Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens

Kengo Kubota, Hiroyuki Imachi, Shuji Kawakami, Kohei Nakamura, Hideki Harada, Akiyoshi Ohashi

Research output: Contribution to journalArticlepeer-review

36 Citations (Scopus)


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 languageEnglish
Pages (from-to)54-59
Number of pages6
JournalJournal of Microbiological Methods
Issue number1
Publication statusPublished - 2008 Jan


  • 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)


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