On the issue of visualization of parasporal bodies and spores

Abstract

Despite the rapid development of molecular genetic methods of research, which have literally overturned our understanding of microorganisms, the classical methods of microbiology still do not lose their relevance. Especially it concerns questions when it is necessary to establish the presence of organelles or toxin-forming ability in a bacterium. In the present article the possibilities of visualisation of parasporal bodies on the example of B. thuringiensis by means of Cumassi Brilliant Blue staining are considered. B. thuringiensis strains IPM-3 (H-1), IPM-4 and IPM-4, as well as B. anthracis ∆ Ames strain (negative control) were used in this work. From single colonies of cultures grown on Hottinger's agar, smears were prepared as is customary in laboratory practice, then stained with a mixture consisting of 0.133% Cumassi Brilliant Blue (G) in 50% acetic acid solution and microscoped. Examination of smears showed that the best visualisation of parasporal bodies is achieved when a two-day culture is used, and dye exposure for 30 minutes. In this case visualisation of parasporal bodies in the form of dark-blue structures on the background of light-blue cells and shiny oval spores in all fields of view is achieved. The proposed staining method allows for relatively simple visualization of parasporal bodies and spores, which is especially important when differentiating B. anthracis from other representatives of the Bacillus cereus complex. In addition, visualization of spores in the case of B. anthracis allows for evaluation of the quality of sporulation, making the proposed method a safe competitor to existing ones.