Premium
Application of a direct fluorescence‐based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water
Author(s) -
Savichtcheva Olga,
Okayama Noriko,
Ito Tsukasa,
Okabe Satoshi
Publication year - 2005
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20608
Subject(s) - bacteroides fragilis , bacteroides , incubation , fluorescence in situ hybridization , biology , feces , microbiology and biotechnology , anaerobic exercise , gram staining , viability assay , 16s ribosomal rna , human feces , fish <actinopterygii> , bacteria , biochemistry , physiology , fishery , cell , genetics , gene , chromosome , antibiotics
To evaluate the viability and survival ability of fecal Bacteroides spp. in environmental waters, a fluorescence‐based live/dead staining method using ViaGram™ Red + Bacterial gram stain and viability kit was combined with fluorescent in situ hybridization (FISH) with 16S rRNA‐targeted oligonucleotide probe (referred as LDS‐FISH). The proposed LDS‐FISH was a direct and reliable method to detect fecal Bacteroides cells and their viability at single‐cell level in complex microbial communities. The pure culture of Bacteroides fragilis and whole human feces were dispersed in aerobic drinking water and incubated at different water temperatures (4°C, 13°C, 18°C, and 24°C), and then the viability of B. fragilis and fecal Bacteroides spp. were determined by applying the LDS‐FISH. The results revealed that temperature and the presence of oxygen have significant effects on the survival ability. Increasing the temperature resulted in a rapid decrease in the viability of both pure cultured B. fragilis cells and fecal Bacteroides spp. The live pure cultured B. fragilis cells could be found at the level of detection in drinking water for 48 h of incubation at 24°C, whereas live fecal Bacteroides spp. could be detected for only 4 h of incubation at 24°C. The proposed LDS‐FISH method should provide useful quantitative information on the presence and viability of Bacteroides spp., a potential alternative fecal indicator, in environmental waters. © 2005 Wiley Periodicals, Inc.