

SPORE STAINING 147 



Note. This method gives excellent results with B. megatherium but is not so 

 good for B. anthracis : absolute alcohol is a better decolourizing agent for the latter. 

 Decolourization is in fact the difficult part of double staining, but after a few 

 trials the extent to which decolourization must be pushed to decolourize the bacilli 

 while leaving the spores stained can be determined for different organisms. 



B. Mceller's method. 1. Make a cover-glass film. Dry. Fix in absolute 

 alcohol for 2 minutes, then in chloroform for 2 minutes. Dry. 



2. Drop a few drops of a 1 in 20 aqueous solution of chromic acid on the 

 film and leave for 4 or 5 minutes. Wash in water. 



3. Stain in carbol-fuchsin in the warm as described above (A). Wash, 

 in water. 



4. Decolourize for a few seconds in a 5 per cent, solution of sulphuric acid 

 and complete the decolourization in absolute alcohol. 



5. 6, 7. Wash. Stain in blue. Mount. 



C. Aladar-Aujeszky's method. 1. Make a cover-glass film. Dry in 

 the air. 



2. Dip the preparation for 2 to 4 minutes into a porcelain capsule con- 

 taining a 0'5 per cent, solution of pure hydrochloric acid which has been 

 heated but not boiled. 



3. Wash freely in water. Dry. Fix in the flame. 



4. Stain with carbol-fuchsin in the warm by heating until steam rises : 

 as the stain evaporates add a fresh supply. 



5. Decolourize rapidly in a 4 per cent, solution of sulphuric acid. 



6. Wash. Stain in blue. Mount. 



D. Orszag's method. 1. Place a small drop of the following mixture on 

 a cover-glass. 



0'5 per cent, aqueous solution of sodium sajicylate, - - 4 parts. 

 5 per cent, aqueous solution of acetic acid, 1 part. 



Make an emulsion of the organisms in this solution. Dry. Fix the film in 

 the flame. 



2. Stain with carbol-fuchsin in the warm for 2 minutes. 



3. Decolourize with a 1 per cent, aqueous solution of sulphuric acid. 



4. Wash. Counterstain with blue. Mount as before. 



E. Thesing's method. 1. Prepare a cover-glass film. Dry. Fix in the 

 flame. 



2. Place a drop of a 1 per cent, aqueous solution of platinum chloride on 

 the film. Heat to boiling. 



3. Wash in a large quantity of water. Dry. 



4. Stain with carbol-fuchsin as in the foregoing methods. 



5. Decolourize with 33 per cent, alcohol. 



6. Wash. Dry. Counterstain with blue. Mount. 



SECTION II. THE STAINING OF CAPSULES. 



Some micro-organisms are surrounded by a bright hyaline area called a 

 capsule which can be demonstrated by certain staining devices. When these 

 are employed the organism is deeply stained, while the capsule surrounding 

 it is pale with a feebly stained margin. 



A. 1. Having dried and fixed a film, stain for 1 minute in carbol-fuchsin. 

 2. Wash. Treat for 20-30 seconds with water containing 1 per cent, 

 acetic acid. 



