3M Petrifilm™ High-Sensitivity Coliform Count Plates Operating instructions

3M Petrifilm™ High-Sensitivity Coliform Count Plates Operating instructions
Interpretation
Guide
The 3M™ Petrifilm™ High-Sensitivity Coliform
Count Plate is a sample-ready-culture medium
system which contains modified Violet Red
Bile (VRB) nutrients, cold-water-soluble
gelling agent, and a tetrazolium indicator that
facilitates colony enumeration.
HSCC
High-Sensitivity Coliform Count Plate
1
The United States Food and Drug Administration (FDA) Bacteriological Analytical Manual
(BAM) defines coliforms as Gram-negative rods which produce acid and gas from lactose
during fermentation. Gas production is used to differentiate coliform from non-coliform
colonies. Gas trapped around red colonies indicates coliforms on the 3M™ Petrifilm™
High-Sensitivity Coliform Count Plate. Acid production causes the pH indicator to deepen
the gel color to a more pink-red background color.
ISO defines coliforms by their ability to grow in method-specific, selective media. ISO
method 4831, enumerating coliforms by the most probable number (MPN) method,
defines coliforms by their ability to grow and produce gas in the conditions described in
the standard. On the 3M Petrifilm High-Sensitivity Coliform Count Plate, these coliforms
are indicated by red colonies with gas.
Figure 1
Coliform count = 4
Figure 2
Coliform count = 13
It is easy to count coliform colonies on 3M Petrifilm High-Sensitivity Coliform Count Plates. A red indicator dye
in the plate colors Gram-negative colonies and the top film traps gas produced by the coliforms.
When coliforms produce acid, the gel surrounding the colony becomes pinker, as shown in Figure 2.
Look for pink-red zones around the colony to aid in counting. Count red colonies that are associated with gas
bubbles as coliforms.
High-Sensitivity Coliform Count Plate
2
2
3
1
Figure 3
Figure 4
Coliform count = 30
Coliform count = 0
Gas production is used to differentiate coliform from noncoliform colonies. Circles 1, 2 and 3 show how bubble patterns
may vary. The gas bubble in Circle 1 is adjacent to the colony. In
Circle 2, the gas disrupts the coliform colony so that the colony
“outlines” the bubble. In Circle 3, three small gas bubbles circle
the colony. All of these examples are coliforms. Red colonies
which are not associated with gas bubbles should not be
counted as coliforms.
Notice the change in gel color in Figures 4 through 9. As the
coliform count and acid production increases, the color of the
gel deepens from a light orange in Figure 4 to a bright pink-red
in Figure 9. Plating and incubating a negative control will aid in
differentiating changes in gel color.
Figure 5
Figure 6
Coliform count = 90
Estimated coliform count = 320
The countable range on 3M Petrifilm High-Sensitivity Coliform
Count Plate is less than or equal to 150 colonies.
The circular growth area is approximately 60 cm2. 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 60 to determine the estimated
count per plate.
For a more accurate count, further dilution of the sample may be
necessary.
3
Figure 7
Figure 8
Estimated coliform count = 840
Coliform count = too numerous to count (TNTC)
Colonies and gas bubbles may be smaller around the edge
of the inoculated area, as noted in Figure 7. The different gel
appearance around the edge of the inoculum does not affect
colony counts.
A TNTC coliform count will cause the gel to turn a darker pinkred color. Additionally, one may observe many small colonies
and/or many gas bubbles. The higher the count the less
prominent the gas and colonies may be. All three characteristics
are shown in Figure 8.
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.
2
1
Figure 9
Figure 10
Coliform count = TNTC
Coliform count = 2
Figure 9 shows many small colonies and a deepening of the
gel color.
Food particles often are irregularly shaped and are not
associated with gas bubbles. See Circle 1.
For a more accurate count, further dilution of the sample may
be necessary.
Artifact bubbles may result from improper inoculation of the
3M Petrifilm High-Sensitivity Coliform Count Plate. They are
irregularly shaped and not associated with a red colony. See
Circle 2.
4
Reminders for Use
Storage
≤8°C
1
Store unopened 3M Petrifilm HighSensitivity Coliform Count Plate pouches
frozen or refrigerated at temperatures
≤8°C (≤46°F). Use before expiration
date on package. Just prior to use, allow
unopened pouches to come to room
temperatures before opening. Return
unused plates to pouch.
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 resealed pouches
in a cool dry place for no longer than four
weeks.
Inoculation
Remember to inoculate and spread each 3M Petrifilm Plate before going on to the next plate.
3
Place the 3M Petrifilm High-Sensitivity
Coliform Count Plate on flat, level
surface. Lift top film.
4
With 3M™ Electronic Pipettor or
equivalent perpendicular to plate, place 5
mL of sample suspension onto center of
bottom film.
5
Roll top film down onto sample gently to
prevent pushing sample off film and to
avoid entrapping air bubbles. Do not let
top film drop.
6
Place the 3M™ Petrifilm™ High-Sensitivity
Plate Spreader on top film over inoculum.
7
Distribute sample with a gentle
downward pressure on the handle of
the spreader. Do not twist or slide the
spreader.
8
Lift spreader. Wait 2-5 minutes for gel
to form.
High-Sensitivity Coliform Count Plate
5
Use Appropriate
Sterile Diluents
Butterfield’s phosphate buffered
dilution water, 0.1% peptone water,
peptone salt diluent, quarter-strength
Ringer’s solution, dipotassium
hydrogen phosphate, saline solution
(0.85-0.90%), bisulfite-free letheen
broth or distilled water.
9
Incubate plates with clear side up in stacks
of up to 10. It may be necessary to humidify
incubator to minimize moisture loss. See
product instructions for third party
validated methods.
10
3M Petrifilm High-Sensitivity Coliform
Count Plates can be counted 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.
For optimal growth and recovery of
the microorganisms, adjust the pH of
the sample suspension to 6.5-7.5.
Do not use buffers containing citrate,
bisulfite or thiosulfate; 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 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
High-Sensitivity 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.
70-2008-6465-6 (Rev-1017)
6
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