Staining Paraffin Sections 72 



Variations in the composition and purit) ol the component stains 

 ha\e necessitated revisions of schedule to suit the currently available 

 stains. Some workers prefer to differentiate the safranin with very 

 dilute HCl or with acidified alcohol before adding the violet. The 

 acid must subsecjuently be thoroughly washed out with water. 



The gentian violet may be almost fully differentiated in clove 

 oil containing no orange G. The orange can then be added 

 progressively and the violet brought to final differentiation in xylene 

 containing 1/10 to I/4 (by volume) clove oil saturated with orange G 

 or gold orange. 



Quadruple-stain combinations using four coal-tar dyes are avail- 

 able in some excellent commercial slides. Conant (Triarch) uses a 

 combination of safranin, crystal violet, fast green, and gold orange; 

 Johansen (California Botanical Materials Co.) uses safranin, methyl 

 violet 2B, fast green, and orange G. These complex processes yield 

 striking preparations but are probably unnecessarily elaborate for 

 most tasks. The advanced worker can obtain details of procedure 

 from the excellent service leaflets of the above manufacturers and 

 from the Johansen manual (1940) . 



Staining processes using coal-tar dyes are entering a most interest- 

 ing and important phase of development. Many new organic solvents 

 are being produced by synthetic methods. Solvents that have been 

 little more than chemists' curiosities are now being produced in large 

 quantities and are available at reasonable cost. Some illustrations are 

 the higher alcohols, such as the butyl, propyl, and amyl series, ethyl 

 and methyl Cellosolve, trichloroethylene, and many other solvents. 

 The stains themselves are imdergoing constant study and improve- 

 ment. The possibilities of systematic study, or just plain dabbling, 

 should gratify the heart of the most inveterate experimenter. 



IRON HEMATOXYLIN 



The next stain to be considered is known as Heidenhain's, or iron- 

 alum hematoxylin. The history of this stain and the names of several 

 investigators who have contributed to its development may be found 

 in the literature. This stain is primarily a cytological stain, used 

 especially for chromosome studies, but it is useful for studies on the 

 cell wall, plastids, and in some studies in pathological histology. The 

 formidas of the mordant (iron alum) , the stain (hematoxylin) , and 

 the destaining agent are given in the stain formulary. The schedule 

 advocated here and outlined in Staining Chart VI is known as the 

 short schedule, or 4-4 schedule; i.e., 4 hr. in mordant, thorough but 



