366 BACTERIOLYSINS 



hold about 2 milligrams of microorganisms. (See p. 216.) All dilu- 

 tions are made with sterile neutral broth, and not with salt solution. 

 Mixtures are injected intraperitoneally into 250-gram guinea-pigs to 

 determine the dose that will be fatal within twenty-four hours. 



Great care should be exercised in all manipulations to avoid acci- 

 dental infection. The mouth-ends of pipets should be plugged with 

 cotton. Sufficient assistants should be on hand to facilitate the making 

 of injections and the examination of peritoneal exudates with ease, 

 caution, and certainty. All pipets, measuring glasses, test-tubes, and 

 hanging-drop preparations should be immersed after using in 1 per cent, 

 formalin solution before cleaning. In other words, every precaution 

 should be taken to carry out a thorough and conscientious bacteriologic 

 technic. 



Guinea-pig No. 1: Four loopfuls of agar culture emulsified in 4 c.c. 



of bouillon; inject 1 c.c. intraperitoneally (=1 loopful). 

 Guinea-pig No. 2: 1 c.c. of foregoing emulsion + 1 c.c. of bouillon; 



inject 1 c.c. intraperitoneally ( = J^ loopful). 

 Guinea-pig No. 3: 1 c.c. of first emulsion + 4 c.c. of bouillon; inject 



1 c.c. intraperitoneally ( = |- loopful). 

 Guinea-pig No. 4: 1 c.c. of emulsion No. 3 + 1 c.c. of bouillon; 



inject 1 c.c. intraperitoneally (=iV loopful). 



A satisfactory culture is one in which a dose of loopful will prove 

 fatal within twenty-four hours. The immune serum is then titrated with 

 five times this amount of culture, or one loopful. 



Method of Titrating a Bacteriolytic Serum. The serum is inacti- 

 vated by heating to 56 C. for half an hour, and dilutions are made with 

 bouillon in sterile shallow glasses or test-tubes. One loopful of an eigh- 

 teen-hour agar culture of the microorganism is thoroughly emulsified 

 in the diluted serum, and the mixtures are injected intraperitoneally 

 in 250-gram guinea-pigs. Higher dilutions than those given here may 

 be employed until the limit of bacteriolytic activity is reached. 



1. Mix 0.5 c.c. of inactivated serum with 4.5 c.c. of bouillon (1 : 10). 

 Place 2 c.c. of this mixture in a separate test-tube, and add 2 loopfuls of 

 culture. Inject 1 c.c. ( = 0.1 c.c. immune serum). 



2. Mix 2 c.c. of the first dilution (1 : 10) with 18 c.c. of bouillon ( = 

 1 : 100). Place 2 c.c. in a separate tube and add 2 loopfuls of culture. 



, Inject 1 c.c. ( = 0.01 c.c. immune serum). 



3. Mix 1 c.c. of the second dilution (1 : 100) with 4 c.c. of bouillon 

 (= 1 : 500). Place 2 c.c. in a separate tube and add 2 loopfuls of culture. 

 Inject 1 c.c. ( = 0.05 c.c. immune serum). 



