CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



603 



(9) Allow to cool to 60°C. 



(10) Clarify with white of egg. 



(11) Bring the volume to its orginal 

 amount. 



(12) Estimate and make the reaction 

 0.6% acid to phenolphthalein. 



(13) Add to the melted nutrient agar, 

 2.5% glucose. 



(14) Steam 30 minutes. (Note: It is 

 better to rely on one steaming for 

 sterilization than to sterilize by 

 steaming on three successive days.) 



(15) Distribute in quantities of 4.0 cc. 

 into test tubes. 



(16) Keep until required for use in the 

 ice chest. 



(17) Prepare sterile 10.0 cc. centrifuge 

 tubes each containing 2.0 cc. sterile 

 2.0% sodium citrate. 



(18) Have in readiness corks or rubber 

 bungs, contained in alcohol to fit the 

 centrifuge tubes. 



(19) Fill up the centrifuge tubes with 

 human blood sterilely aspirated. 



(20) Replace the wool plugs of the centri- 

 fuge tubes by corks after burning 

 off the alcohol. 



(21) Centrifuge. 



(22) Prepare with sterile precautions: 

 Centrifuged blood fluid at 45°C. 

 (21) 75; melted nutrient agar (13) 

 contained in the test tubes at 45°C. 

 4. 



(23) Roll the test tubes between the 

 hands to mix. 



(24) Test sterility by incubating 48 hours. 

 Sterilization: See step (14) under prep- 

 aration. 



Use: Cultivation of gonococci. 

 Reference: Harvey (1921-22 p. 82). 



1903. Grassberger's Blood Agar 



Constituents : 



1. Infusion agar. 



2. Blood. 

 Preparation: 



(1) Prepare beef infusion agar. 



(2) Add 10.0% NaOH until blue litmus 

 paper is no longer blue. 



(3) Distribute in 20.0 cc. lots. 



(4) Add 20 drops diluted 10.0% soda 

 solution to each flask so as to obtain a 

 normal agar (10.0 cc. N/1 soda solu- 

 tion, for 1000.0 cc. agar). (The 



e.xact amount of soda solution, or 

 dilution of soda solution was not 

 specified.) 



(5) Pour into plates. 



(6) Smear 1.0 cc. of blood over each plate. 

 Sterilization: Not specified. 



Use: Cultivation of influenza bacilli. 



Other investigators employed similar 



media for various purposes. 

 Variants : 



(a) Waksman studied the metabolism of 

 actinomycetes on a medium prepared 

 as follows: 



(1) Boil 500.0 g. veal with 1000.0 cc. 

 tap water for 10 minutes. 



(2) Filter. 



(3) Add 10.0 g. Bacto peptone, 5.0 g. 

 NaCl, and 25.0 g. agar and dissolve. 

 (Heat). 



(4) Adjust to +1.0 (pH = 7.6-7.8). 



(5) Flask and sterilize. 



(6) Cool to 45°-50°C. and add 10.0% 

 (100.0 cc.) sterile whole rabbit 

 blood. Shake. 



(7) Pour, under sterile conditions, into 

 sterile test tubes or Petri dishes. 



(8) Slant tubes. 



(b) Twort and Twort prepared a similar 

 medium as follows: 



(1) Prepare infusion agar and infusion 

 broth. 



(2) Sterilize the broth in a flask. 



(3) Add 5.0% rabbit blood, withdrawn 

 from the vein of the ear with a 

 sterile syringe, to (2). 



(4) Incubate (3) at 37° for one hour, 

 shaking repeatedly to prevent the 

 forming of too much clot. 



(5) Melt infusion agar tubes and cool 

 to 50°C. 



(6) Add about 20 drops of the fluid 

 from (4) to each tube of (5). 



(7) Slant. 



(c) Harvey gave the following method 



of preparation : 



(1) Wash and scrub the finger to be 

 used well with hot soap and water. 



(2) Drop on to the finger absolute 

 alcohol followed by ether to remove 

 the alcohol. 



(3) Congest the pulp of the finger by 

 winding a bandage round the base. 



(4) Prick the congested pulp with a 

 sterile needle. 



