126 HUGH GLASGOW. 



freshly isolated from tissues do not usually react so readily to 

 the agglutination test, the process was finally reversed and the 

 animals were immunized with material direct from the caeca, 

 the crushed ca^ca really representing a greater bulk of bacterial 

 cells than of insect tissue. 



For this purpose the caeca were removed from hibernating 

 insects and crushed very thoroughly by rolling between sterile 

 slides. The bacteria thus liberated were then washed off the 

 slides and collected in sterile vials which were at once immersed 

 in a water-bath and kept at 54 C. for thirty minutes to kill the 

 organisms, it having been found that this temperature was 

 sufficient for the purpose. 



Material prepared in this way was injected intraperitoneally, 

 each animal receiving five graded doses at intervals of a week 

 or ten days, the doses varying from the caeca substance of ten 

 bugs for the first injection to that from thirty insects for the last. 



The immunized animals were killed about ten days after the 

 last injection, the blood was drawn aseptically from the heart, 

 and the serum stored in capillaries having a capacity of two or 

 three drops each. Since the normal serum of many animals is 

 known to agglutinate certain strains of bacteria in low dilution, 

 I did not know what to expect in a case like this, and to guard 

 against any possible error, a normal rat was bled whenever an 

 immunized animal was killed and the sera from the two were 

 checked against one another in every test made. 



A large series of cultures from the caeca of Anasa tristis were 

 tested out against such immune sera, and from the very first of 

 these tests it was evident that the organism in culture was un- 

 doubtedly the identical form in the caeca, for they were agglu- 

 tinated readily by the immune serum of the caecal bacteria in 

 dilutions as high as 1-500. No dilutions higher than this were 

 tried, but from the readiness with which clumping was produced 

 in this concentration, we may infer that the reactions would 

 certainly have been reliable in dilutions twice as great. 



In this work the macroscopic method was relied upon almost 

 entirely, the dilutions being made in small, 6 or 7 mm., test 

 tubes. It was found that while serum from the immunized rat 

 would regularly produce complete clumping in high dilutions in 



