STAINING 193 



until the stain clears, and repeat once more; wash in tap 

 water and then wash in 1 per cent. H2SO4 three times, 

 dropping on plenty of acid, tilting and running this over the 

 slide three times and then pour off and use fresh acid and 

 repeat this once. Wash thoroughly in distilled water, then 

 stain with LofHer's blue one to three minutes. Wash, dry 

 and examine. The spores should be bright red in a blue rod. 



This method will give good results if care is taken to 

 secure cultures of the right age. If the culture is too old 

 the spores will all be free outside the rods, while if too young 

 they will decolorize with the acid. For Bacillus subtilis and 

 Bacterium anthracis, cultures on agar slants forty-eight hours 

 in the 37° incubator are just right. For the spores of Bacillus 

 tetani, the culture should be three days old, but may be as 

 old as a week. 



Staining of "Acid-fast" Bacilli. — Bacterium tuberculosis, 

 Bacterium of Johne's disease, "grass" and "butter bacilli," 

 Bacterium leprce. Bacterium smegmatis. 

 Gabbet's method: 



1. Prepare the film as usual. 



2. Stain with carbol-fuchsin as given above for spores. 



3. Wash with tap water. 



4. Decolorize and stain at the same time with Gabbet's 



blue, 2 or 3 minutes. 



5. Wash, dry and examine. 



The sulphuric acid in Gabbet's blue removes the carbol- 

 fuchsin from everything except the " acid-fast" bacteria, 

 which remain red, and the blue stains the decolorized bacteria 

 and nuclei of any tissue cells present. 



Ziehl- Neelson method: 



1, 2, 3, as in Gabbet's method. 



4. Decolorize with 10 per cent. HCl until washing with 



water shows only a faint pink color left on slide:. 



5. Wash thoroughly. 



6. Stain with LofHer's blue 1 or 2 minutes. 



7. Wash, dry and examine. 



The results are the same as with Gabbet's method. 

 13 



