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    17.2.05 AOAC Official Method 986.32 Aerobic Plate Count in Foods

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    JDLatorre
    This document describes AOAC Official Method 986.32 for performing an aerobic plate count in foods using a hydrophobic grid membrane filter method. It involves the following steps: 1. Using a membrane filter imprinted with a hydrophobic grid pattern to divide the surface into compartments for counting bacterial colonies. 2. Preparing appropriate dilutions of test samples and treating some samples with enzymes to break down compounds interfering with bacterial growth. 3. Filtering the dilutions using a unit with a prefilter to remove food particles, then enumerating the number of grid squares occupied by colonies to calculate bacterial concentration.

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    17.2.05 AOAC Official Method 986.32 Aerobic Plate Count in Foods

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    JDLatorre
    405 views3 pages
    This document describes AOAC Official Method 986.32 for performing an aerobic plate count in foods using a hydrophobic grid membrane filter method. It involves the following steps: 1. Using a membrane filter imprinted with a hydrophobic grid pattern to divide the surface into compartments for counting bacterial colonies. 2. Preparing appropriate dilutions of test samples and treating some samples with enzymes to break down compounds interfering with bacterial growth. 3. Filtering the dilutions using a unit with a prefilter to remove food particles, then enumerating the number of grid squares occupied by colonies to calculate bacterial concentration.

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    normatividad microbiologica para alimentos

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    AOAC986_32

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    This document describes AOAC Official Method 986.32 for performing an aerobic plate count in foods using a hydrophobic grid membrane filter method. It involves the following steps: 1. Using a membrane filter imprinted with a hydrophobic grid pattern to divide the surface into compartments for counting bacterial colonies. 2. Preparing appropriate dilutions of test samples and treating some samples with enzymes to break down compounds interfering with bacterial growth. 3. Filtering the dilutions using a unit with a prefilter to remove food particles, then enumerating the number of grid squares occupied by colonies to calculate bacterial concentration.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    405 views3 pages

    17.2.05 AOAC Official Method 986.32 Aerobic Plate Count in Foods

    Uploaded by

    JDLatorre
    This document describes AOAC Official Method 986.32 for performing an aerobic plate count in foods using a hydrophobic grid membrane filter method. It involves the following steps: 1. Using a membrane filter imprinted with a hydrophobic grid pattern to divide the surface into compartments for counting bacterial colonies. 2. Preparing appropriate dilutions of test samples and treating some samples with enzymes to break down compounds interfering with bacterial growth. 3. Filtering the dilutions using a unit with a prefilter to remove food particles, then enumerating the number of grid squares occupied by colonies to calculate bacterial concentration.

    Copyright:

    © All Rights Reserved
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    Download as pdf or txt
    of 3

    17.2.

    05
    AOAC Official Method 986.32
    Aerobic Plate Count in Foods
    Hydrophobic Grid Membrane Filter Method
    First Action 1986
    Final Action 1987
    A. Principle

    Hydrophobic grid membrane filter (HGMF) uses membrane filter


    imprinted with hydrophobic material in grid pattern. Hydrophobic
    lines act as barriers to spread of colonies, thereby dividing membrane filter surface into separate compartments of equal and known
    size. Number of squares occupied by colonies is enumerated and
    converted to most probable number (MPN) value of organisms by
    using formula given [see D(a)].

    ing 0.45 m membrane filter. Store up to 1 week at 46C or up to


    3 months at 18C.
    (m) Diastase stock solution.Dilute 10 g diastase (Sigma,
    No. A3176 or equivalent) to 100 mL with tris buffer, pH 7.0. Warm to
    35C if necessary to aid solution. Filter through Whatman No. 1 paper
    (or equivalent) to remove insoluble material; then filter-sterilize using
    0.45 m membrane filter. Store up to 1 week at 46C or up to 3 months
    at 18C.
    (n) Hemicellulase stock solution.Dilute 10 g hemicellulase
    (Sigma, No. H2125 or equivalent) to 100 mL with acetate buffer,
    pH 5.5. Warm to 35C if necessary to aid solution. Filter through
    Whatman No. 1 paper (or equivalent) to remove insoluble material;
    then filter-sterilize using 0.45 m membrane filter. Store up to 1 week at
    46C or up to 3 months at 18C.

    B. Apparatus, Culture Media, and Reagents

    (a) HGMF.Membrane filter has pore size of 0.45 m and is imprinted with nontoxic hydrophobic materials in grid pattern. ISO-GRID
    (QA Life Sciences, Inc., 6645 Nancy Ridge Dr, San Diego, CA 92121,
    USA) or equivalent meets these specifications.
    (b) Filtration units for HGMF.Equipped with 5 m mesh prefilter
    to remove food particles during filtration. One unit is required for each
    sample. ISO-GRID (QA Life Sciences, Inc.) or equivalent meets these
    specifications.
    (c) Pipets.1.0 mL serological with 0.1 mL graduations, 1.1 or
    2.2 mL milk pipets are satisfactory. 5.0 mL serological with 0.1 mL
    graduations.
    (d) Blender.Waring, or equivalent multispeed model, with
    low-speed operation at 10 00012 000 rpm, and 250 mL glass or metal
    blender jars with covers. One jar is required for each sample.
    (e) Vacuum pump.H2O aspirator vacuum source is satisfactory.
    (f) Manifold or vacuum flask.
    (g) PeptoneTween 80 (PT) diluent.Dissolve 1.0 g peptone
    and 10.0 g Tween 80 in 1 L H2O. Dispense enough volume into dilution bottles to give 90 1 mL or 99 1 mL after autoclaving
    15 min at 121C.
    (h) Tryptic soy-fast green agar (TSFA).15.0 g tryptone, 5.0 g
    Phytone (or soytone), 5.0 g NaCl, 0.25 g fast green FCF (CI No.
    42053), and 15.0 g agar diluted to 1 L with H2O. Heat to boiling. Autoclave 15 min at 121C. Temper to 5055C. Aseptically adjust pH to
    7.3 0.1. Dispense ca 18 mL portions into 100 15 mm Petri dishes.
    Surface-dry plated medium before use.
    ( i ) T r i s b u f f e r. 1 . 0 M . D i s s o l v e 1 2 1 . 1 g
    tris(hydroxy-methyl)amino methane in ca 500 mL H2O. Adjust solution to desired pH with concentrated HCl and dilute to 1 L with H2O.
    Store at either room temperature or 46C.
    (j) Acetate buffer.1.0M. Dissolve 60 mL glacial acetic acid in ca
    500 mL H2O. Adjust solution to desired pH with 5M NaOH and dilute
    to 1 L with H2O. Store at 46C.
    (k) Amylase stock solution.Dilute 10 g -amylase (Sigma Chemical Co., No. A6814 or equivalent) to 100 mL with tris buffer, pH 7.0.
    Warm to 35C if necessary to aid solution. Filter through Whatman
    No. 1 paper (or equivalent) to remove insoluble material; then filter-sterilize using 0.45 m membrane filter. Store up to 1 week at 46C
    or up to 3 months at 18C.
    (l) Cellulase stock solution.Dilute 10 g cellulase (Sigma,
    No. C0901 or equivalent) to 100 mL with acetate buffer, pH 5.0. Warm
    to 35C if necessary to aid solution. Filter through Whatman No. 1 paper (or equivalent) to remove insoluble material; then filter-sterilize us-

    Table 986.32

    Enzyme treatments for foodsa

    Food

    Enzyme

    Skim milk

    None

    Raw milk

    None

    Fluid dairy products other than skim milk

    Trypsin

    Ice cream: without stabilizers

    Trypsin

    containing gums

    Hemicellulase

    containing cellulose derivatives

    Cellulase

    Spray-dried milks

    Trypsin

    Cheeses

    Trypsin

    Spray-dried cheese powders

    Cellulase or proteaseb

    Sour cream

    Diastase

    Yogurt

    Trypsin

    Butter

    None

    Margarine

    None

    Egg: liquid or powder

    Trypsin

    Raw beef, pork, poultry

    Trypsin

    Cooked meat or poultry

    Trypsin

    Flour

    None

    Rice

    None

    Chocolate

    Amylase

    Breakfast cereals

    Cellulase

    Cake mixes

    Amylase

    Fruit puree (e.g., fig paste)

    Pectinase

    Raw vegetables

    None

    Lecithin

    Lecithinase

    Food colorings

    None

    Gums

    Hemicellulase

    Citrus juices

    Pectinase

    Infant formula

    Trypsin

    Sodium caseinate

    Protease

    Nut meats

    None

    Shrimp

    None

    Oysters

    Trypsin

    Based on analysis of 1 mL of 1:10 dilution. Foods tested at dilutions of 1:100


    or higher do not usually need enzyme treatment.
    Varies, depending on individual product.

    2000 AOAC INTERNATIONAL

    Figure 986.32AFiltration unit.

    (o) Trypsin stock solution.Dilute 10 g trypsin to 100 mL with tris


    buffer, pH 7.6. Warm to 35C if necessary to aid solution. Filter through
    Whatman No. 1 paper (or equivalent) to remove insoluble material;
    then filter-sterilize using 0.45 m membrane filter. Store up to 1 week at
    46C or up to 3 months at 18C.
    (p) Lecithinase (phospholipase A2) stock solution.Dilute
    commercial enzyme solution (Sigma, No. P6534 or equivalent) to
    25 units/mL with tris buffer, pH 8.0. Filter-sterilize using 0.45 m
    membrane filter. Store up to 1 week at 46C or up to 3 months at
    18C.
    (q) Pectinase stock solution.Use commercial enzyme solution of
    pectinase from Aspergillus niger, containing 36 units/mg protein, dissolved in 40% glycerol (Sigma, No. P9932 or equivalent). Filter-sterilize using 0.45 m membrane filter. Store up to 1 week at 46C
    or up to 3 months at 18C.
    (r) Protease stock solution.Use commercial enzyme solution of
    protease from Bacillus subtilis, containing 715 units/mg protein
    (Biuret) in aqueous solution (Sigma, No. P8775 or equivalent). Filter-sterilize using 0.45 m membrane filter. Store up to 1 week at 46C
    up to 3 months at 18C.
    C. Test Sample Preparation

    (a) Liquid egg.Thoroughly mix test sample with sterile spoon or


    spatula and prepare 1:10 dilution by aseptically weighing 11 g egg material into sterile wide-mouth glass-stoppered or screw-cap bottle; add
    99 mL PT diluent, (g), and 1 tbsp of sterile glass shot. Thoroughly agitate 1:10 dilution to ensure complete solution or distribution of egg material in diluent by shaking each container rapidly 25 times, each shake
    being an up-and-down movement of ca 30 cm, time interval not exceeding 7 s. Let bubbles escape. Transfer representative portion from 1:10
    dilution for higher serial dilutions as needed. If enzyme treatment is
    needed (see Table 986.32), combine 5 mL of 1:10 dilution with 1 mL
    enzyme stock solution. Incubate 2030 min at 3537C in H2O bath.
    Correct for additional dilution factor by filtering 1.2 mL of enzyme-treated test portion.
    (b) Other liquid test samples.Mix contents of laboratory sample container thoroughly. To prepare 1:10 dilution, aseptically transfer 10 mL test portion into 90 mL PT diluent, B(g). Mix by shaking
    bottle 25 times through 30 cm arc in 7 s. Transfer representative portions from 1:10 dilution as needed. If enzyme treatment is needed
    (see Table 986.32), combine 5 mL of 1:10 dilution with 1 mL enzyme

    stock solution. Incubate 2030 min at 3537C in H2O bath. Correct


    for additional dilution factor by filtering 1.2 mL of enzyme-treated
    test portion.
    (c) Whole egg powder.Thoroughly mix test sample with sterile
    spoon or spatula and prepare 1:10 dilution by aseptically weighing
    11 g egg material into sterile wide-mouth glass-stoppered or
    screw-cap bottle; add 99 mL PT diluent, B(g), and 1 tbsp sterile glass
    shot. Thoroughly agitate 1:10 dilution to ensure complete solution or
    distribution of egg material in diluent by shaking each container rapidly 25 times, each shake being an up-and-down movement of ca
    30 cm, time interval not exceeding 7 s. Let bubbles escape. Transfer
    representative portion from 1:10 dilution for higher serial dilutions
    as needed. If testing 1:10 dilution is necessary, prepare 1:100 dilution and combine 10 mL of 1:100 dilution with 1 mL trypsin stock
    solution, B(o). Incubate 2030 min at 3537C in H2O bath. Filter
    entire 11 mL volume to test 1:10 dilution.
    (d) Other foods.To prepare 1:10 dilution, aseptically weigh
    10 g test portion into sterile blender jar. Add 90 mL PT diluent, B(g),
    and blend 2 min at low speed (10 00012 000 rpm). Transfer representative portion from 1:10 dilution for higher serial dilutions as
    needed. If enzyme treatment is needed (see Table 986.32), combine
    5 mL of 1:10 dilution with 1 mL enzyme stock solution. Incubate
    2030 min at 3537C in H2O bath. Correct for additional dilution
    factor by filtering 1.2 mL of enzyme-treated test portion.
    D. Analysis

    Select appropriate dilution for analysis, depending on desired


    counting range. Ordinarily, 1:100 dilution is satisfactory, producing
    counting range of 100/g or mL to 500 000/g or mL. Use 1:10 dilution
    if very low counts are expected.
    (See Figures 986.32A and B.) Turn on vacuum source. Place sterile filtration unit on manifold or vacuum flask. Open clamp A. Rotate back funnel portion C. Aseptically place sterile HGMF on
    surface of base D. Rotate funnel forward. Clamp shut by sliding jaws
    L of stainless steel clamp over entire length of flanges B extending
    from both sides of funnel C and base D, and rotating moving arm K
    into horizontal (locked) position.
    Aseptically add ca 1520 mL sterile H2O to funnel. Pipet required
    volume of appropriate dilution into funnel. Apply free end of vacuum tubing E to suction hole F to draw liquid through prefilter
    mesh G. Aseptically add additional 1015 mL sterile H2O to funnel
    and draw through mesh as before. Close clamp A to direct vacuum to
    base of filtration unit and draw liquid through HGMF.
    Open clamp A. Rotate moving arm K of stainless steel clamp into
    unlocked (ca 45 angle) position and slide jaws L off of flanges B.
    Rotate back funnel C. Aseptically remove HGMF and place on sur-

    Figure 986.32BFiltration unit clamp


    2000 AOAC INTERNATIONAL

    face of predried TSFA, B(h), plate. Avoid trapping air bubbles between filter and agar.
    (a) Raw milk, pasteurized milks and creams, and egg powders.Incubate 48 3 h at 32C. Colonies will be various shades of
    green. Count all squares containing one or more colonies (positive
    squares) except if a single colony has clearly spread to adjacent
    squares, count it as one positive square. Convert positive square
    count to MPN with the formula, MPN = {N loge [N/(N x)]}, where
    N = total number of squares and x = number of positive squares. Multiply by reciprocal of dilution factor and report as MPN of total
    bacteria / g or mL.
    (b) Liquid egg.Incubate 3 days (72 3 h) at 32C. Proceed as in (a).
    (c) All other foods.Incubate 48 3 h at 35C. Proceed as in (a).
    Reference: JAOAC 69, 671(1986).

    2000 AOAC INTERNATIONAL

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