376 Mr. A. H. Caulfeild. Inhibitive and Fixation [Aug. 24, 



periodically the hsemolysin to pooled complement of two or more guinea-pigs. 

 Then twice the strength (half the dilution) producing complete haemolysis in 

 this standardisation is used in the tests. "With this amount of haeinolysin, 

 which it is convenient to designate as two units, complement is titrated before 

 each protocol. From these results twice the strength (half the dilution) of 

 complement is used. The sheep-corpuscle suspension of 1/20 is finally 

 prepared by washing three times in 80 c.c. centrifuge tubes. This amount of 

 washing has seemed sufficient to prevent any interference by remaining traces 

 of sheep serum. 



(3) Antigen. — The inhibitive reaction has, so far, been tried only with an 

 alcohol-ether extract of tubercle bacillus freshly cullured on glycerine broth. 

 It has been prepared as follows: — After the tubercle mass obtained by 

 filtering the glycerine broth cultures is thoroughly washed with saline 

 solution and allowed to dry at room temperature, it is transferred to wide- 

 mouthed 2-litre bottles. The tubercle mass is then covered with a mixture 

 of absolute alcohol (Kahlbaum's) and ether sulph. CP. (Kahlbaum's) in 

 equal parts, the amount of solvent being 10 to 20 times the weight of 

 tubercle mass. The bottles are shaken frequently during the 5 to 10 days 

 allowed for extraction. Loss of the acid-fast character has in some of the 

 preparations been complete, in others this has been but slight. This has 

 seemed to depend chiefly upon the age of the strain and the capacity for 

 rapid growth. The solution thus obtained has been evaporated at varying 

 rates in vacuo over sulphuric acid, and also under a stream of CO2. The 

 yields of 100 c.c, no matter how prepared, have been very close, being in 

 the neighbourhood of 0'2 grm. The solid substance thus obtained is 

 re-dissolved in the same solvent as a 2-per-cent. solution. Both the original 

 and stock solution are passed through double layers of hardened filter 

 paper. 



Very considerable differences have been found between the different 

 antigens. Those which have in general given the best results have been 

 obtained from long cultured and rapidly growing strains. It has seemed 

 that an antigen, which in its antigenic dilution gives with suitable sera the 

 most satisfactory complement fixation, is also the most suitable for the 

 demonstration of the inhibitive reaction. Differences in the various prepara- 

 tions have been shown to exist between the range of dilutions that are 

 necessary, on the one hand to produce the full anti-complementary effect 

 with saline, and on the other to act efficiently in antigenic dilutions with 

 sera containing sensitizers (amboceptors). Work in sufficient detail has not 

 been done to correlate completely these end results to the variations in 

 preparation or use of the antigen. Other things being equal, the more non- 



