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APPENDIX: LABORATORY METHODS 



virulence of the culture is checked by injecting three doses into mice 

 in triplicate with 0.5 cc. of dilutions of the 4^2 hour culture made in 

 peptone solution. Fluctuations in the virulence and amount of growth 

 of the culture from time to time may require variations in the dilu- 

 tions, and those used in a given test are determined upon the basis of 

 previous tests. The dilutions employed have thus varied from 1 : 100 

 million, 1:300 million, and 1:1,000 million to 1:250 million, 1:750 

 million, and 1 : 2,500 million for Type I ; and 1 : 90 million, 1 : 270 

 million, and 1:900 million to 1:125 million, 1:375 million, and 

 1:1,250 million for Type II. Dilutions for other types are deter- 

 mined by experience. Virulence test dilutions are made in sterile test 

 tubes. 



(2) Test Dose of Culture: The test dose of culture used in deter- 

 mining the protective power of serums is 0.5 cc. of a dilution of the 

 4>Y2 hour culture made in peptone solution. The dilution employed 

 depends upon fluctuations in growth and virulence (mentioned above) 

 and must be determined by experience. For Type I tests, the dilu- 

 tion used has varied from 1:1,000 to 1:2,500; for Type II from 

 1 : 9,000 to 1 : 12,500. The test dilution of culture is usually made in a 

 sterile 125 cc. or 250 cc. Erlenmeyer flask, the amount depending 

 upon the number of mice to be injected. A loopful of the test dilution 

 is streaked on a blood-agar plate as a purity test and, in addition, 

 three mice are injected with the test dose of culture alone. 



Illustrations of the method of making culture dilutions in typical 

 tests, for a total of 132 mice, are shown in the following tables: 



This is the test dilution of culture. 



