3
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Sample Preparation
1. Use appropriate sterile diluents: Butterfield’s phosphate buffered dilution water
5
, 0.1% peptone water
5
, peptone salt
diluent
6
, buffered peptone water
6
, quarter-strength Ringer’s solution
6
, dipotassium hydrogen phosphate
6
, saline
solution (0.85-0.90%), bisulfite-free letheen broth or distilled water.
See section “
Specific Instructions for Validated Methods
” for specific requirements.
Do not use diluents containing citrate, bisulfite or thiosulfate with 3M Petrifilm SEC Plates; they can inhibit
growth.
If citrate buffer is indicated in the standard procedure, substitute with one of the buffers listed above,
warmed to 40-45°C (104-113°F).
2. Blend or homogenize sample.
3. For optimal growth and recovery of microorganisms, adjust the pH of the sample suspension to 6.5 - 7.5. For acidic
products, adjust the pH with 1
N
NaOH. For alkaline products, adjust the pH with 1
N
HCl.
Plating
1. Place the 3M Petrifilm SEC Plate on a flat, level surface.
2. Lift the top film and with the pipette perpendicular to the inoculation area dispense 1 mL of sample suspension onto
the center of bottom film.
3. Roll the top film down onto the sample to prevent trapping air bubbles.
4. Place the 3M™ Petrifilm™ Spreader with the flat side down on the center of the 3M Petrifilm SEC Plate. Press gently
on the center of the 3M Petrifilm Spreader to distribute the sample evenly. Spread the inoculum over the entire 3M
Petrifilm SEC Plate growth area before the gel is formed. Do not slide the 3M Petrifilm Spreader across the film.
5. Remove the 3M Petrifilm Spreader and leave the 3M Petrifilm SEC Plate undisturbed for at least one minute to
permit the gel to form.
Incubation
Incubate 3M Petrifilm SEC Plates in a horizontal position with the clear side up in stacks of no more than 20 plates.
Incubate 3M Petrifilm SEC Plates 24 hours ± 2 hours at 42ºC ± 1ºC. Several incubation times and temperatures can
be used depending on current local reference methods, some of which are listed in the section below titled “
Specific
Instructions for Validated Methods
”.
Interpretation
1. 3M Petrifilm SEC Plates can be counted using a standard colony counter or other illuminated magnifier. Do not count
colonies on the foam dam since they are removed from the selective influence of the medium. Do not count artifact
bubbles that may be present.
E. coli
produce dark green to light-green colonies. Colonies may have gas bubbles
associated with them. Count all green to blue-green colonies as
E. coli
whether there are gas bubbles present or not.
W
WARNING
Do not use the 3M Petrifilm SEC Plate for the detection of
E. coli
O157. Because most
E. coli
O157 strains are
atypical, for example, they are glucuronidase negative, they will not produce a blue-green color, and will not be
interpreted as
E. coli
on the 3M Petrifilm SEC Plates.
2. The circular growth area is approximately 20 cm
2
. Estimates can be made on 3M Petrifilm SEC Plates containing
greater than 150 colonies by counting the number of colonies in two or more representative squares and
determining the average number per square. Multiply the average number by 20 to determine the estimated count
per 3M Petrifilm SEC Plate.
3. When colonies are present in large numbers, 3M Petrifilm SEC Plates will cause the entire growth area to become
gray or blue and either or both of the following characteristics: many small, indistinct colonies and/or many gas
bubbles. When this occurs, record results as too numerous to count (TNTC). When an actual count is required, plate
at a higher dilution.
4. Where necessary, colonies may be isolated for further identification. Lift the top film using proper testing technique
and pick the colony from the gel. Test using standard procedures.
5. If the 3M Petrifilm SEC Plates cannot be counted within 1 hour of removal from the incubator, they may be stored for
later enumeration by freezing in a sealable container at temperatures lower than or equal to negative 15°C (5°F) for
no longer than one week.
For further information refer to the “3M™ Petrifilm™ Select
E. coli
Count Plate Interpretation Guide.” If you have
questions about specific applications or procedures, please visit our website at www.3M.com/foodsafety or contact
your local 3M representative or distributor.
