276 APPENDIX 



cent agar makes the diflfusion of the acid which is formed 

 more difficult. 



Three pounds of chopped beef are allowed to stand 24 

 hours with 2 liters of water. The meat infusion is boiled 

 one hour and filtered. Twenty gm. of Witte's peptone, 

 20 gm. of nutrose, and 10 gm. of NaCl are then added, 

 and the mixture boiled another hour. After filtration and 

 the addition of 60 gm. of agar the mixture is boiled for 3 

 hours, made alkaline and filtered. In the meantime 300 

 c.c. of litmus solution (Kahlbaum) are boiled for 15 minutes 

 with 30 gm. of lactose. Both solutions are then mixed 

 and the mixture, which is now red, made faintly alkaline 

 with 10 per cent soda solution. To this feebly alkaline 

 mixture 4 c.c. of hot sterile 10 per cent soda solution are 

 added and 20 c.c. of a sterile solution (i to 1000) of crystal 

 violet (Hochst B.). 



ENDO MEDIUM 



Make 3 per cent agar as follows: 



1. To I liter of cold water add 30 gm. of powdered agar 

 by sifting successive small portions upon the surface and 

 allowing them to settle. 



2. Add 10 gm. of Witte's peptone and 5 gm. of Liebig's 

 meat extract. 



3. Heat in double boiler until the ingredients are thoroughly 

 dissolved. 



4. Neutralize the sodium carbonate, using litmus as an 

 indicator, and then add 10 c.c. of a 10 per cent solution 

 of sodium carbonate. 



5. Store the medium in flasks in lots of 100 c.c. or in larger 

 known quantities, the flasks being large enough for the 

 other ingredients to be added later. 



6. Sterilize 2 hours in streaming steam. 



It is essential to use powdered agar and cold water, 

 since the agar settles more readily in cold water. 



