Volodymyr HLEBENIUK, Natalia ALEKSEEVA, Olena HAVRYLINA, Maryna BILAN, Oleksiy TKACHENKO, Natalia KOZAK

Morphological Forms of Mycobacterium bovis Under Conditions of Long-Term Storage at Low Above-Zero Temperature (3°C)

Mycobacterium bovis cultures stored for a long time at a low above-zero temperature (3°C) are characterized by pronounced changes in their biological properties that occur during numerous passages in an artificial culture medium, as a result of which frequency of formation of small forms of bacteria and their adaptation to the conditions of their existence increase. Membrane filters with pore sizes of 0.1 and 0.05 µm let through filterable and L-forms of mycobacteria, which were cultivated at 3°C for a long time, and their cultures acquire the ability to show variability in tinctorial properties. It has been proved that dissociative elementary bodies are cultivated simultaneously with other morphological forms, preserving a possibility of reversion to acid fast-negative forms of the causative agent of tuberculosis, which is possibly determined by specificity of the life cycle of mycobacteria. Elementary bodies are found in subcultures of mycobacteria that were multiply passaged in a solid culture medium, generating rod-shaped acid fast-negative dissociative forms, which is due to the ability of small forms to adapt to a selective culture medium over time after multiple passages. Therefore, elementary bodies do not generate descendants of similar morphology but form acid fast-negative rod-shaped variants of mycobacteria instead. The study of archival strains of dissociative mycobacteria has shown that the frequency of in vitro isolation of filterable forms depends on the storage time of mycobacterial cultures and the frequency of isolation of cultures from filtrates does not depend on the number of passages in a solid culture medium. A low above-zero temperature of cultivation (3°C) results in a more frequent isolation of mycobacterial cultures from the filtrate, while the highest frequency of isolation of filterable forms of M. bovis was recorded after a single guinea pig passage. Electron microscopy has clearly demonstrated that L-forms of mycobacteria isolated from the control culture filtrate (F-0.05) are represented by thick rods that have semitransparent borders and uneven edges.

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