CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



503 



(2) Weigh the kettle used in (1) and 

 its contents. 



(3) Heat at 45°C. for an hour. 



(4) Boil for 30 minutes. 



(5) Make up the loss by weight by the 

 addition of water. 



(6) Strain thru cheese cloth. Squeeze 

 by twisting the cloth or use the 

 meat press. 



(7) If used at once dissolve 1.0% pep- 

 tone and NaCl in (6). 



(8) Dissolve 1.5% agar in (7) by heat- 

 ing in the autoclave at 10 to 15 

 pounds pressure for 30 minutes or 

 by boiling over the free flame. 



(9) If boiled over a ree flame make up 

 the loss in weight by the addition 

 of water. 



(10) Adjust the reaction to +0.2% to 

 phenolphthalein . 



(11) Cool to 50°C. and add one egg. 



(12) Test the reaction and readjust if 

 necessary. 



(13) If more than 0.2% normal soda is 

 required per liter, heat again for 

 ten minutes. 



(14) Filter thru cotton. 



(15) Distribute in tubes or flasks. 



(16) Sterilize at 15 pounds pressure for 

 30 minutes. 



(g) Stafseth (Huddleson, Hesley and 

 Torrey used the following medium 

 for isolation and cultivation of Bac- 

 terium abortus (Bang): 



(1) Grind fresh fat free beef liver in a 

 meat chopper to a plastic mass. 



(2) Mix (1) with 500.0 cc. of tap water. 



(3) Place in flowing steam for 20 

 minutes. 



(4) Remove the lid, and stir with a 

 glass rod in order to mix thoroly. 



(5) Continue the heating in flowing 

 steam for Ij hours. 



(6) Remove and filter thru a wire 

 screen. (This infusion may be 

 sterilized and stored until ready 

 for use, or used at once.) 



(7) Mix 500.0 cc. of (6) with 500.0 cc. of 

 tap water, 20.0 g. washed agar, 

 10.0 g. Bacto peptone and 5.0 g. 

 NaCl, and place in a covered 

 container. 



(8) Place in flowing steam for 30 

 minutes. 



(9) Adjust to pH = 7.0. 



(10) Add 1.0% dissolved egg albumin. 



(11) Mix well and place in flowing 

 steam for 90 minutes. 



(12) Decant the liquid from the clot. 



(13) Remove small clumps of albumin 

 by filtering thru a discarded pres- 

 sure cooker. Place glass wool, 

 previously washed with dilute 

 acid, in the barrel of the filter and 

 a 30 mesh copper screen funnel for 

 collecting large coagulated par- 

 ticles. 



(14) Readjust to pH = 7.0 if necessary. 

 The reaction should fall to pH 6.6 

 following sterilization. 



(15) Sterilize at 15 pounds pressure for 

 30 minutes. 



(16) For isolation work add sufficient 

 quantity of a saturated aqueous 

 solution of gentian violet so that 

 the concentration be 1:10,000. 



References: White (1917 p. 49), Bronfen 

 brenner and Schlesinger (1918 p. 125) 

 (1918-19 p. 219), Heller (1921 p. 460) 

 Goss, Barbarin and Haine (1921 p. 615) 

 Harvey (1921-22 p. 69), Heller (1922 p. 9) 

 Klimmer (1923 p. 201), Park, Williams 

 and Krumwiede (1924 p. 131), Huddleson, 

 Hasley and Torrey (1927 p. 356). 



1670. Richardson's Mucosa Infusion Agar 



Constituents : 



1. Distilled water 500.0 cc. 



2. Mucosa of hog intestine 250.0 cc. 



3. Peptone 5.0 g. 



4. NaCl 2.5 g. 



5. Agar 3.8 g. 



Preparation : 



(1) Take small intestine of a hog in 

 fresh condition without cutting, and 

 wash thoroly with running water 

 until the water runs perfectly clear. 



(2) Lay the intestine open and scrape 

 off the mucosa with a glass slide 

 (150.0 g. of mucosa is easily obtained 

 from one hog). 



(3) To 250.0 cc. of the mucosa add 

 500.0 cc. of distilled water. ) 



(4) Boil for 30 minutes. Cool. "^ 



(5) Boil again then neutralize and boil 

 once more. 



(6) Filter. 



