Interpretation Guide

The 3M™ Petrifilm™ Environmental Listeria (EL) Plate is a sample-ready culture medium containing selectiveagents, nutrients, a cold-water-soluble gelling agent, and a chromogenic indicator that facilitates colony detection.Petrifilm EL Plates are used for the detection and/or enumeration of Listeria in environmental samples. PetrifilmEL Plates detect the following Listeria species: Listeria monocytogenes, Listeria innocua, Listeria grayi/murrayiand Listeria welshimeri, but do not differentiate these organisms from one another.

Environmental conditions and sanitizers may stress and/or injure microorganisms. Buffered peptone water (BPW)is used as a repair broth in conjunction with the Petrifilm EL Plate. Repair in BPW is not an enrichment step.

The Petrifilm EL Plate method may be used as a qualitative, semi-quantitative or quantitative test. For further directions, please refer to the Reminders for Use section.

Qualitative interpretation: Listeria detected on this plate

Semi-quantitative interpretation: Listeria level shouldbe recorded as categories that are meaningful to yoursampling location and your individual plant standards (e.g.,low, medium, high, or acceptable and unacceptable).

Quantitative interpretation: Listeria colonies on thisplate: 11. Please refer to the "Quantitative Sampling"section of this guide for calculating the quantity of Listeriaper environmental sample.

3Petrifilm™

Environmental Listeria PlateThis guide familiarizes you with results on 3M™ Petrifilm™ Environmental Listeria Plates.For more information, contact the official 3M Microbiology representative nearest you.

1. This picture shows a typical Petrifilm EL Plate withListeria colonies. Interpret or count all red-violetcolonies as Listeria. If any colonies are grey or light pinkat 26 to 29 hours, then continue incubating. Colonies thatare initially grey or light pink and deepen to red-violetduring incubation should be interpreted as Listeria.Colonies that remain grey or light pink at the maximumincubation time (30 hours) should not be interpreted asListeria. Do not consider or count colonies on the foamdam since they are removed from the selective influenceof the medium.

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3M™ Petrifilm™Environmental Listeria Plate

Qualitative interpretation: Listeria not detected on this plate

Semi-quantitative interpretation: Listeria level should berecorded as categories that are meaningful to your samplinglocation and your individual plant standards (e.g., low, medium,high, or acceptable and unacceptable).

Quantitative interpretation: Listeria colonies on this plate: <1.Please refer to the "Quantitative Sampling" section of this guide forcalculating the quantity of Listeria per environmental sample.

2. This Petrifilm Plate has no colonies after 30 hours of incubation.The test is complete.

Qualitative interpretation: Listeria detected on this plate

Semi-quantitative interpretation: Listeria level should berecorded as categories that are meaningful to your samplinglocation and your individual plant standards (e.g., low, medium,high, or acceptable and unacceptable).

Quantitative interpretation: Listeria colonies on this plate: 84.Please refer to the "Quantitative Sampling" section of this guide forcalculating the quantity of Listeria per environmental sample.

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3. The Petrifilm EL Plate is selective and differential for Listeria,which form red-violet colonies.

3M™ Petrifilm™Environmental Listeria Plate

Qualitative interpretation: Listeria detected on this plate

Semi-quantitative interpretation: Listeria level should berecorded as categories that are meaningful to your samplinglocation and your individual plant standards (e.g., low, medium,high, or acceptable and unacceptable).

Quantitative interpretation: Estimated Listeria colonies onthis plate: est. 4600. When large numbers of Listeria are present,estimate by determining the count per square of two or morerepresentative squares. Determine the average per square and thenmultiply by 42. The inoculated area of the Petrifilm EL Plate isapproximately 42 cm2.

Qualitative interpretation: Listeria detected on this plate

Semi-quantitative interpretation: Listeria level should berecorded as categories that are meaningful to your samplinglocation and your individual plant standards (e.g., low, medium,high, or acceptable and unacceptable).

Quantitative interpretation: Listeria on this plate is toonumerous to count (TNTC, approximately 104 shown in this image).

5. When colonies are present in large numbers, Petrifilm ELPlates may have many small, indistinct colonies and/or apink-brown color throughout.

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6. Background color may vary due to the presence of dust, soil,grit, or other sediment from the environment sampled ordepending on the sample collection device and/or the brandof buffered peptone water (repair broth). Interpret or countthe red-violet colonies as Listeria.

Qualitative interpretation: Listeria detected on this plate

Semi-quantitative interpretation: Listeria level shouldbe recorded as categories that are meaningful to yoursampling location and your individual plant standards (e.g.,low, medium, high, or acceptable and unacceptable).

Quantitative interpretation: Listeria colonies on thisplate: 11. Please refer to the "Quantitative Sampling"section of this guide for calculating the quantity of Listeriaper environmental sample.

4. Since the Petrifilm EL Plate may be interpreted in three ways,no counting range is suggested. When colonies are crowded,interpret the result (qualitative or semi-quantitative) orestimate the count (quantitative) as described below.

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Collect environmental samples usinga swab, sponge or other collectiondevice moistened with ≤ 10 mL.

The moistening agent can be sterilewater, buffered peptone water (BPW)or neutralizing buffer such as LetheenBroth or Dey/Engley (DE)Neutralizing Broth.

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Mix, stomach or vortex thecollected sample with BPW forapproximately one minute. Allowthe sample to remain at roomtemperature, 20-30˚C, for 1 hourup to a maximum of 1.5 hours.This is required for repair ofinjured Listeria.

Store unopened pouches at ≤8°C(≤46°F). Use before expirationdate on package. In areas of highhumidity, it is best to allowpouches to reach roomtemperature before opening.

To seal opened pouch, fold endover and tape shut.

To prevent exposure tomoisture, do not refrigerateopened pouches. Store resealedpouches in a cool, dry place for nolonger than one month. Avoidexposing plates to temperature>25°C (>77°F) and/or the relativehumidity is > 50%.

Place Petrifilm Plate on levelsurface. Lift top film.

With 3M™ Electronic Pipettor orequivalent pipettor heldperpendicular to Petrifilm Plate,place 3 mL of sample onto thecenter of bottom film.

Petrifilm

3Petrifilm™ Environmental Listeria Plates Reminders for Use

Petrifilm

Petrifilm

Storage

Sample Preparation

Inoculation

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7 8 9 Roll the top film down onto thesample to prevent trapping airbubbles.

For detailed WARNINGS, CAUTIONS, DISCLAIMER OF WARRANTIES / LIMITED REMEDY, LIMITATION OF 3M LIABILITY,STORAGE AND DISPOSAL information, and INSTRUCTIONS FOR USE see Product’s package insert.

Aseptically add 5 mL sterile (20-30˚C)buffered peptone water (BPW) (repair broth) to the collected sample.

Remember to inoculate and spread each Petrifilm plate before going on to the next plate.

Do not use the PetrifilmEnvironmental Listeria Plate withUniversity of Vermont medium(UVM), Fraser broth, Listeriaenrichment broth (LEB) or bufferedListeria enrichment broth (BLEB).

<70¡F

Incubation Interpretation

Incubate plates with clear side up instacks of up to 10 for 28h ± 2h at35°C ± 1°C or 37°C ± 1°C. It maybe necessary to humidify incubatorto minimize moisture loss.

Petrifilm Environmental ListeriaPlates can be counted orinterpreted using a standard colonycounter or other illuminatedmagnifier. Do not count colonies onthe foam dam since they areremoved from the selectiveinfluence of the medium.

11 12Gently place the plastic spreader on the top film over the inoculum.Do not press, twist or slide thespreader. Lift spreader. Wait at least10 minutes to permit the gel toform.

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Colonies may be isolated for furtheridentification. Lift top film and pickthe colony from the gel.

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The Petrifilm Environmental Listeria Plate method can be used as a qualitative, semi-quantitative or quantitative test.

For a qualitative test, recordresults of the plate as detected ornot detected based on thepresence or absence of red-violetcolonies.

You may wish to choose aqualitative test if a yes/no answer issufficient and appropriate for yourreporting.

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DetectedNot detected

Optional

For a quantitative test, count andrecord all red-violet colonies.

You may wish to choose aquantitative test if you take differentactions based upon the numberpresent.

Listeria colonies onthis plate: 16

For a semi-quantitative test,record results based on the relativelevel of red-violet colonies present.

You may wish to choose a semi-quantitative test if you take differentactions depending on the relativelevel present, and if recording anactual number is not required.

Listeria level should be recorded ascategories that are meaningful to yoursampling location and your individualplant standards (e.g., low, medium,high, or acceptable and unacceptable).

Please refer to the "QuantitativeSampling" section of this guide forcalculating the quantity of Listeria perenvironmental sample.

Quantitative Sampling & Interpretation

3M™ Petrifilm™Environmental Listeria Plate

If your facility chooses to use the 3M Petrifilm Environmental Listeria Plate in a quantitative manner, pleaserefer to the product package insert, and then calculate the colony forming units (CFU) per area as shown below.You may also want to consider the following points:

• Consistency is the key to obtaining useful information from your environmental monitoring program. Use a consistent procedure each time that you sample. Ideally, use the same type of sampling device, template area, technician and sampling techniques.

• The sampling area size may be based on regulations, internal standards, and/or the location of the monitoring, e.g., you may need to sample a larger area for a finished goods line because the numbers of bacteria are expected to be low.

• More information on environmental sampling can be found at the references listed below, and in the 3M Petrifilm Plates Environmental Monitoring Procedures brochure.

TO DETERMINE the quantity of Listeria per sampled area, you will need to record: 1) area size sampled 2) volume of hydration fluid in the sampling device 3) volume of the buffered peptone water added 4) volume plated5) number of colonies counted

APPLY the following equation or worksheet to determine the CFU/area sampled. Examples are given on the following pages. See Package Insert & Reminders for Use for full details of the method.

You may also determine the result per sample, e.g., CFU/ drain.

OR

Environmental quantitative sampling is consistent with the following references:• Standard Methods for the Examination of Dairy Products, Section 3.7D, American Public Health Association,

Washington D.C., 1992.• Compendium of Methods for the Microbiological Examination of Foods, Section 3.512 and 3.521, American Public

Health Association, Washington D.C., 2001.

A. Total number of mL of BPW + hydration fluid ________B. Number of mL plated_____________________________3C. Divide line A by line B ___________________________D. Number of colonies counted _______________________E. Multiply line C by line D __________________________F. Area sampled ___________________________________G. Divide line E by line F ____________________________

Line G equals CFU/area ____________________________

CFU/area = (Number of colonies x [mL hydration fluid + mL BPW] ÷ 3 mL) ÷ area sampled

3M™ Petrifilm™Environmental Listeria Plate

Example: Sponge Contact Method

Using a sponge moistenedwith 10 milliliters (mL) ofhydration fluid, sample anarea, for example, onesquare foot (1 ft2).

Return the sponge to thesterile container and add5 mL of bufferedpeptone water.

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After repair step, plate 3 mL onto the PetrifilmEnvironmental ListeriaPlate.

After incubation, countcolonies (for thisexample, assume youcount ninety colonies).

Example: Swab Contact Method

Using a swab moistenedwith 1 milliliter (mL) ofhydration fluid, sample anarea, for example, fiftysquare centimeters (50 cm2).

Return the swab to thesterile container and add5 mL of buffered peptonewater.

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After repair step, plate 3 mL onto the PetrifilmEnvironmental ListeriaPlate.

After incubation, countcolonies (for thisexample, assume youcount ninety colonies).

A. Total number of mL of BPW + hydration fluid ____________5 + 10 = 15B. Number of mL plated ________________________________3C. Divide line A by line B _______________________________5D. Number of colonies counted ___________________________ 90E. Multiply line C by line D______________________________450F. Area sampled _______________________________________ 1 ft2

G. Divide line E by line F _______________________________450 CFU/ft2

A. Total number of mL of BPW + hydration fluid ____________5 + 1 = 6B. Number of mL plated ________________________________3C. Divide line A by line B _______________________________2D. Number of colonies counted___________________________ 90E. Multiply line C by line D _____________________________180F. Area sampled _______________________________________ 50 cm2

G. Divide line E by line F _______________________________3.6 or 4 CFU/cm2

Quantitative Interpretation

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