OPSONIC ACTION 483 



Methods of Hsemolytic Tests. A hsemolytic serum is usually pre- 

 pared by injecting the red corpuscles of an animal into the peritoneum 

 of an animal of different species the corpuscles of the ox are most 

 frequently used, and the rabbit is the most suitable animal for injection. 

 The corpuscles ought to be completely freed of serum by repeatedly 

 washing them in sterile salt solution and centrifugalising. An injection 

 of the corpuscles of 5 c.c. of ox's blood followed by two injections, each 

 of 10 c.c., at intervals of ten days, will usually give an active serum. 

 The animal should be killed by bleeding it, aseptically as far as possible, 

 seven to ten days after the last injection ; the serum which separates 

 may be collected in suitable lengths of quill glass-tubing drawn out at 

 the ends, which are afterwards sealed in the flame. To ensure sterility 

 when the serum is to be kept some time, it is advisable to heat it for an 

 hour at 55 C. on three successive days ; we have always found that 

 serum treated in this way remains sterile. It is of course devoid of 

 complement. 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 com- 

 plement must be devoid of haemolytic 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 com- 

 plement. The activity of a serum as complement varies considerably, 

 and each sample must be separately tested. The above will serve as an 

 indication of the fundamental methods ; for further details special 

 papers on the subject must be consulted. 



(b) Opsonic Action. The presence of a substance in an 

 immune-serum which makes the corresponding organism sensitive 

 to phagocytosis was first demonstrated by Denys and Leclef in 

 1895, in the case of an anti-streptococcal serum. They also 

 showed that the serum produced this effect by acting on the 

 organism, not on the leucocytes. It is, however, chiefly to the 

 researches of Wright and his co-workers that this subject has 

 come into special prominence. Wright and Douglas in their 

 first paper showed that the phagocytosis of staphylococci by 



