310 



INFECTION AND IMMUNITY 



Bactericidal Reactions in tlie Test Tube. Bactericidal reactions 

 in the test tubes may be made by mixing in small sterile test tubes, 

 definite quantities of the bacteria with inactivated serum and com- 

 plement, the latter in the form of unheated normal serum. The 

 mixtures, diluted with equal volumes of neutral broth or salt solu- 

 tion, are set away for a definite time three to four hours in an 

 incubator at 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 bac- 

 teria 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 solution, or a twenty-four-hour broth culture properly 

 diluted may be used. Complement is obtained by taking fresh 

 normal rabbit serum and diluting 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 

 340, Fig. 42.) The controls are set up in a similar way, all of them 

 containing a similar quantity of bacterial emulsions, one control 

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

 of salt solution -f- 0.5 c.c. of the diluted complement, and the third 

 control containing inactivated normal serum 1 c.c. -f- 0.5 c.c. of 

 diluted complement. Definite quantities of these mixtures, taken 

 with a standard loop, or preferably with a capillary pipette, are 

 plated in agar immediately after mixing. 



After incubation for two or three hours similar quantities are 

 again measured into tubes of melted agar with the capillary pipette. 

 With a little practice, great accuracy in these measurements can. 

 be acquired. The inoculated agar tubes arc very thoroughly mixed, 

 and plates are poured. At the end of twenty-four hours' incubation, 



