O icial Methods of Analysis of AOAC INTERNATIONAL (22)

Dr. George W Latimer, Jr. (ed.)

[Link]
Published: 2023 Online ISBN: 9780197610145 Print ISBN: 9780197610138

CHAPTER

Subchapter 2 Chilled, Frozen, Precooked, or Prepared

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Foods, and Nut Meats 
[Link] Pages C17-4–C17-32
Published: January 2023

Subject: Analytical Chemistry

Collection: AOAC Publications, O icial Methods of Analysis of AOAC INTERNATIONAL

17.2.01 AOAC O icial Method 966.23

Microbiological Methods

First Action 1966

Final Action 1989

(For the determination of aerobic plate count, most probable number of coliform bacteria, and Escherichia
coli and Staphylococcus in products such as frozen cooked meat, poultry, and vegetable products; cooked
and/or breaded seafood; bakery products; salads; tree nut meats; and ingredients of food laboratory samples
collected during sanitation inspections of food-producing establishments, unless speci c directions are
given for that product.)

A Media and Reagents

Ingredients and reagents used to prepare the following media may be products of any manufacturer if
comparative tests show that satisfactory results are obtained. Use pure carbohydrates suitable for biological
use, ACS reagent grade inorganic chemicals, and dyes certi ed by the Biological Stain Commission for use in
media.

For convenience, dehydrated media of any brand equivalent to formulation may be used. Test each lot of
medium for sterility and growth-promoting qualities of suitable organisms (e.g., inoculate media
containing lactose with coliform bacteria, Staphylococcus media with Staphylococcus, etc.).

Determine pH before autoclaving with pH meter standardized against standard bu ers, 964.24 (see A.1.04).
Adjust pH, when necessary, by adding 1 M NaOH or l M HCl so that stated nal pH results after autoclaving.

Use sterile glass or plastic, 100 × 15 mm, Petri dishes.

 (a) Plate count agar.—See 940.36A(g) (see 17.1.02).

(b) Lauryl sulfate tryptose broth.—Dissolve 20.0 g Trypticase or tryptose (pancreatic digest of casein), 5.0
g NaCl, 5.0 g lactose, 2.75 g K2HPO4, 2.75 g KH2PO4, and 0.1 g sodium lauryl sulfate in 1 L H2O with
gentle heat, if necessary. Dispense 10 mL portions into 20 × 150 mm test tubes containing inverted
10 × 75 mm fermentation tubes. Autoclave 15 min at 121°C. Final pH, 6.8 ± 0.1.

(c) Brilliant green lactose bile (BGLB) broth.—Dissolve 10.0 g peptone and 10.0 g lactose in ca 500 mL H2O.
Add solution (pH 7.0–7.5) of 20 g dehydrated oxgall or oxbile in 200 mL H2O. Dilute to 975 mL and
adjust pH to 7.4. Add 13.3 mL 0.1% solution of brilliant green, and dilute to 1 L with H2O. Filter
through cotton and dispense 10 mL portions into 20 × 150 mm test tubes containing inverted 10 × 75
mm fermentation tubes. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.1.

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(d) Eosin methylene blue agar (Levine).—See 940.36A(d) (see 17.1.02).

(e) Baird-Parker medium (egg tellurite glycine pyruvate agar, ETGPA).—(1) Basal medium.—Suspend 10.0 g
tryptone, 5.0 g beef extract, 1.0 g yeast extract, 10 g sodium pyruvate, 12.0 g glycine, 5.0 g LiCl∙6H2O,
and 20.0 g agar in 950 mL H2O. Heat to bp with frequent agitation to dissolve ingredients
completely. Dispense 95 mL portions into screw-capped bottles. Autoclave 15 min at 121°C. Final pH,
7.0 ± 0.2 at 25°C. Store ≤1 month at 4 ± 1°C.

(2) Enrichment.—Bacto EY tellurite enrichment (BD Biosciences Codi ed Cat. No. 277910, or equivalent)
or prepare as follows: Soak fresh eggs ca 1 min in dilution of saturated HgCl2 solution (1 + 1000,
w/v). Aseptically crack eggs and separate yolks from whites. Blend yolk and physiological saline
solution, 940.36B(c) (see 17.1.02), (3 + 7, v/v) in high-speed blender ca 5 s. To 50 mL egg yolk
emulsion, add 10 mL lter-sterilized 1% potassium tellurite solution (w/v). Mix and store at 4 ± 1°C.

(3) Complete medium.—Add 5 mL warmed enrichment to 95 mL molten basal medium cooled to 45–
50°C. Mix well, avoiding bubbles, and pour 15–18 mL into sterile 100 × 15 mm Petri dishes. Store
plates at room temperature (≤25°C) for ≤5 days before use. Medium should be densely opaque; do
not use nonopaque plates. Dry plates before use by one of the following methods: (a) in convection
p. C17-4 oven or incubator 30 min at 50°C with lids removed and agar surface downward; (b) in forced-
draft oven or incubator 2 h at 50°C with lids on and agar surface upward; (c) in incubator 4 h at 35°C
with lids on and agar surface upward; or (d) on laboratory bench 16–18 h at room temperature with
lids on and agar surface upward.

(4) Interpretation.—Colonies of S. aureus are typically circular, smooth, convex, moist, 2–3 mm in
diameter on uncrowded plates, gray-black to jet-black, frequently with light-colored (o -white)
margin, surrounded by opaque zone (precipitate) and frequently with outer clear zone; colonies
have buttery to gummy consistency when touched with inoculating needle. Occasional nonlipolytic
strains may be encountered which have same appearance, except that surrounding opaque and clear
zones are absent. Colonies isolated from frozen or desiccated foods which have been stored for
extended periods are frequently less black than typical colonies and may have rough appearance and
dry texture.

(f) Trypticase (tryptic) soy broth with 10% sodium chloride.—Add 95 g NaCl to 1 L of solution of 17.0 g
Trypticase or tryptose (pancreatic digest of casein), 3.0 g Phytone (papaic digest of soya meal), 5.0 g
NaCl, 2.5 g K2HPO4, and 2.5 g glucose. Heat gently if necessary. Dispense into 16–20 mm diameter
tubes to depth of 5–8 cm. Autoclave 15 min at 121°C. Final pH, 7.3 ± 0.2.

(g) EC broth.—Dissolve 20.0 g Trypticase or tryptose (pancreatic digest of casein), 1.5 g Bacto bile salt
No. 3 or bile salt mixture, 5.0 g lactose, 4.0 g K2HPO4, 1.5 g KH2PO4, and 5.0 g NaCl in 1 L H2O.
 Dispense 8 mL into 16 × 150 mm test tubes containing inverted 10 × 75 mm fermentation tube.
Autoclave 15 min at 121°C. Final pH, 6.9 ± 0.1.

(h) Brain–heart infusion.—See 967.25A(r) (see 17.9.01). Dispense into bottles or tubes for storage and
autoclave 15 min at 121°C.

(i) Desiccated coagulase plasma (rabbit) with EDTA.—Reconstitute according to manufacturer’s

directions. If not available, reconstitute desiccated coagulase plasma (rabbit) and add Na2H2EDTA to
nal concentration of 0.1% in reconstituted plasma.

(j) Tryptophane broth.—See 940.36A(h) (see 17.1.02) but dispense in 10 mL portions.

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(k) Bu ered glucose broth (MR-VP medium).—See 940.36A(b) (see 17.1.02).

(l) Koser’s citrate broth.—See 940.36A(e) (see 17.1.02).

(m) Butter eld’s Phosphate Bu ered Diluent.—(1) Stock solution.—Dissolve 34.0 g KH2PO4 in 500 mL H2O,
adjust to pH 7.2 with ca 175 mL 1 M NaOH, and dilute to 1 L. Store in refrigerator. (2) Diluent.—Dilute
1.25 mL stock solution to 1 L with H2O. Prepare dilution blanks with this solution, dispensing enough
to allow for losses during autoclaving. Autoclave 15 min at 121°C.

B Preparation of Test Suspensions

(Prepare all decimal dilutions with 90 mL sterile diluent plus 10 mL previous dilution unless otherwise
speci ed. Shake all dilutions 25 times in 30 cm arc. Pipets must accurately deliver required volume. Do not
use to deliver <10% of their total volume. For example, to deliver 1 mL, do not use pipet >10 mL; to deliver
0.1 mL, do not use pipet >1 mL.)

(a) Frozen and/or prepared foods.—Use balance with capacity of ≥2 kg and readability of 0.1 g to
aseptically weigh 50 g unthawed (if frozen) test portion into sterile high-speed blender jar. Add 450
mL diluent, A(m)(2), and blend 2 min. (If necessary to temper frozen test sample to remove 50 g
portion, hold ≤18 h at 2–5°C.) Not >15 min should elapse from time test sample is blended until all
dilutions are in appropriate media.

If entire test sample consists of <50 g, weigh portion equivalent to 1/2 test sample and add volume of sterile
diluent required to make 1:10 dilution. Total volume in blender jar must completely cover blades.

(b) Tree nut meat halves and larger pieces.—Aseptically weigh 50 g test portion into sterile jar. Add 50 mL
0
diluent, A(m)(2), and shake vigorously (50 times through 30 cm arc) to obtain 10 dilution. Let stand
3–5 min and shake just before making serial dilutions and inoculations.

(c) Nut meal.—Aseptically weigh 10 g test portion into sterile jar. Add 90 mL diluent, A(m)(2), and shake
–1
vigorously (50 times through 30 cm arc) to obtain 10 dilution. Let stand 3–5 min and shake to
resuspend just before making serial dilutions and inoculations.

C Aerobic Plate Count

Seed duplicate Petri dishes in dilutions of 1:10, 1:100, 1:1000, etc., using plate count agar, A(a). Ordinarily,
1:100 through 1:10 000 are satisfactory. Place 1 mL appropriate dilution in each plate, and add molten agar
(cooled to 42–45°C) within 15 min from time of original dilution. Incubate 48 ± 2 h at 35°C and count
duplicate plates in suitable range (30–300 colonies). If plates do not contain 30–300 colonies, record
dilution counted and note number of colonies found. Average counts obtained and report as aerobic plate
count/g.
17.2.02 AOAC O icial Method 966.24
Coliform Group and Escherichia coli: Microbiological (MPN) Method

First Action 1966

Final Action 1971

Seed 3-tube most probable number (MPN) series into lauryl sulfate tryptose broth, 966.23A(b) (see 17.2.01),
using 1 mL inocula of 1:10, 1:100, and 1:1000 dilutions, with triplicate tubes at each dilution. [For nut meats
0 –1
(halves and larger pieces), begin MPN determination with 10 dilution; for nut meal, begin with 10
dilution.] Incubate 48 ± 2 h at 35°C for gas formation as evidenced by displacement of liquid in insert tube or

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by vigorous e ervescence when tubes are shaken gently. Examine tubes for gas formation at 24 and 48 h
intervals. Transfer, using 3 mm loop, from gassing tubes to BGLB, 966.23A(c) (see 17.2.01), (omit this
transfer for tree nuts), and EC broth, 966.23A(g) (see 17.2.01), at time gas formation is noted.

Incubate BGLB broth 48 ± 2 h at 35°C. Using MPN Table 966.24A, compute MPN on basis of number of tubes
of BGLB broth producing gas by end of incubation period. Report as MPN of coliform bacteria/ g.

Table 966.24A Most probable numbers (MPN) per 1 g test portion, using three tubes with each of 0.1, 0.01, and 0.001 g portions

Positive tubes

0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN

0 0 0 <3 1 0 0 3.6 2 0 0 9.1 3 0 0 23

0 0 1 3 1 0 1 7.2 2 0 1 14 3 0 1 39
a
0 0 2 6 1 0 2 11 2 0 2 20 3 0 2 64
a a a a
0 0 3 9 1 0 3 15 2 0 3 26 3 0 3 95

0 1 0 3 1 1 0 7.3 2 1 0 15 3 1 0 43

0 1 1 6.1 1 1 1 11 2 1 1 20 3 1 1 75
a a
0 1 2 9.2 1 1 2 15 2 1 2 27 3 1 2 120
a a a
0 1 3 12 1 1 3 19 2 1 3 34 3 1 3 160

0 2 0 6.2 1 2 0 11 2 2 0 21 3 2 0 93
a
0 2 1 9.3 1 2 1 15 2 2 1 28 3 2 1 150
a a
0 2 2 12 1 2 2 20 2 2 2 35 3 2 2 210
a a a
0 2 3 16 1 2 3 24 2 2 3 42 3 2 3 290

0 3 0 9.4 1 3 0 16 2 3 0 29 3 3 0 240
a a
0 3 1 13 1 3 1 20 2 3 1 36 3 3 1 460
a a a
0 3 2 16 1 3 2 24 2 3 2 44 3 3 2 1100
a a a
0 3 3 19 1 3 3 29 2 3 3 53 3 3 3 >1100
a Such highly improbable results suggest that factors were present that interfered with recovery or identification at the
lower dilutions. Therefore, the indicated MPN value could be much lower than the true concentration.
 Incubate EC broth 48 ± 2 h at 45.5 ± 0.02°C in covered water bath. Submerge broth tubes in bath so that
water level is above highest level of medium. Examine tubes for gas formation at 24 and 48 h intervals.
Streak gas-positive tubes on Levine’s eosin methylene blue agar plates, 966.23A(d) (see 17.2.01), and
incubate plates 24 ± 2 h at 35°C.

Pick two or more well-isolated typical colonies from Levine’s eosin methylene blue agar plates and transfer
to agar slants prepared from agar medium, 940.36A(g) (see 17.1.02). Incubate 18–24 h at 35°C. If typical
colonies are not present, pick two or more colonies most likely to be E. coli. Pick ≥2 from every plate.

p. C17-5 Transfer growth from plate count agar slants into following broths for identi cation by biochemical tests:

(a) Tryptophane broth.—Incubate broth, 966.23A(j) (see 17.2.01), 24 ± 2 h at 35°C and test for indole by

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adding 0.2–0.3 mL Kovacs reagent, 967.25B(a) (see 17.9.01), to 24 h culture. Test is positive if upper
layer turns red.

(b) MR-VP medium.—Incubate medium, 966.23A(k) (see 17.2.01), 48 ± 2 h at 35°C. Aseptically transfer 1
mL culture to 13 × 100 mm test tube to test for acetylmethylcarbinol. Add 0.6 mL 5% alcoholic α-
naphthol solution (w/v), 0.2 mL KOH solution (4 ± 10), and few crystals of creatine. Shake and let
stand 2 h. Test is positive if eosin pink develops. Alternatively, see 967.27D(c)(1) (see 17.9.03).

Incubate remainder of MR-VP medium for additional 48 h and test for methyl red reaction by adding ve
drops methyl red solution to culture. Test is positive if culture turns red; negative, if yellow. (Prepare methyl
red solution by dissolving 0.1 g methyl red in 300 mL 90% alcohol and diluting to 500 mL with H2O.)

(c) Koser citrate broth.—Incubate broth, 966.23A(l) (see 17.2.01) 96 h at 35°C and record growth as + or
–.

(d) Lauryl sulfate tryptose broth.—Incubate broth, 966.23A(b) (see 17.2.01) 48 ± 2 h at 35°C. Examine
tubes for gas formation.

(e) Gram stain.—Perform Gram stain on 18 h agar slant [Standard Methods for the Examination of Water
and Wastewater (1992) 18th Ed., American Public Health Association, American Water Works
Association, and Water Pollution Control Federation, Washington, DC, USA]. Coliform organisms will
stain red (negative); Gram-positive organisms will stain blue-black.

(f) Classi cation.—Classify biochemical types as in Table 966.24B.

 a
Table 966.24B Classification of biochemical types

Indole MR VP Citrate Type

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+ + – – Typical E. coli

– + – – Atypical E. coli

+ + – + Typical intermediate

– + – + Atypical intermediate

– – + + Typical Enterobacter aerogenes

+ – + + Atypical Enterobacter aerogenes

a Other groupings may appear; in such cases, cultures are usually mixed. Restreak to determine their purity. Compute MPN of E. coli/g, considering Gram-
negative, nonspore-forming rods producing gas in lactose and producing + + – – or – + – – IMViC patterns as E. coli.
 References:
JAOAC 49, 270, 276(1966); 51, 865, 867(1968); 58, 1154(1975)

17.2.03 AOAC O icial Method 977.27

Bacteria in Foods and Cosmetics: Spiral Plate Method

First Action 1977

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Final Action 1981

A Principle
Bacterial suspension from prepared test portion of food or cosmetic is deposited continuously on surface of
rotating agar plate. Resultant track on surface is in form of Archimedes spiral. Volume is decreased while
dispensing stylus moves from center to edge so that exponential relationship exists between volume
deposited and radius of agar. On incubation, colonies develop along lines where liquid was deposited.
Counting grid is calibrated for test portion volume associated with di erent areas of agar. Number of
colonies per known area is counted and calculated to bacterial concentration.

p. C17-6 B Apparatus

Spiral plating machine.—For use with 150 × 15 mm (100 × 15 mm may be used) Petri dishes and adjusted to
deliver total volume of 0.035 mL/plate. Platform carrying plate is rotated at ca 50 rpm and is connected
mechanically to lead screw driving hollow syringe dispenser. Back ow syringe, 2-way valve, and vacuum
trap control loading and dispensing of test portion, disposal of residual test portion, and rinsing of system.
Liquid is dispensed from back ow syringe through thin wall Te on tubing through stylus to surface of agar
plate.

C Plates
Pour 40–45 mL portions plate count agar, 940.36A(g) (see 17.1.02), into 150 × 15 mm (100 × 15 mm may be
used) Petri dishes; let harden and dry to smooth, even surface.

D Calibration of Spiral Counters

To determine volume associated with di erent parts of counting grid, prepare 11 bacterial suspensions by
6 3
diluting 1:1 from 10 to 10 cells/mL (use nonspreaders). Plate all dilutions in duplicate by both 966.23C (see
17.2.01) and spiral plater, using same medium and incubator. Count spiral plates as in G and divide by
average count/ mL by 966.23C (see 17.2.01) to calculate volume of counted grid area.

number spiral colonies on area

mL in Counted area =
count/mL by 966.23 C (see 17.2.01)
E Preparation of Test Suspension
Weigh 50 g test portion into sterile blender jar, add 450 mL dilution H2O, 940.36A(a) (see 17.1.02), and blend
2 min. If necessary, let settle few minutes before removing portion of supernate for spiral plating. (Presence
of particles may clog tubing.)

Liquids may be used directly or after diluting 1 + 9 with dilution H2O.

F Operation
Check stylus tip angle by letting vacuum hold microscope cover slip against face of stylus tip at 1 mm above
platform. Cover slip should be parallel to rotating platform in all directions. Adjust angle if necessary. Check

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stylus at start position.

Clean stylus tip before use and between plating each test solution by rinsing 1 s with commercial 5.25%
NaOCl solution and then 1 s with sterile H2O. Identify three disposable polyethylene cups and ll with
commercial 5.25% NaOCl solution, sterile H2O, and test solution. Turn vacuum lling valve to “on” and
move test sample holder into position under stylus tip. Lower stylus into NaOCl solution and lift out twice.
Repeat with H2O. Lower stylus into test solution. Draw solution through stylus until continuous column of
liquid is present in tube above vacuum lling valve. With tip of stylus still below surface of test solution,
close vacuum valve. Raise stylus and move test sample holder out of way.

Identify lid of agar plate and remove lid. Place dish on turntable and lower stylus until tip rests freely on
agar surface. Start apparatus and let rotate until stylus is lifted and apparatus stops automatically. Remove
dish and replace cover. Incubate 48 ± 3 h at 35 ± 1°C.

After all test portions have been plated, ush apparatus with NaOCl solution and H2O. When not in use, leave
lled with H2O.

G Counting Spiral Plates

Transparent viewing grid consists of 13.2 cm circle divided into ve areas by four concentric circles
equidistant along diameter [marked 1 (furthest) through 4 (nearest) to center] and into eight 45° wedges or
octants, marked A through H. Thus, each octant is subdivided by four arcs linearly equidistant from each
other. Outer ring of two opposite octants (e.g., A and E) is further subdivided in half by arc in middle
(marked 1/2), and outer ring thus formed is divided in half by line toward center. Additional lines are
provided for use with 10 cm plates.

After incubation, center plate over grid. Choose any octant sector and count colonies from outer edge toward
center until 20 colonies have been counted. Continue counting remaining colonies contained in segment in
which 20th colony was observed. Record this count together with number segment that included 20th
colony (i.e., 1/2, 1, 2, 3, or 4). Count opposite similar segment and add together. If 20 colonies are not
contained in an octant, count all colonies on plate and designate as T (total). If total number colonies
counted exceeds 75 in completing count in segment containing 20th colony, count will generally be low
because of coincidence error associated with crowding of colony. In this case, count circumferentially
adjacent annular segments starting with sector 1 until ≥50 colonies are counted, and complete count of
remaining colonies in segment in which 50th colony was observed.

Divide number of colonies counted (or sum of two sector counts) by corresponding volume sectors counted
in mL to obtain bacterial count/mL. Use as volume that calculated for that sector(s) from calibration, D,
based on standard plate count.
References:

JAOAC 60, 807(1977); 64, 408(1981)

Revised: March 1997

17.2.04 AOAC O icial Method 993.11

Bacterial Counts in Raw and Pasteurized Milk: Reflectance
Colorimetric Method (Omnispec™)

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First Action 1993

4.5
[Applicable to enumeration of 10 CFU (colony-forming units)/ mL bacteria in milk.]

See Tables 993.11A and B for results of the interlaboratory study supporting acceptance of the method.
Table 993.11A Interlaboratory study results for bacterial counts in raw and pasteurized milk by reflectance colorimetric (RC) method

a b
Product Incubation Spike RC

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c
Log mean counts/mL sr sR RSDr, % RSDR, %

UHT, 2% — — >3.000 — — — —

HP1, 2% 48 h, 7°C — 4.825 0.131 0.473 2.71 9.80

HP1, 2% 96 h, 7°C — 6.941 0.130 0.337 1.87 4.85

HP1, 2% — Log 3 4.867 0.130 0.459 2.67 9.43

HP1, 2% — Log 5 6.612 0.136 0.299 2.05 4.52

HP1, 2% — Log 7 6.410 0.144 0.195 2.24 3.04

RW1 48 h, 7°C — 7.468 0.085 0.191 1.14 2.56

RW2 48 h, 7°C — 7.391 0.055 0.211 0.74 2.85

RW3 48 h, 7°C — 7.436 0.099 0.248 1.33 3.33

RW1 96 h, 7°C — 7.690 0.080 0.157 1.04 2.04

RW2 3 h, 35°C — 7.574 0.076 0.133 1.00 1.76

RW1 — Log 7 6.558 0.146 0.259 2.23 3.95

Mean — — 6.706 0.110 0.269 1.73 4.41

a UHT, 2% = 2% milk treated at ultra-high temperature; HP1, 2% = 2% homogenized pasteurized milk, test sample 1; RW1 = raw whole milk, test sample 1; RW2 =
raw whole milk, test sample 2; RW3 = raw whole milk, test sample 3.
b Test sample spiked with Pseudomonas fluorescens.
c Color detection time (CDT) values converted to log10 count/mL using calibration curve calculated from C(e): Y = 8.8674 – 0.3836X, (R = 0.9850, RMSE = 0.1870).

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Table 993.11B Interlaboratory study results for bacterial counts in raw and pasteurized milk by standard plate count (SPC) method

a
Product Incubation SPC

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b c
Spike Log mean, counts/mL sR RSDR, %

UHT, 2% — — <3.000 — —

HP1, 2% 48 h, 7°C — 4.308 1.910 43.72

HP1, 2% 96 h, 7°C — 5.988 1.837 30.68

HP1, 2% — Log 3 5.784 1.959 33.88

HP1, 2% — Log 5 5.987 2.137 35.69

HP1, 2% — Log 7 7.317 1.160 15.85

RW1 48 h, 7°C — 7.482 0.570 7.62

RW2 48 h, 7°C — 7.442 0.598 8.04

RW3 48 h, 7°C — 7.543 0.898 11.90

RW1 96 h, 7°C — 7.684 0.752 9.78

RW2 3 h, 35°C — 7.577 0.816 10.77

RW1 — Log 7 7.156 0.888 12.40

Mean — — 6.756 1.23 20.03

a UHT, 2% = 2% milk treated at ultra-high temperature; HP1, 2% = 2% homogenized pasteurized milk, test sample 1; RW1 = raw whole milk, test sample 1; RW2 =
raw whole milk, test sample 2; RW3 = raw whole milk, test sample 3.
b Test sample spiked with Pseudomonas fluorescens.
c Color detection time (CDT) values converted to log10 count/mL using calibration curve calculated from C(e): Y = 8.8674 − 0.3836X, (R = 0.9850, RMSE = 0.1870).

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 A Principle
Bacteria present in milk test samples reduce triphenyl tetrazolium chloride (TTC) dye in nutrient medium
during incubation. Re ectance colorimeter analyzes test portions at regular intervals for color changes;
when soluble colorless TTC converts to insoluble red form, colorimeter estimates bacterial count.
Calibration values in computer software allow estimation of bacterial count in original test sample on basis
of color detection time (CDT), i.e., incubation time before color change.

B Apparatus and Reagents

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(a) Re ectance colorimeter (RC) system.—Equipped with incubator, colorimeter, and computer with
Omnispec software installed (Omnispec 4000, Wescor, Inc., Logan, UT, USA, is suitable source).

p. C17-7 (b) Medium.—Containing pancreatic digest of casein, 2.5%; yeast extract, 1.25%; glucose, 0.5%; and
triphenyltetrazolium chloride, 0.04% in H2O. Sterilize (0.2 μm lter) 10 mL/tube (Medium “A”).

(c) Microtiter plates, lids, and sealing tape.—(1) Flat-bottom microtiter plates containing 96 wells, with
lids. (2) Plate sealing tape or invisible plastic tape.

(d) Pipettors/tips.—(1) Adjustable pipettor.—50–200 μL. (2) Fixed-volume pipettor.—200 μL. (3) Sterile tips.
—To t (1) and (2).

(e) Reagent reservoirs.—Sterile reagent reservoirs, plastic.

 C Computer-Based Calibration and Control
(a) Colorimeter calibration.—At computer power-up, insert instrument calibration plate (white color
standard). Press enter and instrument measures white color standard and calibrates color scales in
instrument.

(b) Re ectance colorimeter system start-up.—Using software command “Add a Test,” program the
following into computer software: Test Name, RC Plate Count; Color Parameter, a*; Color Endpoint,
p. C17-8 4.00 Baseline [+ –]; Values Increase to Endpoint? Y; Take Sample at Start-Up Time? N; Test
Interval Time, 0 h 30 min; Number of Intervals, 32; Y Intercept, 8.8674; Line Slope; −0.3836
(intercept and slope values may vary with market and be changed after local recalibration);
Reporting Limits, Insert Log values for local standards (i.e., if local lower standard is 10 000

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CFU/mL), then insert 4.000 in –NEG– box (will display in green on monitor and with normal type on
printout). If margin values are 10 000–50 000 CFU/mL, insert 4.700 in :MRGN: box (will display in
yellow on monitor and with bold type on printout). The +POS+ box will automatically show log 4.700
and will program all estimates above log 4.700 to be displayed in red on monitor and with underlined
bold type on printout.

(c) Recalibration of SPC correlation.—Split 12 test samples of raw or pasteurized milk into two test
portions, >30 mL each. Store one set 48 h at 0.0–4.4°C. Incubate other set at selective preliminary
intubation (SPI) time and temperature suitable to allow log 3 increase in CFU/mL (48 h at 7°C for
psychrotrophs or 3–4 h at 35°C for mesophylic organisms). Analyze test samples using reference
pour plate technique, 966.23C (see 17.2.01). Test samples without SPI will range ca 3 logs lower than
SPI samples. Measured values will depend upon initial bacterial levels. Analyze duplicate test
samples in colorimeter. Export RC data into statistical program. Input SPC data and calculate
regression line values. Program new intercept and line slope values into “Edit a Test” program [see
(b)].

(d) System suitability.—For colored substances, make repeated measurements to ensure reproducible
color measurements (Note: absolute color values are not critical in this method.)

(e) Calculation of CFU/mL.—Correlation of CFU/mL with detection time is approximated by linear

regression with a negative slope. CFU/mL is automatically calculated from detection time as follows:

CFU/mL = intercept + (slope × detection time)

Using values entered in C(b), the equation becomes:

CFU/mL = 8.8764 − 0.3836(detection time)

D Preparation of Test Suspension

Shake test samples 25 times in 30 cm arc for 7 s. Let foam subside 30 s before sampling.

E Determination
Pipet 50 µL medium into microtiter plate wells for each test sample tested. Pipet 200 µL each test sample
into separate wells and mix by re lling and discharging contents three times. Prepare in duplicate if desired.
(If test samples become contaminated, mark contaminated well by adding drop of India ink into well;
resample into uncontaminated well.)
Remove backing from seating tape and apply to microtiter plate, sealing tightly around all wells.
Alternatively, replace lid and seal between lid and plate using invisible plastic tape (taking care to avoid
tilting plate and contaminating wells).

Check that incubator is at 30 ± 1°C. Start “Begin a Test” following computer software instructions. Results
are displayed on monitor as CDTs are recorded. After 16 h, results are printed out, either in number (e.g., 1.8
4
× 10 ) or in log10 format (e.g., 4.255) as selected.

Reference:

J. AOAC Int. 77, 623(1994)Crossref

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17.2.05 AOAC O icial Method 986.32
Aerobic Plate Count in Foods: Hydrophobic Grid Membrane Filter
Method

First Action 1986

Final Action 1987

A Principle
Hydrophobic grid membrane lter (HGMF) uses membrane lter imprinted with hydrophobic material in
grid pattern. Hydrophobic lines act as barriers to spread of colonies, thereby dividing membrane lter
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)].
 B Apparatus, Culture Media, and Reagents
(a) HGMF.—Membrane lter has pore size of 0.45 µm and is imprinted with nontoxic hydrophobic
materials in grid pattern. ISO-GRID (Neogen Corp., Lansing, MI, USA) or equivalent meets these
speci cations.

(b) Filtration units for HGMF.—Equipped with 5 µm mesh pre lter to remove food particles during
ltration. One unit is required for each test sample. ISO-GRID (Neogen Corp.) or equivalent meets
these speci cations.

(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.

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(d) Blender.—Waring, or equivalent multispeed model, with low-speed operation at 10 000–12 000
rpm, and 250 mL glass or metal blender jars with covers. One jar is required for each test sample.

(e) Vacuum pump.—H2O aspirator vacuum source is satisfactory.

(f) Manifold or vacuum ask.

(g) Peptone-Tween 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 or 99 ± 1 mL after autoclaving 15 min at 121°C.

(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 121°C. Temper to 50–55°C. 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) Tris bu er.—1.0 M. Dissolve 121.1 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 4–6°C.

(j) Acetate bu er.—1.0 M. Dissolve 60 mL glacial acetic acid in ca 500 mL H2O. Adjust solution to desired
pH with 5 M NaOH and dilute to 1 L with H2O. Store at 4–6°C.

(k) Amylase stock solution.—Dilute 10 g α-amylase (Sigma Chemical Co., No. A6814, or equivalent) to 100
mL with tris bu er, pH 7.0. Warm to 35°C if necessary to aid solution. Filter through Whatman No. 1
paper (or equivalent) to remove insoluble material; then lter-sterilize using 0.45 µm membrane
lter. Store up to 1 week at 4–6°C or up to 3 months at –18°C.

(l) Cellulase stock solution.—Dilute 10 g cellulase (Sigma, No. C0901, or equivalent) to 100 mL with
p. C17-9 acetate bu er, pH 5.0. Warm to 35°C if necessary to aid solution. Filter through Whatman No. 1
paper (or equivalent) to remove insoluble material; then lter-sterilize using 0.45 μm membrane
lter. Store up to 1 week at 4–6°C or up to 3 months at −18°C.

(m) Diastase stock solution.—Dilute 10 g diastase (Sigma, No. A3176, or equivalent) to 100 mL with tris
bu er, pH 7.0. Warm to 35°C if necessary to aid solution. Filter through Whatman No. 1 paper (or
equivalent) to remove insoluble material; then lter-sterilize using 0.45 μm membrane lter. Store
up to 1 week at 4–6°C or up to 3 months at −18°C.

(n) Hemicellulase stock solution.—Dilute 10 g hemicellulase (Sigma, No. H2125, or equivalent) to 100 mL
with acetate bu er, pH 5.5. Warm to 35°C if necessary to aid solution. Filter through Whatman No. 1
paper (or equivalent) to remove insoluble material; then lter-sterilize using 0.45 µm membrane
lter. Store up to 1 week at 4–6°C or up to 3 months at −18°C.
(o) Trypsin stock solution.—Dilute 10 g trypsin to 100 mL with tris bu er, pH 7.6. Warm to 35°C if
necessary to aid solution. Filter through Whatman No. 1 paper (or equivalent) to remove insoluble
material; then lter-sterilize using 0.45 µm membrane lter. Store up to 1 week at 4–6°C or up to 3
months at −18°C.

(p) Lecithinase (phospholipase A2) stock solution.—Dilute commercial enzyme solution (Sigma, No.
P6534, or equivalent) to 25 units/mL with tris bu er, pH 8.0. Filter-sterilize using 0.45 µm
membrane lter. Store up to 1 week at 4–6°C or up to 3 months at −18°C.

(q) Pectinase stock solution.—Use commercial enzyme solution of pectinase from Aspergillus niger,
containing 3–6 units/mg protein, dissolved in 40% glycerol (Sigma, No. P9932, or equivalent).
Filter-sterilize using 0.45 µm membrane lter. Store up to 1 week at 4–6°C or up to 3 months at

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−18°C.

(r) Protease stock solution.—Use commercial enzyme solution of protease from Bacillus subtilis,
containing 7–15 units/mg protein (Biuret) in aqueous solution (Sigma, No. P8775, or equivalent).
Filter-sterilize using 0.45 µm membrane lter. Store up to 1 week at 4–6°C up to 3 months at −18°C.
 C Preparation of Test Suspension
(a) Liquid egg.—Thoroughly mix test sample with sterile spoon or spatula and prepare 1:10 dilution by
aseptically weighting 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 20–30 min at 35–37°C in water bath. Correct for additional dilution
factor by ltering 1.2 mL of enzyme-treated test portion.

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(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 20–30 min at 35–37°C in water bath. Correct for additional dilution factor
by ltering 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
p. C17-10 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 20–30 min at 35–37°C in water
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 (10 000–12 000 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 20–30 min
at 35–37°C in water bath. Correct for additional dilution factor by ltering 1.2 mL of enzyme-treated
test portion.
 a
Table 986.32 Enzyme treatments for foods

Food Enzyme

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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
b
Spray-dried cheese powders Cellulase or protease

Sour cream Diastase

Yogurt Trypsin

Butter None

Margarine None

Egg: liquid or powder Trypsin

Raw beef, pork, poultry Trypsin

 Cooked meat or poultry Trypsin

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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

a 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.
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Varies, depending on individual product.
b
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 ltration unit on manifold or vacuum
ask. Open clamp A. Rotate back funnel portion C. Aseptically place sterile HGMF, B(a), on surface of base D.
Rotate funnel forward. Clamp shut by sliding jaws L of stainless steel clamp over entire length of anges B
extending from both sides of funnel C and base D, and rotating moving arm K into horizontal (locked)

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position.

Figure 986.32A

Filtration unit.

Figure 986.32B

Filtration unit clamp.

Aseptically add ca 15–20 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 pre lter mesh G. Aseptically add
additional 10–15 mL sterile H2O to funnel and draw through mesh as before. Close clamp A to direct vacuum
to base of ltration 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 o of anges B. Rot ate back funnel C. Aseptically remove HGMF and place on surface of predried
TSFA, B(h), plate. Avoid trapping air bubbles between lter and agar.

(a) Raw milk, pasteurized milks and creams, and egg powders.—Incubate 48 ± 3 h at 32°C. 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

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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 32°C. Proceed as in (a).

(c) All other foods.—Incubate 48 ± 3 h at 35°C. Proceed as in (a).

Reference:

JAOAC 69, 671(1986)

17.2.06 AOAC O icial Method 988.18

Aerobic Plate Count: Pectin Gel Method

First Action 1988

Final Action 1990

A Principle
Method uses Redigel™ pretreated dishes containing thin “hardener” layer and liquid medium containing
nutrients with pectin as only gelling agent. Liquid medium, 12–15 mL, is poured into Redigel pretreated dish
and undiluted or diluted test portion is added. Dish is rotated and rocked to mix test portion and medium.
Dishes are then allowed to stand on level surface 30–40 min until medium solidi es. Total process is done
at ambient temperature. Dishes are then incubated and counted.

B Materials
(Note: Before pectin base medium formulated from individual ingredients is used, comparability to
commercially available medium must be demonstrated.)

Pectin gel tubes and dishes.—Autoclaved pectin gel liquid is available in one test or 10 test bottles. Use bottles
of Redigel and Redigel pretreated dishes (3M Microbiology Products, St. Paul, MN, USA), or equivalent that
meet speci cations.
p. C17-11To prepare plate count pectin from individual ingredients, suspend 5.0 g pancreatic digest of casein, 2.5 g
yeast extract, and 1.0 g glucose in 500 mL H2O. Suspend 15 g low methoxyl pectin in 500 mL H2O. Heat
individual mixtures until all ingredients are dissolved. Autoclave solutions 15 min at 121°C. Combine
nutrient and pectin solutions and adjust pH to 7.0 ± 0.1. To prepare Redigel pretreated dishes, prepare
hardener layer mixture of 1% agar with 2% CaCl2 concentration. Sterilize mixture by autoclaving 15 min at
121°C. Aseptically dispense 5 mL portions of mixture into sterile Redigel pretreated dishes.

C Preparation of Test Inoculum

Prepare all decimal dilutions with 90 mL sterile diluent [Butter eld’s Phosphate Bu er, 996.23A(m)(2) (see
17.2.01)] plus 10 mL of previous dilution unless otherwise speci ed. Shake all dilutions 25 times in 30 cm

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arc. Pipets must accurately deliver required volume. Do not use to deliver <10% of their total volume. For
example, to deliver 1 mL, do not use pipet >10 mL; to deliver 0.1 mL, do not use pipet >1 mL.

(a) Dairy products.—Measure (or weigh) 11 mL (or g) test portion and dilute in 99 mL Butter eld’s
Diluent. For solid test materials, blend 2 min at 10 000–12 000 rpm. Prepare additional dilutions so
that total colonies/dish is in 25–250 range. Incubate dishes 48 ± 3 h at 32 ± 1°C.

(b) Nondairy products.—Weigh 50 g test portion into 450 mL Butter eld’s Diluent and blend 2 min at 10
000–12 000 rpm. Prepare further dilutions by dispensing 10 mL test portion into 90 mL diluent so
that total colonies/dish is in 30–300 range. Incubate dishes 48 ± 2 h at 35 ± 1°C.

D Determination
(1) Lift lid of Redigel pretreated dish and pour liquid pectin gel from 1 bottle (12–15 mL) into dish.
Replace lid and swirl dish to cover bottom with pectin gel. Prepare number of dishes needed for test
portions being run (duplicate dishes for each dilution). Dishes must be used within 5 min after liquid
pectin gel is poured.

(2) Add 1 mL inoculum to liquid pectin gel in Redigel pretreated dish. Touch pipet tip once to dry spot on
inside wall of dish (above level of liquid pectin gel) after dispensing test portion to rest point in pipet
tip. Immediately rot ate and rock dish to mix test portion thoroughly with pectin gel. Do not spill
pectin gel over sides of dish. (Note: This step is the primary di erence in procedure between pectin
gel and agar-based media. Do not add inoculum to Redigel pretreated dish and pour pectin gel over
it. This would lock test portion in one small area of dish without separation of individual colonies.)

(3) Let inoculated dishes stand on level surface until pectin gel is solid (ca 30–40 min), and then
incubate 48 ± 2 h at 35 ± 1°C for nondairy products and 48 ± 3 h at 32 ± 1°C for dairy products.

(4) Count duplicate dishes in suitable range (30–300 colonies for nondairy products, 25–250 colonies
for dairy products). If dishes do not contain proper range of colonies, record dilution counted, and
note number of colonies found. Average counts obtained and report as aerobic plate count/g or mL.

Reference:

JAOAC 71, 343(1988)

Revised: March 2002

17.2.07 AOAC O icial Method 990.12
Aerobic Plate Count in Foods: Dry Rehydratable Film (Petrifilm™
Aerobic Count Plate) Method

First Action 1990

Final Action 1994

Results of the interlaboratory study supporting acceptance of the method:

Flour: sr = 0.225; sR = 0.246; RSDr = 5.3%; RSDR = 5.8%

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Nuts: sr = 0.272; sR = 0.674; RSDr = 7.4%; RSDR = 18.4%

Shrimp: sr = 0.540; sR = 0.615; RSDr = 9.8%; RSDR = 11.1%

Spice: sr = 0.274; sR = 0.303; RSDr = 6.0%; RSDR = 6.6%

Turkey: sr = 0.278; sR = 0.348; RSDr = 5.3%; RSDR = 6.6%

Vegetables: sr = 0.310; sR = 0.454; RSDr = 6.3%; RSDR = 9.2%

A Principle
See 989.10A (see 17.3.03).

B Apparatus
See 989.10B(a) and (c)–(e) (see 17.3.03).

C Reagent
Dilution water.—To prepare stock solution, dissolve 34 g KH2PO4 in 500 mL H2O, adjust to pH 7.2 with 1 M
NaOH (ca 175 mL), and dilute to 1 L with water. To prepare bu ered water for dilutions, dilute 1.25 mL stock
solution to 1 L with boiled and cooled water. Autoclave 15 min at 121°C.

D Preparation of Test Suspension

See 966.23B (see 17.2.01).

E Determination
Place dry- lm aerobic count plate on at surface. Lift top lm and inoculate 1 mL test suspension onto
center of lm base. Carefully place top lm down on inoculum. Distribute suspension over prescribed
growth area with downward pressure in center of plastic spreader device (recessed side down). Leave plate
undisturbed 1 min to permit gel to solidify. Incubate plates 48 ± 3 h at 35 ± 1°C.

In incubator, place plates in horizontal position, clear side up, in stacks not exceeding 20 units. Count plates
promptly after incubation period. After incubation is complete, plates may be stored frozen (≤−15°C) up to 7
days. Avoid this as a routine practice.
 Use standard colony counter for counting purposes. Magni er-illuminator may also be used to facilitate
counting. Colonies stain in various shades of red. Count all colonies in countable range (30–300 colonies).

To compute bacterial count, multiply total number of colonies per plate (or average number of colonies per
plate if counting duplicate plates of same dilution) by reciprocal of dilution used. When counting colonies on
duplicate plates of consecutive dilutions, compute mean number of colonies for each dilution before
determining average bacterial count. Estimated counts can be made on plates with >300 colonies and should
be reported as estimated counts. In making such counts, circular growth area can be considered to contain
ca twenty 1 cm squares. To isolate colonies for further identi cation, lift top lm and pick colony from gel.

Reference:

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JAOAC 73, 242(1990)

Revised: March 2002

17.2.07A AOAC O icial Method 2002.07 Detection and Quantification

p. C17-12
of Total Aerobic Microorganisms: SimPlate Total Plate Count–Color
Indicator (TPC–CI) Method

First Action 2002

Final Action 2005

(Applicable to detection and quanti cation of total aerobic microorganism populations in milk chocolate,
cake mix, ground pepper, nut meats, dairy foods, red meats, poultry meats, seafoods, lunch meat, frozen
pot pies, cereals, pasta, egg products, our, hash brown potatoes, vegetables, fruits, and fruit juice.)

Caution: Test portion dilutions and incubated SimPlate devices from food products could contain pathogenic
bacteria if the particular test portion was contaminated. Use standard aseptic microbiological laboratory
techniques, including decontamination of any spills with disinfectant.

See Tables 2002.07A–C for results of the interlaboratory study supporting acceptance of the method.
Table 2002.07A Statistical analysis of interlaboratory results for total aerobic microorganisms by the AOAC 35 and SimPlate 35 methods

a b e f g h
Food Lot N Mean sr RSDr, % r sR

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group
c d c d c d c d c d
AOAC SimPlate AOAC SimPlate AOAC SimPlate AOAC SimPlate AOAC SimPlate
k l
Pepper A 12 6.04 6.13 0.09 0.15 1.50 2.50 0.25 0.43 0.22 0.16
k k
B 13 7.30 7.53 0.11 0.14 1.60 1.80 0.32 0.38 0.35 0.17
k l l
C 14 6.44 6.66 0.11 0.16 1.70 2.30 0.31 0.44 0.34 0.22

Flour Rye 14 5.73 5.83 0.15 0.18 2.70 3.10 0.43 0.50 0.27 0.28
l l
Wheat 11 3.87 3.93 0.13 0.19 3.30 4.80 0.35 0.53 0.30 0.46

White 13 4.13 4.24 0.34 0.27 8.30 6.40 0.96 0.76 0.36 0.45
l l
Nut meats Peanuts 13 4.04 4.24 0.29 0.41 7.30 9.70 0.83 1.15 0.33 0.37
k
Almonds 13 2.42 2.53 0.11 0.23 4.70 9.10 0.32 0.64 0.31 0.29

Hazelnuts 12 3.67 3.72 0.69 0.57 18.80 15.30 1.93 1.59 0.65 0.69
k l
Frozen A 15 2.78 3.01 0.13 0.25 4.30 7.90 0.35 0.69 0.27 0.38
patties
k
B 15 3.84 4.06 0.31 0.17 8.20 4.10 0.88 0.47 0.53 0.50
l l
C 15 2.85 2.86 0.14 0.20 5.00 7.01 0.40 0.57 0.26 0.35

Frozen Peaches 10 3.24 3.37 0.14 0.14 4.30 4.30 0.39 0.40 0.27 0.33
fruits
 k
Strawberries 12 3.96 4.33 0.14 0.12 3.60 2.60 0.40 0.32 0.27 0.21

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k
Blueberries 10 2.85 3.08 0.17 0.15 5.90 4.80 0.47 0.42 0.20 0.28

Fresh Carrots 12 7.27 7.30 0.27 0.31 3.70 4.20 0.75 0.86 0.58 0.47
vegetables
l l
Broccoli 12 7.20 7.22 0.26 0.18 3.60 2.50 0.73 0.50 0.61 0.42
k
Celery 10 7.47 7.40 0.13 0.19 1.70 2.50 0.36 0.53 0.74 0.36

a Number of laboratories with valid data.

b Mean log aerobic microorganisms/g.

c Standard Methods Agar incubated at 35°C.

d SimPlate method incubated at 35°C.

e Repeatability standard deviation.

f Repeatability relative standard deviation.

g Repeatability values, 2.8 × sr.

h Reproducibility standard deviation.

i Reproducibility relative standard deviation.

j Reproducibility values, 2.8 × sR.

k Significantly di erent at p < 0.01.

l Significantly di erent at p < 0.05.

Table 2002.07B Statistical analysis of interlaboratory results for total aerobic microorganisms by the ISO 30 and SimPlate 30 methods
a b e f g h
Food Lot N Mean sr RSDr, % r SR R

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group
c d c d c d c d c d
ISO SimPlate ISO SimPlate ISO SimPlate ISO SimPlate ISO SimPlate IS

Pepper A 14 5.69 5.71 0.13 0.14 2.20 2.40 0.35 0.39 0.41 0.41 7
k l
B 14 7.20 7.29 0.17 0.35 2.40 4.80 0.48 0.98 0.34 0.51 4
l
C 14 6.33 6.36 0.14 0.21 2.20 3.20 0.39 0.58 0.39 0.35 6
l
Flour Rye 14 5.74 5.84 0.22 0.24 3.90 4.00 0.62 0.66 0.41 0.30 7
k
Wheat 14 3.86 3.79 0.13 0.23 3.30 6.00 0.36 0.64 0.35 0.33 9

White 14 4.33 4.46 0.12 0.13 2.70 2.80 0.33 0.35 0.27 0.24 6

Nut meats Peanuts 12 4.31 4.28 0.41 0.41 9.60 9.60 1.15 1.15 0.67 0.67 15
l
Almonds 12 2.29 2.52 0.16 0.23 7.00 9.10 0.45 0.65 0.57 0.43 25

Hazelnuts 11 3.35 3.40 0.77 0.83 23.10 24.50 2.17 2.33 0.86 0.89 25

Frozen A 14 3.16 3.13 0.13 0.14 4.00 4.40 0.36 0.39 0.23 0.23 7
patties
k k
B 13 5.18 5.11 0.06 0.10 1.10 1.90 0.17 0.27 0.23 0.44 4
k
C 15 3.19 3.06 0.06 0.14 2.00 4.50 0.18 0.39 0.59 0.43 17
k l
Frozen Peaches 13 3.70 3.92 0.11 0.21 2.90 5.30 0.30 0.59 0.47 0.32 12
fruits
 l l k
Strawberries 11 3.95 4.13 0.09 0.14 2.30 3.40 0.26 0.40 0.35 0.16 9

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Blueberries 13 2.66 2.78 0.16 0.20 6.10 7.20 0.46 0.56 0.25 0.32 9

Fresh Carrots 11 7.49 7.56 0.30 0.38 3.90 5.00 0.83 1.06 0.58 0.63 7
vegetables

Broccoli 13 7.80 7.89 0.29 0.24 3.70 3.00 0.81 0.66 0.53 0.58 6
k
Celery 11 8.58 8.49 0.08 0.43 0.90 5.00 0.23 1.20 0.52 0.73 6

a Number of laboratories with valid data.

b Mean log aerobic microorganisms/g.
c Standard Methods Agar incubated at 30°C.

d SimPlate method incubated at 30°C.

e Repeatability standard deviation.
f Repeatability relative standard deviation.

g Repeatability values, 2.8 × sr.

h Reproducibility standard deviation.

i Reproducibility relative standard deviation.

j Reproducibility values, 2.8 × sR.

k Significantly di erent at p < 0.01.

l Significantly di erent at p < 0.05.

Table 2002.07C Statistical analysis of interlaboratory results for total aerobic microorganisms by the ISO 30 and AOAC 35 methods
a b e f g h i
Food Lot N Mean sr RSDr,% r sR RSDR, %

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group
c d c d c d c d c d c d
ISO AOAC ISO AOAC ISO AOAC ISO AOAC ISO AOAC ISO AOAC

Pepper A 14 5.71 5.91 0.10 0.09 1.70 1.50 0.28 0.24 0.40 0.46 7.00 7.70
k
B 13 7.20 7.30 0.18 0.11 2.50 1.60 0.50 0.32 0.35 0.36 4.90 4.90

C 14 6.30 6.44 0.14 0.11 2.20 1.70 0.39 0.31 0.39 0.34 6.20 5.30

Flour Rye 13 5.69 5.73 0.12 0.16 2.20 2.70 0.34 0.44 0.36 0.28 6.40 4.70

Wheat 10 3.75 3.83 0.16 0.11 4.20 2.80 0.44 0.31 0.26 0.32 7.00 8.40
k l
White 12 4.33 4.13 0.08 0.35 1.80 8.50 0.21 0.98 0.27 0.37 6.20 9.00
l
Nut meats Peanuts 12 4.31 4.05 0.41 0.30 9.60 7.40 1.15 0.84 0.67 0.34 15.50 8.40
k l
Almonds 12 2.29 2.32 0.16 0.10 7.00 4.20 0.45 0.29 0.57 0.32 25.00 13.00

Hazelnuts 12 3.34 3.62 0.78 1.18 23.30 33.70 2.18 3.29 0.88 1.20 26.20 34.60
l
Frozen A 14 3.19 2.93 0.14 0.14 4.30 4.60 0.39 0.38 0.23 0.28 7.30 9.50
patties
l l k
B 14 5.14 3.79 0.19 0.32 3.70 8.50 0.53 0.90 0.33 0.52 6.40 13.60
l l l
C 15 3.20 2.86 0.07 0.14 2.00 5.00 0.18 0.40 0.57 0.26 17.90 9.00
l l
Frozen Peaches 11 3.86 3.25 0.09 0.17 2.40 5.40 0.26 0.49 0.34 0.27 8.90 8.20
fruits
 k
Strawberries 11 3.95 3.97 0.09 0.14 2.30 3.50 0.25 0.39 0.35 0.28 8.90 7.00

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k
Blueberries 10 2.71 2.85 0.15 0.17 5.60 5.90 0.43 0.47 0.20 0.20 7.30 7.10

Fresh Carrot 11 7.49 7.33 0.30 0.28 3.90 3.70 0.83 0.76 0.58 0.55 7.70 7.50
vegetables
l k
Broccoli 11 7.82 7.15 0.29 0.26 3.70 3.60 0.81 0.73 0.39 0.57 5.00 8.00
l k k
Celery 9 8.69 7.39 0.08 0.12 0.90 1.60 0.23 0.34 0.42 0.74 4.90 10.10
a Number of laboratories with valid data.

b Mean log aerobic microorganisms/g.

c Standard Methods Agar incubated at 30°C.
d Standard Methods Agar incubated at 35°C.
e Repeatability standard deviation.
f Repeatability relative standard deviation.
g Repeatability values, 2.8 × sr.

h Reproducibility standard deviation.

i Reproducibility relative standard deviation.

j Reproducibility values, 2.8 × sR.

k Significantly di erent at p < 0.05.

l Significantly di erent at p < 0.01.

A Principle
SimPlate Total Plate Count–Color Indicator (TPC–CI) method is used for detection and quanti cation of
total aerobic populations. The TPC–CI medium and food mixture is dispensed into a SimPlate device and
incubated for 24–28 h. The medium changes color in the presence of aerobic microorganisms. The total
aerobic microorganisms count is determined by counting the wells with changed color and correlating the
number of positive wells with the number of total aerobic microorganisms found in Table 2002.07D.

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Table 2002.07D SimPlate conversion table

a
No. of positive wells = population

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1=2 31 = 76 61 = 216

2=4 32 = 80 62 = 224

3=6 33 = 84 63 = 232

4=8 34 = 86 64 = 240

5 = 10 35 = 90 65 = 248

6 = 12 36 = 94 66 = 256

7 = 14 37 = 96 67 = 266

8 = 16 38 = 100 68 = 276

9 = 18 39 = 104 69 = 288

10 = 22 40 = 108 70 = 298

11 = 24 41 = 112 71 = 312

12 = 26 42 = 116 72 = 324

13 = 28 43 = 120 73 = 338

14 = 30 44 = 124 74 = 354

15 = 32 45 = 128 75 = 372
 16 = 36 46 = 132 76 = 392

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17 = 38 47 = 136 77 = 414

18 = 40 48 = 142 78 = 440

19 = 42 49 = 146 79 = 470

20 = 46 50 = 150 80 = 508

21 = 48 51 = 156 81 = 556

22 = 50 52 = 160 82 = 624

23 = 54 53 = 166 83 = 738

24 = 56 54 = 172 84 = >738

25 = 58 55 = 178 If there are no positive

26 = 62 56 = 184 wells, and the sponge is positive

27 = 64 57 = 190 population is 1

28 = 68 58 = 196 If there are no positive

29 = 70 59 = 202 wells, and the sponge is negative,

30 = 74 60 = 208 population is <1

a The population reflects the number of microorganisms per plate. To determine the number of microorganisms per g (mL) food product, refer to I(c), Reading
and Interpretation of Results.
B Media and Reagents
(a) Dehydrated TPC–CI medium.—In individually packaged single or multiple test format.

(b) Supplement A (optional).—Add 1.0 mL sterile Supplement A solution per 100 mL sterile deionized H2O.
Alternatively, add 1.0 mL Supplement A to 100 mL deionized H2O and autoclave for 15 min at 121°C.

(c) Butter eld’s Phosphate Bu ered Diluent (BPBD).—See 966.23A(m) (see 17.2.01).

(d) Peptone salt solution.—Dissolve 1.0 g enzymatic digest of casein and 8.5 g NaCl in 1 L deionized H2O.
Autoclave for 15 min at 121°C. Final pH, 7.0 ± 0.2 at 25°C.

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(e) SimPlate devices.—Twenty devices per package.

Items (a), (b), and (e) are available from BioControl Systems, Inc. (Bellevue, WA, USA;
[Link]).

C Apparatus
(a) Incubator.—Maintaining 35–37°C.

(b) Micropipetor.—Accurately dispensing 0.1 and 1.0 mL.

(c) Pipets.—Accurately dispensing 1.0 and 10 mL.

(d) Blender/stomacher.—Waring, or equivalent, for blending test portions; IUL Instruments masticator
(IUL, S.A., Barcelona, Spain; Tel: +34-93-274-0232; [Link]), or equivalent, for
macerating test portions. Note: A blender is used if testing in accordance with the AOAC method; a
stomacher is used if testing in accordance with the ISO method.

D General Instructions
Do not use expired media. Store reconstituted medium between 15 and 25°C in the dark and use within 12 h.
Dispose of medium in a decontamination container, and sterilize before discarding.

E Preparation of Test Suspension

(a) Weigh 50 g test portion into 450 mL sterile diluent, e.g., BPBD (AOAC method) or peptone salt
solution (ISO method). This is a 1:10 dilution. Macerate or blend to homogenize.

(b) If alternative test portion size is speci ed in testing procedure, prepare 10% (w/v) suspension.

(c) If necessary, prepare 10-fold serial dilutions appropriate for the anticipated population of the test
portion.
 F TPC–CI Test Procedure, Single Test Medium
(a) For 1.0 mL test portion size.—Resuspend powdered medium with 9.0 mL sterile deionized water
containing 1 mL Supplement A per 100 mL water. Add 1.0 mL prepared test portion and mix well. Do
not count this reconstitution as a dilution.

(b) For 0.1 mL test portion size.—Resuspend powdered medium with 9.9 mL sterile deionized water
containing 1 mL Supplement A per 100 mL water. Add 0.1 mL prepared test portion and mix well. This
is an additional 1:10 dilution from E.

Note: The nal volume of test portion/medium mixture in the container should be 10 ± 0.2 mL.

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(c) Remove lid from SimPlate device and transfer test portion/medium mixture onto center of plate.
Immediately replace lid. Continue with H(a).

G TPC–CI Test Procedure, Multiple Test Medium

(a) Empty contents of one container into 100 mL sterile deionized water containing 1 mL Supplement A
per 100 mL water. Shake to completely dissolve.

(b) Remove lid from SimPlate device. Pipet prepared test suspension onto center of plate. If prepared test
suspension size is 1.0 mL, overlay test suspension with 9.0 mL medium. Do not count this media
addition as a dilution.

(c) For 0.1 mL prepared test suspension, overlay with 9.9 mL medium; this is an additional 1:10 dilution
of test suspension from E.

Note: The nal volume of test suspension/medium mixture on the plate should be 10 ± 0.2 mL.

(d) Immediately replace the lid. Continue with H(a).

H Test Procedure for Single and Multiple Tests

(a) Gently swirl to distribute test suspension/medium mixture into all wells. Hold plate with both hands,
tilted slightly to help distribute medium into wells.

(b) Pour o excess medium by holding lid against plate on either side of sponge cavity. Tip plate toward
you to allow liquid to drain into sponge. Observe background color of wells. Background is de ned as
color of the test suspension/medium mixture inside the wells before incubation.

p. C17-16 (c) If testing in accordance with AOAC/BAM/USDA methods, incubate SimPlate devices in an upright
position in the dark for 24–28 h at 35 ± 1°C (32 ± 1°C for dairy products).
I Reading and Interpretation of Results
(a) After incubation, observe color change of liquid in wells. Disregard particulate matter if present.
Count number of wells showing a color change from the background color. The most common color
change produced by microorganisms is pink, but orange, peach, red, brown, and white may also be
observed.

(b) To determine the population, perform the following calculations: (1) Count the number of positive
wells on the plate; (2) use Table 2002.07D to determine total number of microorganisms per plate.

(c) To calculate number of microorganisms per gram (mL), multiply the count in I(b)(2) by the
appropriate dilution factor (see E and F for Single test, or E and G for Multiple test).

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Reference:

J. AOAC Int. 86, 257(2003)Crossref

17.2.08 AOAC O icial Method 995.21

Yeast and Mold Counts in Foods: Hydrophobic Grid Membrane Filter
(ISO-GRID) Method Using YM-11 Agar

First Action 1995

Final Action 1999

(Applicable to enumeration of total yeasts and molds in all foods.)

See Table 995.21A for results of the interlaboratory study supporting acceptance of the method.
Table 995.21A Interlaboratory study results for enumeration of yeast and mold in foods by ISO-GRID and reference methods

a
Food Lot Method Log10 mean, count/g or mL Precision estimates

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r R sr sR RSDr, % RSDR, %

Garlic A ISO-GRID 1.23 0.95 1.12 0.34 0.40 27.49 32.09

powder Reference 1.08 0.90 1.23 0.32 0.44 29.12 40.19

B ISO-GRID 1.20 0.76 1.01 0.27 0.36 22.35 30.03

Reference 1.20 0.98 1.20 0.35 0.43 28.95 36.10

C ISO-GRID 1.18 0.53 1.01 0.19 0.36 16.11 30.42

Reference 1.20 1.34 1.48 0.48 0.53 40.00 44.18

Overall ISO-GRID 1.20

Reference 1.18

Raw ground A ISO-GRID 3.61 0.76 1.06 0.27 0.38 7.50 10.60

beef Reference 3.59 0.81 1.06 0.29 0.38 8.23 10.61

B ISO-GRID 3.91 0.95 1.32 0.34 0.47 8.70 11.99

Reference 3.84 0.64 1.01 0.23 0.36 5.94 9.36

C ISO-GRID 4.43 1.09 1.68 0.39 0.60 8.82 13.60

Reference 4.28 0.81 1.46 0.29 0.52 6.88 12.03

 Overall ISO-GRID 3.99

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Reference 3.90

Walnuts A ISO-GRID 3.57 0.67 0.73 0.24 0.26 6.77 7.15

Reference 3.68 0.53 0.53 0.19 0.19 4.93 5.07

B ISO-GRID 4.08 0.28 0.64 0.10 0.23 2.47 5.59

Reference 4.23 0.22 0.45 0.08 0.16 1.90 3.81

C ISO-GRID 3.66 0.62 0.70 0.22 0.25 5.97 6.74

Reference 3.79 0.59 0.59 0.21 0.21 5.43 5.43

b
Overall ISO-GRID 3.76

Reference 3.90

Flour/meal A ISO-GRID 3.36 1.20 1.20 0.43 0.43 12.80 12.80

Reference 3.52 1.01 1.01 0.36 0.36 10.23 10.23

B ISO-GRID 4.63 0.59 1.18 0.21 0.42 4.45 9.05

Reference 4.76 0.76 0.92 0.27 0.33 5.66 6.97

C ISO-GRID 3.38 1.01 1.18 0.36 0.42 10.57 12.46

Reference 3.20 0.73 1.09 0.26 0.39 8.19 12.23

Overall ISO-GRID 3.80

 Reference 3.83

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Orange juice A ISO-GRID 3.11 0.53 0.64 0.19 0.23 5.91 7.19

Reference 3.32 0.42 0.64 0.15 0.23 4.39 6.85

B ISO-GRID 7.20 1.04 1.18 0.37 0.42 5.16 5.87

Reference 6.66 1.12 2.10 0.40 0.75 6.06 11.31

C ISO-GRID 6.00 0.73 1.74 0.26 0.62 4.39 10.26

Reference 5.45 1.29 1.54 0.46 0.55 8.35 10.08

b
Overall ISO-GRID 5.45

Reference 5.15

Yogurt A ISO-GRID 6.54 0.48 1.37 0.17 0.49 2.51 7.44

Reference 6.75 0.31 1.82 0.11 0.65 1.59 9.58

B ISO-GRID 6.46 0.45 1.85 0.16 0.66 2.46 10.25

Reference 6.61 0.34 2.38 0.12 0.85 1.81 12.85

C ISO-GRID 6.72 0.59 1.48 0.21 0.53 3.07 7.83

Reference 6.66 0.62 1.93 0.22 0.69 3.25 10.36

Overall ISO-GRID 6.60

Reference 6.67
a Precision estimates were calculated a er conversion of the data to log10; r = 2.8 × sr; R = 2.8 × sR; sr = repeatability standard deviation; S R = reproducibility
 standard deviation; RSDr= repeatability relative standard deviation; RSD R = reproducibility relative standard deviation.

b Di erence between the methods is statistically significant at the 5% level.

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A Principle
Method uses membrane lter imprinted with hydrophobic material in grid pattern. Hydrophobic lines act as
barriers to spread of colonies, thereby dividing membrane lter surface into separate compartments of
equal and known size. Squares occupied by colonies are counted and converted to most probable number
(MPN) value of organisms.

B Apparatus, Media, and Reagents

(a) Hydrophobic grid membrane lter (HGMF).—Pore size, 0.45 µm; imprinted with nontoxic hydrophobic

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material in grid pattern.

(b) Filtration units for HGMF.—With 5 µm mesh pre lter to remove food particles during ltration. Use
one unit/test.

(c) Pipets.—1.0, 5.0, and 10.0 mL, serological with 0.1 mL graduations; 1.1 or 2.2 mL milk pipets may also
be used.

(d) Blender.—Multi-speed with low-speed operation at 10 000–12 000 rpm with 250 mL glass or metal
blender jars with covers. Use one jar/test.

(e) Vacuum pump.—H2O aspirator vacuum source is satisfactory.

(f) Manifold or vacuum ask.

(g) Peptone diluent.—Dissolve 1.0 g peptone (gelatin hydrolysate peptone) in 1 L H2O. Dispense peptone
diluent into dilution bottles to obtain 90 ± 1 or 99 ± 1 mL after autoclaving 15 min at 121°C.

(h) Peptone-Tween 80 (PT) diluent.—Dissolve 1.0 g peptone (gelatin hydrolysate peptone) and 10.0 g
Tween 80 in 1 L H2O. Dispense PT diluent into dilution bottles to obtain 90 ± 1 or 99 ± 1 mL after
autoclaving 15 min at 121°C.

(i) YM-11 agar.—Dilute 20.0 g soy peptone, 20.0 g tryptone, 5.0 g D-glucose, 5.0 g NaCl, 2.4 g anhydrous
K2HPO4, 0.03 g trypan blue, 0.1 g chloramphenicol, and 15.0 g agar to 1 L with H2O. Heat to boiling
while stirring. Autoclave 15 min at 121°C and then temper to 45–50°C in water bath. Temper
chlortetracycline-HCl supplement, (j), to 45–50°C. Add 20 mL supplement to 1 L YM-11 medium and
mix well. Aseptically pour su cient volume of complete YM-11 agar into small weighing boat or
Petri dish to obtain ≥3 mm layer of agar. Let agar solidify.

Check pH using at-surface combination electrode. Adjust pH, if necessary, to 7.0 ± 0.2 by adding sterile 1 M
NaOH or 1 M HCl, as required. Aseptically pour another small portion of medium and check pH as before.
Continue adjustments until pH is within speci ed range.

Dispense ca 18–20 mL agar into 15 × 100 mm Petri dishes. Let agar solidify. Surface-dry medium before use
by inverting partly open Petri dishes and placing them in incubator 15–20 min at 35°C.
 (j) Chlortetracycline–HCl supplement.—Dissolve 0.5 g chlortetracycline∙HCl in 100 mL H2O. Stir without
heating until dissolved. Filter-sterilize solution. Unused portion of supplement can be stored ≤2
months at 4–6°C, if protected from evaporation.

p. C17-18 (k) Papain stock solution.—16 000 Food Chemicals Codex papain units/mg. Reconstitute 10.0 g sterile
papain powder with 100 mL sterile H2O. Unused portion of solution can be stored frozen ≤3 months.

(l) Hemicellulase stock solution.—1500 hemicellulase units/g. Reconstitute 20.0 g sterile hemicellulase
powder with 100 mL sterile H2O. Unused portion of solution can be stored frozen ≤3 months.

Items (a), (b), and (i)–(l) are available from Neogen Corp. (Lansing, MI, USA).

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C Preparation of Test Suspensions
(Note: See Table 995.21B for diluents and enzymes to be used for various foods.)
 a
Table 995.21B Diluents and enzyme treatments for various foods

Food Diluent Enzyme

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Flour/meal Peptone None

Black pepper Peptone None

Onion powder Peptone Papain

Garlic powder Peptone Papain

Soy beans Peptone None

Pinto beans Peptone None

Rice Peptone None

Co ee beans Peptone None

Cocoa beans Peptone None

Cream cheese Peptone Hemicellulase

b
Other cheeses PT Papain

Sour cream Peptone Papain

Vegetable juices Peptone None

Fruit juices Peptone None

Raw tomato Peptone None

 Frozen strawberries Peptone None

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Fresh blackberries Peptone None

Fruit preserves Peptone None

Walnuts Peptone Papain

Peanuts Peptone Papain

Raw ground beef PT None

Summer sausage PT None

Roast beef PT None

a Based on analysis of 1 mL 1:10 dilution. Foods tested at dilutions of 1:100 or higher do not usually need enzyme treatment. See Table 986.32 (see 17.2.05) for
recommended enzyme treatments of foods not listed in this table.
b Peptone–Tween 80 diluent, B(h).
 (a) Liquid egg.—Thoroughly mix test sample using sterile spoon or spatula. Aseptically weigh 11 g egg
material into sterile wide-mouth, screw-top bottle. Add 99 mL PT diluent, B(h), and 1 tbsp of sterile
glass shot. Thoroughly agitate 1:10 dilution suspension to ensure complete distribution of egg
material in diluent by shaking each bottle rapidly 25 times in ≤7 s, using an up-and-down movement
of ca 30 cm for each shake. Let bubbles escape. If enzyme treatment is needed (see Table 995.21B),
combine 5 mL homogenate (1:10 dilution) with 1 mL appropriate enzyme stock solution, mix well,
and incubate 20–30 min in water bath at 35–37°C. Correct for additional dilution by ltering 1.2 mL
enzyme-treated test suspension homogenate.

(b) Other liquid products.—Thoroughly mix contents of container. Prepare 1:10 dilution by aseptically
transferring 10 mL test portion into 90 mL peptone diluent, B(g), or 90 mL PT diluent, B(h), (see

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Table 995.21B) in sterile wide-mouth, screw-top bottle. Mix by shaking bottle 25 times through 30
cm arc in ≤7 s. If enzyme treatment is needed, combine 5 mL homogenate (1:10 dilution) with 1 mL
appropriate enzyme stock solution, mix well, and incubate 20–30 min in water bath at 35–37°C.
Correct for additional dilution by ltering 1.2 mL enzyme-treated test suspension homogenate.

(c) Whole egg powder.—Thoroughly mix with sterile spoon or spatula. Dilute 1:10 by aseptically weighing
11 g egg material into sterile wide-mouth, screw-top bottle. Add 99 mL PT diluent, B(h), and 1 tbsp
of sterile glass shot. Shake rapidly as in (a) to ensure complete distribution of egg material in diluent.

Prepare 1:100 dilution by aseptically transferring 10 mL homogenate (1:10 dilution) into 90 mL PT diluent.
Combine 10 mL of 1:100 dilution with 1 mL papain stock solution, B(k), mix well, and incubate 20–30 min in
water bath at 35–37°C. Filter entire 11 mL volume of enzyme-treated 1:100 dilution.

(d) Other powders.—Thoroughly mix with sterile spoon or spatula. Prepare 1:10 dilution by aseptically
weighing 10 g test portion into sterile wide-mouth, screw-top bottle. Add 90 mL peptone, B(g), or
PT diluent, B(h), (see Table 995.21B). Shake each bottle rapidly 25 times in ≤7 s, using an up-and-
down movement of ca 30 cm for each shake. Let bubbles escape. If enzyme treatment is needed,
combine 5 mL homogenate (1:10 dilution) with 1 mL appropriate enzyme stock solution, mix well,
and incubate 20–30 min in water bath at 35–37°C. Correct for additional dilution by ltering 1.2 mL
enzyme-treated test suspension homogenate.

(e) Other foods.—Prepare 1:10 dilution by aseptically weighing 10 g test portion into sterile blender jar.
Add 90 mL peptone, B(g), or PT, B(h), diluent (see Table 995.21B), and blend 2 min at low speed (10
000–12 000 rpm). If enzyme treatment is needed, combine 5 mL homogenate (1:10 dilution) with 1
mL appropriate enzyme stock solution, mix well, and incubate 20–30 min in water bath at 35–37°C.
Correct for additional dilution by ltering 1.2 mL enzyme-treated suspension homogenate.

D Analysis
See Figures 986.32A and B (see 17.2.05) for ltration unit and ltration unit clamp.

Turn on vacuum source. Place sterile ltration unit on manifold or vacuum ask. Open clamp A. Rotate back
funnel portion C. Aseptically place sterile lter, B(a), on surface of base D. Rotate funnel forward. Clamp
shut by sliding jaws L of stainless steel clamp over entire length of anges B that extend from both sides of
funnel C and base D, and rotating moving arm K into horizontal (locked) position.

Aseptically add ca 15–20 mL sterile H2O to funnel. Pipet 1.0 mL homogenate (1:10 dilution) or appropriate
volume of enzyme-treated homogenate into funnel. Apply free end of vacuum tubing E to suction hole F to
p. C17-19draw liquid through pre lter mesh G. Aseptically add additional 10–15 mL sterile H2O to funnel and draw
through mesh as before. Close clamp A to direct vacuum to base of ltration unit and draw liquid through
lter.
Open clamp A. Rotate moving arm K of stainless steel clamp into unlocked (ca 45° angle) position and slide
jaws L o anges B. Rotate back funnel C.

Place lter on surface of predried YM-11 plate, B(i). Avoid trapping air bubbles between lter and agar.
Incubate plates 50 ± 2 h at 25 ± 1°C.

Count all squares containing one or more colonies (positive squares). Colonies are usually some shade of
blue. Examine lter using illuminated magni cation as some colonies may be only pinpoint in size. To
con rm that lter does not contain any positive squares, hold up Petri dish and examine “horizon” of lter
for raised “bumps” or areas that re ect light di erently. These may be either very small or very pale
colonies. While hydrophobic lines act as barriers to spread of colonies, some fast-growing molds produce
too much mycelium to remain completely con ned within a single square (spreader). In case of spreader,

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only count the square of origin, which usually has the densest or tallest growth. Do not count squares into
which colony has spread.

Count squares containing one or more colonies as described above. Convert total number of positive squares
to MPN index as follows:

MPN = 1600 loge [1600/(1600 − x)]

where x = number of positive squares.

Multiply MPN by reciprocal of dilution factor, round to two signi cant gures, and report as yeast and mold
count/g or mL.

Reference:

J. AOAC Int. 79, 1069(1996)Crossref

17.2.09 AOAC O icial MethodSM 997.02

Yeast and Mold Counts in Foods and Dried Cannabis Flower: Dry
Rehydratable Film Method (Petrifilm™ Method)

First Action 1997

Final Action 2000

Revised First Action 2021 (for Cannabis Flower, THC >0.3%, Only)

[Applicable to enumeration of total yeasts and molds in foods and dried cannabis ower (THC >0.3%).]

See Tables 997.02A and B for results of the interlaboratory study supporting acceptance of the method.

A Principle
The method uses culture plates of dry medium supplemented with antibiotics, dye to enhance visualization
of growth, and cold-water-soluble gelling agent. Undiluted or diluted suspensions are added to plates at a
2
rate of 1 mL/plate. Suspension is spread over a 30 cm growth area. Gelling agent is allowed to solidify, plates
are incubated, and yeasts and molds are counted.
B Apparatus and Reagent
(a) Yeast and mold (YM) count plates.—Contain nutrients supplemented with chlortetracycline,
chloramphenicol, cold-water-soluble gelling agent, and dye sensitive to presence of phosphatase
(5-bromo-4-chloro-3-indolyl phosphate) that enhances visualization of yeast and mold growth.
The circular growth area of a single plate contains thirty 1 × 1 cm squares outlined on a lm base
(available as 3M™ Petri lm™ Yeast and Mold Count Plates, Cat. No. 6407/6417; 3M Food Safety, St.
Paul, MN, USA).

(b) Plastic spreader.—Provided with Petri lm plates, designed to spread suspension evenly over plate
growth area.

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(c) Pipets.—Serological pipet or pipetting syringe accurately delivering 1.0 mL.

(d) Colony counter.—Standard apparatus, Quebec model preferred, or one providing equivalent
magni cation (1.5×) and visibility.

(e) Blender.—High-speed mechanical blender rotating at 10 000–12 000 rpm or stomacher.

(f) Sterile diluents.—Butter eld’s phosphate-bu ered dilution water or 0.1% peptone water (PW).

C General Instructions
Store unopened 3M Petri lm YM Plate pouches refrigerated or frozen at temperatures ≤8°C (46°F). Just
prior to use, allow unopened pouches to come to room temperature before opening. Return unused 3M
Petri lm YM Plates to pouch. Seal by folding the end of the pouch over and applying adhesive tape. To
prevent exposure to moisture, do not refrigerate opened pouches. Store resealed pouches in a cool, dry place
for no longer than 4 weeks. It is recommended that resealed pouches of 3M Petri lm YM Plates be stored in
a freezer (see product instructions) if the laboratory temperature exceeds 25°C (77°F) and/or the laboratory
is located in a region where the relative humidity exceeds 50% (with the exception of air-conditioned
premises).

After use, plates contain viable yeast and/or mold cultures. Autoclave used plates 15 min at 121°C prior to
discarding.

D Preparation of Test Suspension

Aseptically prepare 1:10 or greater dilution of food samples with sterile diluent. Blend or stomach 2 min and
plate. Prepare additional dilutions as required. For dried cannabis ower (THC >0.3%), weigh out 10 g
sample from test portion into sterile stomacher bag and dilute with 90 mL sterile 0.1% PW. Shake 25 times
to homogenize. Prepare additional dilutions as required.

E Analysis
Place Petri lm YM Count Plate on at surface. Lift top lm, hold pipet perpendicular to plate, and carefully
inoculate 1 mL test suspension onto center of lm base. Place top lm down onto inoculum.

Lift plastic spreader using circular handle. Align center of spreader with approximate center of plate.
Distribute suspension evenly using gentle downward pressure on center of spreader. Do not slide spreader
across lm. Remove spreader and leave plate undisturbed for 1 min to let gel solidify.

Place plates in incubator in horizontal position, clear side up, in stacks not exceeding 20 units. Incubate
plates 5 days at 20–25°C.
Count plates promptly after incubation period. Yeast colonies appear as blue-green or o -white in color and
form small, de ned colonies. Mold colonies are usually blue but may also assume their natural
pigmentation (e.g., black, yellow, green). They tend to be larger and more di use than yeast colonies.

To calculate yeast and mold count, multiply total number of yeast and mold colonies/plate (or average
number of colonies/plate if counting duplicate plates of same dilution) by the appropriate dilution factor.
When counting colonies on duplicate plates of consecutive dilutions, calculate the mean number of colonies
for each dilution before determining average yeast and mold count.

Estimated counts can be made on plates with >150 colonies and should be reported as estimated counts. In
2 2
making such counts, determine average count/1 cm and multiply by 30 (circular growth area is 30 cm ).

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High numbers of yeast colonies may cause entire growth area to turn blue. High numbers of mold colonies
may cause growth area to turn blue, black, yellow, etc. When this occurs, do not make estimated counts, but
further dilute and plate test suspension to obtain a more accurate count.

Revised: March 2002

Table 997.02A Interlaboratory study results for determination of mold count in foods by dry rehydratable film method
a b
Product Mold level Method Mean log10 colony count sr sR RSDr, % RSDR, % r R

c
Orange juice Low PYM 2.50 0.13 0.17 5.05 6.93 0.36 0.49
d
BAM 2.50 0.33 0.38 13.23 15.17 0.94 1.07

High PYM 3.23 0.18 0.37 5.68 11.51 0.52 1.05

BAM 3.21 0.12 0.36 3.66 11.10 0.33 1.01

Hot dog Low PYM 2.35 0.32 0.80 13.67 34.00 0.91 2.26

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BAM 2.20 0.08 0.98 3.44 44.69 0.21 2.78

High PYM 3.09 0.11 0.97 3.58 31.54 0.31 2.76

BAM 3.06 0.19 0.98 6.18 31.89 0.54 2.76

Yogurt Low PYM 2.34 0.16 0.75 6.90 31.81 0.46 2.11

BAM 2.15 0.11 0.92 5.18 42.68 0.31 2.59

High PYM 3.21 0.43 0.50 13.45 15.70 1.22 1.42

BAM 3.00 0.17 0.92 5.50 30.49 0.47 2.59

Ketchup Low PYM 2.17 2.52 2.61 116.00 120.10 7.13 7.38

BAM 1.90 0.27 0.67 13.93 35.08 0.75 1.88

High PYM 2.76 0.42 0.50 15.35 18.01 1.20 1.41

BAM 2.78 0.18 0.80 6.32 30.50 0.50 2.40

Corn meal Low PYM 2.28 0.69 0.76 30.18 33.53 1.95 2.16

BAM 2.29 0.39 0.63 17.13 27.56 1.11 1.78

High PYM 2.50 0.61 0.76 24.54 29.81 1.73 2.11

BAM 2.54 0.51 0.64 20.01 25.10 1.44 1.80

Cake mix Low PYM 1.73 0.30 0.68 17.29 39.06 0.85 1.92

BAM 1.57 0.52 0.82 33.35 52.29 1.48 2.31

High PYM 1.73 0.49 0.78 28.13 44.96 1.37 2.20

BAM 1.71 0.31 0.77 18.26 45.01 0.88 2.18

a r = 2.8 × sr.

b R = 2.8 × sR.

c PYM = Petrifilm Yeast and Mold Count Plate.

d BAM = Method described in U.S. Food and Drug Administrationʼs Bacteriological Analytical Manual, 7th Ed., AOAC
INTERNATIONAL, Rockville, MD, USA.
Table 997.02B Interlaboratory study results for determination of yeast count in foods by dry rehydratable film method
a b
Product Yeast level Method Mean log10 colony count sr sR RSDr, % RSDR, % r R

c
Orange juice Low PYM 1.72 0.48 0.77 28.05 44.98 1.36 2.18
d
BAM 1.72 0.51 0.82 29.41 47.81 1.43 2.33

High PYM 2.93 0.26 0.38 8.98 13.07 0.74 1.08

BAM 2.95 0.15 0.36 5.20 12.32 0.43 1.03

Corn meal Low PYM 1.32 0.98 1.48 73.99 112.00 2.76 4.18

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BAM 1.49 0.81 1.45 54.00 96.88 2.28 4.09

High PYM 1.99 1.16 1.51 58.33 75.70 3.28 4.26

BAM 2.27 1.08 1.32 47.85 58.15 3.07 3.73

Cake mix Low PYM 1.51 0.58 1.11 38.47 73.61 1.64 3.14

BAM 1.23 0.75 1.18 61.10 96.59 2.12 3.35

High PYM 2.09 0.25 1.06 11.79 50.78 0.70 3.00

BAM 2.07 0.40 1.14 19.25 55.01 1.13 3.22

a r = 2.8 × sr.

b R = 2.8 × sR.

c PYM = Petrifilm Yeast and Mold Count Plate.

d BAM = Method described in FDA Bacteriological Analytical Manual, 7th Ed., AOAC INTERNATIONAL, Rockville, MD, USA.

References:

AOAC SMPR 2021.009 Viable Yeast and Mold Count Enumeration in Cannabis and Cannabis Products, [Link]
content/uploads/2021/06/SMPR-2021_009.pdf (accessed April 2021)

J. AOAC Int. 80, 806(1997) (First Action)

J. AOAC Int. 106(2), 412(2023) (Revised First Action) DOI: [Link]

17.2.10 AOAC O icial Method 2002.11 Detection and Quantification of

Yeasts and Molds in Foods: SimPlate Yeast and Mold–Color Indicator
(Y&M–CI) Method

First Action 2002

Final Action 2005

(Applicable to detection and quanti cation of yeasts and molds in chocolate, cake mix, spices, nut meats,
dairy foods, red meats, poultry meats, seafoods, fermented meats, frozen corn dogs, cereal, pasta, egg
products, our, prepackaged fresh salad, frankfurters, vegetables, fruits, and fruit juice.)
Caution: Test portion dilutions and incubated SimPlate devices from food products could contain pathogenic
fungi if the particular test portion was so contaminated. Use standard aseptic microbiological laboratory
technique, including decontamination of any spills with disinfectant.

See Tables 2002.11A–C for results of the interlaboratory study supporting acceptance of the method.

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Table 2002.11A Statistical analysis of interlaboratory results for total fungi recovery by the BAM culture method and the SimPlate Y&M–CI method

a b e f g h i j
Food Lot/level N Mean sr RSD, % r sR RSDR,% R

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group
c d c d c d c d c d c d c
BAM SIM BAM SIM BAM SIM BAM SIM BAM SIM BAM SIM BAM

Frozen Low 8 3.29 3.44 0.17 0.17 5.10 5.00 0.47 0.49 0.21 0.23 6.00 6.50 0.59
corn
dog

Medium 8 3.98 4.02 0.20 0.19 5.00 4.80 0.56 0.54 0.24 0.21 6.00 5.30 0.67

High 8 4.40 4.48 0.11 0.14 2.50 3.10 0.31 0.38 0.18 0.17 4.20 3.90 0.52

Frozen Raspberries 12 4.27 4.37 0.35 0.39 8.20 9.00 0.98 1.10 0.51 0.60 11.90 13.60 1.42
fruit

Blackberries 12 4.32 4.46 0.29 0.29 6.60 6.60 0.80 0.82 0.36 0.39 8.30 8.60 1.00

Strawberries 12 3.70 3.74 0.19 0.25 5.10 6.60 0.53 0.69 0.22 0.36 5.90 9.70 0.61

Cereal Low 11 2.17 2.28 0.18” 0.33 8.00 14.40 0.49 0.92 0.44 0.43 20.20 19.00 1.23

Medium 11 2.59 2.77 0.18 0.21 6.70 7.40 0.49 0.57 0.47 0.46 17.90 16.60 1.30

High 11 3.75 3.87 0.14 0.20 3.70 5.10 0.39 0.55 0.29 0.30 7.80 7.60 0.82
k
Cheese Blue 13 7.77 7.89 0.44 0.25 5.60 3.20 1.22 0.71 0.49 0.36 6.20 4.60 1.36
k
Mozzarella 13 7.26 7.39 0.09 0.15 1.20 2.10 0.25 0.43 0.24 0.27 3.30 3.60 0.68
k l
Cheddar 14 6.09 6.10 0.12 0.22 1.90 3.50 0.33 0.60 0.32 0.65 5.30 10.70 0.90
 Cake Low 12 2.07 1.97 0.45 0.51 21.60 26.00 1.25 1.43 0.49 0.57 23.80 28.90 1.38
mix

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Medium 12 2.50 2.43 0.31 0.41 12.50 17.00 0.88 1.16 0.50 0.53 20.00 22.00 1.40
m
High 12 3.29 3.09 0.22 0.31 6.60 9.90 0.61 0.86 0.29 0.36 8.70 11.60 0.80

Nut Hazelnut 13 2.55 2.69 0.46 0.55 18.10 20.50 1.30 1.54 0.64 0.66 24.90 24.50 1.78
meats
n l
Pecan 12 2.17 2.58 0.11 0.14 5.30 5.50 0.32 0.40 0.17 0.52 7.80 20.20 0.48
n k o
Walnut 12 3.63 4.15 0.27 0.48 7.30 11.60 0.75 1.35 0.32 0.61 8.70 14.70 0.88
a Number of laboratories with valid data.

b Mean log count of total fungi/g.

c U.S. Food and Drug Administrationʼs Bacteriological Analytical Manual culture method.
d SimPlate Yeast and Mold-Color Indicator method.

e Repeatability standard deviation.

f Repeatability relative standard deviation.

g Repeatability values, 2.8 × sr.

h Reproducibility standard deviation.

i Reproducibility relative standard deviation.

j Reproducibility values, 2.8 × sR.

k Significantly di erent repeatability p < 0.05; statistically not di erent p < 0.01.
l Significantly di erent reproducibility p < 0.01.

m Significantly di erent mean log counts p < 0.05; statistically not di erent p < 0.01.
n Significantly di erent mean log counts p < 0.01.
o Significantly di erent reproducibility p < 0.05; statistically not di erent p < 0.01.

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Table 2002.11B Statistical analysis of interlaboratory results for total fungi recovery by the ISO culture method and the SimPlate Y&M–CI method
a b e f g h i j
Food Lot/level N Mean sr RSDr,% r sR RSDR, % R

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group
c d c d c d c d c d c d c
ISO SIM ISO SIM ISO SIM ISO SIM ISO SIM ISO SIM ISO SIM
k
Frozen Low 8 3.29 3.47 0.13 0.19 4.00 5.50 0.37 0.53 0.23 0.22 6.80 6.30 0.63 0.6
corn
dog

Medium 8 3.93 4.10 0.17 0.22 4.30 5.30 0.47 0.61 0.27 0.22 7.00 5.30 0.77 0.6

High 8 4.36 4.44 0.17 0.13 3.80 2.80 0.47 0.35 0.24 0.21 5.60 4.60 0.68 0.5

Frozen Raspberries 12 4.22 4.43 0.29 0.24 6.80 5.40 0.81 0.67 0.51 0.58 12.20 13.10 1.44 1.6
fruit

Blackberries 11 4.34 4.49 0.14 0.16 3.30 3.70 0.40 0.46 0.26 0.35 6.10 7.70 0.74 0.9

Strawberries 12 3.67 3.67 0.16 0.12 4.30 3.30 0.44 0.34 0.26 0.28 7.00 7.70 0.71 0.7
l
Cereal Low 11 2.04 2.08 0.35 0.75 17.10 36.00 0.98 2.10 0.50 0.75 24.60 36.10 1.41 2.1

Medium 11 2.67 2.71 0.14 0.18 5.40 6.50 0.40 0.49 0.25 0.32 9.50 11.70 0.71 0.8
m
High 10 3.81 3.89 0.04 0.13 1.00 3.30 0.11 0.36 0.26 0.33 6.90 8.40 0.73 0.9
n o
Cheese Blue 13 7.75 8.05 0.20 0.21 2.60 2.60 0.56 0.58 0.27 0.52 3.50 6.40 0.76 1.4
n m o
Mozzarella 14 7.24 7.47 0.07 0.20 0.90 2.70 0.19 0.57 0.20 0.39 2.80 5.20 0.56 1.0
p
Cheddar 14 6.15 6.18 0.11 0.11 1.80 1.80 0.32 1.80 0.26 0.62 4.20 10.00 0.72 1.7
 o
Cake Low 12 2.11 1.88 0.38 0.55 18.00 29.20 1.06 1.54 0.38 0.66 18.00 35.10 1.06 1.8
mix

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k
Medium 12 2.57 2.25 0.36 0.51 13.90 22.80 1.00 1.44 0.41 0.56 16.00 24.60 1.15 1.5
k
High 12 3.2 2.98 0.27 0.34 8.40 11.40 0.75 0.95 0.33 0.35 10.20 11.90 0.92 0.9

Nut Hazelnut 13 2.67 2.81 0.33 0.37 12.40 13.10 0.93 1.03 0.71 0.76 26.40 27.00 1.98 2.1
meats
k p
Pecan 13 2.06 2.42 0.21 0.17 9.90 7.00 0.57 0.47 0.32 0.75 15.50 30.90 0.90 2.0
n
Walnut 11 3.51 3.94 0.23 0.39 6.40 9.90 0.63 1.09 0.36 0.58 10.30 14.60 1.01 1.6

a Number of laboratories with valid data.

b Mean log count of total fungi/g.
c International Organization for Standardization culture method.

d SimPlate Yeast and Mold-Color Indicator method.

e Repeatability standard deviation.
f Repeatability relative standard deviation.
g Repeatability values, 2.8 × sr.

h Reproducibility standard deviation.

I Reproducibility relative standard deviation.
j Reproducibility values, 2.8 × sR.

k Significantly di erent mean log counts p < 0.05; statistically not di erent p < 0.01.
l Significantly di erent repeatability p < 0.05; statistically not di erent p < 0.01.
m Significantly di erent repeatability p < 0.01.
n Significantly di erent mean log counts p < 0.01.
o Significantly di erent reproducibility p < 0.05; statistically not di erent p < 0.01.

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p Significantly di erent reproducibility p < 0.01.
Table 2002.11C Statistical analysis of interlaboratory results for total fungi recovery by the ISO method and the BAM method
a b e f g h i j
Food Lot/level N Mean sr RSDr, % r sR RSDR, % R

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group
c d c d c d c d c d c d c
BAM ISO BAM ISO BAM ISO BAM ISO BAM ISO BAM ISO BAM IS

Frozen Low 8 3.28 3.29 0.17 0.13 5.10 4.00 0.47 0.37 0.21 0.23 6.40 6.80 0.59 0
corn
dog

Medium 8 3.98 3.93 0.20 0.17 5.00 4.30 0.56 0.47 0.24 0.27 6.00 7.00 0.56 0

High 8 4.40 4.36 0.11 0.17 2.50 3.80 0.31 0.47 0.18 0.24 4.20 5.60 0.52 0

Frozen Raspberries 12 4.27 4.22 0.35 0.29 8.20 6.80 0.98 0.81 0.51 0.51 11.90 12.20 1.42 1
fruit

Blackberries 12 4.32 4.37 0.29 0.43 6.60 9.80 0.80 1.21 0.36 0.40 8.30 9.10 1.00 1

Strawberries 12 3.70 3.67 0.19 0.16 5.10 4.30 0.53 0.44 0.22 0.26 5.90 7.00 0.61 0

Cereal Low 10 2.14 2.04 0.18 0.18 8.50 8.70 0.51 0.50 0.45 0.48 21.00 23.40 1.26 1
k
Medium 11 2.59 2.67 0.18 0.14 6.70 5.40 0.49 0.40 0.47 0.25 17.90 9.50 1.30 0
l
High 11 3.75 3.82 0.14 0.07 3.70 1.80 0.39 0.19 0.29 0.25 7.80 6.60 0.82 0

Cheese Blue 12 7.68 7.75 0.15 0.21 2.00 2.70 0.43 0.58 0.23 0.28 3.00 3.60 0.64 0

Mozzarella 13 7.26 7.25 0.09 0.07 1.20 0.90 0.25 0.19 0.24 0.21 3.30 2.90 0.68 0

Cheddar 14 6.09 6.15 0.12 0.11 1.90 1.80 0.33 0.32 0.32 0.26 5.30 4.20 0.90 0
 Cake Low 13 2.00 2.05 0.44 0.36 21.90 17.80 1.23 1.02 0.53 0.43 26.40 20.90 1.48 1
mix

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Medium 13 2.46 2.54 0.30 0.35 12.20 13.80 0.84 0.99 0.50 0.41 20.20 16.20 1.39 1

High 13 3.20 3.14 0.22 0.29 6.80 9.40 0.61 0.82 0.42 0.46 13.10 14.70 1.18 1

Nut Hazelnut 13 2.55 2.67 0.46 0.33 18.10 12.40 1.30 0.93 0.64 0.71 24.90 26.40 1.78 1
meats
l
Pecan 13 2.12 2.06 0.12 0.21 5.60 9.90 0.33 0.57 0.27 0.32 12.70 15.50 0.75 0

Walnut 10 3.62 3.62 0.08 0.10 2.10 2.60 0.63 0.52 0.23 0.19 6.20 5.10 0.63 0

a Number of laboratories with valid data.

b Mean log count of total fungi/g.
c U.S. Food and Drug Administrationʼs Bacteriological Analytical Manual culture method.
d International Organization for Standardization culture method.
e Repeatability standard deviation.
f Repeatability relative standard deviation.
g Repeatability values, 2.8 × sr.

h Reproducibility standard deviation.

i Reproducibility relative standard deviation.

j Reproducibility values, 2.8 × sR.

k Significantly di erent reproducibility p < 0.05; statistically not di erent p < 0.01.

l Significantly di erent repeatability p < 0.05; statistically not di erent p < 0.01.
A Principle
SimPlate Yeast and Mold–Color Indicator (Y&M–CI) method is used to detect and quantify yeast and mold
populations. The Y&M–CI medium and food mixture is dispensed into a SimPlate device and incubated for a
minimum of 56 h. The medium changes color in the presence of yeasts and molds. The yeast and mold count
is determined by counting the wells with changed color and referring to Table 2002.11D.

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Table 2002.11D SimPlate conversion table

a
No. of positive wells = population

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1=2 31 = 76 61 = 216

2=4 32 = 80 62 = 224

3=6 33 = 84 63 = 232

4=8 34 = 86 64 = 240

5 = 10 35 = 90 65 = 248

6 = 12 36 = 94 66 = 256

7 = 14 37 = 96 67 = 266

8 = 16 38 = 100 68 = 276

9 = 18 39 = 104 69 = 288

10 = 22 40 = 108 70 = 298

11 = 24 41 = 112 71 = 312

12 = 26 42 = 116 72 = 324

13 = 28 43 = 120 73 = 338

14 = 30 44 = 124 74 = 354

15 = 32 45 = 128 75 = 372
 16 = 36 46 = 132 76 = 392

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17 = 38 47 = 136 77 = 414

18 = 40 48 = 142 78 = 440

19 = 42 49 = 146 79 = 470

20 = 46 50 = 150 80 = 508

21 = 48 51 = 156 81 = 556

22 = 50 52 = 160 82 = 624

23 = 54 53 = 166 83 = 738

24 = 56 54 = 172 84 = >738

25 = 58 55 = 178 If there are no positive

26 = 62 56 = 184 wells, and the sponge is positive,

27 = 64 57 = 190 population is 1

28 = 68 58 = 196 If there are no positive

29 = 70 59 = 202 wells, and the sponge is negative,

30 = 74 60 = 208 population is <1

a The population reflects the number of microorganisms per plate. To determine the number of microorganisms per g (mL) food product, refer to I(c), Reading
and Interpretation of Results.
B Media and Reagents
(a) Dehydrated Y&M–CI medium.—Individually packaged single or multiple test format with
supplement(s), available from BioControl Systems, Inc. (Bellevue, WA, USA;
[Link]).

(b) SimPlate devices.—In packs of 20 (BioControl).

(c) Peptone water diluent, 0.1% (used in BAM method).—Dissolve 1.0 g peptone in 1 L deionized water.
Autoclave at 121°C for 15 min. Final pH is 7.0 ± 0.2.

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(d) Peptone salt solution (used in ISO method).—Use 1.0 g enzymatic digest of casein and 8.5 g sodium
chloride. Suspend ingredients in 1 L deionized water. Autoclave at 121°C for 15 min. Final pH is 7.0 ±
0.2.

C Apparatus
(a) Incubator.—Maintaining 25°C in dark environment.

(b) Micropipetor.—Accurately dispensing 0.1 and 1.0 mL.

(c) Pipets.—Glass or plastic sterile pipets, 1 mL with 0.01 mL graduations; and 10 mL with 0.1 mL
graduations.

(d) Stomacher/masticator.—IUL Instruments (IUL, S.A., Barcelona, Spain; Tel: +34-93-274-0232;

[Link]), or equivalent, for macerating test portions.

D General Instructions
Do not use expired medium. Store reconstituted medium between 15 and 25°C in the dark and use within 12
h. Dispose of medium in decontamination container, and sterilize before discarding.

E Preparation of Test Suspension

(a) Weigh 50 g test portion into 450 mL sterile diluent [e.g., 0.1% peptone water (BAM method) or
peptone salt solution (ISO method)]. This is a 1:10 dilution. Macerate mixture.

(b) If alternative test portion size is speci ed in testing procedure, prepare 10% (w/v) suspension.

(c) If necessary, prepare 10-fold serial dilutions appropriate for anticipated population of test portion.

F Y&M–CI Test Procedure, Single Test Medium

(a) For 1.0 mL test suspension.—Suspend powdered medium with 9.0 mL sterile deionized water
containing 1 mL Supplement A per 100 mL water. Add 1.0 mL prepared test suspension and mix well.
Do not count this reconstitution as a dilution.

(b) For 0.1 mL test suspension.—Suspend powdered medium with 9.9 mL sterile deionized water
containing 1 mL Supplement A per 100 mL water. Add 0.1 mL prepared test suspension and mix well.
This is an additional 1:10 dilution from E.

The nal volume of test suspension/medium mixture in the container should be 10 ± 0.2 mL.
 Note: For raw meats and spices, add 0.1 mL Supplement M to hydrated medium. For undiluted fruit juice
containing vitamin C or dry pet food, add 0.1 mL Supplement V. For foods containing spreading mold
populations, add 0.1 mL Supplement D. Supplements are available from BioControl Systems, Inc. The nal
volume of the test suspension/medium mixture in the container should be 10 ± 0.2 mL.

(c) Remove lid from the SimPlate device and transfer test suspension/medium mixture onto center of
plate. Immediately replace lid. Continue with H.

G Y&M–CI Test Procedure, Multiple Test Medium

(a) Empty contents of one container, B(a), into 100 mL sterile deionized water containing 1 mL

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Supplement A per 100 mL water. Shake to completely dissolve.

p. C17-25Note: For raw meats and spices, add 1.0 mL Supplement M to hydrated medium. For undiluted fruit juice
containing vitamin C or dry pet food, add 1.0 mL Supplement V. For foods containing spreading mold
populations, add 1.0 mL Supplement D. Supplements are available from BioControl Systems, Inc.

(b) Remove lid from SimPlate device. Pipet prepared test suspension onto center of plate. If prepared test
suspension size is 1.0 mL, overlay test suspension with 9.0 mL medium. Do not count this media
addition as a dilution.

(c) For 0.1 mL of prepared test suspension, overlay with 9.9 mL medium; this is an additional 1:10
dilution of test suspension from E.

The nal volume of test suspension/medium mixture on the plate should be 10 ± 0.2 mL.

Immediately replace lid. Continue with H.

H Test Procedure for Single and Multiple Tests

(a) Gently swirl to distribute test suspension/medium mixture into all wells. Hold plate with both hands
tilted slightly to help distribute medium into wells.

(b) Pour o excess medium by holding lid against plate on either side of sponge cavity. Tip plate toward
you to allow liquid to drain into sponge. Observe background color of wells. Background is de ned as
color of test suspension/medium mixture inside wells.

(c) Do not invert the SimPlate device. Incubate at room temperature (22–25°C) for 56–72 h in the dark.
If test sample being analyzed is known to contain slow-growing fungal populations, such as certain
mold genera, incubation time may be extended to a total of 72 h. If Supplement V is used, incubate
SimPlate devices for 72 h in the dark.

I Reading and Interpretation of Results

(a) If reading is taken between 56 and 63 h after inoculation, place SimPlate device on illuminated
background to help visualize positive results. After 63 h this is not necessary.

After incubation, observe color change of liquid in wells. Disregard particulate matter if present. Count
number of wells showing color change from background color. The common color changes produced by
microorganisms are orange, peach, red, and white. Note: For mixtures containing Supplement V, count only
the number of wells that uoresce blue by holding a UV light (365 nm wavelength) approximately 5 cm (2
in.) above the SimPlate device. Do not count non uorescent wells that only exhibit a color change.
 (b) To determine the population, calculate as follows: (1) Count the number of positive wells on the
plate; (2) use Table 2002.11D to determine the total population per plate.

(c) To calculate the number of fungi/g, multiply the number in I(b)(2) by the appropriate dilution factor
(see E and F for single test or E and G for multiple test).

Reference:

J. AOAC Int. 86, 296(2003)Crossref

17.2.11 AOAC O icial MethodSM 2014.05

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Enumeration of Yeast and Mold in Foods, Selected Surfaces, and Dried
Cannabis Flower: 3M™ Petrifilm™ Rapid Yeast and Mold (RYM) Count
Plate

First Action 2014

Final Action 2017

Revised First Action 2022 (for Cannabis Flower, THC > 0.3%, and Selected Surfaces Only)

[Applicable to enumeration of yeast and mold in the following high-water activity matrixes: yogurt, frozen
bread dough, fermented salami, sour cream, ready-made pie, raw frozen ground beef patties (77% lean),
ready-to-eat deli sandwiches, and sliced apples; low-water activity matrixes: raw almonds, dehydrated
soup, and dried cannabis ower (THC > 0.3%); and environmental surfaces: stainless steel, sealed concrete,
and rubber.]

Caution: After use, diluents and 3M Petri lm RYM Plates may contain microorganisms that may be potential
biohazard as several foodborne molds can produce toxic metabolites known as mycotoxins. If further
identi cation of mold species is required, appropriate personal protective equipment (PPE) should be used
when top lm is retracted and exposure to spores or mycotoxins may occur. When testing is complete,
follow current industry standards for disposal of contaminated waste. Consult material safety data sheet for
additional information and local regulations for disposal. For information on potential biohazards, refer to
Biosafety in Microbiological and Biomedical Laboratories, 6th Ed., Section VIII-B: Fungal Agents.

3M Petri lm RYM Plates contain chloramphenicol and chlortetracycline, potent broad spectrum antibiotic
drugs commonly used in yeast and mold enumeration. The drugs, when used in humans, are associated with
many toxic e ects. Care should be taken to avoid coming into direct contact with the gel on the plates.

See Tables 2014.05A and B for a summary of results of the interlaboratory study supporting acceptance of
the method. Results for each collaborating laboratory’s aerobic plate count analysis for each matrix is
shown in Table 2014.05C.

See Table 2014.05D for results of matrix studies for cannabis ower supporting extension of the method.

See Table 2014.05E for results of matrix studies for selected environmental surfaces supporting extension
of the method.

See additional tables in the Journal of AOAC INTERNATIONAL for detailed results of the interlaboratory study
[J. AOAC Int. 98, 767(2015) DOI: 10.5740/jaoacint.15-006].
A Principle
3M Petri lm RYM Plate is a sample-ready culture medium system, which contains nutrients supplemented
with antibiotics, cold-water-soluble gelling agent, and indicator system that facilitates yeast and mold
enumeration. 3M Petri lm RYM Plates are used for enumeration of yeast and mold in as little as 48 h in the
food and beverage industries and 60 to 72 h in the cannabis industry. 3M Food Safety is certi ed to ISO
(International Organization for Standardization) 9001 for design and manufacturing.

B Apparatus and Reagents

(a) 3M Petri lm Rapid Yeast and Mold Count Plate.—Cat. Nos. 6475/6477 (3M Food Safety, St. Paul, MN,

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USA).

(b) Sterile diluents.—0.1% Peptone water (PW) or Butter eld’s phosphate bu er diluent (BPBD).

(c) Pipets.—Capable of 1000 µL or serological pipet.

(d) Sterile pipet tips.—Capable of 1000 µL.

(e) Stomacher.—Seward or equivalent.

(f) Filter stomacher bags.—Seward or equivalent.

(g) 3M Petri lm Flat Spreader.—Cat. No. 6425.

(h) 3M Swab Sampler with 10 mL Letheen broth.—Cat. No. RS96010LET or equivalent.

(i) Incubators.—Capable of maintaining 25 ± 1°C and 28 ± 1°C and having solid front to maintain dark
interior.

(j) Refrigerator.—Capable of maintaining 2–8°C, for storing the 3M Petri lm RYM Plates.

(k) Standard colony counter or illuminated magni er.

C General Instructions
(a) Store unopened 3M Petri lm RYM Plate pouches refrigerated or frozen (–20 to 8°C/–4 to 46°F). Just
prior to use, allow unopened pouches to come to room temperature before opening (20–25°C/<60%
relative humidity; RH). Return unused 3M Petri lm RYM Plates to pouch. Seal by folding end of
pouch over and applying adhesive tape. To prevent exposure to moisture, do not refrigerate opened
pouches. Store resealed pouches in cool dry place (20–25°C/<60% RH) for no longer than 4 weeks. It
is recommended that resealed pouches of 3M Petri lm RYM Plates be stored in freezer if the
laboratory temperature exceeds 25°C (77°F) and/or the laboratory is located in a region where RH
exceeds 60% (with the exception of air-conditioned premises).
To store opened pouches in freezer, place 3M Petri lm RYM Plates in sealable container.
Post-incubation 3M Petri lm RYM Plates can be stored at –10 to –20°C for up to 7 days.

(b) Follow all instructions carefully. Failure to do so may lead to inaccurate results.
D Sample Preparation
(a) Aseptically prepare 1:10 dilution of each test portion.

(1) Dairy products.—Pipet 11 mL or weigh 11 g sample into 99 mL sterile 0.1% PW. Shake 25 times to
homogenize.

(2) All other foods.—Weigh out 25 g sample from test portion into sterile stomacher bag and dilute
with 225 mL 0.1% PW; stomach at high speed to homogenize.

(3) Dried cannabis ower (THC >0.3%).—Weigh out 10 g sample from test portion into sterile
stomacher bag and dilute with 90 mL sterile 0.1% PW. Shake 25 times to homogenize.

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(4) Environmental surfaces.—Mix or shake swab in Letheen broth vigorously.

(b) Prepare 10-fold serial dilutions in 0.1% PW or BPBD. Environmental surface samples may be plated
directly as needed.

(c) Place 3M Petri lm RYM Plate on at, level surface for each dilution to be tested.

(d) Lift top of lm. Dispense 1 mL of each dilution onto center of bottom lm of each plate.

(e) Roll lm down onto sample.

(f) Place 3M Petri lm Flat Spreader on center of plate. Press gently on center of spreader to distribute
sample evenly. Spread inoculum over entire 3M Petri lm RYM Plate growth area before gel is
formed. Do not slide spreader across lm.

(g) Remove spreader and leave plate undisturbed for at least 1 min to permit gel to form.

(h) Incubate 3M Petri lm RYM Plates at 25 or 28°C in horizontal position with clear side up in stacks of
no more than 40.

(1) For food or environmental samples.—Enumerate plates after 48 h of incubation. If colonies

appear faint, allow for additional 12 h of incubation time for enhanced interpretation. If 60 h
time point for interpretation is not convenient, extending incubation time to 72 h is acceptable
alternative.

(2) For dried cannabis ower.—Enumerate plates at 60 to 72 h of incubation.

(i) 3M Petri lm RYM Plates can be counted using standard colony counter with the use of backlight or
illuminated magni er to assist with estimated enumeration. Do not count colonies on foam dam
since they are removed from the nutrient medium.

(j) Yeast colonies appear raised and small with de ned edges. Colonies may appear pink/tan or
blue/green in color.

(k) Mold colonies appear at with dark center and di used edges. Colonies may appear blue/green to
variable upon prolonged incubation. See Table 2014.05F for yeast and mold appearance.

2
(l) The circular growth area is approximately 30 cm . Plates containing >150 colonies can be either
estimated or recorded as too numerous to count (TNTC). Estimation can only be done by counting
the number of colonies in one or more representative squares and determining the average number
per square. The average number can be multiplied by 30 to determine the estimated count per plate.
If a more accurate count is required, the sample will need to be retested at higher dilutions. When
 the sample contains substantial amounts of mold, depending on the type of mold, the upper
countable limit may be at user discretion.

(m) Samples may occasionally show interference on the 3M Petri lm RYM Plates, for example:

(1) Uniform blue background color (often seen from organisms used in cultured products). These
should not be counted as TNTC.

(2) Intense pinpoint blue specks (often seen with spices, granulated products, or dried cannabis
ower).

(n) Report nal results as colony-forming units/gram (CFU/g).

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(o) If required, colonies may be isolated for further identi cation by direct microscopy or biochemical
analysis. Lift top lm and pick colony from gel.
Table 2014.05A. Interlaboratory study results of 3M Petrifilm RYM versus FDA-BAM and ISO 21527 methods for frozen raw ground beef patties

Contam. 3M Petrifilm RYM method FDA-BAM/ISO 21527 P- Di erence of Reverse transformed mean

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a d e
level methods value means di erence
b c
N Mean sr sR N Mean sr sR

25°C, 48 Control 11(0) <1.00 — — 11(0) <1.00 — — — — —

h

Low 11(0) 2.12 0.41 0.41 11(1) 2.07 0.36 0.38 0.5323 0.05 14.34

Medium 11(0) 3.52 0.10 0.10 11(0) 3.47 0.09 0.11 0.1637 0.05 360.10

High 11(0) 4.65 0.13 0.14 11(0) 4.59 0.10 0.14 0.2266 0.06 5763.84

25°C, 60 Control 11(0) <1.00 — — 11(0) <1.00 — — — — —

h
f
Low 11(0) 2.14 0.36 0.37 11(1) 2.07 0.36 0.38 0.3773 0.07 20.55

Medium 11(0) 3.52 0.10 0.10 11(0) 3.47 0.09 0.11 0.1573 0.05 360.10

High 11(0) 4.65 0.14 0.15 11(0) 4.59 0.10 0.14 0.1750 0.06 5763.84

28°C, 48 Control 11(0) <1.00 — — 11(0) <1.00 — — — — —

h
f
Low 11(0) 2.17 0.29 0.30 11(1) 2.07 0.36 0.38 0.1391 0.10 30.42

Medium 11(0) 3.53 0.10 0.10 11(0) 3.47 0.09 0.11 0.0824 0.06 437.23
f
High 11(0) 4.67 0.08 0.11 11(0) 4.59 0.10 0.14 0.0966 0.08 7869.00
 28°C, 60 Control 11(0) <1.00 — — 11(0) <1.00 — — — — —
h

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f
Low 11(0) 2.16 0.29 0.29 11(1) 2.07 0.36 0.38 0.1843 0.09 27.05

Medium 11(0) 3.53 0.09 0.10 11(0) 3.47 0.09 0.11 0.1095 0.06 437.23
f
High 11(0) 4.67 0.08 0.11 11(0) 4.59 0.10 0.14 0.1088 0.08 7869.00

a Samples were analyzed by harmonized FDA-BAM Chapter 18 and ISO 21527 methods using 0.1% peptone as the sample diluent.
b N = Number of laboratories that reported complete results. Outliers are in parentheses.

c Log10 yeast and mold CFU/g.

d Significant di erence (P <0.05).

e Results presented as CFU/g.

f Results indicate that the candidate method is more repeatable than the reference methods. sr = Repeatability standard deviation; sR = reproducibility standard
deviation.
Table 2014.05B. Interlaboratory study results of 3M Petrifilm RYM versus FDA-BAM and ISO 21527 methods for raw almonds

Contam. 3M Petrifilm RYM method FDA-BAM/ISO 21527 P- Di erence of Reverse transformed mean

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a d e
level methods value means di erence
b c
N Mean sr sR N Mean sr sR

25°C, 48 Control 12(0) <1.00 — — 12(0) <1.00 — — — — —

h
f
Low 14(0) 1.45 0.17 0.26 14(0) 1.55 0.19 0.34 0.4165 0.10 –7.30

Medium 14(1) 2.12 0.26 0.39 14(0) 2.21 0.20 0.24 0.3322 0.09 –30.36

High 14(2) 3.00 0.18 0.49 14(1) 3.08 0.12 0.31 0.2833 0.08 –202.26

25°C, 60 Control 12(0) <1.00 — — 12(0) <1.00 — — — — —

h

Low 14(0) 1.53 0.23 0.28 14(0) 1.55 0.19 0.34 0.8391 0.02 –1.60

Medium 14(0) 2.20 0.21 0.27 14(0) 2.21 0.20 0.24 0.7789 0.01 –3.69

High 14(2) 3.04 0.18 0.41 14(1) 3.08 0.12 0.31 0.5418 0.04 –105.79

28°C, 48 Control 12(0) <1.00 — — 12(0) <1.00 — — — — —

h
f
Low 14(0) 1.58 0.16 0.21 14(0) 1.55 0.19 0.34 0.7381 0.03 2.54
f
Medium 14(0) 2.17 0.17 0.29 14(0) 2.21 0.20 0.24 0.6139 0.04 –11.73

High 14(2) 3.01 0.17 0.45 14(1) 3.08 0.12 0.31 0.3904 0.07 –178.97
 28°C, 60 Control 12(0) <1.00 — — 12(0) <1.00 — — — — —
h

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f
Low 14(0) 1.60 0.17 0.20 14(0) 1.55 0.19 0.34 0.5474 0.05 4.33
f
Medium 14(0) 2.21 0.17 0.23 14(0) 2.21 0.20 0.24 0.9483 0.00 0.00

High 14(2) 3.03 0.18 0.42 14(1) 3.08 0.12 0.31 0.4687 0.05 –130.75
a Samples were analyzed by harmonized FDA-BAM Chapter 18 and ISO 21527 methods using 0.1% peptone as the sample diluent.
b N = Number of laboratories that reported complete results. Outliers are in parentheses.

c Log10 yeast and mold CFU/g.

d Significant di erence (P <0.05).

e Results presented as CFU/g.

f Results indicate that the candidate method is more repeatable than the reference methods. sr = Repeatability standard deviation; sR = reproducibility standard
deviation.
Table 2014.05C. Results of aerobic plate count for collaborating laboratories

Lab Frozen raw ground beef, CFU/g Raw almonds, CFU/g

2 1
1 3.8 × 10 6.0 × 10
3 2
2 1.1 × 10 6.0 × 10
1
3 <10 3.0 × 10

4 Not reported Not reported

3 1
5 2.8 × 10 2.8 × 10
1 1

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6 8.0 × 10 2.2 × 10
2 2
7 9.1 × 10 1.6 × 10

8 Not reported Not reported

2 2
9 9.0 × 10 2.0 × 10
3 2
10 1.3 x 10 4.0 × 10
1
11 >2500 1.0 × 10
1
12 Not reported 7.0 × 10
1 1
13 9.5 × 10 1.0 × 10
2 2
14 7.3 × 10 2.3 × 10
2 1
15 3.7 × 10 8.0 × 10
Table 2014.05D. Matrix studies: 3M Petrifilm RYM Plate vs Dichloran Rose Bengal Chloramphenicol (DRBC) agar–di erence of means

c d e
Matrix Contamination level 3M Petrifilm RYM DRBC DOM SE 90% CI 95%

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a b f g
Mean sr Mean sr LCL UCL LCL UCL

3M Petrifilm RYM Plate 25°C at 72 h

h
Cannabis flower Low 3.281 0.094 3.688 0.218 –0.407 0.106 –0.577 –0.236 –0.629 –0.185

Med 3.816 0.042 3.906 0.047 –0.09 0.028 –0.12 –0.06 –0.129 –0.051

High 5.139 0.063 5.145 0.204 –0.005 0.064 0.143 0.132 –0.184 0.174
i
Cannabis flower Low 2.850 0.222 3.151 0.211 –0.301 0.121 –0.468 0.047 –0.403 –0.199

High 5.435 0.180 5.539 0.143 –0.105 0.075 –0.264 0.055 –0.379 –0.222

3M Petrifilm RYM Plate 25°C at 60 h

h
Cannabis flower Low 2.788 0.217 3.151 0.211 –0.363 0.113 –0.427 –0.299 –0.446 –0.280

High 5.392 0.166 5.539 0.143 –0.147 0.072 –0.302 0.007 –0.348 0.054

3M Petrifilm RYM Plate 28°C at 72 h

h
Cannabis flower Low 3.181 0.067 3.688 0.218 –0.507 0.082 –0.682 –0.332 –0.736 –0.279

Med 3.697 0.039 3.906 0.047 –0.209 0.035 –0.284 –0.135 –0.306 –0.112

High 5.065 0.208 5.145 0.204 –0.08 0.130 –0.172 0.013 –0.200 0.041
i
Cannabis flower Low 2.838 0.214 3.061 0.226 –0.222 0.127 –0.493 0.048 –0.575 0.130
 High 5.450 0.176 5.539 0.143 –0.089 0.049 –0.194 0.016 –0.225 0.047

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3M Petrifilm RYM Plate 28°C at 60 h
h
Cannabis flower Low 2.817 0.204 3.061 0.226 –0.244 0.114 –0.486 –0.001 –0.560 0.072

High 5.432 0.188 5.539 0.143 –0.107 0.054 –0.221 0.007 –0.256 0.042

a Mean of five replicate portions, a er logarithmic transformation: Log10[CFU/g + (0.1)f].

b sr = Repeatability standard deviation.

c DOM = Di erence of means.

d SE = Standard error on the mean di erence for paired analysis.

e CI = Confidence interval for DOM.

f LCL = Lower confidence limit for DOM.

g UCL = Upper confidence limit for DOM.

h Samples tested at North Coast Laboratories.

i Samples tested at Aurum Labs.
Table 2014.05E. Matrix study: 3M Petrifilm RYM Plate vs BAM Ch. 18 results

e f g
Matrix Contam. 3M Petrifilm FDA/BAM Ch. DOM SE 90% CI 95% CI

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a d
level RYM 18
b c h i
Mean sr Mean sr LCL UCL LCL UCL

3M Petrifilm RYM Plate (25°C, 48 h)

j
Stainless steel/Aspergillus flavus ATCC 9643 Low 1.004 0.000 1.124 0.164 – 0.073 – 0.037 – 0.084
0.120 0.276 0.323

Med 2.054 0.061 2.445 0.055 – 0.037 – – – –

0.391 0.461 0.321 0.478 0.304

High 3.044 0.076 3.254 0.105 – 0.031 – – – –

0.211 0.278 0.144 0.298 0.123

Rubber/Penicillium chrysogenum ATCC Low 1.745 0.066 1.745 0.066 0.000 0.021 – 0.045 – 0.059
10106 0.045 0.059

Med 2.794 0.109 2.811 0.115 – 0.054 – 0.096 – 0.131

0.018 0.132 0.166

High 3.320 0.088 3.440 0.173 – 0.057 – 0.001 – 0.038

0.121 0.242 0.279
k
Sealed concrete/Candida lusitaniae QL Low 1.752 0.256 1.722 0.250 0.029 0.145 – 0.339 – 0.433
15166-2 0.281 0.374

Med 2.691 0.182 2.623 0.190 0.068 0.058 – 0.193 – 0.230

0.056 0.094
 High 3.748 0.146 3.727 0.143 0.021 0.063 – 0.156 – 0.197
0.114 0.155

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3M Petrifilm RYM Plate (25°C, 60 h)

Stainless steel/Aspergillus flavus ATCC 9643 Low 1.004 0.000 1.124 0.164 – 0.073 – 0.037 – 0.084
0.120 0.276 0.323

Med 2.086 0.030 2.445 0.055 – 0.027 – – – –

0.359 0.416 0.301 0.434 0.284

High 3134 0.078 3.254 0.105 – 0.039 – – – –

0.120 0.203 0.037 0.228 0.012

Rubber/Penicillium chrysogenum ATCC Low 1.772 0.083 1.745 0.066 0.027 0.034 – 0.099 – 0.121
10106 0.044 0.066

Med 2.786 0.093 2.811 0.115 – 0.056 – 0.094 – 0.130

0.026 0.146 0.182

High 3.405 0.061 3.440 0.173 – 0.056 – 0.085 – 0.121

0.035 0.155 0.191

Sealed concrete/Candida lusitaniae QL Low 1.761 0.265 1.722 0.250 0.038 0.149 – 0.356 – 0.452
15166-2 0.280 0.376

Med 2.690 0.179 2.623 0.190 0.068 0.059 – 0.193 – 0.231

0.058 0.096

High 3.742 0.140 3.727 0.143 0.015 0.059 – 0.141 – 0.179

0.111 0.150

3M Petrifilm RYM Plate (28°C, 48 h)

Stainless steel/Aspergillus flavus ATCC 9643 Low 1.004 0.000 1.124 0.164 – 0.073 – 0.037 – 0.084
0.120 0.276 0.323

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Med 2.105 0.050 2.445 0.055 – 0.034 – – – –
0.339 0.411 0.268 0.433 0.246

High 3.109 0.111 3.254 0.105 – 0.037 – – – –

0.145 0.225 0.066 0.249 0.042

Rubber/Penicillium chrysogenum ATCC Low 1.761 0.062 1.745 0.066 0.016 0.035 – 0.090 – 0.113
10106 0.059 0.081

Med 2.781 0.100 2.811 0.115 – 0.047 – 0.069 – 0.099

0.030 0.129 0.159

High 3.312 0.101 3.440 0.173 – 0.070 – 0.022 – 0.067

0.128 0.278 0.323

Sealed concrete/Candida lusitaniae QL Low 1.788 0.232 1.722 0.250 0.066 0.055 – 0.184 – 0.220
15166-2 0.053 0.088

Med 2.723 0.144 2.623 0.190 0.100 0.055 – 0.217 – 0.252

0.017 0.053

High 3.627 0.301 3.727 0.143 – 0.097 – 0.106 – 0.168

0.100 0.306 0.368

3M Petrifilm RYM Plate (28°C, 60 h)

Stainless steel/Aspergillus flavus ATCC 9643 Low 1.004 0.000 1.124 0.164 – 0.073 – 0.037 – 0.084
0.120 0.276 0.323

Med 2.164 0.085 2.445 0.055 – 0.042 – – – –

0.281 0.370 0.192 0.397 0.165
 High 3.154 0.103 3.254 0.105 – 0.030 – – – –
0.100 0.164 0.036 0.183 0.017

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Rubber/Penicillium chrysogenum ATCC Low 1.766 0.119 1.745 0.066 0.021 0.049 – 0.126 – 0.158
10106 0.083 0.115

Med 2.772 0.105 2.811 0.115 – 0.045 – 0.057 – 0.086

0.040 0.136 0.166

High 3.341 0.119 3.440 0.173 – 0.074 – 0.058 – 0.105

0.099 0.256 0.303

Sealed concrete/Candida lusitaniae QL Low 1.788 0.232 1.722 0.250 0.066 0.055 – 0.184 – 0.220
15166-2 0.053 0.088

Med 2.735 0.149 2.623 0.190 0.112 0.058 – 0.236 – 0.273

0.011 0.049

High 3.723 0.168 3.727 0.143 – 0.039 – 0.078 – 0.102

0.005 0.087 0.111
2
a All surfaces are artificially contaminated, 100 cm test areas.
b Mean of five replicate portions, a er logarithmic transformation: Log10[CFU/g + (0.1)f] where f is the smallest reportable result.

c sr = Repeatability standard deviation.

d FDA/BAM Ch. 18, Yeasts, Molds, and Mycotoxins.

e DOM = Di erence of means between candidate and reference methods.
f SE = Standard error.
g CI = Confidence interval.
h LCL = Lower confidence limit for DOM.
i UCL = Upper confidence limit for DOM.
j ATCC = American Type Culture Collection, Manassas, VA, USA.
k QL = Q Laboratories Culture Collection, Cincinnati, OH, USA.

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Table 2014.05F. Appearance of yeast and mold on 3M Petrifilm RYM Plates

Yeast colonies Mold colonies

Small Large

Defined edges Di used edges

Pink/tan to blue/green in color Blue/green to variable upon prolonged incubation

Appear raised (3-dimensional) Appear flat

Uniform color Dark center with di used edges

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References:

J. AOAC Int. 98, 767(2015) (First Action) DOI: 10.5740/jaoacint.15-006

J. AOAC Int. 106, 389(2023) (Matrix extension to selected surfaces) DOI: [Link]

J. AOAC Int. 106, 401(2023) (Matix extension to dried cannabis flower) DOI: [Link]

AOAC SMPR 2021.009 (Yeast and Mold Count Enumeration in Cannabis and Cannabis Products) [Link]
content/uploads/2021/06/SMPR-2021_009.pdf

17.2.12 AOAC O icial Method SM 2015.13

Enumeration of Aerobic Bacteria in Food and on Selected Surfaces:
3MTM PetrifilmTM Rapid Aerobic Count (RAC) Plate

First Action 2015

Final Action 2018

Revised First Action 2022 (for Selected Surfaces Only)

[Applicable to the enumeration of aerobic bacteria from raw ground beef, raw ground pork, raw ground
turkey, chicken carcass rinsate, fresh swai, fresh tuna, fresh tiger shrimp, raw easy-peel shrimp, cherry
tomato wash, frozen blueberries, Mediterranean apricots, creamy salad dressing, fresh pasta, vanilla ice
cream, instant nonfat dry milk (NFDM), pasteurized skim milk, stainless steel, sealed concrete, and rubber.]

Caution: After use, the diluents and 3M Petri lm RAC Plates may contain microorganisms that may be a
potential biohazard. When testing is complete, follow current industry standards for disposal of
contaminated waste. Consult Material Safety Data Sheet for additional information and local regulations for
disposal.

To reduce risks associated with bacterial infection and workplace contamination, perform 3M Petri lm RAC
Plate testing in properly equipped laboratory under control of skilled microbiologist. The user must train
personnel in current proper testing techniques—for example, good laboratory practices, ISO 17025, or ISO
7218.

See Tables 2015.13A and B for results of the interlaboratory study supporting acceptance of the method.

See Table 2015.13C for results of the matrix study supporting extension of the method.
A Principle
The 3M Petri lm RAC Plate is a sample-ready culture medium system that contains nutrients, cold-water-
soluble gelling agent, and indicator system that facilitates aerobic bacterial enumeration. 3M Petri lm RAC
Plates are used for the enumeration of aerobic bacteria in as little as 24 h for most food matrixes. 3M Food
Safety is certi ed to ISO (International Organization for Standardization) 9001 for design and
manufacturing.

B Apparatus and Reagents

(a) 3M Petri lm RAC Plate.—Cat. Nos. 6478/6479 (3M Food Safety, St. Paul, MN, USA).

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(b) Sterile diluent.—Butter eld’s Phosphate-Bu ered Diluent (BPBD).

(c) Pipets.—Capable of pipetting 1000 μL or serological pipet.

(d) Sterile pipet tips.—Capable of 1000 μL.

(e) Stomacher.—Seward or equivalent.

(f) Filter stomacher bags.—Seward or equivalent.

(g) 3M Petri lm Flat Spreader.—Cat. No. 6425.

(h) 3M Swab Sampler with 10 mL Letheen broth.—Cat. No. RS96010LET or equivalent.

(i) Incubators.—Capable of maintaining 32 ± 1°C and 35 ± 1°C and having solid front to maintain dark
interior.

(j) Refrigerator or freezer.—Capable of maintaining temperature between –20 and 8°C for storing
unopened 3M Petri lm RAC Plates.

(k) Freezer.—Capable of maintaining temperature at less than –15°C for storing 3M Petri lm RAC
pouches after incubation.

(l) Standard colony counter or illuminated magni er.

C General Instructions
(a) Storage conditions.—Store 3M Petri lm RAC Plates at –20 to 8°C. After opening 3M Petri lm RAC
Plate pouches, seal pouch, and store at ambient temperature, <60% relative humidity. Post-
incubation 3M Petri lm RAC Plates can be stored at less than –15°C for up to 1 week.

(b) Spreader.—Place 3M Petri lm Flat Spreader on center of plate when preparing sample aliquot to
prevent trapping air bubbles.

(c) Follow all instructions carefully. Failure to do so may lead to inaccurate results.
D Sample Preparation
(a) Aseptically prepare 1:10 dilution of each test portion.

(1) Dairy products.—Pipet 11 mL or weigh 11 g sample into 99 mL sterile BPBD.

(2) All other foods.—Weigh 50 g test portion into sterile stomacher bag and dilute with 450 mL
BPBD; blend or homogenize per standard.

(3) Environmental surfaces.—Mix or shake swab in Letheen vigorously.

(b) Prepare 10-fold serial dilutions in BPBD. Environmental surface samples may be plated directly as
needed.

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(c) Place 3M Petri lm RAC Plates on at, level surface for each dilution to be tested.

(d) Lift lm. With the pipet perpendicular, dispense 1 mL of each dilution onto center of bottom lm of
plate.

(e) Roll lm down onto sample.

(f) Place 3M Petri lm Flat Spreader on center of plate. Press gently on center of spreader to distribute
sample evenly. Spread inoculum over entire 3M Petri lm RAC Plate growth area before gel is
formed. Do not slide spreader across lm.

(g) Remove spreader and leave plate undisturbed for at least 1 min to permit gel to form.

(h) Incubate 3M Petri lm RAC Plate at either 32 ± 1°C (seafood and dairy products) or 35 ± 1°C (all other
foods and environmental surfaces) in horizontal position with clear side up in stacks of no more
than 20 (for dairy products) or 40 (for all other foods). Enumerate plates after 24 ± 2 h of incubation
(or 48 ± 3 h in the case of dairy powders, including whey powder). 3M Petri lm RAC Plates can be
counted using standard colony counter with use of backlight or illuminated magni er to assist with
estimated enumeration.

(i) Enumerate all colonies regardless of size, color, or intensity.

2
(j) Circular growth area is approximately 30 cm . Plates containing >300 colonies can be either
estimated or recorded as too numerous to count (TNTC). Estimation can be done only by counting
the number of colonies in one or more representative squares and determining the average number
per square. The average number can be multiplied by 30 to determine the estimated count per plate.
If a more accurate count is required, the sample may need to be retested at higher dilutions.

(k) Report nal results as colony-forming units per gram or milliliter (CFU/g or CFU/mL).
Note: If there are two dilutions within the countable range, use the following calculation to
determine the nal count:

N = ΣC/ (1.1 × d)

where N = number of colonies per milliliter or per gram of product; ΣC = sum of all colonies on both
plates; and d = dilution from which rst counts were obtained.

(l) Food samples may occasionally show interference on 3M Petri lm RAC Plates—for example:

(1) Uniform blue background color (often seen from organisms used in cultured products). These
should not be counted as TNTC.
 (2) Intense pinpoint blue specs (often seen with spices or granulated products).

(m) When necessary, colonies may be isolated for further identi cation test using standard procedures.
Lift top lm and pick colony from gel.

Revised: October 2018; October 2019 [D(11): Deleted “Average the counts between the replicate plates” for clarity];
May 2022 (Matrix extension to selected environmental surfaces)

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Table 2015.13A. Interlaboratory study results of 3M Petrifilm RAC Plate vs FDA BAM Chapter 3 method for raw easy-peel shrimp

Matrix 3M Petrifilm RAC Plate FDA BAM Chapter 3 Di erence Di erence Reverse- Reverse-

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of means of means transformed transformed
a b c
Lot N Mean sr sR Lot N Mean sr sR 95% LCL, di erence of di erence of
d,e
log10 log10 UCL mean, means LCL,
CFU/g CFU/g CFU/g UCL

Raw Low 16 2.96 0.132 0.280 Low 16 3.02 0.218 0.356 0.06 –0.11, 0.24 139.47 0.77, 1.72
easy-
peel Medium 16 4.29 0.202 0.215 Medium 16 4.23 0.095 0.298 –0.06 –0.18, 0.06 –2424.10 0.67, 1.15
shrimp
32•C High 16 5.56 0.110 0.248 High 16 5.76 0.097 0.214 0.20 –0.01, 0.42 214352.79 0.97, 2.61

Raw Low 16 2.80 0.121 0.335 Low 16 3.02 0.218 0.356 0.22 –0.03, 0.48 422.68 0.92, 3.03
easy-
peel Medium 16 4.22 0.172 0.273 Medium 16 4.23 0.095 0.298 0.01 –0.08, 0.11 539.37 0.83, 1.28
shrimp
35•C High 16 5.67 0.141 0.174 High 16 5.76 0.097 0.214 0.09 –0.09, 0.26 105217.30 0.82, 1.83

a N = Number of laboratories that reported complete results.

b sr = Repeatability.

c sR = Reproducibility.

d LCL, UCL = 95% Lower and upper confidence limits, respectively.

e A 95% confidence interval that contains the point 0 indicates no statistical significant di erence between methods.
Table 2015.13B. Interlaboratory study results of 3M Petrifilm RAC Plate vs SMEDP Chapter 6 method for pasteurized skim milk and instant NFDM

Matrix 3M Petrifilm RAC Plate SMEDP Chapter 6 Di erence Di erence Reverse- Reverse-

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of means of means transformed transforme
a b c
Lot N Mean sr sR Lot N Mean sr sR 95% LCL, di erence of di erence o
d,e
log10 log10 UCL mean, means LCL,
CFU/g CFU/g CFU/g UCL

Pasteurized Low 13 2.51 0.131 0.310 Low 13 2.47 0.123 0.301 –0.04 –0.08, 0.01 24.56 0.83, 1.03
skim milk
Medium 13 3.53 0.180 0.242 Medium 13 3.48 0.119 0.264 –0.05 –0.13, 0.03 346.20 0.75, 1.08

High 13 4.63 0.136 0.232 High 13 4.58 0.116 0.196 –0.05 –0.11, 0.01 4936.41 0.78, 1.00

Instant Low 15 2.42 0.096 0.126 Low 15 2.34 0.129 0.179 –0.08 –0.16, 0.01 42.05 0.69, 1.02
NFDM
Medium 15 3.04 0.059 0.148 Medium 15 2.98 0.104 0.195 –0.06 –0.14, 0.01 153.18 0.73, 1.02

High 15 4.26 0.174 0.190 High 15 4.19 0.185 0.197 –0.07 –0.14, 0.01 2806.94 0.71, 1.00

a N = Number of laboratories that reported complete results.

b sr = Repeatability.

c sR = Reproducibility.

d LCL, UCL = 95% Lower and upper confidence limits, respectively.

e A 95% confidence interval that contains the point 0 indicates no statistical significant di erence between methods.
Table 2015.13C. Matrix study: 3M Petrifilm RAC Plate vs BAM Ch. 3

e f g
Matrix/organism Contamination 3M Petrifilm RAC FDA/BAM Ch. DOM SE 90% CI 95% CI

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a d
level Plate 3
b c h i
Mean sr Mean sr LCL UCL LCL UCL

j
Stainless steel/Listeria innocua (ATCC Low 1.609 0.297 1.531 0.244 0.078 0.078 – 0.244 – 0.294
33090) 0.088 0.139

Med 2.144 0.156 2.093 0.133 0.051 0.057 – 0.173 – 0.210

0.072 0.109

High 3.123 0.107 3.191 0.095 – 0.017 – – – -0.021

0.067 0.103 0.032 0.114

Rubber/Klebsiella aerogenes (ATCC Low 1.823 0.056 1.803 0.101 0.020 0.047 – 0.119 – 0.150
35029) 0.080 0.110

Med 2.862 0.042 2.819 0.070 0.043 0.041 – 0.130 – 0.156

0.044 0.070

High 3.871 0.033 3.801 0.087 0.070 0.029 0.008 0.131 – 0.150
0.010

Sealed concrete/Klebsiella oxytoca Low 1.659 0.243 1.788 0.110 – 0.136 – 0.160 – 0.247
(ATCC 43165) 0.129 0.419 0.506

Med 2.136 0.017 2.141 0.042 – 0.016 – 0.028 – 0.038

0.005 0.038 0.048

High 3.105 0.055 3.124 0.062 – 0.008 – – – 0.003

0.019 0.036 0.002 0.041
2
a All surfaces are artificially contaminated, 100 cm test areas.
b Mean of five replicate portions, a er logarithmic transformation: Log10[CFU/g + (0.1)f] where f is the smallest reportable result.

c sr = Repeatability standard deviation.

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d FDA/BAM Ch. 3, Aerobic Plate Count.

e DOM = Di erence of means between candidate and reference methods.

f SE = Standard error on mean di erence.
g CI = Confidence interval.

h LCL = Lower confidence limit for di erence of means.

i UCL = Upper confidence limit for di erence of means.
j ATCC = American Type Culture Collection, Manassas, VA, USA.
References:

J. AOAC Int. 99, 664(2016) (First Action) DOI: 10.5740/jaoacint.15-0260

J. AOAC Int. 106, 165(2023) (Matrix extension) DOI: [Link]

17.2.13 AOAC O icial Method 2018.02

Enumeration of Yeast and Mold in Select Foods MC-Media Pad™ Yeast
and Mold (YM) Device

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First Action 2018

[Applicable to the enumeration of yeast and mold from breaded chicken nuggets, dry pet food, orange juice
concentrate, yogurt, and cake mix.]

See Table 2018.02 for a summary of results of the collaborative study.

A Principle
The MC-Media Pad YM is intended to enumerate viable yeast and mold in select foods in as little as 48 h.
The MC-Media Pad YM uses a special medium composition and unique redox indicator for detection. Once
the liquid sample is inoculated onto the test pad, the sample di uses through the whole pad by capillary
action. The medium reconstitutes automatically. If target organisms are present, they grow red colored
colonies on the test pad.

B Apparatus and Reagents

(a) MC-Media Pad YM.—100 Test pads (25 pads/pouch, four pouches/box; MilliporeSigma, Cat. No.
1323030001).

(b) Sterile diluent.—0.1% Peptone water and phosphate bu ered saline (PBS).

(c) Pipets.—Capable of pipetting 1000 μL, or a serological pipet.

(d) Sterile pipet tips.—Capable of 1000 μL.

(e) Laboratory paddle-blender.—Seward 400, or equivalent. (f) Filter stomacher bags.—Seward, or

equivalent.

(g) Incubators.—Capable of maintaining 25 ± 1°C.

(h) Refrigerator.—Capable of maintaining 2–15°C, for storing the MC-Media Pads.

(i) Standard colony counter or illuminated magni er.

(j) Top-loading balance.—Capable of weighing 1–2000 g.

 C General Instructions
(a) Read the instruction manual carefully before use.

p. C17-31 (b) Store the MC-Media Pads at 2–15°C. After opening the aluminum bag, unused pads should be
stored in the aluminum bag sealed with tape and kept in a cool (2–15°C) environment. After opening,
use all pads within 1 month.

(c) Do not expose unused pads to sunlight or ultraviolet light.

(d) Do not use a discolored or damaged pad.

(e) A wrinkle on the test pad should not a ect detection.

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(f) Small fragments of fabric on or around the test pad should not a ect detection.

(g) Do not use the pads after the expiration date. The quality of an expired product is not guaranteed.

(h) The measurement range should be <200 CFU/pad. If more than 200 CFU/pad are counted, further
dilution is recommended.

(i) The nature of food (high-viscosity liquids, e.g., raw egg; syrups; or the presence of food dye or
strongly colored products such as tomato concentrate, cocoa powder, or ground spices) may a ect
test use or results. In that case, the cause needs to be eliminated. Typically, a further 1:10 dilution will
reduce the viscosity or background color su ciently, whereas sedimentation or ltration may
remove (colored) particulates.

(j) The used kit must be decontaminated by autoclaving or boiling and disposed according to local
regulations for waste.

D Safety Precautions
After use, the diluents and MC-Media Pads may contain microorganisms that may be potential biohazards.
When testing is complete, follow current industry standards for the disposal of contaminated waste. Consult
the Safety Data Sheet for additional information and comply with local regulations for disposal. To reduce
the risks associated with fungal infection and workplace contamination, perform MC-Media Pad testing in a
properly equipped laboratory under the control of a skilled microbiologist.

E Sample Preparation
(a) Solid foods.—Place a 25 g test portion in a ltered laboratory blender bag and add 225 mL 0.1%
peptone water. If necessary, adjust the pH to neutral. Homogenize with a laboratory blender.
Table 2018.02 Interlaboratory study results of MC-Media Pad YM versus FDA BAM Chapter 18

MC- Pad YM FDA BAM Ch. 18

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Media
a b c
Matrix Lot N Mean, sr sR Lot N Mean, sr sR Di erence Di erence
d
log10 log10 of means of means
e
CFU/g CFU/g 95% LCL ,
f
UCL

Orange juice concentrate Uninoculated 11 0.000 0.00 0.00 Uninoculated 11 0.00 0.00 0.00 0.00 0.00, 0.00
48 h incubation
Low 11 1.704 0.13 0.41 Low 11 1.893 0.11 0.33 –0.19 –0.04, –
0.34

Medium 11 2.486 0.05 0.20 Medium 11 2.615 0.05 0.26 –0.10 –0.01, –
0.20

High 11 3.326 0.13 0.20 High 11 3.367 0.09 0.17 –0.04 –0.15, 0.07

Orange juice concentrate Uninoculated 11 0.000 0.00 0.00 Uninoculated 11 0.000 0.00 0.00 0.00 0.00, 0.00
72 h incubation
Low 11 1.996 0.10 0.22 Low 11 1.893 0.11 0.33 0.10 –0.14, 0.22

Medium 11 2.555 0.09 0.25 Medium 11 2.615 0.05 0.26 –0.06 –0.20, 0.08

High 11 3.505 0.10 0.13 High 11 3.367 0.09 0.17 0.14 0.05, 0.23
a N = Number of laboratories that reported complete results.
b sr = Repeatability standard deviation.

c sR = Reproducibility standard deviation.

d A di erence of means <0.5 indicates no statistically significant di erence between methods.

e LCL = Lower confidence limit.
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UCL = Upper confidence limit.
f
 (b) Liquids.—Liquid samples can be applied directly. If necessary, adjust the pH to neutral (pH 7.0 ± 0.2).
For all products, if necessary, make 10-fold serial dilutions with 0.1% peptone water.

(c) Open the aluminum bag and remove MC-Media Pad.

(d) Lift the transparent cover lm and pipet 1.0 mL sample solution onto test pad. It is recommended to
lift the cover lm diagonally for easy and secure resealing.

(e) Close the cover lm and lightly press the edges of the lm to seal.

(f) Incubate the test pad at 25 ± 1°C for 48–72 h.

(g) After incubation, count all reddish-colored colonies. Yeasts will appear as circular reddish colored

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colonies. Molds will appear as di use and fuzzy round reddish colored colonies.

(h) If a large number of colonies are di cult to count, estimate by counting colonies in one grid square
and multiplying by 20. If more than 104 microbes are grown, the entire test pad may appear stained,
and it may appear that no individual colonies were formed. If this occurs, dilute the sample further
and retest.

Reference:

J. AOAC Int. 102, 1138(2019)Crossref

DOI: 10.5740/jaoacint.18-0262
Posted: August 2019

17.2.14 AOAC O icial Method 2019.02

Enumeration of Total Aerobic Count in Select Foods
®
MC-Media Pad Rapid Aerobic Count (RAC)

First Action 2019

[Applicable to the enumeration of total aerobic count in raw chicken breast, raw ground pork, cream cheese,
yogurt drink, parsley, vegetable juice, prawns, tuna pâté, sandwiches, and pasta salad.]

See Tables 2019.02A and B for summary of results of the collaborative study. See Tables 2019.02C–G in J.
AOAC Int. [J. AOAC Int. 103, 1318(2020) DOI: 10.1093/jaoacint/qsaa040] for detailed results of the
collaborative study.

A Principle
The MC-Media Pad RAC is intended to enumerate viable total aerobic bacteria in select foods in as little as
24 h. The MC-Media Pad RAC uses a special medium composition that reconstitutes automatically and a
unique redox indicator for detection. Once the liquid sample is inoculated onto the test pad, the sample
di uses through the whole pad by capillary action. If target organisms are present, colonies appear red on
the test pad.
B Apparatus and Reagents
(a) MC-Media Pad RAC.—100 test pads (25 pads/pouch, four pouches/box). Available from
MilliporeSigma No. 132302.

(b) Sterile diluent.—Butter eld’s Phosphate Bu er (BPB).

(c) Pipets.—Capable of pipetting 1000 μL or a serological pipet.

(d) Sterile pipet tips.—Capable of 1000 μL.

(e) Laboratory paddle blender.—Seward 400 or equivalent.

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(f) Filter stomacher bags.—Seward or equivalent.

(g) Incubators.—Capable of maintaining 35 ± 1°C and 30 ± 1°C (ISO).

(h) Refrigerator.—Capable of maintaining 2–15°C, for storing media pads.

(i) Standard colony counter or illuminated magni er.

(j) Top-loading balance.—Capable of weighing 1–2000 g.

C General Instructions
(a) Read instruction manual carefully before use.
Table 2019.02A Collaborative study results of MC-Media Pad RAC vs USDA MLG 3.02

MC-Media Pad RAC USDA MLG 3.02

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a d
Matrix Lot n Mean, log10 sr sR n Mean, log10 sr sR Di erence of Di erence of means, 95%
b,c
CFU/g CFU/g 10 means LCL, UCL

Raw ground pork Low 9 3.438 0.08 0.12 9 3.423 0.16 0.18 0.015 –0.038, 0.068
(24 h)

Medium 9 4.768 0.07 0.14 9 4.754 0.09 0.16 0.014 –0.013, 0.042

High 9 5.313 0.12 0.17 9 5.322 0.08 0.16 –0.008 –0.054, 0.037

Raw ground pork Low 9 3.465 0.09 0.13 9 3.423 0.16 0.18 0.042 –0.018, 0.101
(48 h)

Medium 9 4.782 0.08 0.14 9 4.754 0.09 0.16 0.029 0.005, 0.052

High 9 5.354 0.11 0.15 9 5.322 0.08 0.16 0.032 –0.001, 0.066
a n = Number of laboratories that reported complete results.
b Mean log10 candidate method – mean log10 reference method.

c A 95% confidence interval for the true mean di erence within ±0.5 indicates equivalence of the two methods.
d 95% lower (LCL) and upper confidence limits (UCL).
Table 2019.02B Collaborative study results of MC-Media Pad RAC vs SMEDP

MC-Media Pad RAC SMEDP

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a d
Matrix Lot n Mean, log10 sr SR n Mean, log10 sr sR Di erence of Di erence of means, 95%
b,c
CFU/g CFU/g means LCL, UCL

Yogurt drink (24 Low 9 2.311 0.29 0.29 9 2.331 0.09 0.12 –0.020 –0.053, 0.013
h)
Medium 9 3.766 0.08 0.12 9 3.740 0.06 0.12 0.026 0.008, 0.043

High 9 4.266 0.10 0.16 9 4.266 0.09 0.15 –0.001 –0.032, 0.030

Yogurt drink (48 Low 9 2.329 0.12 0.12 9 2.331 0.09 0.12 –0.002 –0.029, 0.025
h)
Medium 9 3.785 0.07 0.11 9 3.740 0.06 0.12 0.045 0.028, 0.062

High 9 4.278 0.10 0.16 9 4.266 0.09 0.15 0.012 –0.023, 0.047

a n = Number of laboratories that reported complete results.

b Mean log10 candidate method – mean log10 reference method.

c A 95% confidence interval for the true mean di erence within ±0.5 indicates equivalence of the two methods.

d 95% lower (LCL) and upper confidence limits (UCL).

 (b) Storage conditions.—Store media pads at 2–15°C. After opening aluminum bag, store unused pads in
aluminum bag sealed with tape and keep in a cool (2–15°C) environment. After opening, use all pads
within 1 month.

(c) Do not expose unused pads to sunlight or ultraviolet light.

(d) Do not use a discolored or damaged pad.

(e) A wrinkle on the test pad should not a ect detection.

(f) Small fragments of fabric on or around the test pad should not a ect detection.

(g) Do not use the pads after the expiration date. The quality of an expired product is not warranted.

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(h) Measurement range is less than 300 CFU/pad. If more than 300 CFU/pad are counted, further
dilution is recommended.

(i) Nature of food (high viscosity or presence of food dye) may a ect test usage or results. In that case,
the cause needs to be eliminated by dilution or other means.

(j) Decontaminate used kit by autoclaving or boiling and dispose of according to local regulations for
waste.

D Safety Precautions
After use, diluents and media pads may contain microorganisms that may be a potential biohazard. When
p. C17-32testing is complete, follow current industry standards for disposal of contaminated waste. Consult Safety
Data Sheet for additional information and comply with local regulations for disposal. To reduce risks
associated with bacterial infection and workplace contamination, perform media pad testing in a properly
equipped laboratory under the control of a skilled microbiologist.

E Sample Preparation
Prepare samples depending on which reference method is being followed. See below for the validated
reference methods to which the MC-Media Pad RAC test pad was compared.

(a) Microbiology Laboratory Guidebook (MLG) 3.02.—Place a 50 g test portion in a ltered laboratory
blender bag and add 450 mL BPB. Homogenize with a laboratory blender.

(b) Standard Methods for the Examination of Dairy Products (SMEDP) Chapter 6.—Place an 11 g or 11 mL test
portion into a ltered laboratory blender bag and add 99 mL BPB. Homogenize with a laboratory
blender.

(c) 966.23 (see 17.2.01).—Parsley (50 g), vegetable juice (50 mL), cooked prawns (50 g), tuna pâté (50
g), egg salad sandwich (50 g), and deli pasta salad (50 g). Place a 50 g test portion in a ltered
laboratory blender bag and add 450 mL BPB. Homogenize with a laboratory blender.

(d) ISO 4833:2013.—Raw chicken breast (10 g), raw ground pork (10 g), cream cheese (10 g), yogurt drink
(10 mL), parsley (10 g), vegetable juice (10 mL), cooked prawns (10 g), tuna pâté (10 g), egg salad
sandwich (10 g), and deli pasta salad (10 g). Place a 10 g test portion in a ltered laboratory blender
bag and add 90 mL of maximum recovery diluent (MRD). Homogenize with a laboratory blender.

For all products, if necessary, make 10-fold serial dilutions with BPB or MRD.
 (1) Open aluminum bag and remove MC-Media Pad.

(2) Lift transparent cover lm and pipet 1.0 mL of sample solution onto test pad (it is recommended to
lift the cover lm diagonally for easy and secure resealing).

(3) Close cover lm and lightly press edges of lm to seal.

(4) For preparation of samples according to the USDA MLG and SMEDP methods, incubate MC-Media
Pad RAC test plate at 35 ± 1°C for 24–48 h.

(5) For preparation of samples according to the ISO method, incubate MC-Media Pad RAC test plate at
30 ± 1°C for 72 ± 3 h.

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(6) After incubation, count all reddish-colored colonies.

(7) If the large number of colonies are di cult to count, estimate by counting colonies in one grid
4
square and multiplying by 20. If more than 10 microbes are grown, entire test pad may appear
stained and it may appear that no individual colonies were formed. If this occurs, dilute sample
further and retest.

Reference:

J. AOAC Int. 103, 1318(2020) (First Action)Crossref

DOI: 10.1093/jaoacint/qsaa040
Posted: April 2019 (Pre-publication); October 2020 (Post-publication)