Pieuro-P 'neumonia of Cattle. 191 



Alcoholic Extracts of Diseased Tissue used as Antigen. 



Saline extracts of diseased tissue having proved unsatisfactory 

 for antigen purposes, alcoholic extracts were next prepared and 

 tested. Thin slices of inflamed sub-epidermal tumour tissue 

 taken from Cow 6, when the animal was destroyed, had been 

 dried in the incubator at 37°C. for some days, and preserved 

 in the dry state in a closely stoppered bottle. Of this dried 

 tissue, 5 grams were taken, and finely pulverised in a mortar 

 with the addition of a small quantity of powdered glass. 50 

 c.c.s. of alcohol were then added, the whole placed in a tightly 

 stoppered bottle, and placed in a mechanical shaking apparatus 

 and thoroughly shaken for 12 hours. It was then stored in the 

 ice chest for 12 days, being shaken up at least once a day. The 

 fluid was then poured off into centrifuge tubes, and whirled in 

 the centrifuge. The supernatent fluid was then carefully pipetted 

 off without disturbing the deposit. 1 c.c. of this alcoholic ex- 

 tract was diluted with 9 c.c.s. of 0.9 per cent, saline solution, the 

 dilution being made slowly in order to obtain the maximum 

 amount of , turbidity. This antigen dilution was then titrated in 

 order to ascertain its anticomplementary and complementary 

 units. 



An important point of technique in the preparation of this 

 antigen for test purposes is the method of diluting the alcoholic 

 -extract with saline solution. 



If the alcoholic extract and saline solution are mixed quickly 

 a slightly turbid mixture results which gives only slight fixation 

 when tested with known positive sera. On the other hand, when 

 the alcoholic extract and saline solution are mixed slowly, an 

 extremely turbid mixture is obtained, the amount of turbidity 

 being in direct proportion to the time allowed for mixing. The 

 mixtures possessing the maximum amount of turbidity have been 

 found on testing to give the maximum amount of fixation. 



The alcoholic extract of sub-epidermal tumour tissue was 

 tested with a number of known positive and known negative sera, 

 and after having been tested with 24 different sera, it was found 

 that the test result in each case was in agreement with the post- 

 mortem findings when the animals supplying the test sera were 

 slaughtered and examined. 



Thus it is evident that complement fixing antibodies are pre- 

 sent in the sera of animals affected with contagious pleuro-pneu- 

 monia, and the complement fixation test provides us with a means 

 of differentiating between infected and non-infected animals. 



