High-throughput Investigation of Germination of Individual Bacillus Thuringiensis HD-1 Spores by Differential Interference Contrast Microscopy Imaging

Bacillus thuringiensis (Bt), a unique bacterium which forms parasporal crystal protein and spore in parallel, is the most widely used environmentally in compatible biopesticide worldwide. Bt spore has a strong resistance to stress and adversity, and germinates rapidly once conditions are favorable for growth. In this work, differential interference contrast (DIC) microscopy was used to monitor the germination of individual Bt HD-1 spores, and the germination process was also investigated with Raman spectroscopy simultaneously. Experiment results showed that the decrease in normalized DIC intensity of Bt spore was consistent with the release of pyridine-2, 6-pyridine dicarboxylic acid (dipicolinic acid [DPA]) determined by Raman spectroscopy. Temporal resolution of 6 seconds could well describe the dynamic process of spore germination. Bt spore showed similar germination kinetics triggered either by 10 mmol/L alanine at 37 degrees C in 25 mmol/L Tris-HCl buffer (pH 8.3) or in 25 mmol/L Hepes buffer (pH 7.4). Most spores did not germinate triggered by dodecylamine but germinated rapidly triggered by exogenous Ca-DPA. These results indicated that DIC microscopy imaging could be used to observe germination of individual Bt spores in real-time quantitatively, and help to the understanding of mechanisms of Bt spore germination and its heterogeneity.