Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens |
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Authors: | Kubota Kengo Imachi Hiroyuki Kawakami Shuji Nakamura Kohei Harada Hideki Ohashi Akiyoshi |
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Institution: | Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Japan. |
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Abstract: | Several enzymatic permeabilization protocols (utilizing lysozyme, proteinase K, achromopeptidase, or recombinant pseudomurein endopeptidase PeiW]) were evaluated for application of in situ hybridization with horseradish peroxidase-labeled oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) to methanogens. In this study, twelve methanogens were selected that have typical cell surface structures: pseudomurein, surface layer, methanochondroitin and sheath. Among the treatments tested, PeiW treatment was observed to be the most effective one, although methanogens having a sheath were stained heterogeneously and methanogens having methanochondroitin were not permeabilized. On the other hand, lysozyme, proteinase K, and achromopeptidase treatments were ineffective or caused cell-lysis, resulting in weak or no signals. Applicability of PeiW treatment was further evaluated using an anaerobic granular sludge sample. The detection rate of Archaea by CARD-FISH increased remarkably after the treatment. Based on the results obtained in this study, we propose PeiW treatment as a novel permeabilization method for CARD-FISH application to methanogens. |
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