64 CLINICAL BACTERIOLOGY. 



tained either macroscopically or microscopically in one of 

 the three following ways : 



1 . To fresh, sterile bouillon, in carefully measured amount 

 (p. 65), is added one drop of immune serum, and the mix- 

 ture is inoculated with cholera-bacilli, typhoid-bacilli, etc., 

 and then placed in the thermostat at 37 C. (98.6 F.). 

 After the lapse of from four to seven hours the first clumps 

 appear in the culture, which after from twelve to twenty- 

 four hours presents a typical appearance. The bacteria are 

 seen deposited at the bottom of the test-tube as small floc- 

 culi, in a certain degree precipitated, while the overlying 

 bouillon is perfectly clear. In order that the reaction may 

 not be overlooked, it is advisable to inspect the fluid quite 

 frequently if possible, from hour to hour. 



2. To a twenty-four-hour-old bouillon-culture immune 

 serum is added, as in the previous procedure, and the mix- 

 ture is placed in the thermostat at 37 C. (98.6 F.) for 

 from one to eight hours. The originally turbid culture 

 soon clears up, and a flocculent precipitate forms. 



3. To demonstrate the phenomenon of agglutination mi- 

 croscopically it is best to employ young bouillon-cultures 

 not more than twenty-four hours old. In the employment 

 of older cultures there is danger of mistaking for actual 

 agglutination the clump-formation that not rarely takes 

 place spontaneously, especially on the surface of the fluid. 

 The observer assures himself previously through control- 

 preparations that the bacilli in the bouillon employed are 

 actively motile, and especially that they are distinctly sepa- 

 rated one from another. Then to 10, 30, 40, 100 (repre- 

 senting 5 cu. cm.), 200 (about 10 cu. cm.), 1000 (about 50 

 cu. cm.) or even a larger number of drops of this bouillon in 

 sterile Petri dishes is added one drop of immune serum, 

 and microscopic preparations are made of the various mix- 

 tures. If the serum exhibit agglutinating activity, a num- 

 ber of confluent masses or islands of bacteria become visible, 

 as a rule. In these the microbes are absolutely immobile, 

 whereas in the free intervals, a greater or lesser number are 

 at first still in active movement. Should the preparation 

 exhibit molecular movement, it is permitted to stand for 

 from a quarter to half an hour, after which it is again exam- 

 ined. The agglutination is favored by evaporation and by 

 the presence of oxygen. To prevent evaporation, the ex- 

 amination may be made in hanging drop, although this is 



