Chapter XIII —143— Vital Stainin g 



minutes, the liquid was centrifuged, the sediment examined under 

 the microscope and the concentration of the dye in the liquid 

 measured by the colorimeter. It was thus possible to plot in 

 terms of pU the quantity of dye retained by the yeast. A study 

 of the curves showed that the lower limit of pU at which accumu- 

 lation of neutral red begins is much lower than 7. It is at pB. 

 5.6. But at that point the staining of the vacuole is too weak to 

 be visible under the microscope and only the measurements give 

 evidence of the absorption of the dye. This is appreciable only 

 beginning with a pU of 6.0. From a value of 5.6, the absorption 

 gradually increases and the curve rises to a maximum at pR 7.6. 

 From this point the curve descends. The quantity of neutral red 

 absorbed at the maximum pB. value is found to be 0.0042 g. for 



Amount absorbed in grs. per cent of fresh yeast 



0.5 g. of yeast (suspended in a 100 c.c. solution) , which gives a con- 

 centration of 0.84 g. of neutral red for 100. g. of fresh yeast. 



In the experiment with cresyl blue the lower limit at which 

 accumulation of the dye begins is also at pH 5.6 but the curve does 

 not redescend until about pH. 10. The maximum quantity of 

 cresyl blue absorbed is 0.0041 g. for 0.4 g. of yeast which makes a 

 concentration of 1 g. of cresyl blue for 100 g. of fresh yeast. The 

 curve for Nile blue is very different, for the lower limit is at pK 

 2.4 (absorption begins in a very acid medium) and steadily in- 

 creases to pK 10, at which point the curve flattens out. The maxi- 

 mum quantity of Nile blue absorbed by 0.5 g. of yeast in the 

 presence of 0.005 g. of Nile blue is 0.015 g. which makes a con- 

 centration of dye of 3 g. for 100 g. of fresh yeast. 



These rather disconcerting results demand explanations. Two 

 facts are indeed surprising: first the descending portion of the 

 curve for neutral red after the maximum pK value is reached ; sec- 



