110 



in a refrigerator over night to allow the cream to rise and the suspended 

 matter to settle. The skimmed milk shall be siphoned off into a flask for 

 use. It will be found more convenient, however, to allow the milk to stand 

 in a separatory funnel. Should the milk be too acid the reaction shall be 

 corrected to +1 by the addition of normal sodium hydrate. It is then ready 

 to be tubed and sterilized. Litmus milk shall be prepared as above, with 

 the addition of sterile 1 percent azolitmin. As it is impossible to make 

 each lot of litmus milk with the same shade of color, it is recommended 

 that a control tube be always exposed with the inoculated tubes for the 

 purposes of comparison. 



III. MEDIA FOR PRELIMINARY ENRICHMENT OR THE PRE- 

 SUMPTIVE TEST. 



A. Lactose Broth. (Standard Methods A. P. H. A., 1920). Sugar 

 broths shall be prepared in the same general manner as nutrient broth with 

 the addition of 0.5 percent of the required carbohydrate just before sterili- 

 zation. The removal of muscle sugar is unnecessary as the beef extract 

 and peptone are free from any fermentable carbohydrates. The reaction 

 of sugar broths shall be a faint pink with phenol red or, if on titration 

 with phenolphthalein the reaction is not already between neutral and +1, 

 adjust to neutral. Sterilization shall be in the autoclave at 15 pounds 

 (120 C.) for 15 minutes after the pressure reaches 15 pounds, provided 

 the total time of exposure to heat is not more than one-half hour; other- 

 wise a 10 percent solution of the required carbohydrate shall be made in 

 distilled water and sterilized at 100 C. for iy 2 hours, and this solution 

 shall be added to sterile nutrient broth in amounts sufficient to make a 

 0.5 percent solution of the carbohydrate and the mixture shall then be 

 tubed and sterilized at 100 C. for 30 minutes, or it is permissible to add 

 by means of a sterile pipette directly to a tube of sterile neutral broth 

 enough of the carbohydrate to make the required 0.5 percent. The tubes so 

 made shall be incubated at 37 C. for 24 hours as a test for sterility. 



B. Lactose (Peptone) Bile. The lactose bile medium consists 

 of sterilized undiluted fresh ox gall (or a 10 percent solution of dry fresh 

 ox gall) to which has been added 1 percent of peptone and 1 percent of 

 lactose. The addition of peptone is important. 



C. Lactose Bile Salt Broth. (After Savage). 



Sodium taurocholate 5 grammes 



Lactose 5 grammes 



Peptone 20 grammes 



Water 1000 c. c. 



These constituents are heated together until the solids are dissolved. 

 The mixture is filtered, and sufficient neutral litmus solution is added to 

 give a distinct color. The medium is then distributed into Durham's fer- 

 mentation tubes and sterilized by steaming for twenty minutes on three 

 successive days. 



The sodium taurocholate prevents the growth of many saprophytic 

 bacteria. 



