title: Rapid detection of respiring Escherichia coli O157:H7 in apple juice, milk, and ground beef by flow cytometry.
abstract: BACKGROUND
  Rapid and simple methods to detect viable pathogenic microbes in foods and drinks are required. Flow cytometry was used for the rapid detection of respiring Escherichia coli O157:H7 cells in apple juice, milk, and ground beef.
  
  
  METHODS
  CTC (5-cyano-2,3-ditolyl tetrazolium chloride) was used to estimate the respiratory activity of bacteria. Fluorescein isothiocyanate (FITC)-labeled anti-E. coli O157:H7 direct antibody (FA) was used for the specific detection of target cells. Food samples were inoculated with starved E. coli O157:H7 and E. coli K-12 cells, and analyzed by both fluorescent microscopy and flow cytometry after double staining with FA and CTC.
  
  
  RESULTS
  Respiring E. coli O157:H7 cells in food samples showed strong fluorescence of both FA (green) and CTC (red); thus, they could be clearly and specifically distinguished from respiring E. coli K-12 or inactive cells. A good correlation was achieved in flow cytometric analysis between the numbers of inoculated viable E. coli O157:H7 and those detected in milk and apple juice. The detection threshold for this flow cytometry for E. coli O157:H7 in milk, apple juice, and ground beef was 10(3) cells/ml (milk and apple juice) or 10(3) cells/g (ground beef) of sample when the total bacterial number in the sample was 10(6) cells/ml.
  
  
  CONCLUSIONS
  Respiring E. coli O157:H7 in food samples can be detected specifically within a few hours. Flow cytometry with FA-CTC double staining can be used to examine food contamination with various pathogenic microbes demonstrating physiologic activity through the use of a suitable fluorescent antibody.
authors.affiliation: Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
authors.forename: Nobuyasu
authors.initials: N
authors.lastname: Yamaguchi
authors.forename: Makoto
authors.initials: M
authors.lastname: Sasada
authors.forename: Mio
authors.initials: M
authors.lastname: Yamanaka
authors.forename: Masao
authors.initials: M
authors.lastname: Nasu
mesh_terms: D000818:Animals; D001628:Beverages / Q000382:microbiology*; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D027845:Malus / Q000737:chemistry; D008460:Meat / Q000382:microbiology*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D013778:Tetrazolium Salts / Q000378:metabolism
chemical_list: D013778:Tetrazolium Salts; C075453:5-cyano-2,3-ditolyltetrazolium; D016650:Fluorescein-5-isothiocyanate