652 APPENDIX: LABORATORY METHODS 



trol serum and injected into the test mice, will on the average kill ap- 

 proximately 40 per cent of the mice injected. This test dose shall be not 

 more than 0.5 cc. of a 1 : 100 dilution. With larger amounts of serum a 

 higher percentage of mice is expected to live, and with smaller amounts 

 of serum, a lower percentage of mice is expected to live. The culture to 

 be used is one found by experience to be satisfactory for use for test 

 with the control serum. 



c. The test. At least two complete tests, preferably made on differ- 

 ent days, using 10 mice on each serum dilution, must be made for each 

 unknown serum. 



(1) Mice: 16 to 20 gram white mice are used for the test. Ten 

 mice are injected on each serum dilution. 



(2) Serum: The serum is diluted, at room temperature, with 

 physiological salt solution, never using less than 1 cc. volume in mak- 

 ing dilutions, except if economy requires, in the first dilution of the 

 control serum, when 0.5 cc. may be used. Accurate quantitative pi- 

 pettes, with a separate pipette for each dilution, are employed. 



(3) Control serum: (see Unit of serum, ante). It is essential that 

 the control serum P-ll be used in at least 2 dilutions, 300 and 400 

 units for Type I and 150 and 200 units for Type II, namely 0.5 cc. 

 of 1:150 and 0.5 cc. of 1:200 for Type I, and 0.5 cc. of 1:75 and 

 0.5 cc. of 1:100 for Type II, with each comparative test. On the 

 average, in at least two tests, 60 per cent of the mice must survive in 

 the 300 unit dilution and over 50 per cent die in the 400 unit dilution 

 in Type I, and 60 per cent of the mice survive in the 150 unit dilu- 

 tion and over 50 per cent die in the 200 unit dilution in Type II. 



The dilution of the unknown serum depends upon the relative po- 

 tency. Suggestion: A primary titration may be made using mice in 

 triplicate on dilutions representing 100, 200, 400, 800 units, etc., 

 when testing unconcentrated serum, or 1,000, 2,000, 3,000, 4,000 

 units, etc., in refined and concentrated serums or globulins. After es- 

 tablishment of the approximate unit value of the serum, dilutions in 

 the appropriate zone with an increment of 25 to 50 per cent are 

 tested with ten mice to each dilution. 



(4) Culture: (see Test dose of culture, ante). 



(5) Method of Injection: In actual test, 0.5 cc. of serum dilution 

 is drawn into a 1 cc. tuberculin syringe (fitted with a No. 23 gauge, 

 % inch needle) and then 0.5 cc. of the culture dilution is drawn into 

 the same syringe and the mixture injected immediately (within 5 

 seconds). A different syringe must be used for each dilution of serum. 

 For each dilution of serum tested a separate container shall be used 



