STAINING METHODS 23 



of it. The latter is claimed to give deeper staining of both granules 

 and body of the cells without lessening the contrast between them. 



3. Drain without washing. 



4. Treat 1 min in a modified Lugol's solution (iodine, 2 g; KI, 3 g; dis- 

 tilled water, 300 ml). 



5. Wash briefly in tap water. 



6. Blot with filter paper, and examine. 



Results : INIetachromatic granules, black ; bars of diphtheria cells, dark 

 green to black; body of cells, light green. 



Ljuhinsky Stain 

 (from Blumenthal and Lipskerow, 1905) 



Original formula Emended form ida 



Solution A Solution A 



Pyoktanin (Merck) 0.25 g Methyl violet 2B or crystal vio- 



5 % acetic acid 100 ml let (85 % dye content) 0.25 g 



Glacial acetic acid 5 ml 



Distilled water 95 ml 



Solution B Solutio7i B 



Vesuvin 0.1 g Bismarck brown Y 0.1 g 



Distilled water 100 ml Distilled water 100 ml 



Staining schedule: 



1. Make smears as usual and fix with gentle heat. 



2. Stain 30 sec to 2 min in solution A. 



3. Wash in tap water. 



4. Stain 30 sec mth solution B. 



5. Wash in tap water. 



6. Dry, and examine. 



Results: Metachromatic granules, dark blue or black; rest of cell> 

 reddish or yellowish. 



STAINING THE DIPHTHERIA ORGANISM— ALTERNATE PROCEDURES 



Neisser^s Diphtheria Stain 

 Neisser (1903) 



Solution 1 Solution 2 



Methylene blue (dye content not Crystal violet (dye content not 



specified; probably 90 %) 1 g specified; probably 85 %) . . . . 1 g 



Alcohol (e.g., 95 %) 20 ml Alcohol (e.g., 95 %) 10 ml 



Acetic acid (glacial) 50 ml Distilled water 300 ml 



Distilled water 1000 ml Solution 3 



Mix, and agitate until dye is dissolved Chrysoidin 1 or 2 g 



Hot water 300 ml 



Filter after dissolving 



