Pleuro- Pneumonia of Cattle. 199 



decreasing doses from 0.5 c.c. downwards. The smallest amount 

 of complement necessary to produce complete haemolysis in 1/4 

 hour is noted. This amount varies in different complement 

 samples, but is usually approximately 0.15 c.c. of a 1 in 10 dilu- 

 tion. The estimation of the minimum haemolytic dose (M.H.D.) 

 of complement must be decided with absolute accuracy, any quan- 

 tity in excess of the absolute M.H.D. being sufficient to render 

 the reading of the final test exceeding difficult, if not impos- 

 sible. The necessity for only adding the absolute M.H.D. of com- 

 plement to the final test, will be obvious when the action of con- 

 glutinin is considered. 



When the M.H.D. of complement has been determined, the 

 haemolytic system is standardised, and against this standardised 

 haemolytic system the other ingredients of the test are titrated 

 in order to determine the proper quantity of each to employ. 



The antigen is titrated to ascertain its anticomplementary unit. 

 The smallest amount which inhibits haemolysis is the anti-com- 

 plementary unit. Half that amount, or even less, is used in the 

 final test. 



After the titre of the antigen has been determined in the 

 above-mentioned manner, and when carrying out subsequent 

 tests in which fresh complement of an unknown titre has to be 

 used, it is convenient, when ascertaining the M.H.D. of com- 

 plement, to titre it in the presence of the unit of antigen previ- 

 ously determined. 



Each bovine test serum is similarly titrated in order to deter- 

 mine the maximum amount which does not inhibit haemolysis. 

 It was thought at first that a definite amount of test serum could 

 be taken as a standard unit, and, provided the quantity of the 

 standard unit was small enough, it would render a titration un- 

 necessary. It was found, however, that the test sera differed 

 very much in their behaviour towards complement, and that for 

 accuracy in the final result it was necessary to titrate each serum 

 separately. The controls in this titration are most important, 

 and the unit of bovine serum to be determined is the largest 

 amount which allows complete haemolysis to occur in the tube 

 containing it in the same minimum time as complete haemolysis 

 takes place in a tube containing only saline solution plus the 

 titrated units of complement and sensitised red blood cell sus- 

 pension. 



The amount of each test serum to be used in the final test 

 must be so determined that the amount employed is slightly less 



