190 G. G. Heslop: 



slightly from the method employed for the preparation of the 

 saline extracts of lung tissue, and was as follows : — 10 grams 

 of the inflamed subepidermal tumour tissue was cut into small 

 pieces, and ground up. in a mortar with a little sterile sea sand. 

 40 c.c.s. of 0.5 per cent, carbolic acid in 0.9 per cent, saline solu- 

 tion was then added, and the mixture placed in a tightly stop- 

 pered bottle, and shaken in a mechanical shaker for five hours. 

 It was then filtered through gauze to remove the coarser par- 

 ticles, and then placed in the ice chest for a week, being shaken up 

 occasionally. It was finally allowed to sediment, and the super- 

 natent fluid decanted. This fluid was filtered through filter paper, 

 and again placed in the ice chest for a further 48 hours. The 

 supernatent fluid was then carefully pipetted off without dis- 

 turbing the slight deposit in the bottle. 1 c.c. of this fluid was 

 then diluted with 9 c.c.s. of 0.9 per cent, saline solution, and was 

 titrated to determine the anticomplementary unit. This 1 in 10 

 dilution was used in the test. 



When tested with positive sera Nos. 11 and 25, this saline ex- 

 tract gave a negative reaction, but when tested with Serum No. 

 30 (from Experimental Calf 1), a positive complement fixation 

 was recorded. This was the first positive complement fixation 

 result recorded, although Serum No. 30 had been tested with 

 practically every antigen preparation which had been previously 

 tried. 



It should be pointed out that Experimental Calf No. 1 had 

 been specially immunised by several subcutaneous inoculations 

 of virulent material ; therefore, the serum of Calf 1 would be 

 expected to be highly specific, and not properly comparable with 

 the serum of an animal affected with the disease naturally ac- 

 quired. 



As extracts of sub-epidermal tumour tissue apparently offered 

 the most favourable chances of obtaining a successfully reacting 

 antigen, a number of saline extracts were prepared from sub- 

 epidermal tumour tissue, taken from behind the shoulder of 

 Experimental Cow 6 (who was destroyed owing to the exten- 

 sive infiltrating oedema which had developed as a result of the 

 inoculation of pure virus behind the shoulder, and from which 

 the animal was dying). This extract gave a strongly positive 

 result when tested with Serum No. 30, and gave some fixation 

 with positive sera Nos. 33, 36, 37 and 39, but with positive sera 

 Nos. 34, 35, 35a, 35b, and 38, the results were negative; haemo- 

 lysis taking place in the antigen containing tubes just as readily 

 as in the serum control tubes which did not contain antigen. 





