CULTURE MEDIA FOR CULTIVATIOX OF MICROORGANISMS 



773 



2398. Frothingham's Coagulated Blood 

 Serum 



Constituents: 1. Serum 

 Preparation : 



(1) Collect blood in large jars which can 

 be closed tightly. Close the jars. 



(2) When the blood has begun to clot, 

 I pass a sterile glass rod around the 



clot between its surface and the wall 

 of the jar. This permits the clot to 

 sink. 



(3) Close the jars and place in an ice box 

 for 24 to 48 hours. 



(4) Draw off the serum with a sterile 

 pipette and distribute into sterile 

 test tubes. 



(5) Place in a blood serum sterilizer and 

 heat to 68 or 70°C. for one hour on 

 five successive days. 



(6) Place the tubes in the apparatus for 

 solidifying blood serum and heat to 

 75 or 80°C. until the serum is co- 

 agulated. 



Sterilization: Sterilization given with 

 method of preparation. 



Use: Culture medium for parsitic forms. 



Variants: The following methods of prep- 

 aration have been given by the following 

 investigators: 



(a) Frothingham heated the serum col- 

 lected as indicated above, at 90 to 

 95°C. for an hour or more, and then 

 steamed on each of 2 successive days 

 for 15 to 30 minutes. 



(b) Michel cultivated diphtheria bacilli 

 on the following medium: 



(1) Obtain horse or beef serum under 

 sterile conditions. 



(2) Distribute into sterile test tubes. 



(3) Solidify in a Koch's apparatus at a 

 temperature of 85°C. 



(c) Wurtz. 



(1) Collect blood from the slaughter 

 house under aseptic conditions in 

 petri dishes, Erlenmeyer flasks or 

 jars. 



(2) When the blood has clotted pipette 

 off the serum by means of a sterile 

 pipette. 



(3) Heat for 3 hours at 58°C. on each of 

 three successive days to sterilize 

 (Tyndall method). 



(4) Distribute as desired. 



(5) Coagulate by heating between 90 

 and 100°C. 



(d) Gliicksmann cultivated diphtheria 

 bacilli on a medium prepared as 

 follows: 



(1) Collect beef blood under aseptic 

 conditions from a slaughter house 

 into a sterile four liter cylinder. 



(2) Allow to stand in the ice box for 4 

 or 5 days at 1° to 8°C. 



(3) Pipette the serum under aseptic 

 conditions into sterile test tubes in 

 4.0 cc. lots. 



(4) Solidify by heating in a slanted 

 position at a temperature of 70 to 

 72°C. for about one hour. 



(5) Incubate 2 or 3 days to test 

 sterility. 



(e) Smith cultivated tubercle bacilli on 

 the following medium: 



(1) Bleed a dog under chloroform and 

 draw the blood from a femoral 

 artery under aseptic conditions 

 thru sterile tubes directly, or into 

 sterile flasks. 



(2) Draw the serum from the clots with 

 sterile pipettes and either distrib- 

 ute at once into sterile test tubes 

 or else store with 0.25 to 0.3% 

 chloroform. 



(3) Slant the tubes and heat to 75° to 

 76°C. to coagulate, requiring about 

 three hours. No other steriliza- 

 tion or heating is required. If 

 chloroform has been added it is 

 necessary to place the tubes for an 

 hour or longer in a water bath at 

 55-60°C. or under the receiver of an 

 air-pump to drive off the antiseptic. 



(4) Keep in cold closed space and in an 

 inclined position before in- 

 oculation. 



(5) Use a ground glass stopper instead 

 of the ordinary cotton plug to 

 close the tubes. 



(f) Thoinot and Masselin. 



(1) Obtain blood under as nearly 

 aseptic conditions as possible in 

 large glass jars. 



(2) Store in a cool place for 24 to 48 

 hours to allow the blood to clot. 



(3) Remove the serum from the clot 

 by means of a sterile pipette. 



(4) Distribute into sterile tubes. 



