SECTION TWO 



TETRACHROME STAIN 



(After MacNeal) 



For differentiation of types of leucocytes in smears 



Staining solution: 

 Dissolve 0-3 gm. of the dry stain in 100 ml. of pure methyl alco- 

 hol by heating to 50° C. on a water bath. Shake well, and leave 

 for four or five days with occasional shaking; then filter. 



Technique: 



1. Very thin, freshly prepared blood films are dried quickly at 

 room temperature and stained immediately one to three minutes 

 with I ml. of the staining solution prepared as above ; then add 

 2 ml. buffer solution pH 6-8 and allow to stand for about five 

 minutes. 



2. Pour off the stain and wash with buffer solution pH 6-8 until 

 the thin portions of the stained film are pink. 



3. Blot carefully and examine. 



Note: If it is desired to keep the films for any length of time 

 before staining, they should be fixed immediately while still wet, 

 with pure methyl alcohol for one minute. 



Results: 



Erythrocytes are stained yellowish red; polymorphonuclear 

 neutrophiles : dark blue nuclei, reddish mauve granules, pale pink 

 cytoplasm. Eosinophilic leucocytes : blue nuclei, red to orange-red 

 granules, blue cytoplasm. Basophilic leucocytes : purple or dark 

 blue nuclei, dark purple to black granules. Lymphocytes: dark 

 purple nuclei, sky-blue cytoplasm. Platelets: violet to purple 

 granules. 



Reference: MacNeal, W. J. (1922). 



THIONIN (Ehrlich) 

 For mucin 



Solution required: 



Thionin (Ehrlich), saturated, 



aqueous . . . . . . 0-5 ml. 



Distilled water . . . . . . 10 ml. 



^ 437 



