title: Level 2 validation of a flow cytometric method for detection of Escherichia coli O157:H7 in raw spinach.
abstract: The Bacteriological Analytical Manual (BAM) method currently used by the United States Food and Drug Administration (FDA) to detect Escherichia coli O157:H7 in spinach was systematically compared to a new flow cytometry based method. This Food and Drug Administration (FDA) level 2 external laboratory validation study was designed to determine the latter method's sensitivity and speed for analysis of this pathogen in raw spinach. Detection of target cell inoculations with a low cell count is critical, since enterohemorrhagic strains of E. coli require an infective dose of as few as 10 cells (Schmid-Hempel and Frank, 2007). Although, according to the FDA, the infectious dose is unknown (Food and Drug Administration, 1993). Therefore, the inoculation level into the spinach, a total of 2.0±2.6 viable E. coli O157 cells, was specified to yield between 25% and 75% detection by the new method, out of 20 samples (10 positives and 10 negatives). This criterion was met in that the new method detected 60% of the nominally positive samples; the corresponding sensitivity of the reference method was 50%. For both methods the most likely explanation for false negatives was that no viable cells were actually introduced into the sample. In this validation study, the flow cytometry method was equal to the BAM in sensitivity and far superior in speed.
authors: U.S. Food and Drug Administration, National Center for Toxicological Research, Division of Systems Biology, Jefferson, AR 72079, USA. Electronic address: anna.williams@fda.hhs.gov.
authors: Anna J
authors: AJ
authors: Williams
authors: U.S. Food and Drug Administration, National Center for Toxicological Research, Division of Systems Biology, Jefferson, AR 72079, USA. Electronic address: willie.cooper@fda.hhs.gov.
authors: Willie M
authors: WM
authors: Cooper
authors: U.S. Food and Drug Administration, National Center for Toxicological Research, Office of Scientific Coordination, Jefferson, AR 72079, USA. Electronic address: christine.summage-west@fda.hhs.gov.
authors: Christine V
authors: CV
authors: Summage-West
authors: U.S. Food and Drug Administration, National Center for Toxicological Research, Office of Scientific Coordination, Jefferson, AR 72079, USA. Electronic address: lillie.sims@fda.hhs.gov.
authors: Lillie M
authors: LM
authors: Sims
authors: U.S. Food and Drug Administration, Arkansas Regional Laboratory, Division of Microbiology, Jefferson, AR 72079, USA. Electronic address: robert.woodruff@fda.hhs.gov.
authors: Robert
authors: R
authors: Woodruff
authors: U.S. Food and Drug Administration, Arkansas Regional Laboratory, Division of Microbiology, Jefferson, AR 72079, USA. Electronic address: jessica.christman@fda.hhs.gov.
authors: Jessica
authors: J
authors: Christman
authors: Ted Moskal, Life Sciences Consultant, 515 W. Matthews Ave., Jonesboro, AR 72401, USA. Electronic address: tmoskal62@gmail.com.
authors: Ted J
authors: TJ
authors: Moskal
authors: Vivione Biosciences, LLC, Pine Bluff, AR 71602, USA. Electronic address: sramsaroop@vivionebiosciences.com.
authors: Shawn
authors: S
authors: Ramsaroop
authors: U.S. Food and Drug Administration, National Center for Toxicological Research, Division of Microbiology, Jefferson, AR 72079, USA. Electronic address: john.sutherland@fda.hhs.gov.
authors: John B
authors: JB
authors: Sutherland
authors: U.S. Food and Drug Administration, National Center for Toxicological Research, Division of Systems Biology, Jefferson, AR 72079, USA. Electronic address: pierre.alusta@fda.hhs.gov.
authors: Pierre
authors: P
authors: Alusta
authors: U.S. Food and Drug Administration, National Center for Toxicological Research, Division of Systems Biology, Jefferson, AR 72079, USA. Electronic address: jon.wilkes@fda.hhs.gov.
authors: Jon G
authors: JG
authors: Wilkes
authors: U.S. Food and Drug Administration, National Center for Toxicological Research, Division of Systems Biology, Jefferson, AR 72079, USA. Electronic address: dan.buzatu@fda.hhs.gov.
authors: Dan A
authors: DA
authors: Buzatu
mesh_terms: D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005434:Flow Cytometry / Q000592:standards*; D005516:Food Microbiology / Q000379:methods*; D018724:Spinacia oleracea / Q000382:microbiology*; D014481:United States; D014486:United States Food and Drug Administration
keywords: Escherichia coli O157; Flow cytometry; Food pathogens; Microorganisms; Public health