320 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



(7) Filter thru wet filter paper when 

 cold to remove fat. 



(8) Add salt and enough water to bring 

 the volume to 1 liter. 



(9) Adjust so that 20 to 30.0 cc. of a 

 decinormal NaOH solution will be 

 required to bring one liter of the 

 medium to the neutral point of 

 phenolphthalein. 



(10) Clarify and filter. 

 Sterilization: Method not given. 

 Use: Indol production. 

 Reference: Peckham (1897 p. 554). 



1121. Harvey's Trypsinized Meat and 

 Kidney Solution 



Constituents : 



1. Water 1000.0 cc. 



2. Meat 500.0 g. 



3. Kidney, rabbit 

 Preparation : 



(1) Mince finely, fat-free lean meat. 



(2) Add 500.0 g. to 1000.0 cc. tap water. 



(3) Heat the mixture 20 minutes at a 

 temperature not exceeding 50°C. 



(4) Skim off fat floating on the surface. 



(5) Raise the temperature rapidly to 

 boiling point. 



(6) Boil 10 minutes. 



(7) Make faintly alkaline to litmus. 



(8) Add trypsin solution to 2 per cent. 



(9) Place in incubator in an open vessel, 

 4 days. 



(10) Filter thru well-wetted, thick filter 

 paper. 



(11) Bring the volume to 1000.0 cc. by 

 the addition of water. 



(12) Make neutral to phenolphthalein at 

 room temperature. 



(13) Store the sterile medium in sterile 

 flasks under layer of paraffin to 

 which sodium formate has been 

 added to the extent of 1 per cent of 

 the total volume of the medium. 



(14) Test the sterility of the medium 

 before use by anaerobic culture. 

 Incubate at least 7 days. 



(15) Add before use l/16th part of fresh 

 sterile rabbit kidney to 5.0 cc. me- 

 dium. With kidney added, incuba- 

 tion may be aerobic. Use as soon 

 as possible after the addition of the 

 kidney. 



Sterilization: Filter (12) thru a Berkefeld 

 and a Doulton candle in series. 



Use: Cultivation of B. tetani and other 

 anaerobic organisms. Author reported 

 that B. sporogenes was inhibited. 



Variants: Harvey prepared a similar me- 

 dium as follows: 



(1) Mince finely fat-free beef or rabbit 

 flesh. 



(2) Add 500.0 g. to 1000.0 cc. water con- 

 taining 5.0 g. sodium carbonate. 



(3) Place in incubator 20 hours. 



(4) Make faintly alkaline to litmus. 



(5) Add trypsin solution to 2 per cent. 



(6) Place in the incubator for a further 

 20 hours. 



(7) Filter thru well-wetted, thick filter 

 paper. 



(8) Make faintly acid to litmus. 



(9) Steam or boil 45 minutes. 



(10) Filter thru well-wetted, thick filter 

 paper. 



(11) Bring the volume up to 1000.0 cc. 



(12) Make neutral to phenolphthalein. 



(13) Steam 30 minutes. 



(14) Filter, while hot, thru well-wetted 

 thick filter paper. Occasionally it 

 may be necessary to filter thru a 

 Doulton candle. 



(15) Distribute the filtrate into test 

 tubes. 



(16) Add just before use l/16th part of 

 fresh sterile rabbit kidney and use as 

 soon as possible after the addition of 

 the kidney. 



Reference: Harvey (1921-22 p. 115). 



1122. Celozzi's Placenta Digest Blood 

 Solution 



Constituents : 



1. Ringer-Locke solution 1000.0 cc. 



2. Placenta 500.0 g. 



3. Glucose (0.5%) 5.0 g. 



4. Glycerol (2.0%) 20.0 g. 



5. Blood 

 Preparation : 



(1) Thoroly wash fresh placenta and pass 

 thru a meat chopper. 



(2) Mix two parts by weight of Ringer- 

 Locke solution with one part (1). 



(3) Adjust the reaction to that of human 

 blood and add 1.0% pancreatin and 

 0.5% chloroform. 



