Fluorescence microscopic observations of microorganisms in soil
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Microscopic observation of soil thin sections is potentially an important approach to trace the fate of external bacterial cells following soil inoculation and to determine the spatial distribution of indigenous bacterial populations in the soil. Various fluorescence microscopy techniques and their applications are reviewed with special emphasis on microbial distributions within soil microenvironments. Commonly used fluorochromes and factors affecting the staining of soil microorganisms are discussed.
The distribution of inoculated E. coli cells and indigenous bacteria in a silt loam soil was studied using conventional epifluorescence microscopy and confocal laser scanning microscopy (CLSM). A sandy soil was used for comparison of the influence of soil texture on staining quality. Seven fluorochromes and three resins were tested for their suitability for preparing high quality soil thin sections. Scotchcast resin 3™ was chosen as the best embedding medium after a comparison of important features with LR white resin and Nanoplast™ resin. Inoculated bacterial cells, stained with fluorescein isothiocyanate, 5-(4,6-dichlorotriazinyl)aminofluorescein and eosin Y, were distinguished only with difficulty from background fluorescence in the silt loam soil, but were distinguished clearly in the sandy soil due to the non-fluorescence of sand particles. Ethidium bromide, 4',6-diamidino-2-phenylindole and calcofluor white M2R stained inoculated cells clearly against the soil and resin background, although different levels of primary and induced fluorescence existed. Inoculated cells were mainly distributed among soil aggregates and some were located inside soil aggregates. Few indigenous cells were seen in the microscopic field of investigation and they were low in fluorescent intensity. This result was not unexpected due to the fewer and smaller cells (compared to inoculated cells), possible capsular layers around the cells or blocked contact of the fluorochromes with bacterial cells in uninoculated soil. Changing of the solution composition and pH during staining did not significantly improve image quality. Incubation of soil with 0.2% glucose for 24 h before fixation was not successful in enhancing the observation of indigenous cells. Satisfactory results were obtained from both epifluorescence microscopy and CLSM, with the best stain and resin combination being ethidium bromide and Scotchcast resin.
Document number: osu1283178984
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=osu1283178984
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