Seven-hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample.
OBJECTIVE
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.


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 x100 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.


CONCLUSIONS
A FISHFC method capable of evaluating Enterobacteriaceae contamination in food and environmental water within a single working day was developed.
Ootsubo
M
M
Research and Development Department, Hokkaido Industrial Technology Center, Hakodate, Hokkaido, Japan. ohtsubo@techakodate.or.jp
Shimizu
T
T
Tanaka
R
R
Sawabe
T
T
Tajima
K
K
Ezura
Y
Y
D015169:Colony Count, Microbial; D004755:Enterobacteriaceae / Q000235:genetics / Q000302:isolation & purification*; D004784:Environmental Monitoring / Q000379:methods; D005516:Food Microbiology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008856:Microscopy, Fluorescence; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013997:Time Factors; D014871:Water Microbiology
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S