THE AGGLUTINATION REACTION 297 



115C. for not more than fifteen minutes, and are then tested for sterility 

 by incubation at 37 C. for forty-eight hours. 



They are inoculated with a few drops each from a twenty- to twenty- 

 four-hour old bouillon culture of the bacillus (B. typhosus or B. para- 

 typhosus, etc.). 



The culture used should be one which has been subcultivated daily 

 in bouillon for one or two weeks (or longer). This continued subculti- 

 vation has the effect of increasing its agglutinability and diminishing 

 any tendency to spontaneous agglutination. 



At the end of twenty to twenty-four hours' growth at 37 C. the 

 flasks are well shaken, and to each is added 0.1 per cent. (1 c.c. per liter) 

 of commercial (40 per cent.) formalin. They are again shaken and 

 placed in a cold chamber in the dark at about 2 C. 



At intervals on the same day and on subsequent days for four or 

 five days the flasks are again thoroughly shaken and replaced at once 

 in the cold chamber. 



After three or four days they will be found to be absolutely sterilized. 

 Should it happen that the bacterial suspension is not entirety homo- 

 geneous it may be shaken for some hours in a mechanical shaker, or 

 may finally be filtered through sterile cotton-wool. 



(4) To emulsify a culture of the plague bacillus or any other micro- 

 organism that displays a strong tendency to undergo "spontaneous" 

 agglutination, distilled water or 1 : 1000 salt solution should be used. 



(5) In the case of a culture of tubercle bacillus, the growth can be 

 resolved into its elements by prolonged trituration in normal salt solution, 

 and any residue or unresolved clumps removed by centrifugalization. 

 A less laborious and dangerous method is to use the tubercle powder of 

 Koch, which is obtained by reducing dried tubercle cultures to a fine 

 powder by machinery. The powder may be made up into a suitable 

 suspension by rubbing it in a mortar with normal salt solution. 



(6) When it is necessary to work with highly dangerous microorgan- 

 isms, or to operate from day to day with the same bacterial suspension, 

 one may employ suspensions that have been heated for one hour to 60 

 C., or suspensions in salt solution to which 1 per cent, formalin has been 

 added. These will keep well in the refrigerator, but the sediment of 

 dead bacteria must be well shaken before it is used. 



(b) Serum. Serum should preferably be fresh, clear, and free from 

 corpuscles. 



