PREPARATION OF MEDIA ii«^9 



Media for H2S production. In previous editions, four media have 

 been listed containing lead or iron salts, designed to show blackening 

 when hj^drogen sulfide is produced. As the present procedure given 

 in Leaflet V calls for lead acetate test-strips in the mouths of the 

 tubes, these media are no longer recommended for routine use. 

 Those who wish to use such media are referred to the papers of 

 Bailey and Lacy (1927) and of Wilson (1923), who describe lead and 

 iron salt media, respectively; or they may consult the manual of the 

 Difco Laboratories, who manufacture dehydrated media for the 

 purpose in question. 



Churchman's gentian violet agar for selective bacteriostasis. To 

 ordinary beef- extract -peptone agar add a definitely determined 

 amount of crystal violet of about 85% dye content. If the medium 

 is to be used to inhibit Gram-positive organisms and permit the 

 growth of Gram-negatives the dye concentration should be about 

 1 :100,000. If it is to be used for differentiation between the Gram- 

 positives its concentration should be between 1 :400,000 and 1 :800,000; 

 if for differentiation between Grajn-negatives it should be between 

 1:1,000 and 1:40,000. In either of the two latter cases the exact 

 concentration depends upon which particular bacteria it is desired 

 to inhibit and which to permit to grow. 



C. Media for Special Groups of Aerobes 



1. BASAL MEDIA 



Douglas trypsin broth {Hartley) (L&S No. 1123). Mix 150 g. of 

 lean minced horse meat with 250 ml. tap water and heat at 80°C in 

 a steamer. Add 250 ml. of an 0.8% Na2C03 (anhydrous) and cool to 

 45°C. Add 5 ml. of chloroform and 5 ml. of pancreatic extract pre- 

 pared as directed by Cole and Onslow (1916) and Douglas (1922). 



Preparation of pancreatic extract: To 1000 g. minced fresh pig pancreas (free from 

 fat) add 3000 ml. distilled water and 1000 ml. 95% ethyl alcohol. Place in a large 

 clean bottle; shake repeatedly; and allow to stand 3 days at room temperature. Strain 

 through gauze and filter through paper. (Filtration is slow.) Add 1 ml. cone. HCl. 

 to each 1000 ml. of filtrate. This causes a cloudy precipitate which settles in a few 

 days and can be filtered off. The liquid keeps indefinitely if placed in a stoppered 

 bottle; no additional antiseptic is needed. 



Estimation of activity: Centrifuge fresh milk and discard the cream; add 1% CaCl,. 

 Make a series of dilutions (1:100, 1:200. 1:500, 1:1000, 1:2000, 1:-1000, etc.) of the 

 pancreatic extract, and place in tubes, 1 ml. to the tube. To each tube add 1 ml. of 

 the milk. Place in a water bath at 50°C for 30 min. The highest dilution of trypsin 

 which causes clotting is a measure of its potency. Alcoholic pancreatic extract usually 

 causes clotting at 1:1000; Bacto-trypsin at 1:5000. 



