Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation

Diseases of Aquatic Organisms

R. J. Pascho¹  J. E. Ongerth¹ 

关键词: Renibacterium salmoninarum;    Flow cytometry;    Viability assay;    Propidium iodide;    Chlorine;   

DOI  :  10.3354/dao041181

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

ABSTRACT: The slow growth of Renibacterium salmoninarum limits the usefulness of culture as a research tool. Development of a 2-color flow cytometric assay to quantify the proportions of live and dead R. salmoninarum in a test population isdescribed. Bacteria were simultaneously stained with fluorescein isothiocyanate-conjugated immunoglobulin and exposed to the exclusion dye propidium iodide. Propidium iodide red fluorescence profiles of control groups of untreated and killed R.salmoninarum were compared with those for bacteria exposed to chlorine. Bacterial inactivation was based on mean red fluorescence intensity, and analyzed by high-red fluorescence intensity (HRFI) and curve subtraction (CS) analyses. When theconcentration of R. salmoninarum was 8.65 x 10⁶ bacteria ml^-1 and the bacteria exposed to chlorine at 1 mg l^-1 for periods from 1 to 20 min (high-Rs assessment), the mean red fluorescenceintensity of the profile for each chlorine-exposure group was higher than that for the untreated control (p < 0.0001). When the concentration of R. salmoninarum was reduced to 1.76 x 10⁶ bacteriaml^-1 and exposed to 0.8 mg l^-1 free chlorine level for periods from 20 s to 5 min (reduced-Rs assessment), the mean red fluorescence intensities of the exposure groups were higher than that for the untreated control only when theR. salmoninarum was exposed to chlorine for at least 1 min (p ≤ 0.01). On the basis of red fluorescence intensity, the proportion of dead cells generally increased with the duration of chlorine exposure. Whereas the rates of inactivationderived from the HRFI and CS analyses did not correlate with the duration of exposure in the high-Rs assessment (r² ≤ 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r²≥ 0.92). Because of the rapid loss of culturable R. salmoninarum in both assessments following chlorine exposure, neither the duration of exposure nor the inactivation estimates correlated with bacteriological culture (r² ≤ 0In both assessments, there was a correlation between the estimates of inactivation based upon HRFI and CS analyses (r² > 0.99). These results suggest that flow cytometry can be used as a supplementary or alternative method to bacteriologicalculture for monitoring the inactivation of R. salmoninarum.

【 授权许可】

Unknown