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

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

doi:10.1016/j.mimet.2007.10.006

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 journal › Article

32 Citations (Scopus)

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
Pages (from-to)	54-59
Number of pages	6
Journal	Journal of Microbiological Methods
Volume	72
Issue number	1
DOIs	https://doi.org/10.1016/j.mimet.2007.10.006
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)

Access to Document

10.1016/j.mimet.2007.10.006

Fingerprint Dive into the research topics of 'Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens'. Together they form a unique fingerprint.

Cite this

Kubota, K., Imachi, H., Kawakami, S., Nakamura, K., Harada, H., & Ohashi, A. (2008). Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens. Journal of Microbiological Methods, 72(1), 54-59. https://doi.org/10.1016/j.mimet.2007.10.006

@article{e59db9dd7fdb4f8a8bc3106c0fd1c909,

title = "Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens",

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.",

keywords = "Catalyzed reporter deposition, Fluorescence in situ hybridization, Methanochondroitin, Methanogens, Pseudomurein, Pseudomurein endopeptidase PeiW, Sheath, Surface layer",

author = "Kengo Kubota and Hiroyuki Imachi and Shuji Kawakami and Kohei Nakamura and Hideki Harada and Akiyoshi Ohashi",

year = "2008",

month = jan,

day = "1",

doi = "10.1016/j.mimet.2007.10.006",

language = "English",

volume = "72",

pages = "54--59",

journal = "Journal of Microbiological Methods",

issn = "0167-7012",

publisher = "Elsevier",

number = "1",

}

TY - JOUR

T1 - Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens

AU - Kubota, Kengo

AU - Imachi, Hiroyuki

AU - Kawakami, Shuji

AU - Nakamura, Kohei

AU - Harada, Hideki

AU - Ohashi, Akiyoshi

PY - 2008/1/1

Y1 - 2008/1/1

N2 - 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.

AB - 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.

KW - Catalyzed reporter deposition

KW - Fluorescence in situ hybridization

KW - Methanochondroitin

KW - Methanogens

KW - Pseudomurein

KW - Pseudomurein endopeptidase PeiW

KW - Sheath

KW - Surface layer

UR - http://www.scopus.com/inward/record.url?scp=37349048083&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=37349048083&partnerID=8YFLogxK

U2 - 10.1016/j.mimet.2007.10.006

DO - 10.1016/j.mimet.2007.10.006

M3 - Article

C2 - 18022717

AN - SCOPUS:37349048083

VL - 72

SP - 54

EP - 59

JO - Journal of Microbiological Methods

JF - Journal of Microbiological Methods

SN - 0167-7012

IS - 1

ER -