484 PATHOGENIC BACTERIA IN MILK 



II. Remove carefully with a small sterile spoon (it is best to use a 

 platinum spoon) the fat from the top and place it in a sterile covered 

 watch-glass or Petri dish. 



III. Pour off the fluid layer forming the middle stratum, then like- 

 wise, preserve the sediment in a sterile vessel. 



IV. The cream and the sediment may be mixed or examined 

 separately. In very exact examinations it is best to inoculate a 

 mixture of cream and sediment of each sample of milk into a guinea- 

 pig or several of these animals. This is rarely done, as it requires 

 too many experimental animals and too much really unnecessary 

 labor. 



As a rule, several cover-glasses are prepared from the mixed creams 

 and sediments, and they are examined by the Ziehl-Neelson method 

 of staining with carbol-fuchsin solution. The samples which contain 

 acid-fast bacilli are diluted with sterile physiologic salt solution and 

 inoculated subcutaneously into guinea-pigs, best in the neighborhood 

 of some lymph gland (inguinal glands) with the suspected material. 

 In experiments for the detection of live tubercle bacilli in milk, 

 butter, etc., the inoculations should always be made subcutaneously, 

 not intrapejritoneally, because in the latter case guinea-pigs generally 

 develop pseudotuberculous lesions in the presence of the acid-fast 

 bacilli of Moller, Rabinowitsch and others, even when tubercle 

 bacilli are absent. 



For the microscopic examination of butter for acid-fast bacilli 

 the following procedure is recommended by Roth : 



I. Two to 4 grams of butter are placed in a sterile centrifuge tube, 

 which is filled about three-quarters full with sterile, distilled water. 



II. This tube so prepared is placed in a water bath and kept at 

 50 C. until all of the butter is melted. 



III. The tube is now closed with a cork or glass stopper and well 

 shaken for some time to mix the fat and water thoroughly. It is now 

 placed into the incubator in an inverted position, i. e., with the cork 

 or glass stopper down and the pointed end of the centrifuge tube up. 

 After all the fat has risen to what is now the upper end, the tube 

 is left in a cool place until the butter fat has again solidified. 



IV. The tube is now inverted, the cork or stopper opened, the 

 wash water which now contains most of the bacteria is poured into 

 another sterile centrifuge tube, and this is again centrifuged. 



V. The sediment is then used for cover-glass preparations, and 

 these, when air dry, are washed for a short time in an absolute 

 alcohol-ether mixture. 



VI. The cover-glass preparations are then stained and decolorized 

 in the usual manner. 



Typhoid Bacillus. The bacilli which cause the human disease 

 typhoid fever are voided in enormous numbers during and after the 

 course of the affection with the feces. By getting into wells supplying 

 farms and dairies from vaults, cesspools, and other sources, or by 



