BACTERIOLOGICAL TECHNIQUE 25 



XXI. ROTITS METHOD OF EXAMINING BUTTER FOR 

 TUBERCLE BACILLI AND OTHER BACTERIA IN 

 COVER-GLASS SPECIMENS. 



1. About two to four grammes of the suspected butter is placed in 

 a test-tube with a small spatula or a glass rod, the test-tube filled 

 about three-quarters full with water, placed in a water bath at 50 C., 

 and heated until the fat is thoroughly melted. 



2. Place a cork in the test-tube and shake well a few times in 

 order to separate any Tubercle bacilli, etc., from the fatty substance. 



3. Place the test-tube cork downwards in the warm bath for 

 fifteen minutes, until the fat is again thoroughly dissolved. 



4. Place the test-tube in a cool place, so that the butter fat again 

 solidifies. 



5. The fluid contents are either placed in the centrifugal apparatus 

 or set aside until the sediment forms. 



6. Some of the sediment is placed on a cover-glass, air dried, 

 slightly heated, laid in a mixture of one of ether and three of alcohol, 

 removed, dried, and fixed in the flame. 



7. The specimen is stained according to the ordinary process for 

 Tubercle bacilli (see 14). 



' To isolate bacteria by this process from the butter a sterilized 

 platinum loop is dipped in the fluid, and gelatine cultures instituted.' 



XXII. METHOD OF STAINING CAPSULE BACTERIA 

 IN COVER-GLASS SPECIMENS. 



Treat the fixed preparation as follows : 



1. Stain with 2 per cent, gentian violet watery solution, and 



intensify the staining action by heating until vapour 

 arises. 



2. Wash with water, and dry. 



3. Decolorize the capsules with 2 per cent, acetic acid in water, 



six to ten seconds. 



4. Wash with water. 



5. Mount preferably in water, as the high refractive index of 



balsam renders the capsule less visible. 



Fuchsin can also be used in place of gentian violet. 



Many species of bacteria which were supposed not to possess a 

 capsule show them very distinctly by this method. 



It is of especial value in differentiating the capsule of Bacillus 

 anthracis according to 4 Johne.' 



