Contagious Fleuro- Pneumonia of Cattle. 189 



Attempts to expedite the reaction by heating the tubes in a 

 water bath at various temperatures between 45 °C. and 55 °C. 

 have not been successful, and the reactions obtained after incu- 

 bation at such temperatures have not been so satisfactory, even 

 at the end of 48 hours, as the reactions obtained with the same 

 sera heated at 37° C. Incubation for 48 hours at room tempera- 

 ture does not give as satisfactory results as incubation for 48 

 hours at 37°C. 



To prevent the growth of contaminating micro-organisms in 

 the tubes during the 48 hours' incubation period required for 

 the test, carbol saline solution was used as the diluting fluid. 

 This solution, which consists of 0.5 per cent, carbolic acid in 0.9 

 per cent, saline solution, has been found to be effective for the 

 purpose intended, while at the same time it does not appear to 

 exert any unfavourable action in the test. 



The cultures used in these macroscopic agglutination tests 

 were cultures of the organism of contagious pleuro-pneumonia, 

 grown in Martin's broth ox serum. ^ (Reaction PH=8.4 approxi- 

 mately), and subcultured every 7-8 days, until at least the 

 eighteenth subculture stage had been reached. Each subculture 

 at the time it was used in a test was from six to eight days old. 

 In each test the culture used was a live culture which showed 

 a uniform though faint opalescence. 



One c.c. of culture (which quantity was taken as the standard 

 amount) was measured into each of a series of agglutination 

 tubes. To each tube there was added a graded quantity of serum 

 diluted with carbol saline so that the combined amounts of serum 

 and carbol saline in each tube was equal to the amount of culture 

 contained in the same tube. Each tube therefore contained 2 

 c.c.s. of fluid. 



In this manner, serum dilutions of 1 in 20, 1 in 40, 1 in 80, 

 1 in 100, 1 in 133, 1 in 200, and 1 in 400 were prepared and tested. 



The following table will explain the method of setting up the 

 test :— 



Tube. 



Culture. 



Saline. 



Serum. 



Contents. 



Dilution, 



1 



Ic.c. 



•9 



•1 



2c.c. 



1 in 20 



■2 - 



Ic.c. 



•95 



•05 



2c.c. 



1 in 40 



2 



Ic.c. 



•975 



•025 



2c.c. 



1 in 80 



4 



Ic.c. 



•98 



- ^02 



2c.c. 



1 in 100 



5 - 



Ic.c. 



•985 



•015 



2c.c. 



1 in 133 



6 - 



Ic.c. 



•99 



•01 



2c.c. 



1 in 200 



■ 7 . 



Ic.c. 



•995 



•005 



2c.c. 



1 in 400 



*8 . 



— 



■ . 1-9 



•1 



2c.c. 







*9 



Ice. 



Ic.c. 



— 



- 2c.c. 



— 



6. Martin's peptone broth to which has been added 7.5 per cent, fresh un- 

 heated ox serum sterilised by filtration. 

 * Controls. 



