134 CLINICAL BACTERIOLOGY AND H^MATOLOGV 



as to contain 5 M.H.D. per c.c. Perhaps the same amount of double 

 strength antigen, and mix the two just before use : each c.c. will 

 now contain 2^ M.H.D. of complement and the proper amount of 

 antigen. The latter is usually i in 25 to i in 30, so that the double- 

 strength dilution is i in 12^ to i in 15. 

 Thus if you want to prepare 100 c.c. of mixture, and there is i M.H.D. 



of complement in '2 of a i in 25 solution, take x 5 x 50 = 2 c.c. 



of serum and 48 c.c. of saline. If your antigen works at i in 25, take 

 2-4 of heart extract and 1-6 of cholesterin solution and mix, then mix 

 quickly with 46 c.c. of saline. Mix the two solutions just before use. 



The patient's serum is collected in the usual way and heated for 

 thirty minutes in a water-bath at 55 C. 



The Test. If this is to be fully quantitative five test-tubes, each 

 containing i c.c. of antigen-complement mixture, are required for each 

 serum. To these -i, -05, '025, -012, and -006 of serum are added. 

 Mclntosh and Fildes do this by a drop method, but I think most 

 workers will prefer to take ! c.c. of undiluted, i in 2, i in 4, i in 8, 

 and i in 16, thus keeping the total volume constant. It is not neces- 

 sary to make more than one or two dilutions in most cases, except 

 where the effect of treatment is being followed. A sixth tube contain- 

 ing a solution of complement as in the antigen tubes, but without 

 antigen, is also required : this is because some sera are anticomple- 

 mentary in the absence of antigen, which would of course vitiate the 

 test. (In this tube I use only i M.H.D. of complement instead of 2.\ : 

 this should give partial haemolysis, and allows slight amounts of anti- 

 complement to be recognized with some exactness.) 



Having put up these dilutions, incubate i hour in an ordinary incu- 

 bator (I incubate twenty minutes in a water-bath), add *5 c.c. of 

 sensitized corpuscles, and mix. Repeat the incubation in a water- 

 bath, and after twenty minutes remove the tubes, allow to settle, and 

 read off the results. 



The results are recorded thus. Complete inhibition (no haemolysis) 

 is represented by 4, lesser amounts of haemolysis by 3, 2, and i, and 

 complete haemolysis by o. A negative Wassermann is* recorded as 

 ooooo, a positive one by 40000 or in stronger degrees by 44420, 

 44444, etc. Slight inhibition in the first tube only is of doubtful 

 import. 



Controls. Three are used : (i) i c.c. of the antigen-complement mix- 

 ture and -5 c.c. of the sensitized corpuscles. This should give com- 

 plete haemolysis. 



(2) i c.c. of the mixture of diluted complement and -5 c.c. of sensi- 

 tized corpuscles. This also should give complete haemolysis. 



(3) r 5 c<c - f the sensitized corpuscles. Here there should be no 

 haemolysis. 



Cerebro-spinal Fluid. This is tested in exactly the same way as 

 serum, but is not heated (it contains no complement), and twice the 

 volume is used in each tube. 



