188 G. G. H estop: 



This conclusion has now to be considerably modified and 

 amended when viewed in the light of the results obtained in the 

 further experiments carried out during the present year. 



If a first or second subculture of the organism of contagious^ 

 pleuro-pneumonia is used in an agglutination test with the serum 

 of an animal known to be affected with contagious pleuro-pneu- 

 monia, agglutination of the culture cannot be observed macro- 

 scopically at any time up to 40 hours after mixing the culture 

 and serum together. This fact has been repeatedly established 

 by experiments conducted by the writer, and it was upon the 

 results of those experiments that the above quoted conclusion, 

 was arrived at. Further experimentation has demonstrated, how- 

 ever, that agglutination of a culture of the organism of con- 

 tagious pleuro-pneumonia by such a serum will occur, provided 

 the culture used is an old laboratory strain which has been sub- 

 jected to repeated cultivation through several generations of sub- 

 cultures, the subcultures being made at intervals of from six to 

 eight days. 



If, for instance, an eighteenth or twentieth subculture, six to 

 eight days old, is taken, and to it is added various dilutions 

 of a known positive serum, agglutination and sedimentation of 

 the culture will occur, and the super natent fluid will lose its 

 opalescence and become clear. In the lower dilutions agglu- 

 tination can also be observed with the same culture and a known. 

 negative serum, but, while agglutination takes place with a knowa 

 positive serum in a dilution as high as 1 in 400, which dilution 

 contains only 0.005 c.c. of pure serum, no dilution of a known 

 negative serum higher than 1 in 80, which dilution contains 0.025 

 c.c. of pure serum, has been found capable of producing agglu- 

 tination of the same amount of culture. Thus there is a con- 

 siderable difference in the end point values of positive and nega- 

 tive sera respectively, the difference being sufficiently great tO' 

 preclude any possibility of errors in reading and recording the 

 results of the reactions. 



The agglutination reaction in contagious pleuro-pneumonia 

 proceeds slowly at incubator temperature, and apparently it is 

 complete only after 48 hours in the incubator at 37°C. After 

 24 hours' incubation little, if any, agglutination is apparent with 

 any dilution from 1 in 20 upwards of a known positive serum' 

 taken from an acute case of contagious pleuro-pneumonia, 

 whereas after 48 hours, marked agglutination can usually be 

 observed in a dilution of 1 in 400 or even higher. 



