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17.3.10
AOAC Official Method 2003.01
Enumeration of Enterobacteriaceae
in Selected Foods
Petrifilm™ Enterobacteriaceae Count Plate Method
First Action 2003
Final Action 2006
(Applicable to enumeration of Enterobacteriaceae organisms in
cheddar cheese, milk, flour, frozen prepared meals, frozen broccoli,
and nut pieces.)
See Tables 2003.01A and 2003.01B for the results of the
interlaboratory study supporting acceptance of the method.
A. Principle
Method uses bacterial culture plates of dry medium with pH
indicator and cold-water-soluble gel. Undiluted or diluted test
portions are added to plates at 1.0 mL per plate. Pressure applied to
plastic spreader placed on overlay film spreads the test portion
evenly over 20 cm2 growth area. Gelling agent is allowed to solidify,
and plates are incubated for 24 ± 2 h at 37° ± 1°C and then counted.
B. Apparatus and Reagents
(a) Petrifilm Enterobacteriaceae (EB) Count Plates.—Plates,
available from 3M Microbiology Products (3M Center, St. Paul, MN
55144, USA), contain violet red bile glucose (VRBG) nutrients, pH
in di ca tor, cold-wa ter-sol u ble gel ling agent, and tetrazolium
indicator dye.
(b) Plastic spreader.—Provided with Petrifilm plates; has
smooth flat side designed to spread test portion evenly over plate
growth area.
(c) Pipets.—1.0 and 10.0 mL serological pipets with 0.1 mL
graduations. (Calibrated electronic pipettor and tips, or equivalent,
may be used to deliver 1.0 mL.) Pipets must accurately deliver
required volume. Do not use pipets 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.
(d) Colony counter.—Standard apparatus, Quebec Model,
avail able from many suppliers, or one providing equivalent
magnification and visibility.
(e) Dilution water.—Prepare stock solution as follows: Dissolve
34 g KH2PO4 in 500 mL water, adjust to pH 7.2 with 1M NaOH (ca
175 mL), and dilute to 1 L with water. Prepare buffered water for
dilutions by diluting 1.25 mL stock solution to 1 L with boiled and
cooled water. Autoclave 15 min at 121°C.
(f) Sterile sodium hydroxide solution.—1M. Dissolve 40 g
NaOH in water and dilute to 1 L water. Autoclave 15 min at 121°C.
(g) Blender.—High-speed (16 000–18 000 rpm) with sterile jar.
(h) Incubator.—Maintaining 37° ± 1°C.
(i) Balance.—2000 ± 0.1 g capacity.
C. Sample Preparation
Aseptically weigh 11.0 g unthawed test portion into blender jar,
B(g). Add 99 mL di lu tion wa ter, B(e ), and blend a t
16 000–18 000 rpm for 2 min to homogenize. If entire test sample is
<50 g, weigh portion of test sample and add dilution water to make
1:10 dilution. Adjust pH of diluted test portion to 6.5–7.5 with 1M
NaOH, B(f), ca 0.1 mL/g test portion. Do not use diluents containing
citrate, bisulfite, or thiosulfate as they can inhibit growth. Prepare all
decimal dilutions with 90 mL dilution water plus 10 mL from
previous dilution. Mix all dilutions by shaking 25 times through
30 cm arc in 7 s.
D. Analysis
Place dry Petrifilm EB plate, B(a), on flat surface. Lift top film
and inoculate 1 mL test suspension onto center of film base.
Carefully roll top film down onto inoculum. Distribute test portion
over 20 cm2 growth area with downward pressure on center of
plas tic spreader de vice, B(b), flat side down. Leave plate
undisturbed for minimum of 1 min to permit gel to solidify. Incubate
plates for 24 ± 2 h at 37° ± 1°C. In incubator, place plates in
horizontal position, clear side up, in stacks not exceeding 20 plates.
Count plates promptly (within 2 h) after incubation period.
Count Petrifilm EB plates on standard colony counter, B(d), or
other illuminated magnifier. Count all red colonies producing gas
regardless of acid production. Also count all nongassing colonies
with yellow acid zones. Select plates with 10–150 colonies. If no
plate has at least 10 qualifying colonies, record the exact raw count
on the least dilute test suspension. If all the plates have counts >150,
make estimates on plates by counting the number of colonies in one or
more representative squares, determining the average number per
square, and multiplying the average number by 20. If plates are too
crowded for estimated counts, report the count as too numerous to
count (TNTC). Do not count colonies on the foam dam because they
are removed from the selective influence of the medium. Do not count
artifact bubbles that may be present.
Reference: J. AOAC Int. 86, 802(2003).
Posted: April 2006
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 Table 2003.01A. Repeatability performance of Petrifilm Enterobacteriaceae Count Plate, violet red bile glucose (VRBG), and most probable number (MPN) methods for
detection of Enterobacteriaceae in foods
Petrifilm Enterobacteriaceae Count Plate VRBG MP N
a b a b a b
Food Lev el n M ean sr RSDr, % r n M ean sr RSDr, % r n M ean sr RSDr, % r
Cheese Uninoculated 0 0 9 2 .0 9 0 .3 5 1 6 .7 5 0 .9 8
c d e
Low 12 3 .0 2 0 .1 8 5 .9 6 0 .5 1 12 3 .0 3 0 .0 6 1 .9 4 0 .1 7 11 3 .1 2 0 .3 1 9 .7 9 0 .8 6
c d
M ed i u m 12 4 .0 7 0 .1 4 3 .3 3 0 .3 8 12 4 .0 6 0 .1 1 2 .6 9 0 .3 1 12 4 .2 3 0 .3 8 8 .8 9 1 .0 5
c d ,f
High 10 4 .9 1 0 .0 5 0 .9 2 0 .1 3 12 4 .9 3 0 .1 3 2 .5 9 0 .3 6 9 4 .8 2 0 .2 6 5 .3 1 0 .7 2
Mi l k Uninoculated 0 0 7 1 .6 7 0 .3 1 1 8 .6 0 0 .8 7

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c,g d ,f
Low 11 2 .4 9 0 .0 6 2 .5 7 0 .1 8 12 2 .2 7 0 .5 0 2 2 .1 9 1 .4 2 12 1 .9 3 0 .1 2 6 .0 0 0 .3 2
c d ,f
M ed i u m 12 3 .3 4 0 .1 0 3 .0 0 0 .2 8 12 3 .2 7 0 .2 1 6 .4 6 0 .6 0 12 2 .6 5 0 .4 2 1 5 .7 9 1 .1 7
c d ,f
High 11 4 .3 4 0 .1 0 2 .3 7 0 .2 9 11 4 .1 9 0 .2 0 4 .6 9 0 .5 5 11 3 .2 4 0 .6 3 1 9 .3 4 1 .7 5
d
Flour Uninoculated 12 2 .7 0 0 .2 5 9 .3 3 0 .7 1 12 2 .9 8 0 .2 8 9 .2 3 0 .7 8 12 2 .4 4 0 .4 7 1 9 .2 0 1 .3 1
c
Low 12 2 .7 3 0 .3 2 1 1 .7 8 0 .9 1 12 2 .9 5 0 .3 0 1 0 .2 8 0 .8 6 12 2 .2 4 0 .4 0 1 8 .0 2 1 .1 3
c
M ed i u m 11 2 .8 3 0 .3 8 1 3 .3 1 1 .0 6 12 3 .0 6 0 .2 8 9 .1 5 0 .7 9 12 2 .2 0 0 .5 1 2 3 .3 6 1 .4 4
g d
High 12 2 .8 9 0 .2 6 8 .9 0 0 .7 2 12 3 .1 6 0 .2 8 8 .8 4 0 .7 9 12 2 .8 7 0 .5 2 1 8 .1 3 1 .4 6
Prepared meals Uninoculated 0 0 9 1 .4 7 0 .3 4 2 3 .0 4 0 .9 5
c d
Low 11 2 .6 1 0 .1 3 4 .8 1 0 .3 5 11 2 .5 2 0 .1 4 5 .6 9 0 .4 0 9 2 .4 2 0 .4 2 1 7 .4 3 1 .1 8
g d ,f
M ed i u m 11 3 .5 1 0 .0 8 2 .2 1 0 .2 2 12 3 .3 0 0 .1 4 4 .3 5 0 .4 0 12 3 .3 4 0 .3 8 1 1 .4 2 1 .0 7
g d ,f
High 11 4 .4 7 0 .0 8 1 .7 4 0 .2 2 12 3 .9 8 0 .2 1 5 .2 4 0 .5 9 8 4 .3 0 0 .5 2 1 2 .2 1 1 .4 7
Frozen broccoli Uninoculated 0 0 11 1 .3 2 0 .3 4 2 6 .0 6 0 .9 6
Low 10 1 .6 0 0 .3 2 1 9 .9 1 0 .9 0 5 1 .3 6 0 .3 2 2 3 .6 2 0 .9 1 11 1 .5 8 0 .3 6 2 3 .0 2 1 .0 2
c,g d ,f
M ed i u m 12 2 .6 6 0 .1 4 5 .1 5 0 .3 9 11 2 .0 2 0 .2 9 1 4 .2 8 0 .8 1 11 2 .1 1 0 .4 5 2 1 .1 8 1 .2 5
c,g
High 11 3 .4 4 0 .2 9 8 .4 9 0 .8 2 10 2 .8 4 0 .5 5 1 9 .3 8 1 .5 5 10 2 .7 1 0 .4 8 1 7 .7 3 1 .3 4
Nuts Uninoculated 10 2 .7 4 0 .2 1 7 .5 3 0 .5 8 10 2 .8 9 0 .2 4 8 .4 5 0 .6 9 9 2 .6 7 0 .3 4 1 2 .7 6 0 .9 5
d
Low 10 3 .5 0 0 .2 9 8 .4 0 0 .8 3 9 3 .6 4 0 .4 4 1 2 .0 6 1 .2 4 9 3 .4 8 0 .5 3 1 5 .3 1 1 .4 9
M ed i u m 7 4 .0 9 0 .5 0 1 2 .1 0 1 .4 0 7 4 .1 3 0 .4 7 1 1 .3 8 1 .3 2 5 3 .8 2 0 .6 8 1 7 .8 9 1 .9 1
d
High 7 4 .2 0 0 .2 9 6 .9 2 0 .8 2 7 4 .1 4 0 .4 9 1 1 .8 6 1 .3 9 7 3 .8 3 0 .8 5 2 2 .1 3 2 .3 7
a
Number of laboratories with positive results.
b
Log10 Enterobacteriaceae count/g.
c
Significantly different from MPN method (p < 0.05).
d
Significantly better repeatability than for the MPN method.
e
Significantly better repeatability than for the Petrifilm plate method.
f
Significantly better repeatability than for the VRBG method.
g
Significantly different from VRBG method (p < 0.05).
 Table 2003.01B. Reproducibility performance of Petrifilm Enterobacteriaceae Count Plate, VRBG, and MPN methods for detection of Enterobacteriaceae in foods
Petrifilm Enterobacteriaceae Count Plate VRBG MP N
a b a b a b
Food Lev el n M ean sr RSDR, % r n M ean sr RSDR, % r n M ean sr RSDR, % r
Cheese Control 0 0 9 2 .0 9 0 .9 8 4 6 .7 3 2 .7 3
Low 12 3 .0 2 0 .2 0 6 .6 9 0 .5 7 12 3 .0 3 0 .1 0 3 .4 3 0 .2 9 11 3 .1 2 0 .3 4 1 0 .8 0 0 .9 4
M ed i u m 12 4 .0 7 0 .1 4 3 .4 4 0 .3 9 12 4 .0 6 0 .1 2 2 .9 3 0 .3 4 12 4 .2 3 0 .3 8 8 .9 8 1 .0 6
High 10 4 .9 1 0 .2 4 4 .8 9 0 .6 8 12 4 .9 3 0 .2 9 5 .8 4 0 .8 1 9 4 .8 2 0 .2 7 5 .6 0 0 .7 6
Mi l k Control 0 0 7 1 .6 7 0 .5 8 3 4 .6 1 1 .6 2
Low 11 2 .4 9 0 .4 4 1 7 .6 7 1 .2 4 12 2 .2 7 0 .5 0 2 2 .1 9 1 .4 2 11 1 .9 3 0 .5 3 2 7 .6 0 1 .4 9
M ed i u m 12 3 .3 4 0 .1 8 5 .2 7 0 .5 0 12 3 .2 7 0 .3 9 1 1 .8 7 1 .0 9 11 2 .6 5 0 .7 3 2 7 .7 3 2 .0 5
High 10 4 .3 4 0 .1 8 4 .1 7 0 .5 1 10 4 .1 9 0 .3 2 7 .5 7 0 .8 9 9 3 .2 4 0 .9 3 2 8 .7 7 2 .6 1
Flour Control 12 2 .7 0 0 .3 3 1 2 .1 5 0 .9 2 12 2 .9 8 0 .3 7 1 2 .3 4 1 .0 4 12 2 .4 4 0 .4 7 1 9 .2 0 1 .3 1
Low 12 2 .7 3 0 .3 7 1 3 .4 6 1 .0 4 12 2 .9 5 0 .4 4 1 4 .9 9 1 .2 5 12 2 .2 4 0 .4 5 2 0 .2 1 1 .2 7
M ed i u m 11 2 .8 3 0 .4 1 1 4 .3 5 1 .1 5 12 3 .0 6 0 .4 2 1 3 .7 0 1 .1 8 12 2 .2 0 0 .6 3 2 8 .7 2 1 .7 7
High 12 2 .8 9 0 .2 9 1 0 .1 0 0 .8 2 12 3 .1 6 0 .3 7 1 1 .7 4 1 .0 4 12 2 .8 7 0 .6 5 2 2 .5 2 1 .8 1
Prepared meals Control 0 0 9 1 .4 7 0 .3 4 2 3 .0 4 0 .9 5
Low 11 2 .6 1 0 .1 7 6 .4 3 0 .4 7 11 2 .5 2 0 .2 8 1 1 .2 3 0 .8 0 9 2 .4 2 0 .4 2 1 7 .4 3 1 .1 8
M ed i u m 11 3 .5 1 0 .1 0 2 .7 2 0 .2 7 12 3 .3 0 0 .2 8 8 .3 9 0 .7 8 12 3 .3 4 0 .6 4 1 9 .3 0 1 .8 0
High 11 4 .4 7 0 .2 1 4 .7 1 0 .5 9 12 3 .9 8 0 .3 7 9 .2 2 1 .0 3 8 4 .3 0 0 .5 2 1 2 .2 1 1 .4 7
Frozen broccoli Control 0 0 11 1 .3 2 0 .5 1 3 8 .4 3 1 .4 2
Low 10 1 .6 0 0 .4 3 2 6 .9 9 1 .2 2 5 1 .3 6 0 .3 5 2 6 .0 3 1 .0 0 11 1 .5 8 0 .4 8 3 0 .2 7 1 .3 4
M ed i u m 12 2 .6 6 0 .4 0 1 4 .9 8 1 .1 2 11 2 .0 2 0 .6 1 3 0 .3 0 1 .7 2 11 2 .1 1 0 .7 8 3 6 .7 2 2 .1 7
High 11 3 .4 4 0 .5 5 1 6 .1 4 1 .5 6 10 2 .8 4 0 .6 8 2 3 .8 7 1 .9 1 10 2 .7 1 0 .8 7 3 2 .0 7 2 .4 3
Nuts Control 10 2 .7 4 0 .4 8 1 7 .4 7 1 .3 5 10 2 .8 9 0 .4 2 1 4 .7 0 1 .2 0 9 2 .6 7 0 .3 4 1 2 .7 6 0 .9 5
Low 10 3 .5 0 0 .6 8 1 9 .3 3 1 .9 1 9 3 .6 4 0 .7 1 1 9 .5 7 2 .0 1 9 3 .4 8 0 .6 5 1 8 .5 8 1 .8 1
M ed i u m 7 4 .0 9 0 .6 6 1 6 .2 0 1 .8 7 7 4 .1 3 0 .6 8 1 6 .5 0 1 .9 2 5 3 .8 2 0 .6 9 1 8 .0 2 1 .9 3
High 7 4 .2 0 0 .4 7 1 1 .1 8 1 .3 2 7 4 .1 4 0 .5 5 1 3 .3 7 1 .5 6 7 3 .8 3 1 .0 9 2 8 .4 0 3 .0 5
a
Number of laboratories with positive results.
b
Log10 Enterobacteriaceae count/g.

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