Research features of sporulation and hermination processes in representatives of the genus Bacillus

Abstract

The formation of spores by bacilli is considered as a strategy of survival in adverse environmental conditions. The understanding of the sporulation process as an important phase of the life cycle of bacilli began with the development of microscopic methods. The complete cytological model of sporulation has been developed by electron, fluorescence microscopy and genetic research methods. Electron microscopy allows to establish the stage of spore formation (from I to VII). To quantify the course and determine the synchronicity of the spore formation process, light microscopy methods are more suitable. Spores are well detected in culture's native preparation by phase contrast microscopy starting from stage prespores (IV) and interference contrast microscopy starting from septation stage (II). Microscopy of fixed stained preparations provides information about the presence, size, shape and location of spores. The use of some staining methods allows to differentiate spores at the stages of dormancy and germination (Wirtz-Conklin spore stain) or to distinguish mature endospores from young ones (Peshkov's method). Methods of staining spores are based on the use of high temperatures and mordants, which provide loosening of strong shells of spores and facilitate the penetration of the dye into their thick structure. At the decolorization step, the dye is removed from the vegetative cell due to weak binding to its structures, but it cannot be removed from the spores. The counter-staining step provides staining of the vegetative cell in a color different from the color of the spores.

To identify and quantify the process of germination of spores use different methods based on the detection of physical, biological and chemical characteristics of this process. The transition from the phase of light dormant spores to the phase of dark germinating spores can be observed visually using phase-contrast microscopy. A simple way to study the process of germination of spores is to measure the optical density of the spore suspension in the wavelength range from 400 to 1000 nm (usually at 600 nm). But this method has a number of disadvantages. There are methods to avoid them. One of them is the determination of the activity of marker enzymes:  proteases, extracellular hydrolases or esterases, which contribute to the differentiation of cells into vegetative form. An important biochemical marker of spore germination is the release of DPA (dipicolinic acid). It can be quantified by colorimetric, chromatographic, fluorescence spectroscopic, radiometric methods or dye displacement assay using a dual colorimetric-luminescent sensor system. A simple method to detect germination at an early stage is to determine the loss of heat resistance spores. Another effective way to study germination is to use specific dyes that bind to the nucleic acids of the spore nucleus as a result of impaired permeability of the inner membrane, degradation of DNA-binding proteins and cortex of the spores. The ATP bioluminescence method, based on the use of the luciferin / luciferase reaction, is a fast and sensitive way to detect germination. The combination of several methods allows characterizing the germination process more thoroughly and from different angles. Studies that have shown a correlation between sporulation and the subsequent behavior of spores suggest the need for further study of these processes in an inseparable relationship.

Key words: Bacillus, sporulation, germination, microscopy, biomarkers of bacterial spore germination.

DOI: 10.5281/zenodo.6634727