



MORPHOLOGY 309 



4. Spengler's method. 



1. Stain the films by gently warming them in Ziehl's solution. 



2. Treat for a few seconds with picric-alcohol. 



Saturated aqueous solution of picric acid, - - - 60 c.c. 



95 per cent, alcohol, 40 ,, 



3. Wash three times in 60 per cent, alcohol. 



4. Decolourize rapidly (about 20 seconds) in 1 in 6 nitric acid, then in 60 per cent, 

 alcohol. 



5. Treat again with picric-alcohol. Wash. Dry. Mount. 



5. Frankel's method. 



1. Stain the films in the warm for 5 minutes with aniline-fuchsin (prepared in the 

 same way as aniline-violet using an alcoholic solution of fuchsin instead of alcoholic 

 gentian- violet). 



2. Transfer the films direct to the following solution for 1 minute. 



90 per cent, alcohol, 50 c.c. 



Aniline water. 30 ., 



Pure nitric acid, - 20 ,, 



Saturated alcoholic solution of methylene blue, - Q.S. to obtain a 



deep blue colour. 



3. Wash in distilled water. Dry. Mount. 



6. Herman's method. 

 Prepare the following solutions : 



A. Krystal violet, 1 gram. 

 95 per cent, alcohol, 30 c.c. 



B. Ammonium carbonate, - 1 gram. 

 Distilled water, - 100 c.c. 



Immediately before use, pour three parts of solution B into a watch-glass, add 

 one part of solution A and mix intimately. 



1. Heat the staining bath until it just begins to boil and leave the films in it for 

 a minute. 



2. Transfer the films to a 10 per cent, solution of nitric acid for 4 or 5 seconds. 



3. Wash in absolute alcohol to complete the decolourization. 



i> 



FIG. 196. Tubercle bacilli in sputum : Herman's method. 

 (Oc. 2, obj. Ath, Zeiss.) 



4. Transfer the films for 30 seconds to the following solution : 



Eosin, - 1 gram. 



60 per cent, alcohol, 



5. Wash very rapidly in alcohol. Dry. Mount. The tubercle bacilli are stained 

 violet and the background bright pink. 



