570 BIOLOGY OF PNEUMOCOCCUS 



neutralizing antigen is made by adding 0.4 per cent formalin to an 

 eighteen-hour serum-broth culture of virulent pneumococci of the 

 type desired. It is necessary to allow a sufficient period of contact 

 with formalin to elapse in order to kill the organisms. The proper 

 dilution of the neutralizing culture is determined by a preliminary 

 test with the control serum. Mice are injected intraperitoneally 

 with 0.5 cubic centimeter of the serum-neutralizing culture mixture 

 and with 0.5 cubic centimeter of a 1 to 200 dilution of a three 

 to six-hour culture made from an eighteen-hour transplant of a 

 mouse passage culture. Mice used to test the virulence of the or- 

 ganism are also injected with 10 6 , 10" T , 10~ 8 , and 10 9 cubic centi- 

 meter of the living culture, and blood-agar plates are made of 

 these dilutions for counting the number of pneumococci present. 

 The potency of a serum is determined by comparing the highest 

 dilution of serum that will protect for twenty-four hours at least 

 two of the three mice injected with the highest dilution of the con- 

 trol serum conferring a similar degree of protection. 



One of the many difficulties encountered in mouse protection 

 tests has been a lack of uniform results with samples of serum 

 tested in different laboratories, and even when tested repeatedly in 

 the same laboratory. At least one source of the discrepancies has 

 been the fact that too few mice were used on each dose of serum or 

 culture. Recognition of this fault resulted in various changes in the 

 test, and the Park and Cooper 1053 modification of the Felton tech- 

 nique has served as a basis for the mouse protection test commonly 

 used in this country at the present time. 



In February, 1933, the Committee on Standardization of Anti- 

 pneumococcic Serum of the Biological Section of the American 

 Drug Manufacturers' Association suggested a method based on 

 the Park-Cooper modification of the mouse protection test. The 

 method was revised in December, 1933, and in its essentials has 

 been approved by the National Institute of Health which supplies 

 a standard bivalent serum for comparison in Type I and Type II 

 protection tests. In general, the method conforms to that of Fel- 

 ton already described. For establishing the unit value of an un- 



