INNOCULATION METHOD 165 



taining water at 80 to 90 C. The mixture of alcohol and 

 ether boils at once and after ten to fifteen minutes the cover 

 slips are removed and washed with absolute alcohol. The 

 films are then stained with carbol-fuchsine and counterstained 

 with methyline blue according to the Ziehl-Neelson method 

 which is as follows: 



(1) Stain in hot carbol-fuchsine for five to ten minutes, being 

 careful to avoid over-heating. 



(2) Decolourise by dipping in 25 per cent sulphuric acid. 



(3) Wash in water. 



(4) Wash in alcohol until no more stain is removed. 



(5) Wash in water. 



(6) Counterstain for one minute with methylene blue. 



(7) Wash in water, dry, and mount. 



Delepine found that when this method of preparation was 

 carefully followed, very clear films were obtained and no dif- 

 ficulty was caused by other acid fast bacilli when sufficient 

 attention was paid to the morphological characteristics of the 

 organisms. 



Inoculation Method. The inoculation method is the only 

 one that can be relied upon for the detection of very small 

 numbers of B. tuberculosis in milk, but the time required to 

 obtain reliable results is not less than three weeks as com- 

 pared with the few hours required for the completion of the 

 microscopical method. It is good routine practice to make 

 microscopical preparations of all sediments obtained by cen- 

 trifugalisation and to inoculate those yielding negative or doubt- 

 ful results. 



To prepare the sediment, 100 c.cms. of milk are centrifu- 

 galised at 2000 to 3000 revolutions per minute for at least 

 thirty minutes, and, after removing the cream layer with a 

 sterile spatula or spoon, the separated milk is drawn off through 

 a small bore glass tube attached to a suction pump until about 

 4 c.cms. of milk remain. This milk is thoroughly mixed with 

 the deposit and subsequently used for the inoculation of two 

 animals. If the milk is known to be " clean " the milk may 



