CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



Preparation : 



(1) Mince fresh fat free ox heart muscle 

 and suspend in twice its weight of 

 distilled water in an open can in a 

 steamer. 



(2) Raise the temperature gradually to 

 70°-75°C. and hold for three hours. 



(3) Raise the temperature to 100°C. and 

 keep at this temperature for 

 15 minutes. 



(4) Pour the infusion and meat on a 

 wetted butter muslin and filter until 

 clear. 



(5) This constitutes the stock infusion 

 and may be sterilized or used im- 

 mediately to prepare a 4.0% agar 

 solution. 



(6) Prepare an infusion as above, steps 

 (1) and (2). 



(7) Filter until clear thru the meat 

 particles suspended on well wetted 

 butter muslin. 



(8) Prepare an equal volume of 4.0% 

 agar solution in the stock infu- 

 sion (5). 



(9) Cool (8) to 70°C. and mix with (7). 



(10) Heat in the steamer for one hour. 



(11) Allow to set and stand over night. 



(12) Melt the following morning and 

 strain through lint. 



(13) Suspend the residual meat and fine 

 coagulum from (4) and (7) in a 

 quantity of N/100 HCl equal to the 

 weight of the original raw meat. 



(14) Raise to 100°C. and autoclave at 

 130°C. (25 lbs. pressure) for 30 

 minutes. 



(15) Allow to cool to 37°C., incubate 24 

 hours, to test sterility. 



(16) Add 2.0% of sterile pancreatic ex- 

 tract and incubate for 5 to 15 hours. 



(17) Add 0.8% anhydrous NaoCOs in the 

 form of a sterile 32.0% solution. 



(18) Allow digestion to continue until a 

 Sorensen figure of not less than 20 

 is produced. (Titrate the digest in 

 the presence of neutralized formalin, 

 using phenolphthalein as an indi- 

 cator, and express the result in cc. 

 of N/10 NaOH required to neu- 

 tralize the amino acids in 10.0 cc. 

 of the filtered digest.) 



(19) Add 2.0% of N/10 HCl and auto- 

 clave. 



(20) Filter while hot through paper. 



(21) Add 0.25% NaCl, 0.02% KCl, 0.01% 

 CaClj and the desired amount of 

 (20) to (12), 



(22) Adjust to pH = 7.2. 



(23) Tube. 



Sterilization: Autoclave at 120°C. for 



20 minutes. 

 Use: Cultivation of meningococci. 

 Reference: Murray and Ayrton (1924-25 



p. 49). 



2068. Hottinger's Agar (Park, Williams and 

 Krumwiede) 



Constituents : 



1. Water 1500.0 cc. 



2. Meat 750.0 g. 



3. NaaCOa 3.0 g. 



4. Agar 1.5% 



Preparation : 



(1) Free meat from fascia and cut in 

 finger thick pieces. 



(2) Heat 1500.0 cc. of water to boiling. 



(3) Drop 750.0 g. of (1) into (2), piece 

 by piece, stirring constantly. 



(4) Boil strongly and remove from the 

 fire. 



(5) Remove the meat and run thru a 

 chopping machine. 



(6) Cool the water to 37°C. and add 

 1.5 g. NaaCOa per liter. 



(7) Put the chopped meat in 2 liter 

 Erlenmeyer flasks, 550.0 g. per flask. 



(8) Add (6) to each flask, filling the 

 flask to the neck. 



(9) Add 3.0 g. pancreatin, 10.0 cc. chloro- 

 form and 10.0 cc. toluol to each flask. 



(10) Cork tightly and shake well. 



(11) Incubate at 37°C. over night. 



(12) Shake the next day and add more 

 pancreatin unless the fluid shows a 

 yellow color and particles of meat 

 look smaller. 



(13) Continue digestion for four or five 

 days at room temperature, or for 

 two or three days in the incubator. 

 The meat should be finely divided 

 at the end of this time. 



(14) Decant the liquid thru cheese cloth. 



(15) Add an equal volume of water to the 

 residue, shake well and again decant. 



(16) Place the meat on cheese cloth and 

 allow to drain. 



(17) Boil the filtrate for a few minutes. 



