STAINING METHODS 13 



The formula for the Giemsa stain is : Azur II eosine (Griib- 

 ler), 3 grams; Azur II (Griibler), 0.8 gram; acetone free 

 methyl alcohol, preferably Merck's reagent, 375 grams; Merck 

 reagent glycerine, 125 grams. 



Sections of lice and sections of human and animal tissues 

 were stained by the modification of Giemsa's method described 

 by one of us in work on Rocky Mountain Spotted Fever (Wol- 

 bach, 1919). 



Louse and mammalian tissues were fixed in Zenker's fluid 

 saturated with corrosive sublimate and with the addition of 

 5 per cent of glacial acetic acid. The acetic acid was used in 

 order to prevent the staining of mitochondria, although slightly 

 better results may be obtained without it. While we do not be- 

 lieve that the presence of stained mitochondria would be a 

 source of error we preferred to obviate one source of query in 

 the discussion of results. 



After embedding in paraffin and sectioning, the slides were 

 treated by the usual methods except that an extra step was 

 taken to insure complete removal of the iodin employed to 

 remove corrosive sublimate crystals deposited in the tissues. 

 After the iodin, the sections were treated with 0.5 per cent 

 solution of sodium hyposulphite for ten to fifteen minutes, and 

 then thoroughly washed in running water followed by distilled 

 water. 



The sections were stained in a slightly alkaline mixture. The 

 distilled water used in the solution employed for staining sec- 

 tions should be free from acid to begin with and will have the 

 right reaction after the addition of from two to four drops of 

 0.5 per cent solution of sodium bicarbonate to one hundred 

 cubic centimeters of water. It may be necessary to determine 

 the exact amount of alkali by trial. The addition of methyl 

 alcohol to the staining solutions, three to four cubic centimeters 

 to each hundred cubic centimeters of water, retards the pre- 

 cipitation of the dye and yields more intense staining. 



The formula for the stain which has given the most uniformly 

 good results is: Distilled water, 100 cubic centimeters; 0.5 per 

 cent sodium bicarbonate, two to four drops; reagent methyl 



