Guillierrnond - Atkinson — 140 — Cytoplasm 



This is, then, a valuable method of following under the micro- 

 scope the entire life history of the vacuoles during cellular growth 

 and we shall see further on the uses to which it may be put. 



A fact becomes evident from these investigations, namely, that 

 vital staining with neutral red is impossible if the medium is too 

 acid. The starting point at which staining occurs lies between the 

 pR values of 5.5 and 7, according to the type of cell in question. For 

 roots of phanerogams it is, for example, 5.5, for the Saprolegniaceae 

 6.5, for yeasts and Oidium lactis, 7. This is an essential fact pre- 

 viously brought out by Irwin and Pischinger whose work was 

 later confirmed by Bailey and Zirkle, Genaud, Chadefaud and 

 others. 



It is curious to find that, with the exception of the Saproleg- 

 niaceae, most of the fungi are distinguished from all the other 

 plants by their behavior in the presence of neutral red. Cultivated 

 in media to which this dye has been added, they develop very readily 

 but are never stained. We wondered why fungi which accumulate 

 the dye so easily in their vacuoles when placed between slide and 

 cover slip in a solution of neutral red, do not ever accumulate it 

 while they are growing. 



The species of Saprolegnia, in the conditions under which we 

 cultivated them, never appreciably modify the pB. value of the- 

 medium although other fungi make it more acid. The failure of the 

 latter to be stained could therefore be attributed to a rapid acidi- 

 fication of the medium. This hypothesis, which we accepted at first 

 and in which Becker and Skupienski later concurred, is, how- 

 ever, not to be retained, for acidification of the medium may be 

 considerably retarded and it is found that the fungi, even at a pH 

 favorable to staining, do not take up neutral red. Recent research 

 which we have carried out with Gautheret on Oidium lactis and 

 various molds, has shown that these fungi accumulate neutral red 

 only when they have ceased to grow. These same investigations 

 also proved that when the spores of these fungi are sown in a 

 medium to which neutral red has been added, they take up the 

 dye, at first, and then become destained as soon as they begin to 

 grow. If the cells of Saccharomyces ellipsoideus are sown, for ex- 

 ample, in a moist chamber on an aqueous medium containing 1% 

 peptone and 1 % glucose at a pB. of 7.5-8 to which has been added 

 0.005% neutral red and enough agar to hold them in place so 

 that they can be found under the microscope and if the cells are 

 examined microscopically, it is found that all the cells accumulate 

 the dye in their vacuoles even between slide and cover glass. The 

 accumulation is at its maximum at the end of half an hour, then, 

 after about three hours (two hours for some yeasts), the cells lose 

 their color and it is only then that they begin to bud. The loss of 

 color is brought about by a process which is the converse of that 

 by which staining was accomplished. The homogeneously stained 

 vacuolar sap loses its color and there are seen to appear in the 

 vacuoles, intensely stained granules which little by little lose their 

 color and disappear (Fig. 90). 



