STAINING OF PARAFFIN SECTIONS 285 



To proceed with staining, the slides are phieed first in xylol and 

 brought down, as before, through aniline oil to water. The slides are 

 then placed in a 4 per cent iron alum solution for at least an hour 

 for thorough mordanting. Then the slides are washed thoroughly 

 in water and placed in the hematoxylin stain for one to several 

 hours. They will appear deep black if the staining has been effec- 

 tive and no details are at all evident upon microscopic examination. 

 After rinsing in water they are ])laced in a 2 per cent iron alimi 

 solution for extracting excess stain, and after a minute or so exam- 

 ined under the microscope. It is an advantage to have the extrac- 

 tion take ])lace slowly. They should he rinsed, examined, and 

 returned if the stain is still too dee]). The de-staining may also 

 be carried out under the microscope in a glass Petri dish containing 

 the 2 per cent alum. The chromatic network in the nucleus should 

 appear sharply outlined. If one has a metaphase stage in mitosis, 

 the chromosomes should ajipear clear-cut. When the extraction 

 has gone far enough, it can l)e stopped by washing the slides in 

 water to remove e\'ery trace of the free alum solution. Then the 

 slides are "run up" through oO i)er cent alcohol to 70 ])er cent 

 alcohol. They can be counterstained in a solution of orange G 

 or eosin in 70 per cent alcohol for thirty to fifty seconds, as in the 

 preceding techniques. They are then transferred to 95 per cent 

 alcohol, aniline oil, and xylol, as before, and later mounted. 



If hematoxylin is not used in a slightly alkaline condition it acts 

 partly as an acid dye, and the cytoplasm is stained reddish and the 

 nuclei are poorly stained. 



The Mallory Connective-tissue Stain. — This stain gives a colorful 

 effect with sections that contain considerable connecti\'e tissue, 

 muscle, and epithelium. Two solutions are needed. Solution A 

 is a 0.5 per cent solution of acid fuchsin in distilled water. Solu- 

 tion B is 0.5 gram aniline blue (water solution); 2 grams orange G; 

 100 cc. of 1 per cent aqueous solution of phosphomolybdic acid. The 

 stain works better with tissues that have been fixed in Zenker's 

 fluid. x\fter the sections have been brought down, as before, from 

 xylol to water, they are placed in Solution A (acid fuchsin) for 

 about four minutes. Then this is drained off" and the slides are 

 placed at once in Solution B for about eight minutes. They are 

 then rinsed several times in 95 per cent alcohol; it is better to use 

 more than one jar of 95 per cent. Then proceed to aniline oil, 

 xylol, and mount as before. Connective tissue, muscle, and epithelium 

 stain differentially, the prominent colors being red, blue, and yellow. 



