IV46-14 MANUAL OF METHODS FOR PURE CULTURE STUDY 



5. Wash briefly in tap water. 



6. Blot with filter paper, and examine. 



Results: Metachromatic granules, black; bars of diphtheria cells, 

 dark green to black; body of cells, light green. 



LJUBINSKY STAIN 



(from Blumenthal and Lipskerow, 1905) 



ORIGINAL FORMULA EMENDED FORMULA 



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 Solution B 



0.1% vesuvin Bismarck brown Y 0.1 g. 



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. with 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 No. 1 Solution No. 2 



Methylene blue (dye content Crystal violet (dye content not 



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 No. 3 



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



Hot water 300 ml. 



Filter after dissolving 



Dried films are stained 10 seconds in a mixture of 2 parts of Solution No. 1 and 1 part 

 of Solution No. 2. Wash. Stain 10 seconds in Solution No. 3. Wash briefly in water, 

 or not at all. Blot dry. 



Bonder's Diphtheria Stain 

 Ponder (1912); Kinyoun (1915) 



Original As modified 

 formula by Kinyoun 



Toluidine blue 0.02 g. 0.1 g. 



Azure I 0.01 g. 



Methylene blue ■ 0.01 g. 



Glacial acetic acid 1 ml. 1 ml. 



Ethyl alcohol (see below) 2 ml. 5 ml. 



Distilled water 100 ml. 120 ml. 



Dissolve the dyes in the alcohol, add the water, then the acid and let stand 24 hours 

 before using. Do not filter. After prolonged standing, action may be intensified by 

 adding 1 or 2 drops of glacial acetic acid. 



