Chapter XIII 



— 141 



Vital Staining 



The oxidation-reduction potential of neutral red scarcely per- 

 mits this destaining to be attributed to a reduction of the dye and 

 various experiments seem to indicate that yeasts do not reduce 

 neutral red. The destaining of the vacuoles can only be explained, 

 therefore, by assuming a destruction of neutral red, or an ex- 

 cretion of it by the yeasts. The following experiment throws light 

 on this problem. One half gram of Saccharomyces cerevisiae at a 

 pH of 8 is sown in a big flask containing the medium described 

 above to which 0.005% of neutral red has been added. A sample 

 of the liquid is taken at regular intervals and centrifuged. The 

 sediment is examined under the microscope each time and the con- 

 centration of neutral red of the liquid is measured by Meunier's 

 photo-electric colorimeter. The experiment proved that, at the 

 end of half an hour, all the cells accumu- 

 lated neutral red in their vacuoles and that 

 the concentration of the dye in the liquid 

 was reduced from 0.005% -0.0015%. There 

 is therefore a very great absorption of the 

 dye by the yeast. At the end of half an 

 hour, the vacuoles begin to lose their stain 

 and the concentration of neutral red in the 

 liquid again increases and finally at the end 

 of an hour all the cells are destained and 

 the concentration of the dye in the liquid 

 has returned very nearly to its original 

 amount. The vacuoles are then complete- 

 ly destained. Now, various experiments 

 having shown that under these conditions 

 there is no absorption by the membranes 

 of the cells, one is obliged to conclude that 

 the yeasts, after having accumulated the 

 neutral red in their vacuoles, excrete it 

 into the medium. So it is only when they 

 are freed of the dye that they begin to bud. 



Saprolegnia behaves differently from the yeasts, since it ac- 

 cumulates neutral red while growing. Nevertheless it seems also 

 to be able to excrete the dye under some conditions. In fact, al- 

 though it always accumulates neutral red during growth, it does 

 not keep it in the vacuoles unless the medium is poor in nutrients 

 (soy bean bouillon with agar). If the culture is grown in a richer 

 medium (peptone) it is found that, after having accumulated the 

 neutral red at the beginning of growth, the cells suddenly lose their 

 color at the end of 48 hours, i.e., at the moment of maximum 

 growth. From this point of view Saprolegnia occupies an inter- 

 mediate position between the fungi on the one hand, which absorb 

 neutral red only when their growth is arrested, and which reject 

 the dye as soon as they begin to develop, and the phanerogams on 

 the other hand, whose cells accumulate neutral red during their 

 growth and retain it in their vacuoles during their entire de- 

 velopment. 



Fig. 90. — Saccharomycea 

 ellipsoideus grown on 1% pep- 

 tone and 1% glucose containing 

 0.005% neutral red. A. 1, 

 First strongly colored precipi- 

 tates are formed in the vacuoles; 

 2, The precipitates dissolve and 

 the vacuole is diffusely stained; 

 4, The color disappears; 5, 

 Budding. B. Similar pheno- 

 mena. 1-3, staining; 4, 5, loss 

 of color. 



