[Determination of DNA content in individual Vibrjo cholerae cells by using flow cytofluorimetry method: comparative analysis of inhomogeneity in cells of strains with various biological properties].

AIM: Comparative analysis of DNA content in individual cells of Vibrio cholerae strains with various biological properties. MATERIALS AND METHODS: 24-hour agar cultures of 2 avirulent (lacking cholera toxin gene) and 2 virulent strains and their subcultures obtained by cultivation in 1% peptone water for 1, 3 and 5 hours were studied. DNA of the killed bacteria was dyed by a mixture of ethidium bromide and mitramycin. Ratio of cells with low, intermediate and high relative DNA content in conditional units of specific DNA fluorescence intensity was determined by flow cytofluorimetry method. The degree of inhomogeneity of the studied microbial population cells was evaluated by DNA histogram variation coefficient value. RESULTS: At the level of major statistical samples of individual V. cholerae cells a principally different reaction pattern of the studied toxigenic and non-toxigenic strains on changes of cultivation conditions was registered. CONCLUSION: Populations of cells of toxigenic V. cholerae strains in contrast to non-toxigenic probably shift to polyploid state during starvation. This phenomenon may turn out to be a differential feature in determination of the risk group (hazard) of a strain.

[Determination of DNA content in individual Yersinia pestis cells by using flow cytofluorimetry method: comparative analysis of inhomogeneity in cultures of strains with various biological properties].

AIM: Comparative analysis of Yersinia pestis strains with various biological properties by DNA content in individual cells. MATERIALS AND METHODS: Virulent strain 231, avirulent strain KM 260 (12) [231], that is its isogenic (no-plasmid) derivative, and vaccine strain EV NIIEG were used. 48-hour agar cultures of the studied strains reproduced at 28 degrees C and their subcultures obtained by cultivation of the initial cultures by aeration on liquid nutrient medium from 37 degrees C were prepared. DNA of the fixed bacteria was dyed by a mixture of ethidium bromide and mitramycin, and then the bacteria were studied by using flow cytofluorimeter for the determination of rates of cells with relatively low or high DNA content in the studied bacterial populations. The degree of inhomogeneity of a bacterial population was evaluated by DNA histogram variation coefficient value. RESULTS: In 6 hours of growth at 37 degrees C in optically non-dense bacterial cultures a high degree of DNA content per cell inhomogeneity was established that is related to the activation of DNA replication process in bacteria. In 48 hours of growth this inhomogeneity completely disappeared in the virulent strain cultures and remained in the avirulent strain cultures of the plague pathogen. Based on the studied parameters the vaccine strain held an intermediate position. CONCLUSION: Further studies of the plague culture DNA content per cell inhomogeneity may become a base for the operative strain differentiation based on pathogenicity level (hazard) for humans, and therefore the requirements for the management of safe working conditions with this microorganism.