17.3.04B AOAC Official Method 2005.03 Detection and Confirmed Quantitation of Coliforms and E.

coli in Foods
SimPlate Coliform and E. coli Color Indicator First Action 2005

E. Preparation of Test Suspensions

(Applicable to detection and quantitation of confirmed total coliforms and E. coli in cake mix, chocolate, condiments, dairy foods, egg products, flour, frozen prepared meals, frozen vegetables, fruits, fruit juices, mushrooms, nut meats, pasta, poultry meats, red meats, seafood, and spices.) See Tables 2005.03A and 2005.03B for the results of the interlaboratory study supporting acceptance of the method.
A. Principle

(a) Weigh 50 g test portion into 450 mL sterile diluent, i.e., Butterfields phosphate buffer. This is a 1:10 dilution. Macerate test portion. (b) If an alternative test portion size is specified in testing procedure or standard, prepare a 10% weight-to-volume suspension. (c) If necessary, prepare 10-fold serial dilutions appropriate for the anticipated population of the product.
F. CEc-CI Test Procedure, Single Test Medium

(a) For 1.0 mL test suspension size.Resuspend powdered medium with 9.0 mL sterile distilled or deionized 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 size.Resuspend powdered medium with 9.9 mL sterile deionized water. Add 0.1 mL prepared test suspension and mix well. This is an additional 1:10 dilution from step E. The final volume of test portion/medium mixture in the container should be 10 0.2 mL. Note: For select raw foods (meats, seafood, and vegetables), add 0.1 mL Supplement R to the rehydrated medium. Supplements are available from BioControl Systems, Inc. The final volume of suspension medium mixture into the container should be 10 0.2 mL. Remove lid from the SimPlate device and transfer test suspension/medium mixture onto center of plate. Immediately replace lid. Continue with step H.
G. CEc-CI Test Procedure, Multiple Test Medium

The SimPlate Coliform and E. coli Color Indicator (CEc-CI) method is used for detection and confirmed quantitation of total coliform and E. coli populations. The CEc-CI medium and test portion mixture is dispensed into a SimPlate device and incubated for a minimum of 2428 h. The medium changes color in the presence of total coliforms. E. coli is detected by the presence of a blue fluorescent color change in the medium when examined under ultraviolet (UV) light (366 mm). The total coliform and E. coli counts are determined by counting the wells with changed color and those with fluorescent wells, respectively, referring to the SimPlate Conversion Table (Table 2005.03C).
B. Media and Reagents

Items (a) and (b) are available from BioControl Systems (12822 SE 32nd St, Bellevue, WA 98005, USA; www.biocontrolsys.com). (a) Dehydrated CEc-CI medium.Individually packaged single test or multiple test format with supplement(s). (b) SimPlate devices.In packs of 20. ( c ) B u t t e r f i e l d s p h o s p h a t e b u f f e r. ( 1 ) S t o c k solution.Dissolve 34.0 g KH2PO4 in 500 mL H2O, adjust to pH 7.2 with ca 175 mL 1M NaOH, and dilute to 1 L with H2O. Store in the refrigerator. (2) Diluent.Dilute 1.25 mL stock solution to 1 L with H2O. Prepare dilution blanks with this solution. Autoclave 15 min at 121C.
C. Apparatus

(a) Empty contents of one multidose container into 100 mL sterile distilled or deionized water. Shake to completely dissolve. (b) Remove lid from SimPlate device. Pipet prepared test suspension onto center of plate. If the prepared test suspension size is 1.0 mL, overlay the test portion 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 step E. The final volume of test suspenson/medium mixture on the plate should be 10 0.2 mL. Note: For select raw foods (meats, seafood, and vegetables), add 1.0 mL Supplement R to the rehydrated medium. Supplements are available from BioControl Systems, Inc. The final volume of suspension medium mixture into the container should be 10 0.2 mL. (d) Immediately replace the lid. Continue with step H.
H. Test Procedure for Single and Multiple Tests

(a) Incubator.Maintaining 3537C in a dark environment. (b) Micropipettor.Accurately dispensing 0.1 and 1.0 mL. (c) Pipets.Glass or plastic sterile, 1 mL with 0.01 mL graduations, and 10 mL with 0.1 mL graduations. (d) Stomacher/masticator.IUL Instruments (Cincinnati, OH, USA; www.iul-inst.com), or equivalent, for macerating test portions. (e) Long wavelength ultraviolet light source (366 nm).
D. General Instructions

(a) Gently swirl to distribute the test suspension into all wells. Hold plate with both hands tilted slightly to help distribute medium into wells. (b) Pour off excess medium by holding lid against plate on either side of sponge cavity. Tip plate toward operator to allow fluid to drain into sponge. Observe the background color of the wells. Background is defined as the color of the test suspension inside the wells. (c) Do not invert the SimPlate device. Incubate at 3537C for 2428 h in the dark.
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Discard the SimPlate device if the wells are crushed or pinched. Do not use expired medium. Store reconstituted medium between 2 and 25C in the dark and use within 12 h. Dispose of used medium and SimPlate devices in a decontamination container and sterilize according to Good Laboratory Practices (U.S. Food and Drug Administration (1997) Code of Federal Regulations, Title 21, Chapter 1, Subchapter A, part 58, U.S. Government Printing Office, Washington, DC, USA).

Table 2005.03A. M ean

a b

Interlaboratory study results for total coliforms by AOAC and SimPlate CEc-CI methods sr
d e

RSDr, % AOAC 0 .2 2 0 .2 5 0 .2 1 6 .1 8 4 .4 3 4 .7 5 2 .9 9 2 2 .7 6 2 1 .7 0 1 1 .1 5 6 .6 5 4 .7 5 1 .6 8 2 .4 8 2 .0 6 3 .3 6 2 5 .2 8 1 6 .9 0 8 .8 3 1 3 .4 4 0 .9 0 1 .3 3 2 8 .1 6 1 .2 8 1 .7 6 2 8 .9 5 1 .2 6 1 .3 3 6 .2 6 0 .3 7 0 .7 2 0 .1 3
l

r S IM 0 .6 1 0 .4 8 0 .6 3 0 .3 1 0 .4 0 0 .3 1 1 .0 8 1 .1 6 1 .0 4 0 .4 2 0 .3 7 0 .1 5 0 .2 0 0 .2 0 0 .6 1 0 .6 0 0 .4 5 1 .0 0 0 .1 2
l

sR AOAC 0 .6 4 0 .7 1 0 .6 0 1 .5 4 1 .0 1 1 .0 9 1 .1 3 1 .2 4 1 .3 6 0 .9 7 1 .1 5 0 .7 9 0 .7 4 0 .1 1
l

RSDR, % S IM 9 .5 4 7 .5 0 6 .1 8 1 0 .6 9 1 0 .5 8 3 .2 0 3 8 .1 7 2 9 .2 3 1 5 .2 2 1 5 .4 1 1 0 .4 8 1 0 .0 3 3 .6 4 3 .4 8 0 .4 0 0 .6 3 0 .8 3 0 .6 8 3 .3 6 3 4 .0 4 2 2 .2 1 1 2 .4 1 1 0 .8 6 4 3 .9 1 4 1 .1 2 2 2 .7 3 2 4 .3 2 2 0 .4 8 1 2 .0 3 1 5 .3 5 1 0 .9 1 9 .8 9 3 8 .3 0 3 7 .2 8 1 9 .1 6 1 5 .3 3 3 3 .2 2 8 .5 8 0 .6 3 0 .7 6 0 .8 9 0 .3 3 1 .8 1 1 .5 7 1 .4 2 0 .9 8 0 .8 1 0 .9 1 0 .3 0 0 .3 3 0 .3 7 1 .7 0 1 .6 9 1 .2 7 1 0 .0 0 0 .5 7 1 5 .4 0 0 .6 1 AOAC S IM AOAC 0 .9 2 0 .7 6 0 .8 9 2 .0 1 1 .1 8 1 .0 9 1 .7 3 1 .8 8 1 .9 9 1 .4 0 1 .4 9 1 .1 2 1 .1 8 1 .0 0 1 .1 3 1 .7 6 2 .3 3 1 .8 9 0 .3 3 0 .2 7 0 .3 2 0 .7 2 0 .4 2 0 .3 9 0 .6 2 0 .6 7 0 .7 1 0 .5 0 0 .5 3 0 .4 0 0 .4 2 0 .3 6

Food group 2 .2 8 2 .7 1 3 .6 3 2 .5 3 3 .0 3 3 .5 7 1 .7 0 1 .9 1 3 .3 3 2 .2 8 2 .7 3 3 .2 4 2 .9 6 3 .4 4 3 .9 6 1 .7 8 2 .7 1 3 .6 5 3 .5 3 0 .3 2 0 .4 7 2 .4 9 0 .4 6 0 .6 3 1 .6 4 0 .4 5 0 .4 8 4 .0 9 0 .1 3
n m m m

L o t/l e v e l 2 .1 4 2 .7 1 3 .7 0 2 .1 6 2 .7 5 3 .7 2 1 .4 1 1 .6 4 3 .1 2 2 .0 5 2 .6 0 3 .3 3 2 .7 7 3 .2 8 0 .2 6 0 .0 7
k

N 0 .2 2 0 .1 7 0 .2 2 0 .1 1 0 .1 4 0 .1 1 0 .3 9 0 .4 2 0 .3 7 0 .1 5 0 .1 3 0 .0 5 0 .0 7 0 .3 6 1 0 .9 1
k k k n k k k

S IM 9 .5 4 6 .3 3 5 .7 5 2 5 .3 7 1 3 .0 8 1 0 .8 6 2 8 .6 0 2 7 .1 6 1 5 .5 1 1 6 .9 0 1 5 .7 4 8 .4 7 9 .5 9 0 .3 2 0 .2 9
l

AOAC 1 0 .6 3 9 .3 3 0 .2 2 0 .2 7 0 .3 2 0 .1 2 0 .6 5 0 .5 6 0 .5 1 0 .3 5
l l l

S IM 0 .2 2 0 .2 0

S IM

AOAC

S IM

AOAC

Frozen burrito

Low

M edi um 0 .5 5 0 .3 6 0 .3 9 0 .4 0 0 .4 4 0 .4 8 0 .3 5 0 .4 1 0 .2 8 0 .2 6

High

Frozen broccoli

Low

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M edi um

High

Nutmeats

Low

10

M edi um

10

High

10

Fluid milk

Low

M edi um

High

Cheese

Low

M edi um

10

High

Cake mix

Low

M edi um

High

N = Number of laboratories with valid data.

Mean log total coliforms/gram.

SIM = SimPlate method.

AOAC = AOAC culture method.

sr = Repeatability standard deviation.

RSDr = Repeatability relative standard deviation.

r = Repeatability values, 2.8 sr.

sR = Reproducibility standard deviation.

RSDR = Reproducibility relative standard deviation. j R = Reproducibility values, 2.8 sR. k Significantly better repeatability p < 0.01. l

Significantly better reproducibility p < 0.01.

Significantly better mean log counts p < 0.05; statistically not different p < 0.01.

Significantly better repeatability p < 0.05; statistically not different p < 0.01.

Table 2005.03B. M ean N 9 9 9 8 8 8 10 10 10 9 9 9 10 10 10 9 9 8 3 .1 9

n a b

Interlaboratory study results for E. coli by AOAC and SimPlate CEc-CI methods sr
d e

RSDr, % AOAC 0 .4 0 0 .3 5 8 .2 7 4 .4 7 1 1 .8 7 8 .8 6 3 .1 6 5 5 .3 9 2 9 .2 1 1 0 .7 5 3 6 .0 0 2 4 .4 1 8 .7 1 5 .2 6 2 .6 2 5 .2 6 4 8 .8 4 4 3 .2 5 1 6 .2 3 1 9 .3 9 1 .4 5 5 2 .5 3 2 .1 2 2 .2 2 1 .5 1 1 0 0 .9 4 1 .1 3 1 .5 3 2 2 .0 8 0 .4 7 1 .8 1 1 0 .1 0 0 .2 0 0 .7 5 0 .0 7 0 .1 7
m m

r S IM 0 .7 8 0 .5 5 0 .4 3 0 .7 3 0 .6 4 0 .2 8 1 .3 6 0 .9 1 0 .8 0 1 .0 9 1 .2 2 0 .5 4 0 .3 5 1 .0 5 0 .1 2
m

sR AOAC 1 .1 1 0 .9 7 0 .9 8 0 .9 1 0 .6 8 1 .1 2 1 .0 1 0 .9 2 1 .1 8 0 .7 9 1 .2 0 0 .9 0 0 .4 6 0 .5 1 0 .4 8 0 .4 7 0 .4 4 0 .3 2 0 .6 5 0 .4 0 0 .9 6 0 .6 0 0 .6 6 1 .1 4 0 .8 7 0 .5 7 0 .5 4 0 .4 4 0 .7 0 0 .4 9 0 .7 0 4 3 .7 1 1 6 .5 8 5 2 .7 8 2 8 .3 9 2 0 .8 0 5 .2 6 2 .6 2 5 .2 6 4 8 .8 4 5 4 .7 7 1 8 .8 9 0 .4 9 0 .6 9 5 5 .3 9 0 .1 5
m

RSDR, % AOAC 0 .4 0 0 .3 8 9 .0 9 5 .0 9 1 3 .8 7 1 2 .0 5 4 .6 8 1 3 .3 8 1 5 .7 2 8 2 .1 1 7 7 .5 2 2 7 .7 2 4 0 .6 8 2 5 .3 4 2 0 .2 6 2 1 .3 6 1 1 .9 0 2 2 .0 8 1 2 2 .7 7 7 4 .7 4 3 1 .2 3 2 3 .9 9 1 1 .2 2 1 5 .5 4 0 .6 0 0 .4 9 0 .8 5 0 .8 7 0 .4 2 1 .3 6 1 .3 7 1 .2 3 1 .5 9 1 .4 2 1 .3 0 0 .3 5 0 .2 0 0 .4 7 1 .1 3 2 .6 9 1 .6 9 1 4 .1 7 2 0 .3 2 0 .7 8 S IM AOAC S IM 1 .1 1 1 .0 5 0 .9 8 1 .4 8 0 .9 9 1 .3 5 1 .9 3 1 .9 5 1 .9 6 1 .5 2 1 .3 4 1 .3 3 1 .2 2 0 .8 8 1 .8 1 1 .8 6 3 .2 1 2 .4 2

Food group 1 .9 6 2 .3 7 3 .3 0 2 .2 0 2 .5 9 3 .1 7 0 .8 8 1 .1 0 2 .6 5 1 .0 8 1 .7 9 2 .1 9 2 .3 5 2 .7 3 3 .1 6 0 .8 2 1 .7 0 2 .7 7 0 .5 2 0 .5 4
o n

L o t/l e v e l 1 .9 6 2 .4 2 3 .1 7 2 .2 1 2 .6 4 3 .0 6 0 .8 4 0 .9 0 2 .5 2 1 .3 4 1 .8 8 2 .3 4 2 .0 4 2 .6 5 2 .9 3 0 .5 4 1 .4 0 0 .7 6 0 .7 9 0 .4 0 0 .5 4 0 .1 7
k

S IM 0 .2 8 0 .2 0 0 .1 5 0 .2 6 0 .2 3 0 .1 0 0 .4 9 0 .3 3 0 .2 8 0 .3 9 0 .4 4 0 .1 9 0 .1 2 0 .0 7 0 .6 5
k k k k

AOAC 1 4 .1 7 1 4 .3 0 1 1 .2 2 1 4 .7 2 9 .2 3 1 3 .0 9 4 2 .9 7 3 6 .6 2 1 6 .6 4 2 1 .1 3 2 2 .7 5 1 3 .6 7 1 8 .3 1 0 .4 8 0 .3 1 0 .3 5 0 .3 0 0 .5 3 0 .1 7
l

S IM 2 0 .3 2 0 .2 1 0 .3 5 0 .2 7

S IM

AOAC

S IM

AOAC

Frozen burrito 0 .3 5 0 .3 2 0 .2 4 0 .4 0 0 .3 6 0 .3 3 0 .4 2 0 .2 8 0 .4 3 0 .3 2 0 .3 7 0 .2 7

Low

M edi um

High

Frozen broccoli

Low

M edi um

High

Nut meats

Low

M edi um

High

Fluid milk

Low

M edi um

High

Cheese

Low

M edi um

High

Cake mix

Low

M edi um

High

N = Number of laboratories with valid data.

Mean log E. coli/gram.

SIM = SimPlate method.

AOAC = AOAC culture method.

sr = Repeatability standard deviation.

RSDr = Repeatability relative standard deviation.

r = Repeatability values, 2.8 sr.

sR = Reproducibility standard deviation.

RSDR = Reproducibility relative standard deviation.

R = Reproducibility values, 2.8 SR.

Significantly better repeatability p < 0.01.

l Significantly better reproducibilty p < 0.05; not statistically different p < 0.01. m

Significantly better reproducibility p < 0.01.

Significantly better mean log counts p < 0.01.

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Significantly better mean log counts p < 0.05; not statistically different p < 0.01.

Table 2005.03C.

SimPlate Conversion Table

a

I. Reading and Interpretation of Results

Number of positive wells = Population per plate 1=2 2=4 3=6 4=8 5 = 10 6 = 12 7 = 14 8 = 16 9 = 18 10 = 22 11 = 24 12 = 26 13 = 28 14 = 30 15 = 32 16 = 36 17 = 38 18 = 40 19 = 42 20 = 46 21 = 48 22 = 50 23 = 54 24 = 56 25 = 58 26 = 62 27 = 64 28 = 68 29 = 70 30 = 74
a

31 = 76 32 = 80 33 = 84 34 = 86 35 = 90 36 = 94 37 = 96 38 = 100 39 = 104 40 = 108 41 = 112 42 = 116 43 = 120 44 = 124 45 = 128 46 = 132 47 = 136 48 = 142 49 = 146 50 = 150 51 = 156 52 = 160 53 = 166 54 = 172 55 = 178 56 = 184 57 = 190 58 = 196 59 = 202 60 = 208

61 = 216 62 = 224 63 = 232 64 = 240 65 = 248 66 = 256 67 = 266 68 = 276 69 = 288 70 = 298 71 = 312 72 = 324 73 = 338 74 = 354 75 = 372 76 = 392 77 = 414 78 = 440 79 = 470 80 = 508 81 = 556 82 = 624 83 = 738 84 = >738 If there are no positive wells and the sponge is positive, then the population is 1

(a) After incubation, observe color change of fluid in the wells. Disregard particulate matter if present. Count the number of wells showing any color change from the background color. Wells with changed color correspond to the presence of total coliforms. Count the number of colored wells showing white to blue fluorescence by holding a UV light (366 nm) 1530 cm (approximately 6 in.) above the SimPlate device. These wells correspond to the presence of E. coli. (b) To determine the population, perform the following calculations: (1) Count the number of positive wells on the plate; (2) use the SimPlate Conversion Table (Table 2005.03C) to determine the total population per plate. (c) To calculate the number of microorganisms/g of food, multiply the count in I(b)(2) by the appropriate dilution factor (see steps E and F for single test or steps E and G for multiple test). Reference: J. AOAC Int. 88, 1318(2005).

If there are no positive wells and the sponge is negative, then the population is <1

The population reflects the number of microorganisms per plate. To determine the number of microorganisms per g (mL) food product, refer to text, I(c), Reading and Interpretation of Results.

2006 AOAC INTERNATIONAL