3M Petrifilm™ E. coli/Coliform Count Plates Operating instructions

3M Petrifilm™ E. coli/Coliform Count Plates Operating instructions
Interpretation
Guide
The 3M™ Petrifilm™ E. coli/Coliform
Count Plate is a sample-ready-culture
medium system which contains modified
Violet Red Bile (VRB) nutrients, a coldwater-soluble gelling agent, an indicator of
glucuronidase activity, 5-bromo-4-chloro3-indolyl-D-glucuronide (BCIG), and a
tetrazolium indicator that facilitates colony
enumeration. 3M Petrifilm E. coli/Coliform
Count Plates are used for the enumeration
of Escherichia coli (E. coli) and coliforms in
the food and beverage industries.
EC
E. coli/Coliform Count Plate
The United States Food and Drug Administration
(FDA) Bacteriological Analytical Manual (BAM)
define coliforms as Gram negative rods, which
produce acid and gas from lactose fermentation.
Most E. coli (about 97%) produce betaglucuronidase which produces a blue precipitate
associated with the colony indicated by the
blue to red-blue colonies. The top film traps gas
produced by the lactose fermenting coliforms
and E. coli. About 95% of E. coli produce gas, as
indicated by colonies associated with entrapped
gas (within approximately one colony diameter).
Blue colonies without gas are not counted as E.
coli.* Other coliform colonies are red and closely
associated with entrapped gas. The total coliform
count consists of both the red and blue colonies
associated with gas.*
Most E. coli O157 strains are atypical, for example
they are glucuronidase negative; they will not
produce a blue color, and will not be detected on
3M Petrifilm E. coli/Coliform Count Plates.
*Validated through AOAC® Official Methods of AnalysisSM
program. The definition of E. coli and coliform varies by
regional method. In particular, the confirmation of E. coli
may vary by country.
Figure 1
E. coli count = 49 (blue colonies with gas)
Total coliform count = 87 (red and blue colonies
with gas)
All figures have been counted according to AOAC® Official
Methods of AnalysisSM #998.08 and #991.14. The confirmation
of E. coli may vary by country.
Bubbles
The illustrations above show examples of various bubble
patterns associated with gas producing colonies. All should be
enumerated.
E. coli/Coliform Count Plate
2
2
1
3
Figure 2
Figure 3
Total coliform count = 3 (red colonies with gas)
Estimated total coliform count = 150
Food particles are irregularly shaped and are not associated with
gas bubbles.
The recommended counting limit on a 3M Petrifilm E. coli/
Coliform Count Plate is 150 colonies.
Bubble patterns may vary. Gas may disrupt the colony so that
the colony “outlines” the bubble. See Circles 1 and 2. Artifact
bubbles may result from improper inoculation or from trapped
air within the sample. They are irregularly shaped and are not
associated with a colony. See Circle 3.
For a more accurate count, further dilution of the sample may be
necessary.
1
Figure 4
1
Figure 5
No growth = 0
E. coli count = 13 (blue colonies with gas)
Notice the changes in gel color in Figures 4 through 10. As the
E. coli or coliform count increases, the color of the gel turns to
dark red or purple-blue. Background bubbles are a characteristic
of the gel and are not a result of E. coli or coliform growth. See
Square 1.
Total coliform count = 28 (red and blue colonies
with gas)
Do not count colonies that appear on the foam barrier because
they are removed from the selective influence of the medium. See
Circle 1.
3
Front
Lighting
Back
Lighting
1
Figure 6a
2
Figure 6b
Figure 7
E. coli count = 3 (blue colonies with gas)
Estimated E. coli = 17 (blue colonies with gas)
Any blue in a colony (blue to red-blue) with gas indicates the
presence of E. coli. Front lighting may enhance the detection of
blue precipitate formed by a colony. Circle 1 shows a red-blue
colony counted using back lighting. Circle 2 shows the same
colony with front lighting. The blue precipitate is more evident
in Circle 2.
Estimated total coliform count = 150
The circular growth area is approximately 20cm2. Estimates
can be made on plates containing greater than 150 colonies by
counting the number of colonies in one or more representative
squares and determining the average number per square.
Multiply the average number by 20 to determine the estimated
count per plate.
For a more accurate count, further dilution of the sample may be
necessary.
Too Numerous to Count (TNTC)
Figure 8
Figure 9
Total coliform count = TNTC
Total coliform count = TNTC
3M Petrifilm E. coli/Coliform Count Plates with colonies that are
too numerous to count (TNTC) have one or more of the following
characteristics: many small colonies, many gas bubbles and a
deepening of the gel color from red to purple-blue.
High concentrations of E. coli may cause the growth area to turn
purple-blue.
For a more accurate count, further dilution of the sample may be
necessary.
For a more accurate count, further dilution of the sample may be
necessary.
4
Figure 10
Figure 11
Total coliform count = TNTC
Total coliform count = TNTC
High concentration of coliforms may cause a deepening of the
gel color and many small, indistinct colonies.
When high numbers of non-coliform organisms such as
Pseudomonas are present on 3M Petrifilm E. coli/Coliform Count
Plates, the gel may turn yellow.
For a more accurate count, further dilution of the sample may be
necessary.
For a more accurate count, further dilution of the sample may be
necessary.
Reminders for Use
Storage
Inoculation
1 mL
≤8°C
1
Store the unopened pouches of plates at
frozen or refrigerated temperatures ≤8°C
(≤46°F). Use before expiration date on
package. It is best to allow pouches to
reach room temperature before opening.
2
Seal by folding the end of the pouch over
and applying adhesive tape. To prevent
exposure to moisture, do not refrigerate
opened pouches. Store sealed pouches in a
cool dry place for no longer than four weeks.
3
Place 3M Petrifilm E. coli/Coliform Count
Plate on flat, level surface. Lift the top
film and with a 3M™ Electronic Pipettor
or equivalent held perpendicular to plate,
dispense 1 mL of sample suspension onto
center of bottom film.
6
Press firmly on the center of the spreader
to distribute the sample evenly. Spread the
inoculum over the growth area before the
gel is formed. Do not slide the spreader
across the top film.
FLAT
side down
4
Roll the top film down onto sample gently
to prevent pushing sample off film and to
avoid entrapping air bubbles. Do not let
top film drop.
5
E. coli/Coliform Count Plate
With flat side down, place 3M™ Petrifilm™
Spreader on the center of the 3M Petrifilm
E. coli/Coliform Count Plate.
5
Incubation
Use Appropiate
Sterile Diluents
Butterfield’s phosphate buffered
dilution water, 0.1% peptone water,
peptone salt diluent, quarter-strength
Ringer’s solution, saline solution
(0.85-0.90%), bisulfite-free letheen
broth or distilled water.
7
Remove the spreader and leave the plate
undisturbed for one minute to permit the
gel to form.
8
Incubate 3M Petrifilm E. coli/Coliform
Count Plates with clear side up in stacks
of up to 20. It may be necessary to
humidify incubator to minimize moisture
loss. Please refer to the product
instructions for third party validated
methods.
Interpretation
9
For optimal growth and recovery of
the microorganisms, adjust the pH of
the sample suspension to 6.6-7.2.
Do not use diluents containing citrate,
bisulfite or thiosulfate with the 3M
Petrifilm E. coli/Coliform 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.
3M Petrifilm E. coli/Coliform Count Plates
can be counted using the 3M™ Petrifilm™
Plate Reader, on a standard colony counter
or other illuminated magnifier. Colonies may
be isolated for further identification. Lift top
film and pick the colony from the gel.
3M Food Safety offers a full line of
products to accomplish a variety of
your microbial testing needs. For
more product information, visit us
at 3M.com/foodsafety/Petrifilm or
call 1-800-328-6553.
User’s Responsibilities: 3M Petrifilm Plate performance has not been evaluated with all combinations
of microbial flora, incubation conditions and food matrices. It is the user’s responsibility to determine
that any test methods and results meet the user’s requirements. Should re-printing of this Interpretation
Guide be necessary, user’s print settings may impact picture and color quality.
3M Food Safety
3M Center, Building 275-5W-05
St. Paul, MN 55144-1000 USA
1-800-328-6553
3M.com/foodsafety
3M Canada
Post Office Box 5757
London, Ontario N6A 4T1
Canada
1-800-364-3577
E. coli/Coliform Count Plate
For detailed CAUTIONS, DISCLAIMER OF WARRANTIES/LIMITED REMEDY
and LIMITATION OF 3M LIABILITY, STORAGE AND DISPOSAL information
and INSTRUCTIONS FOR USE, see Product’s package insert.
3M and Petrifilm are trademarks of 3M. Used under license in Canada.
Please recycle. Printed in USA. © 3M 2017. All rights reserved.
70-2008-4574-4 (Rev-1217)
6
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