42 o. iSHii 



1 to 5 per cent formalin the agglutination reaction was less than 

 without formalin. However, it has been difficult to standard- 

 ize this test with formalin, because the reaction depends on the 

 strain of microorganism employed. Many baciUi showed agglu- 

 tination in all dilutions used and only a few showed different 

 degrees of agglutination; I found that 0.05 to 0.2 per cent formalin- 

 ized serum gave the best results for most of the strains employed. 



TECHNIQUE 



Since in plam broth or agar cultures spontaneous agglutina- 

 tion interferes with specific agglutination, in such cultures 

 accurate tests are impossible. When spontaneous and non-spon- 

 taneous agglutination types are mixed together, I have observed 

 two types of colonies, (by streak on agar plate) occurring regu- 

 larly throughout the cultures. One type of colony has a smooth 

 surface and regular outline and shows non-spontaneous agglu- 

 tination, while the other type of colony has a rough surface and 

 irregular outline and is spontaneously agglutinating. 



Either type of organism would, however, produce both types 

 of colonies after growing for some time. Some strains changed 

 in a few daj^s, while others remained tioie to type, producing 

 characteristic colonies even after continual transplanting for 

 over a year. Cultures used in the experimental work were 

 obtained before each set of experiments by plating and fishing 

 a characteristic colony into experimental media. 



Only neutral broth cultures gave uniform results. Therefore, 

 18 to 24 hour cultures in this medium at 37°C., could be used as 

 an indicator of spontaneous and non-spontaneous agglutina- 

 tions. 



The method of my investigations has been to mix 0.3 of each 

 suspension of bacterial cultures in each medium, (such as broth, 

 pepton water and so forth) with 0.3 cc. of the seiinn dilution, 

 with formalin or without, or with other experimental fluids in 

 small test tubes, of the usual Wassermann type. The tubes, 

 were left at room temperature (excepting in the experunents 

 on temperature), and the preparations were examined, both 



