ASSIMILATION (IF CARBON 



59 



have exactly the same form. On the other hand, a culture obtained from a 

 single cell is called a pure culture, even though the microorganisms therein 

 contained exhibit diverse forms, since we now know that one and the same 

 species of bacterium or yeast can assume different forms, according to its 

 developmental stage and the influence of the medium in which it is grown. 



The method most frequently used for the production of pure cultures is 

 that of dilution. This method was first used, in its original form, by Lister 1 

 in 1878, to obtain a pure culture of lactic acid bacteria. It was carefully 

 elaborated for yeasts by the Danish bacteriologist, Hansen in 1881/ 



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 

 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 



Fig. 36.— 



Freudenreic h 

 flask. 



Fig. 37. — Moist chamber, or 

 van Tieghem cell, for microscopic 



work, a, cover glass; b, position twenty of the flasks while the other twenty would 



or drop or medium; c, wall of / J 



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



botton/of cWbe / solution in new generation has arisen from only a single cell 



in those flasks where growth does occur. All 



1 Lister, Joseph, On the lactic fermentation and its bearings on pathology. 

 29:425-467. 1878. 



'Hansen, 1896. [See note r, p. 44.]. — Ed. 



Trans. Pathol. Soc. London 



