6o 



PHYSIOLOGY OF NUTRITION 



Let it be supposed that we have a fermenting beer-wort with many different 

 species of yeasts, and that these are to be separated, so that each species may 

 be had in pure culture. After shaking the liquid, several drops are taken up in 

 a sterilized pipette and transferred to a Freudenreich flask (Fig. 36) partly filled 

 with sterilized water. This flask is of glass, with a capacity of 

 from 25 to 30 cc, and is closed by means of a glass cap shaped 

 like a short, inverted thistle-tube, the small opening of which is 

 plugged with cotton. To obtain a uniform distribution of the 

 [f f| yeast cells throughout the liquid, the flask is thoroughly shaken, 

 after which a drop of the contents is transferred, upon the bent 

 end of a platinum wire, to the surface of a microscope cover glass 

 which is marked off into small squares. Here the drop is spread 

 out into a thin layer, and the number of cells present is determined 

 by counting. A van Tieghem cell, or moist chamber, is used for 

 this purpose (Fig. 37). This consists of a slide upon which a glass 

 ring (c) is sealed with vaseline. A small quantity of water {d) is 

 introduced into the chamber so that microorganisms clinging to 

 the under side of the cover glass (a) may not become desiccated. 

 The cross-ruled cover glass is sealed to the glass ring with vaseline, the culture 

 drop hanging from its lower surface (b). The divisions marked upon the 

 cover glass facilitate the counting of the cells under the microscope. 



Suppose that twenty cells are found upon the cover glass. The drop of 

 liquid is again transferred, by means of the platinum hook, to a fresh Freunden- 

 reich flask containing 40 cc. of sterilized water. After vigorous shaking about 1 

 cc. of this liquid is transferred (with a pipette) 

 into each of forty Freudenreich flasks containing 

 sterilized beer-wort. Since the original drop con- 

 tained only twenty cells, we should expect that 

 the yeast would, in all probability, develop only in 

 twenty of the flasks while the other twenty would 

 remain sterile 



Fig. 36. — 



Freudenreich 

 flask. 



o.- 



c- 



- 



,1 



Fig. 37. — Moist chamber, or 



van Tieghem cell, for microscopic 



work, a, cover glass; b, position 



of drop of medium; c, wall of 



It is also highly probable that the chamber made of section of glass 



new generation has arisen from only a single cell -^SSiS s ° lutfo " " 

 in those flasks where growth does occur. All 



this is only highly probable, however, and not definitely established. Hansen 

 employed this method in his work with yeasts. Flasks containing freshly 

 inoculated beer-wort are vigorously shaken and then allowed to stand. The 

 cells sink to the bottom and begin to multiply, so that, after a time, whitish 

 colonies of cells become visible with the unaided eye. If a flask shows but 

 one such colony it follows that only a single cell was introduced, since it is 

 highly improbable that two cells might have settled together after the shaking. 

 If, on the other hand, two or three cells have been introduced into the flask, 

 then two or three colonies, respectively, develop. 



In order to secure pure yeast cultures, solid substrata may also be em- 

 ployed, which make it possible to follow, under the microscope, the development 

 of a colony from a single cell. For this purpose a drop from a young yeast cul- 



