SPORE STAINS 



I0 7 



be red, and the rest of the film colourless or a very 

 light pink.) If satisfactory, pass on to section 7; if 

 unsatisfactory, repeat steps 2 to 5. 



7. Counterstain in weak methylene-blue. (Now 

 spores red, bacilli blue.) 



8. Wash in water. 



9. Dry and mount. 



The spores of different bacilli differ greatly in their 

 resistance to decolourising reagents; even the spores 

 of the same species of organisms vary according to 

 their age. Young spores are more easily decolourised 

 than those more mature. 



Sulphuric acid, i per cent, aqueous solution, and 

 hydrochloric acid, 0.5 per cent, alcoholic (90 per cent.) 

 solution, are useful decolourising reagents. 



3. Moeller's Method. 



1. Prepare and fix films in the usual manner. 



2. Immerse in absolute alcohol for two minutes, then in 

 chloroform for two minutes; wash in water. This dissolves out 

 any fat or crystals that might otherwise retain the "spore" 

 stain. 



3. Immerse in chromic acid, 5 per cent, aqueous solution, for 

 one minute; wash in water. 



4. Pour Ziehl's carbolic fuchsin on the film, warm as in previous 

 methods, and allow it to act for ten minutes. 



5. Wash in water. 



6. Decolourise in sulphuric acid, 5 per cent, aqueous solution, 

 for five seconds. 



7. Wash in water. 



8. Counterstain with Kuehne's carbolic methylene-blue for one 

 or two minutes. 



9. Wash in water. 



10. Dry and mount. 

 (Spores red, bacilli blue.) 



4. Abbott's Method. 



1. Prepare and fix films in the usual manner. 



2. Pour Loeffler's alkaline methylene-blue on the film; warm 

 cautiously over the flame till steam rises and allow the hot 

 steam to act for one to five minutes. 



3. Wash thoroughly in water - 



4. Decolourise in nitric acid, 2 per cent, alcoholic (alcohol 

 80 per cent.) solution. 



