614 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



with two parts sterile melted (2). 

 (Pleuritic or hydrocele fluid may 

 be used instead of ascitic fluid.) 

 (4) Replace the cotton stoppers with 

 sterile corks. 



(b) Harvey prepared a similar medium 

 as follows: 



(1) Mix one part ascitic fluid with two 

 parts infusion agar (see variant 

 (v) medium (1661) for prepara- 

 tion). 



(2) Sterilize in the water bath 30 min- 

 utes at 56°C. on five successive 

 days. 



(3) Test sterility before use by incu- 

 bation 48 hours. 



(c) Schwartz (Stitt) gave the following 

 method of preparation: 



(1) Prepare beef or veal infusion agar 

 in the ordinary manner. 



(2) Adjust (1) to pH = 7.6 phenol- 

 sulphonephthalein being used as 

 an indicator in the hot agar. 



(3) Cool to 50"C. 



(4) Add the whites of three fresh eggs 

 to each liter of agar. 



(5) Start with a low flame, boil for 

 10 minutes. 



(6) Strain thru cloth and filter thru 

 paper. 



(7) Tube in 5.0 to 6.0 cc. quantities 

 and autoclave at 10 pounds pres- 

 sure on 3 successive days. 



(8) Sterilization reduces the pH to 7.4. 



(9) Add sterile ascitic, pleuritic or 

 hydrocele fluid to each tube. One 

 part fluid to two parts agar. 



(10) Seal the tubes with sterile rubber 

 stoppers and slant. 



(11) Cork the tubes and store in the 

 incubator. 



(12) Discard all contaminated tubes. 



(13) Inoculate when the medium is at 

 body temperature. 



(14) After inoculation hold the tube 

 horizontal by the butt with the 

 agar slant up. Pass longitudi- 

 nally thru the Bunsen flame about 

 three or four times and cork 

 quickly. This reduces the pres- 

 sure within the tube. 



(15) The agar tubes should have about 

 0.5 cc. of water of condensation in 

 the lower angle of the slant. 



(d) Mulsow used the following medium 

 for the isolation of gonococci: 



(1) Infuse one pound of ground lean 

 beef in 500.0 cc. of distilled water 

 over night in the ice box. 



(2) Dissolve 10.0 g. peptone in the 

 juice while cool. 



(3) Add 500.0 cc. of a 3.0% agar solu- 

 tion, melted and cooled to 60°C , 

 to (2). 



(4) Adjust to +0.9 to phenolphthalein. 



(5) Before the agar has cooled suffi- 

 ciently to harden, it is heated in 

 the autoclave at 15 pounds pressure 

 for 25 minutes. 



(6) Filter thru moistened cotton and 

 canton flannel. 



(7) Distribute in 100.0 cc. lots in 

 flasks. 



(8) When ready for use, sterile (7) is 

 melted and cooled to 60°C. and 

 added to 50.0 cc. of ascitic fluid. 

 (0.5 g. of any carbohydrate (levu- 

 lose or maltose) may be added 

 along with 1 cc. of a 0.04% solution 

 of brom cresol purple.) 



(9) Pour in sterile plates. 



(10) Final reaction -1.2 to phenol- 

 phthalein or pH = 6.8. 

 References: Scholtz (1899 p. 5), Schwartz 

 and Davis (1920 p. 1125), Harvey (1921- 

 22 p. 83), Stitt (1923 p. 45), Mulsow 

 (1925 p. 421). 



1928. Kiefer's Ascitic Fluid Agar (Abel) 

 Constituents : 

 1- Water lOOO.O cc. 



2. Meat 500.O g. 



3. Agar (3.5%) 35.0 g. 



4. Peptone (5.0%) 5O.O g. 



5. Glycerol (2.0%) 20.0 g. 



6. NaCl (0.5%) 5.0 g. 



7. Ascitic fluid 1000.0 cc. 



Preparation : 



(1) Chop 500.0 g. of fat free meat and 

 add to a liter of water at 50°C. 



(2) Keep at 50°C. for 30 minutes and then 

 boil for 30 to 45 minutes. 



(3) Filter or strain the fluid from the 

 meat. 



(4) Make up the fluid to 1 liter. 



(5) Dissolve 3, 4, 5 and 6 in (4). 



(6) Neutralize. 



(7) Cool to 50°C. 



