CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



795 



and keep at 95° for one hour. Repeat 

 this inspissating at 95°C. for one 

 hour on 2 successive days. Seal 

 tubes by dipping the cotton plug 

 into a hot mixture of 7 parts paraffin 

 and 1 part bees wax and 1 part 

 petrolatum. 



(b) Roddy. 



(1) Infuse 500.0 g. veal in 500.0 cc. of a 

 15.0% solution of glycerol in water 

 for 24 hours. 



(2) Strain off the juice. 



(3) Sterilize the shells of eggs with 

 20.0% phenol. 



(4) Break the eggs into sterile beakers 

 and mix well. Avoid air bubbles. 



(5) Filter thru gauze. 



(6) Add one part of (2) to one part of 

 (5) by volume. 



(7) Add sufficient 1.0% alcoholic gen- 

 tian violet to make a 1:10,000 

 dilution. 



(8) Tube. 



(9) Sterilize in the steam sterilizer or 

 inspissate for 3 successive days at 

 80° for one hour each day. 



(c) Waksman cultivated actinomyces on 



the following medium: 



(1) Prepare 500.0 cc. of meat juice 

 containing 15.0% glycerol. 



(2) Mix 1000.0 cc. beaten eggs, (1) 

 and 1:10,000 gentian violet. 



(3) Tube. 



(4) Slant. 



(5) Coagulate. 



(6) Sterilize in the Arnold at 75 to 

 90° for 3 consecutive days. 



(d) Limousin. 



(1) To 250.0 g. of fresh sterile crushed 

 veal and 212.0 g. of distilled water 

 and 37.5 g. sterile glycerol. 



(2) Place in the ice box over night and 

 filter thru sterile gauze, under 

 aseptic conditions. 



(3) Sterilize the shell of 16 to 20 eggs 

 by immersing in alcohol for 15 

 minutes. 



(4) Open the eggs under aseptic 

 conditions and remove the yolk 

 and white of the eggs. 



(5) Thoroly mix the yolk and white 

 under aseptic conditions. 



(6) Mix 400.0 cc. of (2) and 400.0 cc. 

 of (5) under aseptic conditions. 



(7) Dissolve 0.5 g. gentian violet in 

 50.0 cc. of 95% alcohol. 



(8) Add 1.0 cc. of (7) for each 100.0 

 cc. of (6) to (6) under aseptic 

 conditions. 



(9) Distribute in sterile tubes. 



(10) Heat on 3 successive days as 



follows : 



85° for 30 minutes on the 1st day. 



75° for 30 minutes on the 2nd day 



75° for 30 minutes on the 3rd day. 

 Limousin stated that all apparatus, 

 etc., coming in contact with this 

 medium must be sterile, and prepa- 

 ration carried on under aseptic 

 conditions. 



(e) Despeignes. 



(1) Grind 250.0 g. of veal in a sterile 

 grinder. 



(2) Mix 212.0 cc. distilled water and 

 37.5 g. of sterile glycerol with (1). 



(3) Allow to stand over night and 

 filter thru sterile muslin. 



(4) Sterilize by heating on 8 successive 

 days for one hour at 55 to 57°C. 



(5) Place the necessary number of 

 eggs in alcohol at 70°C. for 15 

 minutes. 



(6) Break the egg and mix the con- 

 tents in a sterile container with a 

 sterile glass rod. 



(7) Filter (6) thru sterile muslin. 



(8) Mix 200.0 cc. of (4) with 400.0 cc. 

 of (7) under aseptic conditions. 



(9) Add 6.0 cc. of a 1.0% solution of 

 gentian violet in 95% alcohol to 

 (8). 



(10) Mix well. 



(11) Distribute in sterile tubes. 



(12) Slant the tubes and heat at 85° 

 for 30 minutes. 



(13) Heat at 75°C. for 30 minutes on 

 the two following days. 



(14) Incubate 3 days at 37°C. to test 

 sterility. 



(f) Stitt. 



(1) Treat 500.0 g. of chopped up meat 

 with 500.0 cc. of 15.0% glycerol 

 solution. 



(2) Keep in the ice chest for 24 hours. 



(3) Filter thru gauze. 



(4) Sterilize the shells of 2 eggs by 

 immersing in 70.0% alcohol for 10 



