CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



633 



this extract being prepared locally 

 from goat's pancreas, according to 

 the method recommended by Cole 

 and Onslow (Lancet (1916, see 

 medium 1130.) Pig's pancreas was 

 not used for various reasons. 



(4) Next 5.0 cc. of the enterokinase solu- 

 tion are added with a view to hasten 

 the trypsinization process. This 

 enterokinase solution is a very dilute 

 watery extract of the duodenal 

 mucous membrane of goats and sheep 

 which has been macerated in chloro- 

 form water. 



(5) Lastly, 1.5 cc. of pure chloroform 

 are added to the flask as a pre- 

 servative and it is plugged with an 

 India rubber cork to prevent the 

 evaporation of the chloroform. 



(6) The flask is then placed in the incu- 

 bator at 37°C., after having shaken 

 it well in order to mix the contents 

 thoroly. 



(7) On the second day the flask will be 

 found to contain a well-settled sedi- 

 ment with a clear supernatant red- 

 dish fluid. The flask is shaken again 

 to resuspend the sediment and put 

 back into the incubator. The shak- 

 ing is repeated on the third day, but 

 after that the flask is allowed to 

 stand undisturbed in the incubator 

 until the eighth day. 



(8) On the eighth day the flask is care- 

 fully removed so as not to disturb 

 the sediment and the clear super- 

 natant fluid is removed with aseptic 

 precautions. In case the sediment 

 has not settled firmly at the bottom, 

 as much of the clear fluid should be 

 removed as possible and the re- 

 mainder filtered thru sterile filter 

 paper, making arrangements to 

 maintain sterility. 



(9) Broth prepared from casein after 

 the method recommended by Cole 

 and Onslow (see medium 1130) is 

 kept ready on hand. 



(10) Tubes, each containing about 4.0 cc. 

 of 3.0% agar, are kept ready steri- 

 lized. Theagar is melted and cooled 

 to a temperature of about 45°C. 



(11) One cc. of the stock broth and 1.0 cc. 

 of the blood fluid are well mixed; 



the tube is sloped, and the agar 

 allowed to cool. 

 (12) Agar slopes thus prepared are trans- 

 parent and have a slightly more 

 golden color than ordinary agar. 

 Sterilization: Final sterilization not spe- 

 cified. 

 Use: Cultivation of B. m/uensae (Pfeiffer). 

 The author reported that agar slopes con- 

 taining the digested blood gave almost 

 as good results as those containing in 

 addition casein digest (stock broth of 

 Cole and Onslow). Many involution 

 forms, however, were developed on this 

 simpler medium, but the addition of 

 sodium phosphate eliminated these. A 

 culture of B. influenzae inoculated on 

 this medium showed a perceptible growth 

 after six hours incubation at 37°C., and 

 after 24 hours incubation a luxuriant 

 growth was obtained. This growth had a 

 characteristic translucency. Individual 

 isolated colonies on this medium meas- 

 ured from 1.0 mm. to 2.0 mm. in 24 hours. 

 Reference: Liston (1918-19 p. 419). 



1982. Bernstein's Basal Blood Agar 



Constituents : 



1. Nutrient agar 150.0 cc. 



2. Blood, beef 10.0 cc. 



Preparation : 



(1) Prepare nutrient agar with 1.0% of 

 one of the added nutrients. 



(2) Draw 400.0 cc. of beef blood directly 



into sterile Erlenmeyer flasks of 

 500.0 cc. capacity containing 35.0 cc. 

 of a 1.0% solution of ammonium 

 oxalate in distilled water. 



(3) Shake for one or two minutes. 



(4) Add 0.5 cc. of 40 volume formalin and 



allow the flask to stand undisturbed 

 for an hour. 



(5) Distribute the blood into sterile 



Erlenmeyer flasks and dilute with 

 twice its volume of sterile (0.9%) 

 saline solution. 



(6) Allow the blood to stand for 24 hours 



to 48 hours at room temperatures 

 before use. 



(7) Seal the flasks and keep on ice until 



ready for use. 



(8) To 15.0 cc. of sterile melted (1), add 

 1.0 cc. of (6). 



(9) Pour into sterile plates. 



