A 5-h screening and 24-h confirmation procedure for detecting Escherichia coli O157:H7 in beef using direct epifluorescent microscopy and immunomagnetic separation.
An antibody-direct epifluorescent filter technique (Ab-DEFT) detected 100% of the raw ground beef samples inoculated with Escherichia coli O157:H7 cells (0.15 cells g-1) and incubated in a prewarmed, modified buffered peptone water (mBPW) non-selective enrichment broth for 5 h at 42 degrees C in an orbital shaking water bath (200 rev min-1). Over 50% of the microscopic fields viewed were positive (1-10 fluorescent cells field-1) in the Ab-DEFT. All positive screening results were confirmed within 24 h by subjecting 1 ml of the mBPW to the Dynabeads anti-E. coli O157 immunomagnetic separation procedure, followed by plating on MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. At this cell concentration, 41.7% of the inoculated samples were detected by the conventional method involving a 24-h selective enrichment. Exposure to viable cells before filtration was minimized by using a 0.58% formaldehyde concentration for 5 min at 50 degrees C (killed > 4.00 logs of E. coli O157:H7 cells) without affecting cell fluorescence.
Restaino
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R & F Laboratories, West Chicago, IL 60185, USA.
Frampton
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Irbe
R M
RM
Allison
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DR
D000818:Animals; D000907:Antibodies, Bacterial; D002417:Cattle; D019453:Escherichia coli O157 / Q000276:immunology / Q000302:isolation & purification* / Q000472:pathogenicity; D005069:Evaluation Studies as Topic; D018189:Immunomagnetic Separation / Q000379:methods*; D008460:Meat / Q000382:microbiology*; D008856:Microscopy, Fluorescence / Q000379:methods*; D013997:Time Factors
D000907:Antibodies, Bacterial