508 THE CHOLERA GROUP 



tube and the process repeated in a third tube, when a nearly pure 

 culture of cholera vibrios will frequently be obtained. The organisms 

 may be plated directly from the enriched growth in the first, second, 

 or, best, from the third tube, and the pure cultures agglutinated with 

 a high potency specific anticholera serum in dilutions from 1 to 500 

 to 1 to 5000. l The nitroso indol test should be made on each of the 

 three Dunham tubes after removal of the organisms, for a positive 

 nitroso indol reaction, while not diagnostic, is very suggestive. 



(6) A small amount of feces or a flake of mucus is emulsified in 

 broth and inoculated on the surface of alkaline agar plates, 2 which 

 have previously been poured and hardened. The very thin trans- 

 parent colonies which develop within twelve to eighteen hours are 

 either transferred to broth and after twelve hours' incubation agglu- 

 tinated, or the colony is emulsified directly in a high potency specific 

 serum diluted five hundred times and a macroscopic or microscopic 

 examination made. Controls are made using either normal serum 

 diluted twenty-five times, or normal salt solution. Cholera vibrios 

 will agglutinate rapidly while the controls remain actively motile. 



3. Agglutination of Organism. (a) A pure culture of cholera vibrios 

 will agglutinate in high dilutions with a high potency specific cholera 

 serum either by the microscopic or macroscopic agglutination method. 

 The macroscopic agglutination test can be made either by preparing 

 successive dilutions of the antiserum in small tubes, 1 to 250 up to 

 1 to 2500, and adding an equal volume of broth culture of cholera 

 vibrios to each, or by making dilutions of the specific serum 1 to 500 

 up to 1 to 5000 in small tubes and emulsifying in each tube a smaK 

 amount of culture from an agar slant. Appropriate controls should 

 be made in either case. A positive diagnosis of cholera vibrios should 

 only be made if agglutination takes place with a specific anticholera 

 serum in a dilution of at least 1 to 500. 



(6) A flake of mucus containing many vibrios is emulsified directly 

 in specific anticholera serum, diluted at least 1 to 500, and a suitable 

 control is made with normal serum. This is best carried out by the 

 microscopic agglutination method. A positive agglutination under 



1 The anticholera serum "is best obtained from rabbits which have been immunized 

 by repeated injections of known cholera vibrios. The titre of the serum should be at 

 least 1 to 4000. A final diagnosis should be made preferably only when the suspected 

 organism agglutinates in a dilution of at least 1 to 2000, although clumping of freshly 

 isolated vibrios at a dilution of 1 to 500 is fairly conclusive. The sera of horses and 

 other large animals are less suitable for agglutination with cholera vibrios; natural 

 antibodies occur which cause clumping in relatively high dilutions. 



2 The necessary degree of alkalinity may be attained by adding 3 c.c. of a 10 per cent, 

 solution of sodium carbonate to each 100 c.c. of neutral (litmus) agar. 



