METHODS OF HEMOLYTIC TESTS 125 



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 intraperitoneal injection 

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

 injections, each of 10 c.c, at intervals of eight days, will usually 

 give an active serum. About a week after the last injection a 

 specimen of blood should be taken from the ear and its titre 

 estimated. If this is satisfactory, the animal should be 

 anaesthetised and bled from the carotid. 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 comple- 

 ment. The test amount of corpuscles is usually 1 c.o. 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 haemolytic 

 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 hemolytic 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 



