76 



might be expected, is less rapid and less strong than on similar 

 media with phosphate. I have not succeeded in making a nutrient 

 medium that was microchemically completely free from P. The 

 chances, indeed, are that on such a medium there is no increase 

 of cells at all. 



Volutin-free cultures were thus made from Ustilago maydes, from 

 Torula monosa, from Saccharomyces cerevisiae and from a Lactose- 

 yeast ^) (also a Torula). 



When transplanting the cells upon a phosphate-containing medium 

 they were seen to be loaded again with volutin within a few hours, 

 in a fluid medium even after a few minutes. This also applies to 

 cultures that had been free from volutin for 8 or 9 months. That 

 the}' have preserved the power to produce volutin is evidenced 

 whenever an opportunity offers. The volutin-free yeast-cells react 

 very rapidly on the addition of a small quantity of phosphate; for 

 instance the addition of 0,04 m.grm KH2PO4 to 10 c.c. of fluid will 

 evolve volutin in several cells. Also from purelj' organic compounds 

 the phosphoric compound required for volutin-formation may be 

 obtained in many cases. When using an albumose prepared from 

 white of egg that contained 0.09 m.grm P. per test-tube, a large number 

 of the cells were loaded with volutin. Volutin is also developed 

 rapidly by the addition of nucleinic-acid-sodium to the culture- 

 medium. As for the amount of fat and glycogen no difference worth 

 mentioning could be made out, on microscopic examination, between 

 phosphate-free and phosphate-containing cultures 



In a Torula monosa culture upon a phosphate-free medium we 

 observed in March 1916 a sudden formation of pigment in the 

 yeast-cells. This could be made out macroscopically as a dark-grey 

 to black coloration, microscopically from the presence of fine, brown 

 pigment-granules in the living-cells, which did not disappear upon a 

 short treatment with alcohol and that could be fixed in Canada- 

 balsam-preparations. The pigment-production was arrested as soon 

 as the pigmented cultures were transplanted on a phosphate-containing 

 medium. It appears, therefore, that it is brought forth only in a 

 phosphate-free medium. For five months this pigmented culture was 

 maintained, then outside influences destroyed it. I never again detected 

 this pigmentation in the same stock of Torula monosa. . 



As appears from the above, the presence of phosphorus-compounds 

 is essential to the formation of volutin. They alone, and not as 

 Henneberg asserts calcium-salts or ammonium-carbonate or sugar, can 



^) Yide Kluyver, 1. c. page 73, 



