Another point in the cultivation of yeasts has been elucidated by a number of workers, chief among whom perhaps is Hansen, namely, methods of obtaining pure cultures. We know, generally speaking, what this term means, and there is no difference in its meaning here to what is understood as its meaning with regard to bacteria. There is, however, some difference in the mode of securing it. It is only by starting with one individual cell that we can hope to secure a pure culture of yeasts. For the study of the morphology of yeasts under the microscope the problem was not a difficult one. It was comparatively easy to keep out foreign germs from a cover-glass preparation enough to perceive germination of spores and growth of mycelium. But when we require pure cultures for various physiological purposes, then a different standard and method are necessary.
| Yeast (Saccharomyces Cerevisiæ) × 1000 | Ascospore Formation in Yeast (The capsule of the parent cell around the spores is invisible) × 1000 |
| Nitrogen Fixing Bacteria from Rootlet-Nodules (Subculture) × 1000 | Bacillus of Tetanus (From broth culture, showing spore formation) × 1000 By permission of the Scientific Press, Limited |
Pasteur and Cohn adopted a practice based upon the fact that when organisms find themselves in a favourable medium they multiply to the exclusion of others to which the medium is less favourable. Hence if an impure mixture be placed under such circumstances there comes a time when those organisms for which the circumstances are favourable multiply to such an extent that they form an almost pure culture. The method is open to fallacy, and will rarely result in a really pure culture; and even if that be secured, it is quite possible that it will be to the exclusion of the desired culture. Hansen has devised a much improved process for securing a pure culture of yeast which depends upon dilution. We believe Lister was one of the first who, in the seventies, introduced some such plan as this. Hansen employed dilution with water in the following manner:
Yeast is diluted with a certain amount of sterilised water. A drop is carefully examined under the microscope, a single cell of yeast is taken, and a cultivation made upon wort. When it has grown abundantly a quantity of sterilised water is added. From this, again, a single drop is taken and added, to, say 20 cc. of sterilised water in a fresh flask. This flask will contain we will suppose ten cells. It is now vigorously shaken, and the contents are divided into twenty portions of 1 cc. each, and added to twenty tubes of sterilised water. It is highly probable that half of those tubes have received one cell each. In the course of a few days it can be seen how far a culture is pure. If only one colony is present, the culture is a pure one, and as this grows we obtain an absolutely pure culture in necessary quantity. Even when the gelatine-plate method is used it is desirable to start with a single cell (Hansen). The advantage of Hansen's yeast method over Koch's bacterial-plate method is that it has a certain definite starting-point. This is obviously impossible when dealing with such microscopic particles as the bacteria proper.
A third matter in the differentiation of yeast species is the question of films. Hansen set to work, after having obtained pure cultures and ascospores, to examine films appearing on the surface of liquids undergoing fermentation. The object of this was to ascertain whether all yeasts produced the same mycelial growth on the surface of the fermenting fluid. To produce these films the process is as follows: Drop on to the surface of sterilised wort in a flask a very small quantity of a pure culture of yeast; secure the flask from movement, and protect it, not from air, which is necessary, but from falling particles in the air. In a short time small colonies appear, which coalesce and form patches, then a film or membrane which covers the liquid and attaches itself to the sides of the flask. By the differences in the films and the temperatures at which they form it is possible to obtain something of a basis for classification. The further advances in a yeast culture and in our knowledge of the agencies of fermentation have, however, tended to show that no strict dividing lines can be drawn. Hansen's researches have, notwithstanding, been of the greatest moment to the whole industry of fermentation. What has been found true in bacteriology has also been demonstrated in fermentation, namely, that though many yeasts differ but little in structure and behaviour, they may produce very different products and possess very different properties. Industrial cultivation of these finer differences in fermentative action has to a large extent revolutionised the brewing industry.
The formation of films is not a peculiarity of certain species, but must be regarded as a phenomenon occurring somewhat commonly amongst yeasts. The requisites are a suitable medium, a yeast cell, a free, still surface, direct access of air, and a favourable temperature. The wort loses colour, and becomes pale yellow. Microscopic differences soon appear between the sedimentary yeast and the film yeast of the same species, the latter growing out into long mycelial forms, the character of which depends in part upon the temperature. This often varies from 3° to 38° C.
A fourth point helpful in diagnosis is the temperature which proves to be the thermal death-point. Saccharomyces cerevisiæ is killed by an exposure to 54° C. for five minutes, and 62° C. kills the spores. As a rule, yeasts can resist a considerably higher temperature when in a dry state than in the presence of moisture.
Lastly, yeasts may be cultivated on solid media. Hansen employed wort-gelatine (5 per cent. gelatine), and found that at 25° C. in a fortnight the growths which develop show such microscopic differences as to aid materially in diagnosis. Saccharomyces ellipsoideus I. exhibits a characteristic network which readily distinguishes it.
There is one other point to which reference must be made. The process of fermentation may be set up by a "high" or a "low" yeast. These terms apply to the temperature at which the process commences. "High" yeasts rise to the surface as the action proceeds, accomplish their work rapidly, and at a comparatively high temperature, say about 16° C.; "low" yeasts, on the contrary, sink in the fermenting fluid, act slowly, and only at the low temperature of 4° or 5° C. This is maintainable by floating ice in the fluid. Formerly all beer was made by the "high" mode, but on the continent of Europe "low" yeast is mostly used, while the "high" is in vogue in England. This latter method is more conducive to the development of extraneous organisms, and therefore risky in all but well-ordered brewing establishments. Whether high and low yeasts consist of one or several species is not known.
Before proceeding to mention shortly some of the commoner forms of yeast we must again emphasise Hansen's method of analysis in separating a species. The shape, size, and appearance of cells are not sufficient for differentiation, because it is found that the same species when exposed to different external conditions can occur in very different forms. Hence Hansen established the analytical method of observing (1) the microscopic appearance, (2) the formation of ascospores, and (3) the formation of films. In addition, the temperature limits, cultivation on solid media, and behaviour towards carbohydrates, are characters which aid in the separation of yeasts. By basing differentiation of species upon these features, the following can be distinguished: