572 THE CHOLERA GROUP 
broth and inoculated on the surface of alkahne agar plates,^ which 
have previously been poured and hardened. The very thin transparent 
colonies which develop within twelve to eighteen hours are either 
transferred to broth and after twelve hours' incubation agglutinated, 
or the colony is emulsified directly in a high potency specific serum 
diluted five hundred times and a macroscopic or microscopic examina- 
tion 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 small 
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. Greig^ has found that cholera- 
like vibrios do not induce agglutinins in rabbits which will agglutinate 
the typical cholera vibrio. 
(b) 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 
these conditions is fairly conclusive. It should be remembered that 
an occasional strain of the cholera vibrio is met with which does not 
agglutinate when freshly isolated; prolonged cultivation in artificial 
media frequently leads to a typical agglutination. 
4. Identification of Cholera Vibrios by the Pfeiffer Phenomenon.— 
If cholera vibrios are introduced directly into the peritoneal cavity 
of an immunized guinea-pig and samples of the peritoneal exudate 
containing vibrios are removed from the peritoneal cavity with a 
capillary pipette after ten minutes, sixty minutes and ninety minutes, 
it will be found that usually after ten minutes, almost invariably 
within an hour, the vibrios will become very much granulated and will 
eventually dissolve. A normal guinea-pig similarly infected intra- 
peritoneally with a mixture of cholera vibrios and immune serum 
will exhibit the same granulation and lysis of the organisms. The 
reaction does not occur when the vibrios alone are introduced into 
the peritoneal ca\dty of a normal pig. It is much simpler to introduce 
the immune serum and vibrios into test-tubes, incubate them at 36° C. 
1 The necessary degree of alkalinity may be attained by adding 3 cc. of a 10 per cent 
solution of sodium carbonate to each 100 cc. of neutral (litmus) agar. 
2 Indian Jour. Med. Research, 1917, 4, No. 4. 
