VII. POTENCY TESTS 649 



which are etherized in 5 or 6 hours, about 0.5 cc. of heart's blood being 

 obtained from each and distributed into two tubes of broth and a blood- 

 agar plate (for purity test) which are incubated overnight. Three of 

 the six tubes showing a good growth of Pneumococcus in pure culture 

 are selected and stored at 15° as stock cultures; after about 3 days (the 

 day before the test) 0.5 cc. to 1.0 cc. are transplanted from each stock 

 culture to fresh tubes of broth, and plates are made for purity. The 

 stock cultures are quickly put back at 15°, to be used for planting the 

 tubes 18 hours before the next passage through mice. The first trans- 

 plants are incubated 16 to 18 hours, transplanted again, and incubated 

 3 to 6 hours, and of these three last transplants, those showing a good 

 growth of pure Pneumococcus, with a turbidity of 200 to 250 parts per 

 million, silica standard, and a pH of 6.6 to 7.0 are pooled and distrib- 

 uted for the test. All dilutions of cultures are made in broth and should 

 not stand diluted for more than an hour at room temperature. 



0.1 cc. of the culture, diluted so that this dose is contained in 0.5 cc. 

 volume (tube 1 in the above table), is given to each mouse, except the 

 cultural check mice. 



b. Serum. 0.2 cc. of each serum (including the control serum) diluted 

 with saline solution so that this dose is contained in 0.5 cc. (2 volumes 

 serum diluted and mixed thoroughly with 3 volumes saline) is given to 

 each mouse, except the cultural check mice. 



c. The test. For each serum tested, at least three mice are injected 

 intraperitoneally with 0.2 cc. of serum, followed in less than 5 minutes 

 by 0.1 cc. of culture intraperitoneally. Six mice should receive the con- 

 trol serum, three at the beginning and three at the end of each test. The 

 mice should weigh 18 to 22 grams. If there is much variation in weight, 

 they should be arranged in three weight groups, and a light, a medium, 

 and a heavy mouse chosen for each serum. The mice are observed for 96 

 hours ; all mice counted as fatalities must be proved, by smear and cul- 

 tures from the heart's blood, to have died with an uncomplicated pneu- 

 mococcal septicemia. Survivals of less than twelve hours should be re- 

 garded as "contaminations." Mice dying of infections or accidents other 

 than pure pneumococcal septicemia, as revealed by necropsy, are re- 

 garded as "contaminations." Contaminations should be regarded as 

 neither deaths nor survivals, but as if not injected. Tests are read 120 

 hours after injection, to allow for the obtaining of results of necropsies 

 on mice dying on the fourth day. The control serum should, on the aver- 

 age, protect at least 50 per cent of the mice. The following data should 

 be included in the protocols of tests: 



