonged exhaustion. The ferment of _mucor_ would probably afford similar indications: it would be very interesting to find out.

[Illustration: 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 place at the bottom. We made a microscopical examination of this deposit, a sketch of which we append (Fig. 37). The dark, double-bordered cells are those which were sown but did not rejuvenesce. We may notice in different places several of these same cells, recognizable by their granular contents, which they are beginning to lose, to make room for germinating cells and joints, often numerous. For instance, in the group at the bottom of our figure one of the cells is in course of rejuvenescence and germination, and has given rise to no fewer than six cells, filaments, or groups of filaments. In different fields of our microscope we met with a crowd of branches, more or less ramified, and chains of cells, of greater or less length, of which we have sketched a few. In proportion as the budding of these branches is repeated, the cells and joints become more readily disunited, grow small, and assume the appearance of _saccharomyces pastorianus_ in ordinary growths, almost as represented in Plate XI. At first, when the old, exhausted cells begin to germinate, their appearance rather resembles that of _dematium pullulans_, as seen in the germination of many of the corpuscles on the surface of clusters of grapes or fruits, or their woody parts, some specimens of which are to be found in Plate IX.

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.[97] Of these various ferments one deserves special mention—namely, the variety termed _saccharomyces pastorianus_. As is the case with all ferments, when we gather it from the deposits produced in must that has been fermented by its action, it is composed entirely of oval or spherical cells or of short joints. When again placed in a similar must it buds, like all the ordinary ferments, and the buds detach themselves from the joints or mother-cells as soon as they have attained the size of these latter, from which time in the new deposit is reproduced the original ferment-form from which it sprung, and so on. Under certain conditions of exhaustion, however, which may be easily obtained, and which we have already accurately described, the cells undergo an absolute change as regards their capabilities of budding and germinating. Each cell, modified in its structure by the conditions we have mentioned, shows a tendency to shoot out all around its surface, with astonishing rapidity, into a multitude of buds, from many of which spring branching chains, covered in parts, and more especially at the internodes, with cells and jointed filaments, which fall off and bud in their turn, soon to present the forms of the yeast deposit. In this way _saccharomyces pastorianus_ seems to afford a kind of bond of union between the race of ferments on the one hand, and certain kinds of ordinary fungoid growths on the other. Of these latter the plant which De Bary has named _dematium_, and which is generally found on the surface of leaves or dead wood, more especially, however, on the wood of the vine at the end of autumn, the time of the vintage, presents a striking example.

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 _dematium_ produce at a certain stage of their vegetation, in addition to aërobian cells and torulæ, other cells and torulæ which are anaërobian, that is, alcoholic ferments.

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 it is difficult to eliminate two sources of error. On the one hand, it is beyond doubt that cells of ferment are actually scattered over everything, and that, consequently, they may easily get into the experimental liquid along with the spores that we sow, and so occasion mistakes. On the other hand, there are a great many fungi which develop budding processes similar to yeast, but incapable of producing fermentation, which yet in some cases spring directly from spores as well as from mycelium, especially we may instance _exoascus_. This last observation is especially applicable to the extraordinarily numerous variety of fungi which rank under the Dematiei and Sphaeriacei, and which I shall term, for convenience of naming, _dematium pullulans_.”[98]

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 infusoria in general. All these foreign organisms would tend to develop just in proportion as the conditions of the media were more or less favourable to their growth, and, in a very few days, our flasks would be filled with swarms of beings which, in most cases, would entirely conceal the facts relating to those forms, the separate study of which it was our object to follow out. We shall have occasion, therefore, to examine, in a subsequent paragraph, the preparation of ferments in a state of purity. At present we may state that yeast, which in its ordinary condition is a mass of cells so liable to change that its preservation in a moist state is impossible, manifesting in the course of a few days during the winter, and in twenty-four hours during the heat of summer, all the signs of incipient putrefaction, thereby losing its distinctive characteristics, is nevertheless capable, when pure, of enduring the highest atmospheric temperatures for whole years without showing the least signs of putrid change or contamination with any other microscopic organisms, and without the cells losing their power of reproduction. In the presence of facts like these, the theory of spontaneous generation must seem chimerical. The hypothesis of the possible transformation of yeast into _penicillium glaucum_, bacteria, and vibrios, or conversely, which the theories of Turpin, H. Hoffmann, Berkeley, Trécul, Hallier, and Béchamp involve, is equally refuted by these facts.

§ 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, as we have shown, it is absolutely necessary for the juice to come into contact with the surfaces of the fruit, so that the ferment may be mixed with it, and so produce subsequent fermentation. Therefore, although we may apply the term _spontaneous ferment_ to the ferments of fruits, we intend that expression to apply in this paragraph solely to those ferments that are generated in a saccharine liquid, in which, by previous boiling, we have destroyed all ferment germs, and which, nevertheless, enters into fermentation after being exposed in free contact with air. In such a case it is entirely from the particles of dust floating in the air that the ferment germs that appear in the liquid are derived. Such are typical _spontaneous_ fermentations, and it is of the ferment so obtained that we are about to speak.

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 brought about several spontaneous fermentations, the germs of which could have been introduced by nothing but the particles of dust in the air. These fermentations, which we were obliged to follow very carefully with the microscope from the time when they first manifested themselves, on account of the transformation that we were seeking, which transformation we thought might possibly be that of the cells of _mycoderma vini_ into cells of ferment, generally gave us during the first days of fermentation the large, elongated, branchy ferment represented in Plate X., which was succeeded by the small ferment represented in Plate XI.[99]

[Illustration: 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 fermentation and increasing the proportion of yeast. On March 19th we made a fresh sketch, which it is not necessary either to reproduce; suffice it to say, that the yeast was now considerably more regular and uniform in appearance.

[Illustration: 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.

The sketch was made directly after the fermentation had commenced. Probably this is simply one of the earlier forms assumed by the _saccharomyces_, or by one of its varieties. It will be seen that the alcoholic ferment is associated with another little filiform ferment, probably the lactic. The spontaneous ferments are almost always impure, a circumstance that may be readily understood if we bear in mind the results described in Chapters III. and IV.

§ 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 from breweries, where and when it would be hard to say. In the case of the first working brewery, the yeast was, no doubt, derived from some spontaneous fermentation, which took place in an infusion of barley that had been left to itself, or, from some natural spontaneous ferment, and nothing could be easier than to realize this fact again. In the brewing industry there are two distinct modes of fermentation:—“high” fermentation and “low” fermentation, some of the distinctive characteristics of which we have pointed out in