PROTOPLASM 53 



The successful staining of living protoplasm with "vital" dyes, which 

 are nontoxic in dilute solution, requires considerable skill. Some of these 

 dyes are indicators of the degree of acidity or alkalinity, since the}^ alter 

 their color in passing through characteristic regions of the pH scale. In 

 this way it has been found in cells of various kinds that the living nucleus 

 is slightly alkaline, with a pH of about 7.5 to 7.6, whereas the cytoplasm 

 is usuall}^ at about 6.7 to 6.9, or slightly acid. Injury causes the acidity 

 to increase, the pH of the cytoplasm falling to 5.2 to 5.5. The cell may 

 later recover, but not if the hydrogen-ion concentration is maintained too 

 long at this level. When an ameba is placed in a dilute solution of 

 methyl red, it becomes pale yellow throughout, showing that the pH 

 in all parts is well above 5.2. If a slight amount of acid is then injected 

 into the cytoplasm near the nucleus, a local reddening of the cytoplasm 

 and then of the nucleus indicates a lowering of the pH to some point 

 below 5.2. Both regions soon recover their yellow color, showing that 

 the protoplasm contains or produces buffering substances tending to 

 maintain its normal reaction in the vicinitj^ of pH 7, the neutral point. 



Living nuclei can be stained with dilute solutions of weakly basic 

 dyes, which enter cells freely, or by acid ones when injected. Protozoa 

 ma}^ live with nuclei and chromosomes stained with neutral red, and 

 certain stages of mitosis in plants can occur with chromosomes colored 

 by Hoffmann's violet or malachite green. Cytoplasm ordinarily does not 

 take the stains markedly, much of the color observed being rather in 

 vacuoles and inclusions. Lipide-soluble dyes appear to stain the cyto- 

 plasm itself in some degree. 



The fixation of tissues with special fluids designed to render their 

 components firm and more resistant to reagents employed in sectioning 

 techniques also has effects on staining. The staining may be greatly 

 improved by previous fixation, but one must always be on guard against 

 interpreting fixation artefacts as natural appearances. After fixation 

 the nucleus acts as an acid and stains markedly with basic coal-tar dyes. 

 This is because the nucleic acid, although combined with other substances 

 in such a way as to render the living nucleus actively alkaline, neverthe- 

 less gives the nucleus a strong potential acidity. Hence when fixed 

 tissues are placed in properly prepared solutions of basic dyes, the nega- 

 tive bonds of the phosphoric acid groups in the nucleic acid unite with the 

 colored cations of the solution. The cytoplasm, on the contrary, com- 

 monly acts as a base in fixed tissues and unites with the colored anions in 

 solutions of acid coal-tar dyes. It is by manipulating a pair of dyes 

 differing in both color and reacting power that double-staining effects are 

 achieved. 



The proteins react as they do in such procedures largely because they 

 are amphoteric, i.e., they have the properties of both bases and acids 



