130 METHODS OF EXAMINING SERUM 



in this way remains sterile. It is, of course, devoid of comple- 

 ment. The test amount of corpuscles is usually 1 c.c. of a 

 5 per cent, suspension of corpuscles in '8 per cent, sodium 

 chloride solution; that is, the corpuscles of 5 c.c. blood are 

 completely freed of serum by repeatedly washing in salt solution, 

 and then salt solution is added to make up 100 c.c. In any 

 investigation it is necessary to obtain the minimum hsemolytic 

 dose (M.H.D.) of the immune-body and of the complement to 

 be used. (It is to be noted that as complement does not 

 increase during immunisation, the hsemolytic dose of the fresh 

 serum will come far short of representing the amount of 

 immune-body present.) In testing the dose of immune-body, 

 the fresh serum to be used as complement must be devoid of 

 hsemolytic action (in the present instance rabbit's serum will be 

 found suitable), and more than sufficient to produce lysis with 

 immune-body is added to each of a series of tubes. Varying 

 amounts of immune-body are added to the tubes, the contents 

 are shaken, made up to 1'5 c.c., and incubated for two hours. 

 The amount of lysis is then noted, and the tubes are placed in 

 a cool chamber till next morning, when a final reading is taken. 

 The smallest amount of immune-body which gives complete 

 lysis is, of course, the M.H.D. : sometimes this may be as low 

 as '001 c.c. for the test amount of corpuscles. When further 

 observations are to be continued on the same day, the reading 

 after incubation must be taken as the working standard. To 

 estimate the M.H.D. of complement, proceed in a corresponding 

 manner ; to each of a series of tubes add several doses of 

 immune-body, and then to the several tubes different amounts 

 of complement. The amount of complement necessary for lysis 

 varies somewhat according to the amount of immune-body 

 used, being smaller with several doses of the latter than with 

 a single dose ; in estimations of the dose of complement, it is 

 accordingly advisable to use the optimum amount of immune- 

 body, in the present instance 3-4 haemolytic doses. The 

 activity of a serum as complement varies considerably, and 

 each sample must be separately tested. 1 The above will serve 

 as an indication of the fundamental methods ; for further 

 details, special papers on the subject must be consulted. 

 Corpuscles treated with sufficient immune-body to produce 



1 Complement is a substance which rapidly (often within twenty-four hours) 

 loses its strength when kept at room temperature. It can, however, be pre- 

 served for a considerable time at or near its original strength if it be kept 

 frozen. Even if this be done, however, the strength of the complementary 

 serum must be titred at the commencement of every experiment in which it 

 is employed. 



