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Three-day nutrient-broth cultures of Vibrio cholerae gave distinct and reproducible results when tested for haemolysis of sheep RBC. As a medium for detecting haemolysis, heart-infusion broth was no better, and brain-heart-infusion broth was less satisfactory. Lack of reproducibility, and difficulty in interpreting results, were experienced when medium containing glycerol was used and when attempts were made to detect haemolysin in young (8-hour or 1-day) cultures. Horse and human RBC were found too fragile for this test.
Strains of V. cholerae could be separated into three biotypes: classical, El Tor, and an intermediate biotype to which many strains of the present pandemic belong. The latter strains are similar to the El Tor biotype but haemolysin negative. Hence, the haemolysin test, if performed by a reliable method, is a valuable epidemiological tool.