TECHNIC OF BACTERIOLYTTC TESTS 373 



transfer it to the second tube of the series; mix as before, and transfer 

 1 c.c. to the third tube; continue in this manner to the last tube r 

 from which, finally, 1 c.c. is discarded. 



Each tube now contains 1 c.c. of fluid, representing dilutions of 1 : 100, 

 1 : 200, 1 : 400, 1 : 800, 1 : 1600, 1 : 3200, and so on up to 1 : 204,800 of 

 the patient's serum. Higher or lower dilutions than these may be em- 

 ployed. It is very desirable that the dilutions be high enough to secure 

 the limit of bactericidal activity, so that the last plates will show an in- 

 crease in the number of colonies. 



Arrange four tubes with the normal control serum, each containing 

 1 c.c. of fluid and representing the first four dilutions, viz., 1 : 100, 

 1 : 200, 1 : 400, and 1 : 800. 



Label each tube carefully with the initials of the patient and the dilu- 

 tion it contains. Add 0.5 c.c. of the diluted bacterial emulsion, and 

 finally 0.5 c.c. of the diluted complement serum, to each tube. 



All manipulations should be made with strict bacteriologic care and 

 with the aid of an assistant, as the introduction of contaminating micro- 

 organisms that may cause spore-formation will considerably vitiate the 

 value of the test. 



The following controls are necessary: 



1. 0.5 c.c. culture directly in a sterile Petri dish + 5 to 10 c.c. of 



neutral agar cooled to 42 C. and thoroughly mixed. This 

 control will show the original number of bacteria contained in 

 the emulsion. Mark as control No. 1. 



2. 0.5 c.c. culture + 1.5 c.c. bouillon. Mark as control No. 2. 



After three hours' incubation this tube receives the usual 

 amount of agar and is plated. It will show to what degree the 

 culture has multiplied in the incubator. 



3. Since the complement serum frequently contains bacteriolysin, 



it is necessary to control this element carefully. To a series of 

 four tubes add the following doses of the serum, diluted 1 : 10: 

 1 c.c., 0.5 c.c., 0.2 c.c., 0.1 c.c. Add 0.5 c.c. culture to each 

 tube, and sufficient bouillon to make the total volume in each 

 tube 2 c.c. 



4. 0.5 c.c. complement + 1.5 c.c. bouillon. To control the sterility 



of the complement serum. 



5. 1 c.c. of patient's serum (1 : 100) + 1 c.c. bouillon. To control 



the sterility of this serum. 



6. 1 c.c. of the control serum (1 : 100) + 1 c.c. of salt solution. To 



control the sterility of this serum. 



