BACTERICIDAL ACTION OF NORMAL SERUM 195 



METHODS 



Bactericidal action of the serum was tested by practically the 

 same method as that used by Neisser and Wechsberg. To each of 

 a series of small test-tubes varying quantities of fresh serum were 

 added, usually 0-3, 0-2, 0-1, 0-05 c.c. To each tube there was then 

 added sufficient 0-8 per cent, sodium chloride solution to make up 

 to 08 c.c., and 0-1 c.c. bouillon was then added. The amount of 

 living organism, as is shown in the tables, varied in different cases. 

 In any case the culture was diluted so that 0*1 c.c. contained the 

 amount to be tested. This was then added to each of the tubes. It 

 is thus evident that each tube will contain 1 c.c., in which there is 

 the same amount of bacteria, but varying amounts of complement. 

 The tubes are then incubated at 37 C. for three hours. At the end 

 of that time 0-025 c.c. is taken from each tube and added to a tube 

 of melted agar. The agar is then plated ; the plates are incubated 

 for forty-eight hours, and the colonies counted after that time. 



In the experiments regarding the modification of the 

 bactericidal properties produced by an emulsion of one 

 bacterium as tested on another, living cultures were at first 

 used ; for example, a minute quantity of living typhoid 

 culture was added, the mixture was incubated for three 

 hours, and thereafter a small quantity of V. Metchnikovi 

 was added. This method was found to be attended with 

 difficulties, as in order to procure definite results it was 

 necessary that the first organism should be completely 

 killed off ; it was accordingly discarded in favour of another, 

 in which the organism whose absorptive properties were to 

 be tested was added in the dead condition, the culture 

 having been killed at a comparatively low temperature, 

 usually about 65 C. The dead organisms were most 

 frequently used as a certain quantity of a twenty-four hours' 

 bouillon culture incubated at 37 C. In testing the absorp- 

 tive properties two or more series of plates were used. In 

 each series varying quantities of complement were added, and 

 the result aimed at was to have no colonies with the largest 



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