VII. POTENCY TESTS 653 



for the culture dilution. Not more than 60 minutes shall elapse be- 

 tween starting and finishing the injections. 



(6) Length of Test: The test is terminated at the end of 96 hours 

 and all mice living at the end of this time are counted as survivals. 

 The hour of death is noted on mice dying before 96 hours. (Ideally, 

 all mice dying on the test should be necropsied and the hearts' blood 

 cultured to rule out deaths due to intercurrent infection. This may be 

 impractical in routine testing, but certainly in each test some mice on 

 important dilutions should be examined post mortem.) 



d. Interpretation of test. The unit value of an unknown serum is esti- 

 mated by direct comparison of the highest or end-point dilution pro- 

 tecting 60 per cent of mice on an average in at least two complete and 

 separate tests with the control serum P-ll end-point dilution equalling 

 300 units per cubic centimeter for Type I and 150 units per cubic 

 centimeter for Type II. Comparison should also be made of the per- 

 centage survival of other dilutions of the unknown and control serum. 



e. Records and reports. Reports of tests shall be in the form of a 

 summary, as outlined on page 661. 



Massachusetts Antitoxin and Vaccine Laboratory 



The method is a modification of the Park-Cooper technique 1054 and 

 of the American Drug Manufacturers' Association test as revised De- 

 cember 29, 1933. The method herein described has been in routine use 

 at the Massachusetts Antitoxin and Vaccine Laboratory since February, 

 1933. 



a. Culture. Virulence. The virulence of the cultures used should be 

 such that no more than 3 to 10 organisms, as determined by 21-hour 

 blood-agar plate counts, are required to kill at least two of three mice 

 within 48 hours after intraperitoneal inoculation. Passages of the cul- 

 tures through mice are made five days each week for maintenance of 

 virulence. 



The culture used for a protection test is obtained in the following 

 manner: A mouse is injected intraperitoneally with 0.5 cc. of an 18-hour 

 broth culture; 6 hours later the mouse is chloroformed and a sample of 

 the heart's blood is inoculated, by means of a sterile capillary pipette, 

 into broth and on a blood-agar plate. After 18 hours growth, if the 

 cultures are pure, a transfer of 1.0 cc. of the broth culture is made to 

 15.0 cc. of broth and a drop of defibrinated normal horse blood added. 

 This culture is allowed to grow for 4l^ hours and is the one used in the 

 test. 



(1) Virulence Test: As a part of the mouse protection test, the 



