II44-10 MANUAL OF METHODS FOR PURE CULTURE STUDY 



Incubate at ST^C for 6 hours, shaking frequently. Add 40 ml. 

 normal HCl and heat in the steamer for 30 minutes. Cool and filter. 

 Adjust to pH 8.0. Distribute as desired. 



Pass steam through the autoclave for one hour then raise the pres- 

 sure slowly to 10 pounds and turn off the steam. For sterilization of 

 larger quantities (one litre in a flask) maintain the pressure at 10 

 pounds for 30 minutes. 



Use: Preparation of diphtheria toxin, for growth of numerous 

 pathogens, and as medium for blood culture. 



KracJce and Teasley medium. Dissolve 500 g. finely ground fat- 

 free heart muscle in 1000 ml. water. Place in ice-box overnight. 

 Filter through four layers of gauze, heat to boiling, and filter through 

 fine wire mesh or copper gauze. 



Mix separately 500 g. finely ground brain in 1000 ml. water. Place 

 in ice-box over night. Filter and heat slowly to boiling; keep stirring. 

 Do not filter after heating. 



Prepare medium as follows : 



75 ml. heart muscle extract 1 g. glucose 



25 ml. brain suspension 1 g. peptone 



0.1 g. sodium citrate (NajCeHsO^+gH^O) 0.5 g. Na^HPO^ (anhydrous) 



Heat until ingredients are in solution, adjust to pH 7.4, autoclave at 

 15 pounds for 15 minutes. 

 Use: Blood culture of pathogens. 



Ascitic fluid agar. Various formulae have been proposed. A simple 

 one is as follows: 



Melt 100 cc. of sterile Douglas' agar, pH 7.4-7.8, in a flask. Cool to about 48° to 

 50°C. With sterile pipette add 20 ml. of sterile, bile-free, ascitic fluid. Pour into 

 tubes or plates and allow to harden. 



Use: Cultivation of pathogenic cocci. 



Loeffler's blood serum. A common formula for this calls for glucose 

 beef-extract broth as its basis. This is prepared as follows: 



Beef extract 3 g. Peptone (Difco or Witte) 10 g. 



Glucose 10 g. NaCl 5 g. 



Distilled water 1000 ml. 



Mix the ingredients and dissolve by warming over a flame. Do not adjust the re- 

 action. Filter through paper. When the broth is cool add one volume to three vol- 

 umes of clear serum of horse, beef, or pig. Tube, 3 to 4 ml. per tube, and place tubes in 

 a slanting position in a pan or rack. Take care to prevent the occurrence of bubbles 

 and frothing. Cover tubes with newspaper. Sterilize in autoclave at 15 pounds for 15 

 minutes without letting the air out, and repeat on two successive days. Or if it is pre- 



