Determination of spore concentration in Bacillus thuringiensis through the analysis of dipicolinate by capillary zone electrophoresis

Abstract

A new capillary zone electrophoresis (CZE) method for the analysis of dipicolinic acid, a specific component found in spores but not in vegetative cells, was used to determine spore concentration in Bacillus thuringiensis according to the relationship between the spore concentration and the content of dipicolinate. The quantitative relationship was established by using purified spores. Electrolyte conditions that affected the separation efficiency of dipicolinate and the reproducibility were investigated. With 10 m M phosphate, 10 m M ethylenediaminetetraacetic acid and 0.25 m M tetradecyltrimethylammonium bromide at pH 6.2 as the carrier electrolyte, dipicolinate can be determined within 8 min at an applied voltage of −25 kV (anode at detector) and a capillary temperature of 25 °C. The method has a high separation efficiency with which the number of theoretical plates is above 300 000 plates m −1. The relative standard deviations for migration time and peak area are less than 0.5% and 2.0%, respectively. The detection limit for dipicolinate was 10 ng ml −1, which corresponds to 7.2·10 5 spores ml −1. The method was used to determine spores in fermentation broths, and the results obtained agreed well with the values obtained by plate counting.