74 



all cells ') nor always in such a quantity as is found in the fixed 

 preparation after staining with methjlene-blue and differentiation in 

 sulphuric acid. Of course it is not outside the probabilities that, 

 coincidentlj with the fixation, compounds are isolated from the living 

 matter, that contribute to the mass of volutin-granules. Allowance 

 should always be made for such possibilities in the examination of 

 fixed substances. It will no longer do, of course, to apply the term 

 vital staining to the staining of living cells with methylene-blue -|- 

 5 perc. formol, as Henneberg does.^) 



Fat may be stained in the yeast-cell at the same time with volutin. 

 We need only apply Sudan III to the preparation treated with 

 methylene-blue and sulphuric acid. As other researchers, among whom 

 Henneberg, have also pointed out, the volutin-granules are, as regards 

 their location, independent of the fat-drops ; it can only be altered 

 passixely by the pressure of glycogen- or fat-dr-ops, by which they 

 may be pushed back to the periphery of the cell. In other respects 

 their independence also appears from the presence of fat and glycogen 

 in cultures where volutin is absent. 



I here wish to emphasize that moulds as well as various other 

 kinds of yeast may be cultivated and multiplied without any 

 volutin being produced in the cell. Earlier researchers were also 

 acquainted with the fact that in the case of insufficient nutrition 

 the amount of volutin in the cell decreases, that some of the cells 

 in the culture may even become completely devoid of volutin. 

 In 1910 Reichenovv ') has also demonstrated in Haematococcus 

 pluvialis, that by not adding phospiiate to the culture-fluid the 

 volutin may be made to disappear ultimately, which, however, 

 results in the death of the haematococci. 



In my own culture-experiments with Ustilaginae and Torulaceae 

 I have likewise watched the effect of the amount of phosphate in 

 the culture-medium and endeavoured to obtain permanent cultures 

 completely free from volutin. To accomplish this effect it will not 

 suffice to omit the phosphate in the agar-cultures because, though 

 in doing so the majority of the cells will indeed be free from 

 volutin, still many volutin-bearers will be seen through the microscope 

 in an alcohol-coverslip- preparation. 



It is remarkable that in an agar-malt medium stained rather deep-red with 

 neutral red, only a very small percentage of the growing cells had, after several 

 weeks, taken up the dye. 



2) Cenlralblatt f. Bact. Bd. XLV, 1916, s. 50; Wochenschrift f. Brauerei 1915, 

 NO. 36—42. 



^) 1. c. page 71. 



