60 HEWSON SWIFT 



sections, it is obvious to anyone who has worked with them that they may 

 differ rather markedly in the staining picture produced. For example, the 

 extremely strong and partially nonspecific binding shown by crystal violet 

 may be compared with the weak staining of the closely related methyl 

 green. The individual behavior of dyes can serve to emphasize that binding 

 is not a function of simple electrostatic forces alone. The binding strength, 

 the number, and distribution of the auxochrome groups, and their disso- 

 ciation characteristics, the ability of the dye to form molecular aggregates, 

 and the effective volume of the dye molecule in penetrating crevices in the 

 tissue — all of these factors are probably involved in reinforcing or counter- 

 acting electrostatic attraction. 



5. Differentiation 



The treatment of tissues after staining is often as important as the stain- 

 ing itself. When the slide is transferred to a rinse to remove the "unbound" 

 dye, it is obviously subjected to a series of additional factors that markedly 

 alter the original dye-nucleic acid equilibrium. The pH of the rinse is of 

 great importance. If lower than the stain solution, in the case of a basic 

 dye, it will reduce the binding affinity and result in loss of much dye pre- 

 viously bound. If higher, it may increase the dye bound since some imbibed 

 dye may be held by the section before it is washed out. If the rinse has the 

 same pH as the staining solution, the equilibrium is still drastically shifted 

 toward dissociated dye, and the tissue will continue to lose stain at a rate 

 determined partly by the dye-nucleic acid bond strength, dye solubility, 

 temperature, and ion concentration. 



Michaelis''^ suggested that alcohol rinses after staining would differen- 

 tiate between bound and unbound dye. This obviously subjects the section 

 to a new set of conditions, among which dye solubility seems to be particu- 

 larly important. Certain dyes, e.g., methyl green and azure B, may be 

 rapidly and completely removed by absolute ethanol so that the use of 

 acetone or higher alcohols has been suggested; for example, ter/-butyl 

 alcohol.*'-^ Where the dye has a particularly low solubility in the rinse, this 

 type of differentiation may not provide removal of all "unbound" dye. In 

 most cases such as with methyl green-stained slides in ter^butyl alcohol, 

 as in buffer solutions, the rinse continues to remove bound dye, so that it is 

 necessary to set a purely arbitrary time limit for reproducible results. 



If the staining picture desired is that at the pH and ionic strength of the 

 stain solution, differentiation in solute minus stain for an arbitrary and 

 controlled period of time seems desirable. Differentiation in nonaqueous 

 media also gives good results, although such treatments may affect the 



" L. Michaelis, Cold Spring Harbor Symposia Quant. Biol. 12, 143 (1947). 

 « M. H. Flax and M. Himes, Physiol. Zool. 25, 297 (1952). 



