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.
Original language | English |
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Pages (from-to) | 54-59 |
Number of pages | 6 |
Journal | Journal of Microbiological Methods |
Volume | 72 |
Issue number | 1 |
DOIs | |
Publication status | Published - 2008 Jan 1 |
Keywords
- Catalyzed reporter deposition
- Fluorescence in situ hybridization
- Methanochondroitin
- Methanogens
- Pseudomurein
- Pseudomurein endopeptidase PeiW
- Sheath
- Surface layer
ASJC Scopus subject areas
- Microbiology
- Molecular Biology
- Microbiology (medical)