274 APPENDIX 



8. Tube and sterilize in an autoclave for 15 minutes 

 at 120 C. (15 pounds). 



Other valuable liver media (for use in the identification 

 of B. sporogenes and other species) are prepared as given 

 below: 



LIVER GELATIN 



1. Proceed as in steps i, 2, 3, in preparing liver broth. 



2. Cool the filtrate to 50 C. Add 10 per cent of sheet 

 gelatin and stir a few minutes until dissolved. 



3. Add i per cent of peptone, i per cent of dextrose 

 and o.i per cent of potassium phosphate. 



4. Stir until the ingredients are dissolved, keeping the 

 temperature below 50 C., and then proceed as in steps 



5, 6, 7, 8. 



LIVER AGAR 



1. Chop 500 gm. of beef liver into small pieces, add 500 

 c.c. of distilled water, and boil slowly for 2 hours, stirring 

 occasionally. 



2. Add 5 gm. of agar (dried at 105 C. for 30 minutes) 

 to 500 c.c. of distilled water and digest for 30 minutes in 

 an autoclave at 120 C. (15 pounds pressure). 



3. After making up the loss by evaporation, pass the 

 liver infusion through a wire strainer, add 500 c.c. of the 

 filtrate to the agar solution and proceed as in steps 4, 5, 



6, 7, 8, in preparing liver broth. 



It is very important to note that liver broth should not 

 be exposed to the high temperature attained in the auto- 

 clave any longer than 15 minutes, as prolonged heating 

 above the boiling-point causes caramelization of the carbo- 

 hydrates, rendering the medium less delicate for bacterial 

 development. For the rejuvenation of attenuated cultures, 

 especially B. sporogenes, the addition of very small pieces 

 of liver tissue, which have been sterilized in Petri dishes 



