Enhanced spore detection using dipicolinate extraction techniques

Abstract

We have developed a method for bacterial endospore detection based on the presence dipicolinic acid (dpa), a substance unique to endospores. Since the sensitivity of this technique correlates directly with the amount of dpa extracted from the spores, we examined several types of extraction techniques for their dpa extraction efficiency. The three main categories investigated are physical, germination, and chemical methods for liberation of dpa from B. subtilis endospores. Attention is concentrated on the speed, efficiency, and simplicity of the extraction techniques for optimization of endospore detection using terbium–dipicolinate photoluminescence. Although methods from all categories succeeded in extracting dpa, the technique utilizing heated dodecylamine (dda) extracted the majority of the available dpa in less than 3min. Application of the dda extraction procedure to the terbium–dipicolinate photoluminescence method in conjunction with an increased detection capability resulted in a two-order of magnitude improvement in endospore detection. This combination of methods resulted the lowest reported limit of detection (LOD) (1000CFU/ml) for a terbium–dipicolinate photoluminescence method in the shortest reported time (5–7min) for the total procedure.