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Method for simple and rapid enumeration of total epiphytic bacteria in the washing solution of rice plants

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The phyllosphere is one of the most common habitats for terrestrial bacteria. However, little is known about the populations of bacteria, including unculturable bacteria, that thrive on plant surfaces. Here, we developed a fluorescent nuclear staining technique to easily and rapidly observe and enumerate populations of total and living epiphytic bacteria, with particular emphasis on the concentration by centrifugation and fixation of the epiphytic bacteria. An investigation on the optimal conditions for centrifugation and fixation revealed that centrifugation at 20 400g for 2 min and fixation with 0.5% glutaraldehyde solution were the optimum conditions for observation of the bacteria. Using this technique, we assessed the populations of the total and living bacteria on the surface of rice plants. When epiphytic bacteria were recovered from rice seeds (Oryza sativa ‘Koshihikari’), the number of total and living bacterial cells was 7.36 and 6.85 log10·g-1 (fresh mass) in the seed washing, respectively. In contrast, the numbers of total and living bacterial cells in the leaf sheath washings were 5.5-5.8 and 5.3-5.7 log10·g-1, respectively. Approximately 5%-30% of the total bacteria in the washing solution of rice plant were culturable. The usefulness of the enumeration method and the amount of bacteria on the plant surfaces are discussed.

La phyllosphère est un des habitats les plus courants des bactéries terrestres. Cependant, on connait peu de chose des populations de bactéries, y compris de bactéries non cultivables, qui prospèrent à la surface des végétaux. Nous avons développé ici une technique de coloration nucléaire fluorescente afin d’observer et de compter facilement et rapidement les populations totales et vivantes de bactéries épiphytes, en mettant l’emphase sur une concentration par centrifugation et une fixation des bactéries épiphytes. La recherche de conditions de centrifugation et de fixation optimales a révélé qu’une centrifugation à 20 400g pendant 2 min et une fixation dans une solution de glutaraldéhyde à 0,5 % étaient les meilleures pour l’observation. Nous avons évalué à l’aide de cette technique les populations de bactéries totales et vivantes à la surface de plants de riz. Lorsque les bactéries épiphytes étaient récupérées des graines de riz (Oryza sativa ‘Koshihikari’), le nombre de bactéries totales et vivantes était respectivement de 7,36 et 6,85 log10·g-1 (masses frais) dans l’eau de rinçage. En comparaison, le nombre de bactéries totales et vivantes issues du rinçage de la gaine des feuilles était de 5,5-5,8 et de 5,3-5,7 log10·g-1, respectivement. Environ 5 %-30 % des bactéries totales de la solution de rinçage des plants de riz étaient cultivables. L’utilité de cette méthode de numération et la quantité de bactéries présentes à la surface des plants sont discutées.

Document Type: Research Article

Publication date: January 1, 2011

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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