656 APPENDIX: LABORATORY METHODS 



TYPE II 



(a), (b), and (c) dilutions as above. 

 6.0 cc. of (c) + 9 cc. saline = 1:1250 (d) 

 4.0 cc. of (d) + 4 cc. saline = 1:2500 (e) 

 2.0 cc. of (d) + 6 cc. saline = 1:5000 (f) 



In each case, only the (d), (e), and (f) dilutions are used for in- 

 jecting mice. 



(2) Unknown Serums: The dilutions used for injecting mice must 

 bear the same ratio to each other as dilutions (d), (e), and (f) of 

 the control serum. As concentrated serums are ordinarily much 

 stronger than the control, obviously the dilutions will be much higher. 

 For example, if a serum being tested for Type I antibody is assumed 

 to be about 10 times as strong as the control serum, the dilutions 

 used for injecting mice will be 1:10,000, 1:20,000, and 1:40,000, 

 these being reached by making dilutions in steps similar to those for 

 the control. 



c. The test. At least two complete tests giving consistent results and 

 preferably made on different days, using 10 mice for each dilution of 

 serum, must be made for final estimation of the potency of an unknown 

 serum. 



(1) The Mice: It is preferable to use mice of a highly inbred, uni- 

 formly susceptible stock, in sound, healthy condition. The weights 

 should lie between 16 and 20 grams each. 



(2) Method of Injection: Serum and culture dilutions are not 

 mixed prior to injection. Two 2 cc. vaccine syringes, fitted with 26- 

 gauge, % inch Schick needles, are used for injecting the culture. Two 

 ordinary 2 cc. hypodermic syringes, fitted with needles as above, are 

 used for each serum. One operator fills the syringes, a second makes 

 the injections, and an assistant handles the mice. The serums are al- 

 ways injected first, starting with the highest dilution, and followed 

 immediately by the test dose of culture. Any mice improperly in- 

 jected are discarded and replaced by others. Four serums (including 

 the standard) and the requisite controls require 132 mice. From 30 

 to 40 minutes are required for injecting this number. 



(3) Length of Test: Observations are made at least twice each 

 day and the approximate hour of death is recorded for mice found 

 dead. The test is terminated at the end of 96 hours and all mice liv- 

 ing at this time are recorded as survivals. 



(4) Necropsies: Necropsies should be performed on a sufficient 



