372 BACTERIOLYSINS 



yields uniform and satisfactory results, and may be employed in making 

 special investigations for determining the bactericidal power of the blood, 

 as after typhoid immunization. 



Technic of the Test. The requisites for success are that all vessels 

 and diluting fluids, as well as the serums employed, should be absolutely 

 sterile. To secure uniform and reliable results it is necessary to famil- 

 iarize oneself with the technic by repeated practice. In order to carry 

 out the steps in the technic according to strict aseptic bacteriologic 

 principles the services of an assistant are required. 



Sterile bouillon is largely used throughout, to maintain proper os- 

 motic conditions. 



With so many tubes and controls, it is important that all tubes and 

 Petri dishes be properly labeled with a wax-pencil to avoid confusion. 



One cubic centimeter of sterile patient's serum and an equal amount 

 from a normal and healthy person to serve as a control are inactivated 

 by heating to 55 or 56 C. for half an hour. These serums are then 

 diluted 1 : 50 by adding 49 c.c. of sterile salt solution to each and mixing 

 thoroughly. 



Complement is prepared by securing 4 to 5 c.c. of sterile rabbit's 

 blood and separating the serum. This serum is chosen because it should 

 be from the same species of animal as that used in producing the immune 

 serum to be tested. In determining the bactericidal titer of human 

 serum either guinea-pig or rabbit complement serum may be used. 

 Dilute 2 c.c. of this fresh serum (not over eighteen hours old) with 18 

 c.c. of sterile normal salt solution (1 : 10). Dose, 0.5 c.c. 



Secure a good twenty-four-hour bouillon culture of typhoid bacilli 

 (grown in the incubator) and dilute 1 : 500 by thoroughly mixing 0.1 

 c.c. of the culture in a flask containing 50 c.c. of sterile normal salt solu- 

 tion. This dilution of culture, when used in constant doses of 0.5 c.c., 

 generally yields satisfactory results, but it may be necessary to try it 

 out beforehand, for the control plates must regularly show a uniformly 

 good growth and contain many thousands of colonies before uniform 

 results can be expected. The bactericidal effect will then be distinctly 

 shown by the reduction, in the proper plates, of this large number of 

 colonies to zero or near it. 



Place 1 c.c. of sterile neutral bouillon in each of 12 test-tubes which 

 have been plugged with cotton, sterilized, and large enough to hold at 

 least from 12 to 15 c.c. Place in the first of these tubes 1 c.c. of the 

 diluted patient's serum and mix thoroughly by alternate sucking up and 

 forcing out of the fluid; then, with the same pipet, draw up 1 c.c. and 



