Determination of the bacterial content of cholera vaccine

Abstract

International and national opacity reference preparations are employed to determine the concentration of vibrios in freshly harvested cultures of Vibrio cholerae for cholera vaccine. The measurements have been the basis for calculating the number of vibrios per milliliter given on the labels of the vaccines. This study showed that suspensions of Inaba 35A3 (In35A3) visually equal to 10 opacity units of the U.S.A. Turbidity Reference (1947) had higher nephelometric measured units and colony-forming units than a visually equivalent suspension of Ogawa 41 (Og41). Maximum differences were 13·3 vs 6·7 neph u and 2·4 vs 0·23 CFU, respectively. Cultivation on alkaline nutrient agar, pH 8·5, or on casamino acid agar, pH 7·4, had very little influence on unitage. However, growth of In35A3 at 40–41 °C significantly decreased the neph u as compared with 35–38 °C grown culture. Inaba V86 (InV86) had optical properties similar to Og41 and both InV86 and Og41 were less affected by the higher growth temperature than In35A3. Nitrogen assays were not sufficiently extensive to determine the relationship of nitrogen content to visual and nephelometric unitage or to CFU. The differences in the optical properties emphasizes the need to establish for each vaccine production strain the relationship between optical readings (visual or instrument) and a constant baseline of reference. Potency is the final criterion.