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 phenomena are to be explained much more simply. The Dutch yeast employed being very impure must have contained traces of foreign ferments, especially of _saccharomyces pastorianus_. Reduced to a dry powder on December 17th, 1872, the two or more varieties of cells comprising it had preserved their vitality in consequence of the plaster, and this vitality had continued at all events until July 25th, 1873. Subsequently, when cultivated in wort, they had multiplied in that medium. The _saccharomyces_ had revived like the rest, but its quantity, compared with the high Dutch yeast, was so small that the microscopical observations made on August 2nd, when the flask was decanted, failed to discover its presence. Between August 2nd and November 15th the high yeast must have perished entirely: the cells of _saccharomyces_, on the contrary, still maintained their vitality, and these alone multiplied in the flask of wort impregnated on November 15th. Here we have an example of the separation of alcoholic ferments, through the unequal resistance they sometimes offer to adverse conditions to which they may be subjected. We may also conclude that if we had prepared a quantity of beer with the “high” yeast, which in our experiment of August 2nd, 1873, seemed to have developed in a state of entire purity, this beer when made and stored in cask or bottle could not have failed to undergo a secondary fermentation, in consequence of a development of _saccharomyces pastorianus_.

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 de la Société chimique_ may be consulted. Now, these various ferments mutually interfere with each other: whereas if _saccharomyces apiculatus_ were there alone it would multiply to a greater extent, and with greater advantage to the fermentation of the must. This result is obtained by filtering the must, as we have already observed.

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.[110]

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 raw yeast in a flask of sweetened water. On the 24th we poured off the liquid, and supplied the deposit with fresh sweetened water. The exterior temperature was 12° C. (54° F.). On the 27th we took some of the deposit and put it into a flask of wort. The following days there was a development of yeast, accompanied by fermentation. We obtained, however, neither the large forms of the ferments of fruits, nor those of the more minute ferments represented in Plate XI. The _saccharomyces pastorianus_, represented in the yeast which we sowed by aged, granular, elongated cells, had, therefore, not revived. Fearing that this result might have been attributable to insufficiency of the exhaustion, which had only lasted for a few days, we raised the temperature of the flask of sweetened water to 25° C. (77° F.), at which we kept it until February 20th. On that day we sowed some of this yeast in wort. There was a very perceptible revival the next day, but it was still impossible to detect with the microscope the forms we have just mentioned, nor did _saccharomyces pastorianus_ appear in fresh, succeeding growths.

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.

We cultivated this yeast (Fig. 58), to some considerable extent, in beer-wort. It produced a peculiar beer, of vinous character, in fact a true _barley wine_. This proves, we may here remark, that ordinary wine, in its flavour and quality, depends to a great extent on the specific nature of the ferments which develop during the fermentation of the vintage; and we may fairly assume that if we were to subject the same must to the action of different ferments we should obtain wines of different characters. With a view to the practical application of this idea, it would be well to undertake new studies in this direction; and the methods of cultivating and managing ferments, explained in this work, would be of great value in such researches.

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.

In the place of flasks we may make use simply of shallow basins, covered with sheets of glass, such as we have already had occasion to describe, for cultivating yeast in wort after it has been for a longer or shorter time in the sweetened water. The success of these methods of purification is mainly due to the fact that wort is highly aerated, and experience shows that the principal disease-ferments of beer are as much checked in their development by the presence of air as they are favoured by its absence, the inverse of which holds good in the case of alcoholic ferments. So true is this that, working with commercial yeast, which is invariably impure, it would be impossible in our opinion to make beer in closed vessels; and, indeed, as a matter of fact, one has never succeeded in doing this, although the attempt has often been made. To do so requires, much more than in methods actually in use, the employment of pure yeast.

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 assure ourselves that this organism has not taken the place of the yeast we are endeavouring to purify. Growths at a very low temperature are of great help in enabling us to get rid of all ferments that are foreign to “low” yeast, and should be resorted to in all cases where this yeast is to be purified.

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 this flask, after fermentation, in an oven at a temperature of 20° or 25° C. (68° to 77° F.). If the beer, in the course of a few weeks, does not thicken, or become covered with efflorescence, if its deposit is microscopically pure, if, in short, it only tastes flat, we may have every confidence in the purity of the yeast which produced it. After we have purified a yeast we are, unfortunately, never sure that it has not undergone some change in the course of the manipulations to which it has been subjected in purification. It is indispensable, therefore, that we should test it, and see if the flavour of the beer produced by it is really the one that we want—viz., that of the beer from which we took the yeast that we submitted to purification.

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.

To what may we attribute the peculiarity of the beer as just described? It is probable that during our processes of purification some ferment had taken the place of the principal yeast. Commercial yeasts, even those with which the brewer is thoroughly satisfied, generally contain various ferments, which are maintained in their relative proportions, or very nearly so, by the uniform conditions under which work is carried on in a brewery; but these proportions, it is obvious, might be very seriously affected by any radical change in the conditions of growth.

Footnote 82:

Extract from a Note which I inserted in 1862 in the _Bulletin de la Société chimique_ of Paris.

Footnote 83:

Schützenberger, in his work on “Fermentation,” following Dr. de Vauréal. Paris, 1875, p. 278. [See pp. 61, 62 English version in International Scientific Series (H. S. King & Co., London, 1876). This appears to be the only reference to this subject in the English copy.—D. C. R.]

Footnote 84:

The principal result of Dr. Rees’ labours consists in the discovery of a sporulation peculiar to yeast cells, that is to say, to a formation in the interior of these cells, and under particular conditions—such as when the growth occurs on slices of cooked potatoes, carrots, &c.—of two, three, or four smaller cells, which, when placed in fermentable liquids, act like the germinating spores of ferments. The mother-cell may be regarded as an _ascus_, and the daughter-cells as _ascospores_, and so the genus _saccharomyces_ may be classified among the group of fungi termed _ascomycetes_. These facts have been frequently confirmed, notably by Dr. Engel, professor of the Faculty of Medicine, at Nancy. Previously to Dr. Rees’ discovery, M. de Seynes (_Comptes rendus_, t. lxvii., 1868) had described an endogenous formation of spores in _mycoderma vini_, particularly in the elongated cells, followed by the rupture of the mother-cell, and subsequent absorption of cell-walls and other contents after the issue of the endospores, which we have just termed _ascospores_. We ourselves had also previously called attention to those refractive corpuscles which appear amongst vibrios as probably being reproductive corpuscles, and we had likewise witnessed the reabsorption of the parts surrounding them. The plate on page 228 of our “Studies on the Silkworm Disease” represents the phenomena in question.

Footnote 85:

See _Comptes rendus de l’Académie des Sciences_, vol. lviii. p. 144.

Footnote 86:

The plates referred to in this paragraph were exhibited at a meeting of the Academy of Sciences, November 18, 1872, and commented upon by the perpetual secretary, M. Dumas.

Footnote 87:

For these observations, we employed small glass cells, which we made out of some St. Gobain glass by punching holes through it, and then cementing on one side one of the little glasses used for covering objects in microscopical examinations. In this manner we made small troughs, in which we placed some wort that had been boiled, and a drop of the water in which grapes had been washed. To prevent evaporation we covered the cells with a sheet of glass. We examined the liquid in these cells by inclining our microscope to the angle required.[88]

We also made use of cells similar to those employed by MM. Van Tieghem and Lemonnier[89] in their researches on _mucorines_ (Fig. 30).

An apparatus similar to that employed by M. Duclaux in 1853[90] would do equally well. We should be able to work with even greater facility if we employed bulbs like some which we ordered in Germany, some twelve years ago, of the well-known glass-blower, Geissler. We have heard that these bulbs now sold by that maker are much used by German microscopists. They consist of a tube blown out into a flat bulb, the sides of which, in the centre, come sufficiently close together to enclose but a very thin layer of liquid, and to admit of microscopical examination. We may fill these tube-bulbs completely with liquid, to the exclusion of air or we may surround the central drop with air.

Footnote 88:

In our essay on acetic fermentation, published in 1864, we have already described this apparatus, which we employed to follow the multiplication of the jointed filaments of _mycoderma aceti_. See PASTEUR, _Etudes sur le vinaigre_, p. 64, Paris, 1868.

Footnote 89:

VAN TIEGHEM and LEMONNIER, _Annales des Sciences naturelles_, 5th series, _Botanique_, t. xvii. 1873.

Footnote 90:

DUCLAUX, _Comptes rendus des séances de l’Académie des Sciences_, t. lvi. p. 1225.

Footnote 91:

In experiments of this kind there is always a slight increase in the volume of air in the jar. This increase may be very perceptible even when the experiment made with fresh grapes, in August, for instance, causes no fermentation due to the action of yeast. After the oxygen of the air has been absorbed and replaced by carbonic acid gas, either by direct oxidation or by the action of moulds, the grapes, although crushed, act like fruits plunged into carbonic acid gas[92], and this effect is even more marked in the case of imperfectly crushed grapes. The reason is, that the crushing is never so perfect as to injure all the cells of the parenchyma. We may easily convince ourselves that the experiment on the liberation of carbonic acid gas and the formation of alcohol by grapes and fruits in general when plunged into carbonic acid succeeds very well in the case of fragments of fruits or grapes, and succeeds better the less the parts are crushed.

Footnote 92:

See paragraph: Fermentation in saccharine fruits immersed in carbonic acid gas, Chap. vi, § 2, p. 266.

Footnote 93:

Dr. Rees has given the name _saccharomyces ellipsoideus_ to the ferment of wine represented in Plates VIII. to XI. of our “Studies on Wine,” which we have termed the ordinary ferment of wine, from its being the most abundant of the ferments found at the end of the fermentation that produces the wine.

Footnote 94:

The alcoholic ferments in general, subjected to these weakening influences, have not all the same power of resistance. That one which seems to possess this power in the highest degree is the _saccharomyces pastorianus_, which ferment we had in view in writing the above.

Footnote 95:

The term _exhaustion_ (_épuisement_), which we have just used, was, perhaps, not altogether felicitously chosen. No doubt we exhaust the cells of yeast when we sow an imponderable weight of them in a large quantity of sweetened water; it might, however, be better to say that in such a case we adopt a particular method of preserving the vitality of the cells, without suffering them to die of exhaustion, or to multiply by budding. We may remark that the yeast, in this case, exists in a state of latent life, which resembles that of cells on the surface of fruit. The cells on the surface of fruits, bunches, or barks, can no more find around them sufficient aliment for their propagation than can our yeast-cells in a great excess of sweetened water. We would not, however, say of the spores on the surface of fruits, or their woods, that they are in a state of exhaustion; the term would be misapplied.

Footnote 96:

See foot note p. 79.—D. C. R.

Footnote 97:

M. Béchamp (_Comptes rendus_, November 18th, 1872) asserts that _the air has no direct influence on the production of ferment or on the process of alcoholic fermentation_. That experienced chemist deduces this erroneous assertion from experiments on sweetened water, to which bunches of grapes, petals of corn-poppies and petals of _robinia pseudo-acacia_ had been added. As may be seen in our “Studies on Wine” (p. 7, 1st edition, 1866), these experiments conducted by M. Béchamp in 1872 were merely a reproduction of those made long before with vine leaves, petals of elder-flowers, leaves of sorrel, &c., by the Marquis de Bullion, Fabroni, and other experimentalists. M. Béchamp has modified his later experiments by not adding the bunches of grapes, leaves, &c., to the sweetened water before having introduced carbonic acid gas into the liquid. Fermentation having still taken place in spite of this change, M. Béchamp wrongly concluded that _air has no direct influence on the production of yeast on an alcoholic fermentation_. The introduction of the carbonic acid gas could not remove all the air imparted to the sweetened water by the objects placed in it, and it was this air which remained adhering to these objects that permitted the production of fermentation. We may avail ourselves of the opportunity here presented to add that, in this same Note of November, 1872, M. Béchamp commences by making various assertions concerning the forms assumed by cells of the alcoholic ferment of the grape when in process of fermentation. This question was discussed by us ten years before, and our conclusions supported by sketches, in a Note which appeared in the _Bulletin de la Société chimique de Paris_, for 1862.

Footnote 98:

The germs of ferments are less widely diffused than M. de Bary supposes, as may be seen from our observations in Chap. III. See, too, our Memoir of 1862, _Sur les Générations dites Spontanées_, p. 49. It is only in a laboratory devoted to researches on fermentation, or places such as vaults, cellars, and breweries, that the air holds appreciably in suspension cells of ferments, ready to germinate in saccharine media. If we except these particular circumstances, ferment is not very largely diffused, save on the surface of fruits and the wood of the trees which bear them, and perhaps, also, on some other plants. The particles of dust held in suspension in any atmosphere whatever rarely produce fermentation in pure must even when we take all possible precautions, so that the action be not overlooked; for true fermentation may be hidden by fungoid growths, when there is much air and but a small quantity of saccharine liquid present.

Footnote 99:

In these experiments the apiculated ferment appeared sometimes, but much less frequently than _saccharomyces pastorianus_. We also met with the ellipsoidal ferment. We should probably have a greater variety of ferments if our experiments could be conducted in the open air, but insects and particles of dust of all kinds brought by the wind render experiments under such conditions difficult and untrustworthy. In a laboratory we have not these difficulties to contend against, but, unfortunately, the operations ordinarily carried on there cause the results of our experiments to be of a less general character than they would be if obtained in free contact with country air.

Footnote 100:

[A rather serious clerical error appears to have here crept into the original, for on referring to Plate I. and the letterpress descriptive of No. 7 (p. 5), we find it applies to a very formidable species of diseased ferment, whereas the author is here speaking of an amorphous deposit, harmless in character, and more or less associated with all yeasts. Doubtless No. 7 should stand No. 6, see p. 6.—D. C. R.]

Footnote 101:

[We would here call the reader’s attention to the following extract from Dr. Graham’s appreciative review of this work in “Nature,” January 11th, 1877. He says: “M. Pasteur seems to be in error in stating (p. 190, Fr. ed.) that the bottom yeast may be distinguished by being less spherical than top yeast. It is true that in London and Edinburgh yeast, the cells will be found usually round; hard water, however, such as that at Burton, or artificially made so, yields yeast in which the cells are distinctly ovoid in appearance, resembling very closely Bavarian bottom yeast.”—D. C. R.]

Footnote 102:

[43° F. to 46° F. or 59° F. to 68° F.]

Footnote 103:

[On this point again Dr. Graham expresses some dissent (“Nature,” loc. cit.): “Here surely M. Pasteur must be thinking rather of the inferior products of the surface fermentation in France and Germany, than of those of England and Scotland.”—D. C. R.]

Footnote 104:

[28·4 c.c. = 1 fl. oz. approximately.]

Footnote 105:

[M. Pasteur has evidently employed the word “caseous” to express the curdy nature of the ferment he is describing, its plasticity and other peculiarities of physical character; but we are, nevertheless, tempted to suggest that he may have had in mind also the peculiar “cheesy” odour given off by these very yeasts, which he refers to in the text as containing a considerable intermixture of “caseous ferment.”—F. F.]

Footnote 106:

The caseous ferment, however, must not be exposed to heat, under the afore-mentioned conditions, when it is too young. At the commencement of its development, for instance, within a few days of having been sown. In such case, it would be in danger of perishing, probably in consequence of the tenderness of its tissues. At the end of a fermentation, and even several months afterwards, it might be safely heated to 50° C. (122° Fahr.) without any harm to it. “Low” yeast also can withstand a temperature of 50° C. in the medium in question.

Footnote 107:

Although we believe that the aërobian ferment of a particular yeast may be produced by a kind of transformation of the cells of the latter, yet we admit that this question is open to some doubt. The facts which we unexpectedly discovered in connection with the _caseous_ ferment should make us extremely careful, and disposed to inquire whether aërobian ferments do not originally, in a state of intermixture, form part of the ferments from which they spring. One reason which might incline us to believe this, is the fact that a ferment sometimes perishes without the appearance of aërobian ferment on the surface. There is nothing very natural indeed in the hypothesis that we advance, which sets aside the supposed intermixture; but, on the other hand, if the aërobian ferment is a particular ferment, simply intermixed with some other variety and developed by change of conditions, how are we to account for its great resemblance in appearance and mode of budding to the ferment on the surface of which it appears? This resemblance, however, might be accounted for very naturally if the two ferments were originally related.

Footnote 108:

We insist on this fact, that Fig. 50 represents the forms on revival of the aërobian ferment of _saccharomyces pastorianus, when this has grown in a mineral medium_. When produced on the surface of fermented wort, the aërobian ferment of which we are speaking presents no peculiarity, nor is there any irregularity in its forms or in its development, and when we proceed to cultivate it in a natural saccharine medium, or in wort, it does not produce any forms of _dematium_, as in the preceding case; but the reason of this is that, in consequence of the nature of the first medium, which is better adapted to its nutrition, it assumes at once, in the second medium, the forms of deposit-yeast in the course of ordinary germination.

Footnote 109:

“La cassure de la levûre.”

Footnote 110:

We have reason to believe that the ratio of the proportions of these ferments depends greatly on the climatic conditions preceding the period of vintage, on the state of dryness or humidity, as well as the temperature at the time of gathering the grapes, and also on the nature of the vines.