CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



787 



(3) Shake thoroly. 



(4) Tube carefully. 



(5) Heat the tubes in a slanted position 

 at a temperature not exceeding 70°C. 

 until coagulation has taken place. 



(6) Paraffin the cotton plugs to keep the 

 medium moist. 



Sterilization: Sterilize (1) by the inter- 

 mittent method in the Arnold sterilizer. 

 Use: Isolation of tubercle bacilli. 

 Reference: Duval (1909 p. 404). 



2429. Ebellng's Serum Tissue Plasma 

 Medium 



Constituents : 



1. Distilled water 90.0 cc. 



2. Plasma, chicken 10.0 cc. 



3. Serum, chicken 



4. Embryonic tissue juice 



5. Acetic acid 1.0% 



Preparation : 



(1) Dilute 10.0 cc. of normal adult 

 chicken plasma with 90.0 cc. of sterile 

 distilled water. Shake thoroly in an 

 Erlenmeyer flask, while adding 1.0 cc. 

 of a 1.0% acetic acid solution drop 

 by drop. 



(2) Allow to precipitate partially in the 

 cold for about an hour. 



(3) Shake the contents of the flask and 

 pour into centrifuge tubes 25.0 cc. 

 in each tube. 



(4) Centrifuge for 10 minutes and decant 

 the supernatant fluid. 



(5) Invert the tubes over a sterile piece 

 of filter paper for complete drainage. 



(6) Make up the precipitate contained in 

 the tubes to 2.5 cc. with sterile dis- 

 tilled water. 



(7) When (6) is thoroly mixed it has the 

 appearance of rich milk. 



(8) Mix i part of (7) with I chicken serum 

 and one volume of embryonic tissue 

 extract, by drawing them up and 

 expelling them from a bulb pipette. 

 Such a preparation has a pH value 

 between 7.0 and 7.3 and coagulates 

 in about 1 minute. 



Sterilization: Method not given. 

 Use: Cultivation of connective tissue. 

 Reference: Ebeling (1921 p. 643). 



2430. Wolbach and Schlesinger's Tissue 

 Plasma Medium 



Constituents : 



1. Plasma, guinea pig. 



2. Brain (testicle) guinea pig. 

 Preparation : 



(1) Obtain guinea pig plasma by bleeding 

 a guinea pig directly from the heart. 

 The anasthetization of the guinea 

 pig must be such as to have no reflex 

 movements of the animal, but still 

 have a vigorous heart action. 



(2) As soon as collected (in a sterile 

 paraffined tube) the blood is placed 

 in an ice and salt mixture and chilled 

 for a period of about 3 minutes. 



(3) Centrifuge surrounded by an ice and 

 salt mixture for a period of 3 to 5 

 minutes. 



(4) Return to the salt and ice mixture 

 (temperature just above the freezing 

 point). 



(5) Collect the supernatant plasma by 

 means of chilled sterile paraffined 

 pipette and transfer to another sterile 

 chilled paraffined tube, and keep in 

 this tube in the ice and salt bath at 

 —5° to — 7°C. If the plasma freezes, 

 thaw out slowly. It may still be 

 used. 



(6) Obtain the tissue bran or testicles for 

 cultivation with aseptic precautions, 

 and keep immersed in sterile Ringer's 

 solution until the plasma is prepared. 



(7) Cut into pieces 0.5 to 1.0 cmm. in 

 size under Ringer's solution. 



(8) Transfer to sterile coverslips and 

 cover immediately with a drop of 

 sterile plasma (5). 



(9) As soon as the plasma has clotted, the 

 coverslips are inverted into hollow 

 ground slides and sealed with sterile 

 vaseline. 



Sterilization: Method not given. 



Use: Cultivation of Dermacentroxenus 

 rickettsi (causing Rocky Mountain Spot- 

 ted Fever) and Rickettsia prowazeki 

 (causing typhus). To make transplants, 

 remove the bits of tissue from the plasma, 

 wash a few minutes in sterile Ringer's 

 solution, transfer to fresh sterile cover- 

 slips and add fresh sterile plasma. 



