648 APPENDIX: LABORATORY METHODS 



virulence of the organism. In other words, the more organisms that are 

 killed, the more actively phagocytic is the blood in the former case, and 

 the less virulent is the organism in the latter case. 



The method is a modification of the one described by Todd. For fur- 

 ther details, the reader should consult the original article by Ward. 



VII. Potency Tests on Antipneumococcic Serum 



United States Hygienic Laboratory, now National Institute of 

 Health (as of January 22, 1926) 



a. Culture. The culture of Type I pneumococcus should be of such 

 virulence that it is fatal to mice, as a rule, in 0.00,000,001 cc. of a 3 to 

 6-hour beef infusion broth culture. Simultaneously with each day's 

 tests, eight mice are inoculated with the culture alone, receiving no se- 

 rum, in order to check the virulence of the culture used. These cultural 

 check mice receive 0.00,000,1 cc, 0.00,000,01 cc, 0.00,000,001 cc, and 

 0.00,000,000,1 cc. of the culture, two mice on each dose. These dilutions 

 are made as follows: 



Either conical flasks or tubes 28 mm. in diameter are used for making 

 these dilutions. Thorough mixing is very essential and this is accom- 

 plished only by a rapid and vigorous blowing and sucking of the mix- 

 ture with a minimal amount of air through the pipette, which is pressed 

 against the bottom of the flask or tube. A separate pipette is used for 

 each dilution. The culture should be checked for purity by plating on 

 blood-agar plates previous to each test. At the Hygienic Laboratory the 

 broth for growing the cultures is made from beef infused in the ice-box, 

 and 2 per cent peptone is used. The hydrogen ion concentration is about 

 pH 7.2. The culture is passed through mice weekly, 0.5 cc. of an 18- 

 hour culture being inoculated intraperitoneally into each of three mice, 



