258 



INFECTION AND IMMUNITY 



37;5° C, and equal quantities from all the tubes are then inoculated 

 into melted agar at 40° C, and plates are poured. Control plates 

 must be made in each case with mixtures of similar quantities of 

 bacteria in salt solution, and similar quantities of bacteria in normal 

 serum. By colony counting after the plates have developed, it is then 

 possible to estimate the degree of bacterial destruction in any of the 

 given dilutions. 



In actually carrying out the test, dilutions of the inactivated serum 

 are first made, ranging from 1 : 10 to 1 : 1,000 and over. An emulsion 

 of bacteria from a twenty-four-hour agar slant is then made in salt solu- 

 tion, or a twenty-four-hour broth culture properly diluted may be used. 

 Complement is obtained by taking fresh normal rabbit serum and dilut- 

 ing it with salt solution 1 : 10 or 1 : 15. Into a series of test tubes, then, 

 1 c.c. of each of the serum dilutions is placed, and to each tube is added 

 0.5 c.c. of the diluted fresh normal rabbit serum (complement) . To these 

 mixtures the bacteria are then added. In adding the bacterial emulsion 

 to these tubes, the writers have found it more accurate to discard the 

 use of the platinum loop and to measure the bacterial emulsion in a 

 marked capillary pipette such as that used in the opsonin test. (See page 

 285, Fig. 68.) The controls are set up in a similar way, all of them con- 

 taining a similar quantity of bacterial emulsion, one control containing 

 1.5 c.c. of salt solution, another control containing 1 c.c. of salt solution + 

 0.5 c.c. of the diluted complement, and the third control containing in- 

 activated normal serum 1 c.c. +0.5 c.c. of diluted complement. Defi- 

 nite quantities of these mixtures, taken with a standard loop, or 

 preferably with a capillary pipette, are plated in agar immediately 

 after mixing. 



BACTERICIDAL TEST IN VITRO 



(To Determine the Bactericidal Power of a Typhoid Immune Serum 

 AGAINST Typhoid Bacilu). 



Plates 



Poured 



After .3 Hrs. 



at 37° C. 



,c. Immune Typh.Ser. 1:200 +0.5 c.c. Typh. Emulsion + 0.5 c.c. Rab. Ser. 1:151 

 •' " 1:400 +0.5 " ''^ " +0.5 I 



+ 0.5 

 1:800 +0.5 

 1:1600 +0.5 

 1:3200 +0.5 

 1:6400 +0.5 

 1:12800 + 0.5 

 1:25600 + 0.5 



+ 0.5 

 + 0.5 

 + 0.5 

 + 0.5 

 + 0.5 

 + 0.5 



::[ 





 Colonies. 



100-1,000 

 Colonies 



More than 



10,000 



Colonies 



la 1 1.5 c.c. NaCI + 0.5 Typh. Emulsion 



Controls 



la 1 1.. 

 lib 1. 



+ 0.5 

 + 0.5 



Plated immediately ) More than 

 after 3 hrs. } 10,000 

 + 0.5 c.c. Rab. Ser. 1:15 " " " " ) Colonies 



