Chapter III — 19 — Physical Properties 



tionably sublethal staining. Nevertheless in cultures to which vital 

 dyes have been added, staining of the cytoplasm is obtained but this 

 staining is purely transitory and is visible only in the earlier hours 

 of culture. The dye, first taken up by the cytoplasm is rapidly 

 excreted into the vacuole which appears to be the region of the 

 cell in which all toxic products accumulate. It is only when the 

 dye has accumulated in the vacuole that the cell begins to grow 

 and multiply. Consequently, only vital staining of the vacuole is 

 compatible with growth and any persistent staining of the cyto- 

 plasm is necessarily sublethal. The same is true for dyes which 

 stain the chondriosomes. The acid dyes, eosine and erythrosine in 

 particular, do not in general stain plants cultivated in media which 

 contain them. 



It seems likely that, ordinarily, living cytoplasm does not have 

 an affinity for dyes. Cytoplasm in healthy cells stains only under 

 special conditions or in cells which have ceased to grow. More 

 often, staining takes place just before their death in cells which 

 are not healthy. In the yeasts, however, Nile blue first accumu- 

 lates in the cytoplasm and is then excreted into the vacuoles. In 

 any case, these dyes, interesting from the point of view of cellular 

 permeability, are of no service in a cytological study of the cyto- 

 plasm except as they stain the chondriosomes. 



Once dead, the cytoplasm becomes, on the other hand, very 

 permeable to all substances dissolved in water. This can be ex- 

 plained by considering that when the cytoplasm is coagulated, the 

 micelles crowded at the periphery, forming the ectoplasmic mem- 

 brane, move away from each other and allow the substances dis- 

 solved in the surrounding medium, especially all the dyes, to pass 

 through freely. 



Consequently, for the study of the cytoplasm, the method of 

 fixation and staining has been resorted to because observation of 

 living material does not suffice. Since the cytoplasm seems to 

 behave like a hydrogel which is very slightly alkaline, as will be 

 shown farther on, its fixation, i.e., its coagulation, can be brought 

 about only by acid dehydrating reagents. The acid reagents usually 

 used are composed of weak acids (acetic, picric), or even strong 

 acids (nitric, trichloracetic), suitably diluted and associated, in 

 most fixatives, with salts of heavy metals (mercury, platinum, 

 osmium). Such fixatives have a pU inferior to 3. Besides these, 

 neutral liquids like alcohol and formalin^ may act as fixatives, for 

 both have the property of precipitating the proteins, the former by 

 its dehydrating action, the latter by combining with the protoplasm 

 to form insoluble compounds still not well defined. 



The most acid fixatives, those with an acetic acid base and 

 fixatives containing alcohol, which has a too strong dehydrating ac- 

 tion, usually cause sudden coagulation, producing in the cytoplasm 

 artificial structures - coarsely granular-reticulate - which, as will be 

 seen, led the early C5d:ologists to attribute to the cytoplasm a reticu- 



^ Formalin is usually acid, almost always containing, furthermore, some fonnic acid. 



