MAI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 101, NO. X, 2018 1 FOOD BIOLOGICAL CONTAMINANTS Performance Validation of the Microbiologique MicrofilmTM Test System for AOAC Research Institute Performance Tested Method SMCertification AOAC Performance Tested Method SM 051702 Abstract ANNA SHAPOVALOVA and HARISH K. JANAGAMA Molecular Epidemiology, Inc., 15300 Bothell Way NE, The Microfilm™ Test System is intended for quantitative Lake Forest Park, WA 98155 microbiology and consists of three types of Microfilms for aerobic LONG VUONG and ALEX FRIEDRICH plate count (Microfilm APC), total coliform and Escherichia coli IEH Laboratories and Consulting Group, Inc., 15300 Bothell count (Microfilm TCEc), and yeast and mold count (Microfilm Way NE, Lake Forest Park, WA 98155 YMC). This study evaluated the performance of the Microfilm DYLAN JOHNSON Test System against International Organization for Standardization Molecular Epidemiology, Inc., 15300 Bothell Way NE, (ISO) methods on 20 food matrixes and 2 environmental surfaces. Lake Forest Park, WA 98155 Ruggedness, robustness, and stability were also determined, LOURDES M. NADALA while inclusivity and exclusivity studies were performed on Molecular Epidemiology, Inc., 15300 Bothell Way N.E., Microfilm TCEc and YMC. An independent laboratory evaluated Lake Forest Park, WA 98155; Microbiologique, the performance on four food matrixes and one environmental Inc., 8215 Lake City Way NE, Seattle, WA 98115 surface. No significant differences and high correlation coefficients VAN NGUYEN were observed between the Microfilm Test System and the Microbiologique, Inc., 8215 Lake City Way NE, Seattle, corresponding ISO methods (ISO 4833-1:2013 for APC, ISO WA 98115 4832:2006 for total coliform count, ISO 16649-2: 2001 for E. coli, ELPIDIO CESAR NADALA, JR and ISO 21527 Part 1 and Part 2 for YMC) in spiked food matrixes Molecular Epidemiology, Inc., 15300 Bothell Way NE, and environmental samples. These results were corroborated by Lake Forest Park, WA 98155 the independent laboratory. Inclusivity and exclusivity studies for MANSOUR SAMADPOUR Microfilm TCEc showed expected results for all theE. coli strains Molecular Epidemiology, Inc. and IEH Laboratories and tested (blue-violet or violet color), while the related coliforms Consulting Group, Inc., 15300 Bothell Way N.E., showed the expected blue-green colonies on the Microfilm. Lake Forest Park, WA 98155 Similarly, all 100 fungal strains tested showed typical growth on Microfilm YMC. Exclusivity testing on Microfilm TCEc and SUBMITTING COMPANY YMC showed no growth of nontarget organisms. Robustness and Microbiologique Inc., 8315 Lake City Way NE, Seattle, ruggedness studies showed no significant differences in mean WA 98115 difference counts at varying incubation temperatures and times. Stability studies on three lots of the Microfilm Test System showed INDEPENDENT LABORATORY that it is stable at 2–25°C for 12 months and at 45°C for 6 weeks. University of Washington, School of Public Health, 4225 Roosevelt Way NE, Suite 100, Seattle, WA 98195 Participants REVIEWERS YI CHEN METHOD AUTHORS U.S. Food and Drug Administration, Center for Food Safety TAM L. MAI and Applied Nutrition, 5100 Paint Branch Pkwy, College Park, Molecular Epidemiology, Inc., 15300 Bothell Way NE, MD 20740 Lake Forest Park, WA 98155 JIM AGIN SHAO-LEI SUNG Q Laboratories, 1400 Harrison Ave, Cincinnati, OH 45214 Microbiologique, Inc., 8215 Lake City Way NE, Seattle, MICHAEL BRODSKY WA 98115 Brodsky Consultants, 73 Donnamore Crescent, Thornhill, ON L3T 4K6, Canada Submitted February 8, 2018. The method was independently tested, evaluated, and certified by Scope of Method the AOAC Research Institute as a Performance Tested Method SM. See http://www.aoac.org/testkits/steps.html for information on certification. Supplemental information is available online at: http://aoac. (a) Target organisms.—Total aerobic bacteria, total publisher.ingentaconnect.com/content/aoac/jaoac coliform/Escherichia coli, yeasts, and molds. Corresponding author’s e-mail: [email protected] (b) Matrixes.—(1) Food.—Meat products [raw beef, 2% DOI: https://doi.org/10.5740/jaoacint.18-0036 fat; raw pork, 20% fat; ready-to eat (RTE) lunch meat], poultry Reproduced from Journal of AOAC International 101 (2018). DOI: https://doi.org/10.5740/jaoacint.18-0036 Lourdes Nadala: Participant of the 15th UM, 1987-1988. 168 169 2 MAI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 101, NO. X, 2018 products (raw chicken, 2% fat; raw turkey, 1% fat), fish and General Information seafood products (raw shrimp, surimi), fruits and vegetable- based products (spinach, lettuce, mung bean sprouts), nut-based Total aerobic count, coliforms, generic E. coli, yeasts, and products (peanut butter, almonds), dairy products (pasteurized molds are routinely enumerated in foods as part of product milk, 2% fat; ice cream), chocolate/bakery products (wheat release criteria or, in the case of swabs, for environmental flour, frost and topping mix), pasta (uncooked noodles), spices monitoring. Tests typically include plating on specific (black pepper, cumin), and animal feed (dry pet food). microbiological media, such as described in the U.S. Food (2) Environmental swabs.—Stainless steel and plastic. and Drug Administration Bacteriological Analytical Manual (c) Summary of validated performance claims.— methods, ISO standards, U.S. Department of Agriculture Performance equivalent to that of the International Organization methods, etc., and ready-to-use films. The Microfilm Test for Standardization (ISO) standards for total aerobic count System provides rapid and easy-to-use enumeration methods (ISO 4833-1:2013, Microbiology of the food chain–Horizontal for aerobic plate count (APC), total coliform count (TCC), method for the enumeration of microorganisms–Part 1: Colony E. coli, yeast, and molds. count at 30°C by the pour plate technique; 1), total coliforms (ISO 4832:2006, Microbiology of food and animal feeding Materials and Methods stuffs–Horizontal method for the enumeration of coliforms– Colony count technique; 2), E. coli (ISO 16649-2:2001, Test Kit Information Microbiology of food and animal feeding stuffs–Horizontal method for the enumeration of ß-glucuronidase-positive (a) Kit name.—Microbiologique Microfilm Test System. E. coli–Part 2: Colony count technique at 44°C using 5-bromo- (b) Cat No.—Microfilm Test System (M-2007): Microfilm 4-chloro-3-indolyl ß–D-glucuronide; 3), and yeasts and molds APC (M-1022), TCEc (M-1021), and Microfilm YM (M-1020). (ISO 21527 Part 1 and Part 2:2008, Microbiology of food and (c) Ordering information.—In the United States and Canada.— animal feeding stuffs–Horizontal method for the enumeration of Tel: 1-888-998-4115, E-mail: [email protected]. yeasts and molds; 4, 5) as reference culture methods. Other.—Tel: +1-206-525-0412. Principle Test Kit Components The Microbiologique Microfilm System is applicable for (a) Microfilm APC.—50 sterile sheets contained in sterile, enumeration of total aerobic bacteria (APC), total coliform/E. coli resealable pouches. (TCEc), or yeast and mold (YMC) in food and environmental (b) Microfilm TCEc.—50 sterile sheets contained in sterile, samples. For solid foods, the sample is first mixed with buffered resealable pouches. peptone water (BPW) or appropriate diluent, then mixed well by (c) Microfilm YMC.—50 sterile sheets contained in sterile, hand-massage or stomacher, and then 1 mL is inoculated onto resealable pouches. each of the three Microfilms that have been pre-impregnated with different media. For food samples with high bacterial load, serial Additional Supplies and Reagents Required But Not dilution may be performed prior to plating on the Microfilms. Supplied With the Kit The inoculated Microfilms are then incubated at 36 ± 1°C for 24 h (APC and TCEc) or 26 ± 1°C for 48 h (YMC) to allow for (a) Rinse/Diluent Solution.—One pouch sufficient for the formation of visible colonies. The colonies are counted, and making 2 L of Rinse solution (may be ordered separately from the count value is used to calculate the original concentration of Microbiologique, Inc.): BPW for Microfilm APC and TCEc, CFUs expressed as CFU/g or CFU/surface swab sample. and Peptone Water Broth, 0.1%, for Microfilm YMC. The System consists of three distinct indicator/selective (b) Swabs.—Sterile, individually wrapped (required for media impregnated into separate support matrixes. Each of environmental sample collection only; Microbiologique- the indicator/selective media contains growth nutrients, recommended swab in packs of 50 may be ordered separately). indicator dyes, and selective reagents needed to support rapid (c) Whirl Pak.—18 ounces, for sample preparation/rinsing. and specific growth for each class of microorganism being (d) Sterile measuring cylinder. enumerated. To calculate the number of CFUs, colonies are (e) Laboratory balance.—2000 g with 0.1 g accuracy. counted as colored spots on the Microfilm. For Microfilm (f) Sterile water. APC, colonies are colored, making them easily visible, (g) Pipet.—200 μL and 1000 μL and corresponding tips. thus facilitating the counting of colonies (Figure 1A). For (h) Fine-tipped marking pen. Microfilm TCEc, the medium contains growth nutrients (i) Gloves.—Nitrile, powder-free. supportive for coliforms and selective agents that inhibit (j) Alcohol lamp (>90% alcohol) or Bunsen burner and the growth of Gram-positive bacteria. Colonies of coliforms stainless steel forceps (round-tipped ). appear bluish-green to green, while E. coli colonies are dark