| Spore is a resting body formed by some bacillus or coccus in the severe environment after complex process.Spore has a very special structure.The rich dipicolinicacid calcium(Ca2+-DPA)in nucleus can enable the spore to survive in the extreme environment,has a strong resistance to high temperature,high pressure,strong acid and strong alkali.As long as the external condition is improved,the spore will germinate into vegetative growth and become the vegetative state cells,which will enable the sporet o lose the stress resistance.To investigate the mechanism of spore germination and to analyze the effects of physical and chemical factors on the germination are important to inhibit and kill the pernicious bacillus in food,medicine and industry.Bacillus thuringiensis(Bt)is one of microbial pesticides mostly widely studied and applied in the world.Bt pesticide has the characteristics of easy mass production,good insecticidal efficiency,no harm to the environment,being safe for human and livestock.To investigate the mechanism of Bt spore germination and the effects of some influence factors on the spores and germination is helpful to understand the sterilizing mechanism on the spore and to improve the efficiency of biopesticide.An image analysis tool-SporeTracker was applied in the phase contrast microscope experiment data processing.Compared with the artificial statistics experimental data,the difference between the two data processing methods and the characteristics of SporeTracker were investigated.The data processing method was optimized by the innovative application of SporeTracker.The germination process of single Bt spore was dynamically monitored through the Differential interference contrast(DIC)microimaging technology combined with tweezers Raman spectroscopy.Using different germination agent and different strains,the effects of glutaraldehyde on Bt spore and spore germination combined with the experimental data,so as to provide a basic scheme for investigating the effects of high temperature,high pressure,strong acid,strong alkali,other physical and chemical factors,also to play an important role in understanding the spore germination and heterogeneity.Therefore,the main contents of this paper included the following:(1)The high-throughput monitoring of Bt spore germination dynamic procedure played an extremely important role in understanding the spore germination and heterogeneity from the single cell level through DIC microscopy combined with tweezers Raman microscopy.The experimental data analysis results showed that there was a big difference between different strains of the same Bt spore.The germination processes of the same strain were different triggered by different germinators.The germination processes of the different strains were also different triggered by the same germinator.(2)SporeTracker was applied in the data processing in Bt spore germination experiment.The results showed that SporeTracker,as an automatized image analysis tool,was a completely feasible tool for DIC experimental image analysis.SporeTracker could quickly mark the spore in the DIC image and draw the brightness change curve with time,and could accurately identify the Tlag value and Trelease values.Although SporeTracke could optimize the experimental data processing methods,it would yet be improved in the field of intelligent identification.(3)The dynamical germination process of Bt spore was analyzed through the DIC microimaging technology combined with tweezers Raman spectroscopy after glutaraldehyde processing.The results showed that when the spores were processed for quite a short time by0.2%glutaraldehyde,the germination of Bt spores was greatly influenced.The germination processes showed great difference triggered by different germinators.The Bt sporeinner membranes and secondary protein structures showed no significant difference after glutaraldehyde processing.However,glutaraldehyde processing could damage the spore germination related proteins,especially germinator receptor,followed by DPA release channel related protein,it had little effect on cortical hydrolase. |
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