The above-mentioned transmission flasks are constructed chiefly with regard to brewery requirements. Only small glasses or bottles are necessary for wine fermentation, as it is then generally a question of sending small quantities of yeast.

5.—Preparation of Spore Cultures.

Spore Cultures of Saccharomyces. Even at the present day the statement sometimes occurs that a spore formation will be produced merely by sowing a little yeast on a moist gypsum block, a potato, or slice of carrot. In so far as the conditions of spore formation are treated, quite incorrect statements are even now not infrequently made, e.g., that the yeast should be well washed beforehand, that the whole process depends upon a starving condition, etc. The old error of the formation of spores at a low temperature is now less frequently met with. Under the circumstances just mentioned it will, however, depend on chance, as regards most species of Saccharomyces, whether they form spores or not.

The essential part of the method does not consist in the use of any particular substratum, such as gypsum blocks, potato or carrot slices. The substratum on which the cultivation proceeds is in the main unimportant. A shallow layer of water in a culture flask, gelatine, etc., may be used with equal advantage, as will be shown later. The chief point lies simply in the use of a moist surface. But it is essential to the method that we should know the best conditions for favouring the function in question. These conditions were ascertained by Hansen and the essentials published in 1883, additions being made in later communications. (A more detailed explanation of the physiology of this function is given in the next section.) According to these investigations a copious formation of spores takes place, if (1) the

growth consists of strong young cells; (2) a high temperature is employed (for most species about 25° C.); and (3) the supply of moist air is plentiful.

The technique of the Hansen method of spore formation is as follows: A flask containing wort is infected with a small quantity of the yeast species in question, shaken up and placed in a thermostat at 25° C.1 In general a quantity of yeast sediment forms in twenty-four hours sufficient to perform the seeding out on the gypsum block. The supernatant fermenting wort is poured off, and a small quantity of yeast taken out by means of a pipette and spread in a thin layer on a dry, sterilised gypsum block in a glass dish (see Fig. 38, p. 65). It is important to make the yeast layer thin; with thick layers the oxygen of the air has no access to the lower cells. As soon as the layer of yeast is spread on the gypsum block, sterile water is poured into the dish until the gypsum block is immersed to about two-thirds of its height.

The addition of water is made by means of the water holder described on page 67 (Fig. 40). During this manipulation the cover of the glass dish must not be raised any higher than is necessary and the whole operation must be done as quickly as possible, because gypsum block cultures are very easily infected from outside. However, if the operation is skilfully done the infection is inconsiderable. As soon as the gypsum block is soaked with water, which is recognised by the glistening of the yeast layer, the culture is set away at the desired temperature.

The use of gypsum blocks was proposed by Engel. Similar substrata were recommended later by others, e.g.,.

1 The culture instead of standing for twenty-four hours at 25° C. may be left for forty-eight hours at the room temperature. If the growth is old it is advisable to freshen it once or twice at the ordinary temperature before it is used. In determining spore curves this has always to be done.

chamotte" blocks by

earthenware cubes by Elion and “ Wichmann. According to experiments made by the author the latter are very inferior to the gypsum blocks, the formation of spores beginning later and the number of sporebearing cells being fewer than when gypsum is employed. The porcelain cubes mentioned were found to be almost as good as the gypsum blocks.

If it is desired to obtain a bacterium-free spore culture of a Saccharomyces according to Hansen, a thin layer of water may be worked with in a Freudenreich or Hansen flask or in a moist chamber, e.g., Ranvier's chamber, with access of air; or a seeding out on gelatine without addition of nutrient substances may be also used. Good results have been obtained from shallow water layers in flasks and moist chambers.

In many cases somewhat more copious formation of spores may be obtained on the gypsum blocks than in thin water layers; if it is desired at the same time to protect the gypsum block culture from infection, it may be placed, according to Schiönning, in a Hansen flask (see Fig. 39, page 66). Sterile water is then added from another Hansen flask, the two side tubes of the flasks being connected. The yeast, on the other hand, is put on the gypsum block by means of a pipette through the neck of the flask.

It has been shown on page 99 that the influence of light may also be employed in the preparation of bacterium-free spore cultures of saccharomycetes.

The spores of saccharomycetes may sometimes be confused with other formations, especially with fat or oil drops, which are frequently found inside yeast cells. (If the preparation is treated with perosmic acid it may be easily ascertained if the bodies in question are of a fatty nature, as they then become brown or black. Fatty particles dissolve also in alcohol and ether, and are again precipitated on the

addition of water.) The practised microscopist will, however, soon learn to distinguish spores from other objects. There is no decisive colour reaction for spores; they are usually stained by means of Ziehl's carbol fuchsine (see page 88) and retain their colour after the preparation has been decolorised with dilute acid. Experiments made by the author have shown further that spores are sometimes not stained by this process, and that on the other hand particles other than spores may be stained. Thus the method is uncertain. The mode of formation, the anatomical structure and germination furnish reliable characteristics for distinguishing whether a particle is a spore or not.

Spore Cultures of Bacteria. There is no perfected method for inducing spore formation in bacteria similar to that which Hansen has described for saccharomycetes. In nearly all species of bacteria, spore formation occurs without using any special method of cultivation, being brought about merely by allowing the cultures to remain after the substratum has become poor in nutriment or has become unsuited for the growth from any other reason (as, e.g., by the accumulation of fermentation products).

Spore Cultures of Moulds. -Zygospore formation is a phenomenon frequently observed in the Mucorineæ (see section III.). The conditions of this are still unknown, and there is therefore as yet no definite method of producing it. Bainier states that Mucor racemosus forms zygospores on gypsum blocks which are placed in dextrose solution. The author has tested this statement, but has obtained no positive result.

In the Mucorineæ and Aspergilleæ sporangia and conidia respectively are always formed when the mycelium grows on the surface of the culture medium, and when the latter is in other respects in a fit condition to act as a food. A mycelium immersed in liquid forms neither sporangia nor conidia.

In Aspergillus, ascospores are only known in those species which are classed under Aspergillus glaucus and A. repens (see Section III.). Although the particular conditions of ascospore culture are not known for these species, yet it is easy to produce them, as such spores always form when the culture is allowed to stand.

In Penicillium glaucum, which likewise comprises several species, a formation of sclerotia precedes the formation of ascospores (see Section III.). These sclerotia were obtained by Brefeld by infecting coarse bread, free from sourness, with conidia, and placing this between two glass plates which were pressed tightly together. Sclerotia developed in the course of three weeks. They were then washed and spread on moist filter paper, after which asci developed in their interior. In this species also the exact conditions are unknown.

6.—Preparation of Film Cultures of Saccharomyces. Since film formation in the saccharomycetes plays a considerable rôle in the characterisation of the species, it will be necessary in many cases to prepare film cultures. The conditions for a vigorous film formation are, according to Hansen, the seeding out of a strong young growth on a favourable culture medium to which air has free access, and the placing of the culture in complete quiet at a moderate temperature. Film cultures are best formed by seeding out the Saccharomyces in an Erlenmeyer or Pasteur flask half filled with wort, this being set away in an undisturbed position at the room temperature. Hansen, Will and others have determined the cardinal points for some species. When the optimum temperature is known this is of course employed.