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
N1 - Funding Information:
We thank Takashi Onodera at Nagaoka University of Technology for his technical assistance and Madan Tandukar at Tohoku University for reading the manuscript. This study was partly supported by research grants from the New Energy and Industrial Technology Development Organization (NEDO), by the Ministry of Education, Culture, Sports, Science and Technology, Japan and by the Japan Society for the Promotion of Science (JSPS). KK and KN were supported by JSPS research fellowships for young scientists.
PY - 2008/1
Y1 - 2008/1
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 -