75 



I could not possibly detect volutin-granules among the gljcogen- 

 drops in the living cell-plasma. It is necessary, therefore, in watching 

 the culture-experiments, to fix coverslip-preparations. With Torula 

 and Ustilago absolute alcohol answered the purpose very well; not 

 quite so well with Saecharomyces in which, as Henneberg ') has also 

 pointed out, the cell-body after fixation in alcohol and colouring in 

 methylene-blue is not sufficiently decolourized by 17o sulphuric acid. 



The culture medium was prepared by adding to the agar chemic- 

 ally pure substances; the agar was extracted for one hour with 

 0.5 7o acetic acid and subsequently washed out with distilled water. 

 No tap-water was used ''). As nutritive substances we first used 

 5 perc. glycose (pro analysi), 0.5 perc. peptone Merck, 0.05 perc. 

 MgSO^ (pro analysi) and a trace of KNO3 (pro analysi). When 

 examining this medium after Neumann') to ascertain whether any 

 phosphorus be present, this will be found to be the case and is 

 chiefly due to the peptone. Neither was the albumose, obtained in 

 the laboratory from the white of a hen's egg, found to be entirely 

 free from phosphorus. When using pure glycocoll or asparagin as 

 N-source, phosphorus is not found anymore macrochemically after 

 Neumann, yet on a microchemical examination the analysis always 

 brings to the front traces of phosphorus ''). 



On the medium carefully prepared with glycocoll or asparagin 

 not a single yeast-cell containing volutin is to be found after some 

 days at the second inoculation from a malt-agar-cuUure of Torula 

 monosa. The growth, however, is slighter than in the phosphate-free 

 cultures with peptone and since in the latter only a few 

 volutin-containing cells were found in a field of thousands of volutin- 

 free cells, they could safely be used for most of our experimenis. 

 Also in these almost phosphate-free peptone-cultures the growth, as 



1) 1. c. page 74. 



2) It should be noted that Reichenow found the methylene blue-sulphuric acid 

 reaction absent in h's experiments with Haematococci, even when simply omitting 

 the phosphate. In the description of the culture-fluids no mention was even made 

 of the use of distilled water Probably the hypho- and blastomycetes studied by 

 me can seize upon smaller quantities of phosphorous-compounds present in the 

 culture fluid than the Haematococci stu<lied by Reichlnow! 



*) After this method the substance to be examined is incinerated with 10 c. c. 

 of distilled nitric acid s. g 1,4 and strong~sulphuric acid aa. For further details 

 we refer to Hoppe Seyler, Hdb d. Physiol, u. Pathol. Ghemischen Analyse p. 359. 

 After incineration and subsequent neutralization phosphoric acid was reacted upon 

 with ammonium-molybdate. 



*) Here part of the nutrient medium was incinerated, the ashes were taken up 

 in a drop of nitric acid and were put on an object-glass. Reaction was then .per- 

 formed under the microscope 



