584 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



(2) Heat at a temperature not exceed- 

 ing 68°C. until the red color of the 

 mixture turns brown. 



(3) Make faintly alkaline to litmus. 



(4) Add after this alkalinization 1.0 

 CO. N/1 sodium hydroxide per liter. 



(5) Steam 60 minutes. 



(6) Separate the clot formed from the 

 sides of the vessel. 



(7) Steam 90 minutes. 



(8) Allow to stand at room tempera- 

 ture 10 minutes. 



(9) Pipette off the fluid and place in a 

 tall cylinder. 



(10) Leave 20 minutes. 



(11) Skim off the fat from the surface. 



(12) Distribute into test tubes. 



(13) Sterilize at 100°C. for three days. 

 Harvey reported that the medium is 

 not filtered at any stage thru any 

 cloth, filter paper or cotton wool. If 

 filtration is needed it should be thru 

 glass wool. A less amount of agar, 

 2. g., 0.5% instead of 1.6% will give a 

 medium in which, if sealed and kept 

 in the incubator, meningococcus and 

 gonococcus sown by the stab culture 

 method will live for two or three 

 months. 



(d) Torrey and Buckell called the follow- 

 ing medium a semi solid agar, but did 

 not mention the addition of agar. 

 Ascitic fluid may be used to ad- 

 vantage in rejuvenating delicate 

 strains of gonococci or recovery of 

 old stock strains. Also in primary 

 fishing of gonococci colonies. 



(1) Mix 500.0 g. of finely chopped fresh 

 beef heart and one whole egg with 

 water. 



(2) Place in a double boiler over a 

 free flame and maintain at 60°C. 

 for 5 minutes. Stir constantly. 



(3) Add 10.0 g. peptone and 5.0 g. 

 NaCl and raise temperature until 

 the mixture assumes a brownish 

 color. 



(4) Adjust reaction to slightly alka- 

 line to litmus using 10.0% NajCO, 

 solution. 



(5) Place (4) in flask or preferably in a 

 coffee pot and heat in Arnold 

 steam sterilizer for an hour. 



(6) Separate clot from the sides of the 

 receptacle and replace in the 

 sterilizer for another hour. 



(7) Clear by centrifugation, or by 

 straining thru wire mesh and then 

 thru glass wool . Or the meat resi- 

 due can be deposited on the glass 

 wool in a funnel and the fluid be 

 allowed to percolate thru it several 

 times. Never use cotton or any 

 other adsorptive materials to 

 clarify. 



(8) Adjust reaction to pH = 6.8. 



(9) Reheat and tube in 7.0 cc. lots. 



(10) Sterilize in Arnold sterilizer. 



(11) If ascitic fluid is to be used, add 

 1.0 cc. to each test tube. 



(e) Park, Williams and Krumwiede pre- 

 pared a similar vitamin agar for the 

 cultivation of meningococci as 

 follows: 



(1) Add 500.0 g. tap water to 500.0 g 

 of chopped fat and tendon free 

 beef heart. 



(2) Add 15.0 g. of peptone, 5.0 g. 

 NaCl, and one well beaten egg to 

 (1). 



(3) Heat in a water bath, double 

 boiler or over the open flame, 

 stirring constantly until the color 

 changes to brown (at about 68° 

 to 70°C.). 



(4) Strain thru a wire sieve or wire 

 gauze. Do not use cheese cloth, 

 cotton or paper. 



(5) Dissolve 15.0 g. of agar in 500.0 cc. 

 tap water. 



(6) Cool(5)to70°C. 



(7) Mix (4) and (6). 



(8) Adjust the reaction to +0.2 

 phenolphthalein or to about pH 

 = 7.4. 



(9) Heat in an autoclave at 15 pounds 

 pressure for 30 minutes. 



(10) Remove the kettle carefully and 

 set aside for sedimentation to take 

 place or run in Sharpies centrifuge. 



(11) If the sedimentation method is 

 used, turn the solid agar out of the 

 kettle in mould form and trim off 

 the layer of sediment and discard 



(12) Melt. 



(13) Tube. 



