The American Brewer Dec. 1934
Longevity Of Yeasts Investigated By Many Authorities
by Leon Milard
Many authorities have investigated the longevity of yeasts. There are numerous reports of old cultures regaining their vigor and recovering their original characteristics through propagation in suitable media after having been kept for many years in liquids completely lacking nutritious bodies and charged with excretory products. Duclaux in his “Treatise on Microbiology” relates observations he made in 1885 on old cultures which Pasteur had used in his work on fermentation; of fifteen cultures, from six to nine year old, he found only three had died. In 1889 similar tests on twenty-six yeasts, from eleven to seventeen years old, resulted in six cases of non-growth. In 1897 in two beers, twenty-two and twenty-three years old, he found yeasts still living and rejuvenated them to normal characteristics by propagating in fresh media. In other tests he reported as having observed living yeasts in the culture medium twenty-five years old.
Duclaux found that the majority of the samples on which his experiments were carried out had been kept in Pasteur flasks not hermetically sealed, having permitted obviously the total loss of CO2 and part of the alcohol derived from the fermentation. These cultures, therefore, were open to oxidation but their taste was unimpaired. The yeasts of these cultures, examined microscopically, showed all the characteristics of old cells—thick walls, granular protoplasm, etc. On studying this curious phenomenon of prolonged resistance of cells in culture media, Duclaux became convinced that the yeasts did not remain inactive in the media in which they had multiplied. Before the exhaustion of nutritive elements constituting their normal food, the cells little by little consumed the glycerine produced by themselves in the fermented media and also attacked the dextrins. Therefore, there had been persistent life among the old cells. These did nor perish through failure of food, a fact which explains the rarity of spores in very old cultures of yeast.
Duclaux likewise has drawn attention to the interesting fact that yeasts, regenerated in fresh media, do not for long survive when this regeneration has been effected in small volumes of liquid, while the same yeasts remain living in their original flask. He is of the opinion that this is due to the fact that yeast, being deprived of all carbohydrate food, attacks the nitrogenous matter left unconsumed and converts some of it into ammonia which, by turning the media alkaline, causes the death of the cells which are not able to live in presence of such a reaction. Duclaux, in fact, previously had found that neutral or acid media in which yeasts of old cultures had been seeded, showed an alkaline reaction some years after this seeding, sometimes a very high one.
Hansen, according to the experiments he made on the same subject, considers solutions of cane sugar without acid as being the most favorable media for the preservation of yeast. By comparing the longevity of certain species, sown at the same time in sugar solution and in beer wort, he found that in the majority of cases the longevity was much greater in the former of these media, all the tests having been made in flasks simply closed with cotton wool. The same tests made on the same yeasts, in closed (sealed) flasks caused the death of all samples after two years’ preservation, while in the flasks closed with cotton wool certain yeasts survived for more than sixteen years.
On the other hand, preservation tests on yeasts in water alone enabled Hansen to ascertain that yeast resists destruction much longer when it is present in a quantity sufficient to make the water cloudy on agitation. When present in very small amounts, and the water does not become obviously turbid on shaking the vitality of the yeast is diminished considerably. From this Hansen concludes that when the yeast is present in sufficient quantity the old cells nourish the young ones and that the yeast escapes death from starvation for a longer time; this observation also had been made by Pasteur in his researches.
Therefore, one may conclude from the observations that the longevity of yeast is the consequence of the presence in the medium of a plant food—not transformable by zymase, such as sugar—which does not ferment directly and is consumed very slowly; and that the yeast seeded in media to which alimentary substances have been added, therefore may survive longer and be found as Duclaux puts it, in much the same conditions as a long-lived plant cultivated in soil. The same author likewise is of the opinion that the extraordinary vitality of beer yeasts appears to be favored by the fact that the media containing them are contained in special flasks, closed by a simple wad of cotton stoppers, which does not prevent slow changing of the air, while protecting the cultures against all infection through dust; and also because these cultures, generally preserved in dark cupboards or in only moderately lighted rooms, are withdrawn from a too marked influence of light. It seems, according to Duclaux, that these are two essential conditions for the long preservation of beer yeasts.
Lastly, the Laboratory of Microbiology of the Institute of the Fermentation Industry at Brussels has made public another account of the surprising longevity of brewery yeasts. The director of the Institute, Marc Van Laer, found among old cultures inherited from his father, the late Henri Van Laer, a certain number of flasks containing beers fermented by various yeasts he had been studying. All these samples were preserved in Van Laer flasks, neck closed by cotton stoppers and provided with a side tube for seeding purposes, closed by rubber tubing with a glass rod in it. Some of these cultures in which the rubber tubing had dried, hardened and become cracked, were covered with mold. The indications on the labels, still in part decipherable, showed that the cultures were made between 1893 and 1897, that is, they were from thirty-six to forty years or age.
When eighteen of these samples were re-seeded in sterile un-hopped wort and placed in an incubator at 25° C., nine developed after periods varying from ten to fifteen days, giving rise to characteristic fermentations. The microscopic examination of the samples before regeneration showed very old cells, thick walls, strongly granular and having ordinary and varied shapes for this kind of cell; stunted or elongated, more or less like Pastorianus with mycelial aspect. After regeneration, these nine samples having produced fermentation, the cells had regained vigor and among the old cells microscopic examination showed others of more satisfactory appearance, normal, round or oval shapes, protoplasm clear and homogeneous, cell wall thinner, vacuoles and buds relatively numerous.
In such samples some sporulating cells were met with. Among others there were rejuvenated cells of the shape of S. Cerevisiae, sometimes showing evidences of branching characteristic of certain types of English yeast from which the cultures probably were derived. Some regenerated cultures developed mold after fermentation. Among those which had escaped infection, one clearly dated 1894, appeared to be a top fermentation yeast of a Belgium brewery.
It would be interesting particularly to study more intensively those of the yeasts which have demonstrated such extraordinary longevity and see if they have preserved intact their special characteristics. This is what the author proposes to do and publish the findings in a future article.