Premium
Seven‐hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample
Author(s) -
Ootsubo M.,
Shimizu T.,
Tanaka R.,
Sawabe T.,
Tajima K.,
Ezura Y.
Publication year - 2003
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1046/j.1365-2672.2003.02051.x
Subject(s) - enumeration , enterobacteriaceae , in situ , sample (material) , food microbiology , microbiology and biotechnology , fluorescence , fluorescence in situ hybridization , biology , environmental science , bacteria , chemistry , chromatography , escherichia coli , mathematics , biochemistry , physics , genetics , combinatorics , chromosome , gene , organic chemistry , quantum mechanics
Aims: A fluorescent in situ hybridization (FISH) technique using an Enterobacteriaceae‐specific probe (probe D) to target 16S rRNA was improved in order to enumerate, within a single working day, Enterobacteriaceae present in food and environmental water samples. Methods and Results: In order to minimize the time required for the FISH procedure, each step of FISH with probe D was re‐evaluated using cultured Escherichia coli . Five minutes of ethanol treatment for cell fixation and hybridization were sufficient to visualize cultured E. coli , and FISH could be performed within 1 h. Because of the difficulties in detecting low levels of bacterial cells by FISH without cultivation, a FISH technique for detecting microcolonies on membrane filters was investigated to improve the bacterial detection limit. FISH with probe D following 6 h of cultivation to grow microcolonies on a 13 mm diameter membrane filter was performed, and whole Enterobacteriaceae microcolonies on the filter were then detected and enumerated by manual epifluorescence microscopic scanning at magnification of ×100 in ca 5 min. The total time for FISH with probe D following cultivation (FISHFC) was reduced to within 7 h. FISHFC can be applied to enumerate cultivable Enterobacteriaceae in food (above 100 cells g −1 ) and environmental water samples (above 1 cell ml −1 ). Conclusions: Cultivable Enterobacteriaceae in food and water samples were enumerated accurately within 7 h using the FISHFC method. Significance and Impact of the Study: A FISHFC method capable of evaluating Enterobacteriaceae contamination in food and environmental water within a single working day was developed.