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June 2, 2005
Milk Safety References
National Conference on Interstate Milk Shipments (NCIMS)
STANDARD PLATE COUNT, COLIFORM, AND SIMPLIFIED COUNT METHODS
[Unless otherwise stated all tolerances are ±5%]
SAMPLES
- __________ Laboratory sample requirements (see CP item 33 & 34)
STANDARD PLATE AND COLIFORM
METHODS DILUTING SAMPLES
- __________ Work Area
- __________ Level plating bench not in direct sunlight
- __________
Sanitized immediately before start of plating
- __________ Selecting Dilutions
- __________ Standard Plate Count
- __________ Plate two decimal dilutions per sample
- __________ Select dilutions to yield one plate with
25–250 colonies
- __________ Raw milk is normally diluted to 1:100 and
1:1000
- __________ Finished products are normally diluted to
1:10 and 1:100
- __________ The above are general guidelines and may have
to be adjusted on a case by case basis
(dilutions below 1:10 not required)
- __________ Coliform Counts
- __________ For milk samples, 1 mL direct and/or decimal
dilutions
- __________ For all other products, distribute 10 mL of a
1:10 dilution among three plates, generally high
fat and viscous products
- __________ Identifying Plates
- __________ Label each plate with sample identification and
dilution
- __________ Arrange plates in order before preparation of dilutions
- __________ Sample Agitation
- __________
When appropriate, wipe top of unopened containers
with sterile, ethyl alcohol saturated cloth
- __________
Before removal of any portion, thoroughly mix contents
of each container
- __________ Shake raw and processed sample containers
(approx ¾ full) 25 times in 7 sec with l ft movement
- __________ Invert filled retail container 25 times, each
inversion a complete down and up motion
- __________
Remove test portion within 3 min of sample agitation
- __________ Sample Measurement, pipets
- __________
Use separate sterile pipets for the initial transfers
from each container
- __________ Pipets in pipet container adjusted without touching
the pipets
- __________
Pipet tip not dragged over exposed exterior of pipets
in container
- __________
Pipet not dragged across lip or neck of sample container
- __________
Pipet not inserted more than 2.5 cm (1") below sample surface (foam avoided
if possible)
- __________
Draw test portion above pipet graduation mark and remove
pipet from liquid
- __________ Pipet aid used, mouth pipetting not permitted
(_________________)
- __________
Adjust test volume to mark with lower side of pipet in
contact with inside of sample container (above the
sample surface)
- __________
Drainage complete, excess liquid not adhering to pipet
- __________
Release test portion to petri dish (tip in contact with
plate, 45° angle) or dilution blank (with lower side of
pipet in contact with neck of dilution blank, or dry
area above buffer where appropriate) with column drain
of 2–4 sec
- __________
Blow out last drop of undiluted sample from pipet
using pipet aid
- __________ Blow out away from main part of sample in plate,
do not make bubbles
- __________
Pipets discarded into disinfectant, or if disposable
into biohazard bags or containers to be sterilized
- __________ Sample Measurement, pipettors (______________________)
- __________
Each day before use, vigorously depress plunger
10x to redistribute lubrication and assure smooth
operation (mechanical pipettors)
- __________
Before each use examine pipettor to assure that no
liquid is expelled from the pipettor nose-cone
(contaminated), if fouling is detected do not use
until cleaned as per manufacturer recommendation
- __________
Use separate sterile tip for the initial transfers
from each container
- __________
Depress plunger to first stop (mechanical pipettors)
- __________
Tip/barrel not dragged across lip or neck of sample
container, and pipettor barrel not allowed within
sample container
- __________
Tip not inserted more than 1 cm below sample surface
(foam avoided if possible)
- __________
With pipettor vertical slowly and completely release
plunger (for electronic pipettors follow manufacturer
instructions)
- __________
Remove tip from sample and depress plunger completely,
re-insert tip into sample and repeat steps f and g, and
then remove tip from liquid
- __________
Touch tip off to inside of sample container above the
sample surface, excess liquid not adhering to tip
(do not lay pipettor down once sample is drawn up, use
vertical rack if necessary)
- __________
Release test portion to petri dish (tip in contact
with plate) by slowly depressing plunger to first stop
allowing about 1 or 2 seconds for complete drainage
- __________
Move tip to a dry spot on plate
- __________ If pipettor only has one (1) stop touch off
- __________ If pipettor has two (2) stops, depress plunger
to second stop and touch off
- __________
Or, dispense test portion to dilution blank (tip in
contact neck of dilution blank, or dry area above
buffer where appropriate) by slowly depressing
plunger to first stop
- __________
If pipettor has two (2) stops, depress plunger to
second stop
- __________
Tips discarded into disinfectant, biohazard bags
or containers to be sterilized
- __________ Dilution Agitation
- __________ Before removal of any portion, shake each dilution
bottle 25 times in 7 sec with a 1 ft movement
- __________ Optionally, use approved mechanical shaker for 15 sec
- __________
Remove test portion within 3 min of dilution agitation
- __________
Dilution Measurement, pipets
- __________
Use separate sterile pipets for the initial transfers
from each container
- __________ Pipets in pipet container adjusted without touching
the pipets
- __________
Pipet tip not dragged over exposed exterior of pipets in
container
- __________
Pipet not dragged across lip or neck of dilution blank
- __________
Pipet not inserted more than 2.5 cm (1") below dilution surface
- __________
Draw dilution portion above pipet graduation mark
and remove pipet from liquid
- __________ Pipet aid used, mouth pipetting not permitted
(_______________)
- __________
Adjust dilution volume to mark with lower side of
pipet in contact with inside of dilution blank neck
- __________
Drainage complete, excess liquid not adhering to pipet
- __________
Gently lift cover of petri dish just high enough to
insert pipet
- __________
Hold pipet at 45° angle to dish with tip touching dish
(or dilution blank neck)
- __________
Release dilution portion to dish (or dilution blank)
with tip in contact with the bottom of the dish (or
dilution blank neck, or dry area above buffer where
appropriate) with column drain of 2–4 sec
- __________
Touch pipet tip once against dry spot on dish bottom
(or dilution blank neck)
- __________
When measuring 0.1 mL, do not re-touch dry area
- __________
Pipets discarded into disinfectant, or if disposable
into biohazard bags or containers to be sterilized
- __________ Dilution Measurement, pipettors (_____________________)
- __________
Use separate sterile tip for the initial transfers
from each container
- __________
Depress plunger to first stop (mechanical pipettors)
- __________
Tip/barrel not dragged across lip or neck of dilution
blank, and pipettor barrel not allowed within dilution
blank
- __________
Tip not inserted more than 1 cm below dilution surface
- __________
With pipettor vertical slowly and completely release
plunger (for electronic pipettors follow manufacturer
instructions)
- __________
Remove tip from dilution and depress plunger completely,
re-insert tip into dilution and repeat steps d and e,
and then remove tip from liquid
- __________
Touch tip off to inside of dilution blank neck or dry
area above buffer where appropriate, excess liquid not
adhering to tip
- __________
Gently lift cover of petri dish just high enough to
insert tip
- __________
Hold pipettor nearly vertical to dish with tip touching
dish
- __________
Release test portion to petri dish (tip in contact with
plate) by slowly depressing plunger to first stop
- __________
Move tip to a dry spot on plate
- __________ If pipettor only has one (1) stop touch off
- __________ If pipettor has two (2) stops, depress plunger
to second stop and touch off
- __________
Tips discarded into disinfectant, biohazard bags/con-
tainers or into spent dilution blanks to be sterilized
- __________ Samples Other than Milk
- __________ Weigh 11g aseptically into dilution blank
- __________ Use dilution blanks heated to 40–45C
- __________
Dry Milk Samples
- __________ Weigh 11g aseptically into dilution blank heated to
40–45C
- __________ Use standard dilution blank
- __________ Or, 2.0% sodium citrate blank (pH<8.0)
for relatively insoluble sample (not to be used with Petrifilm)
- __________ Wet sample completely with gentle agitation (invert)
- __________ Let soak 2 min, then shake 25 times in 7 sec with
l ft movement, use within 3 minutes of agitation
PLATING
- __________ Plating
- __________ Melt agar quickly in boiling water, flowing steam
not under pressure, or microwave oven (use extreme
caution when microwaving)
- __________
Avoid prolonged exposure to high temperatures during
and after melting, establish lab protocol
- __________ Do not melt more than will be used within 3 hours
- __________ Do not melt agar more than once
- __________
Promptly cool melted agar to 45±1C
- __________ Record temperature with other control information
- __________ Temperature control used for each test medium type
- __________ Contains medium identical to type being used
- __________ In container identical to that being used
- __________ Undergoes same heat treatment and cooling as test
medium
- __________ Select number of samples in any series so that all will
be plated within 20 min (pref £ 10) after diluting first sample
- __________
After depositing test portions, promptly pour 10 12 mL
medium into each plate of series, or 15 20 mL for > 1 mL portion/plate or where
agar weight loss is a problem that can not be corrected by other actions
(documentation must be kept to indicate that this is a routine practice)
- __________ Lift cover of petri dish just high enough to pour medium
- __________ As each plate is poured thoroughly and evenly mix
medium and test portion in petri dish
- __________ Multiple plates may be poured and mixed, however,
plates may not be stacked prior to mixing
- __________
Allow to solidify within 10 min on level surface
- __________ For dry milk sample, overlay plate with 3 5 mL PCA
- __________ For coliform count, overlay plate with 3 4 mL VRB
- __________
Invert and incubate within 10 min of medium
solidification
Controls
- __________ Controls
- __________
Check sterility of dilution blanks, medium, petri
dishes, and pipets used for each group of samples
(AM and PM)
- __________
Expose a poured plate with cover completely removed
or pre-hydrated Petrifilm Aerobic Count (PAC) film
(both wet surfaces completely exposed) to air for
15 min during plating, AM and PM
- __________ The air control plate must be the first plate poured
immediately before samples are shaken and must be
located such that it is in the area of the plating
activity (not off to the side)
- __________ If count > 15, take and note corrective
actions
- __________ For PAC films see item 45b7
- __________
Records maintained
- __________
Include information on bench, work sheet or report
sheet(s)
INCUBATION
- __________ Incubation
- __________ Incubate SPC plates at 32±1C for 48±3 hours (dry
milk for 72±3 hours) and incubate coliform plates
at 32±1C for 24±2 hours
- __________ Stack plates no more than 6 high
- __________ Arrange stacks so each is at least
1" from adjacent
stacks and from incubator surfaces
- __________ Place stacks directly over each other on successive
shelves
COUNTING COLONIES
- __________ Counting Aids
- __________ Count colonies with aid of magnification under uniform
and properly controlled artificial illumination with a
hand tally
- __________ Recording Standard Plate Count
- __________ After incubating plates, promptly count all colonies
on selected plates
- __________ Where impossible to count at once, store plates at
0–4.4C for not longer than 24 hr (avoid as a routine
practice)
- __________ Record dilutions used and number of colonies on each
plate counted
- __________ Record results of sterility and control tests
- __________ When possible, select spreader free plates with 25–250
colonies and count all colonies including those of
pinpoint size
- __________ Use higher magnification if necessary to
distinguish colonies from foreign matter
- __________ Examine edge of petri plates for colonies
- __________ If consecutive plates yield 25–250 colonies, count
all colonies on plate(s) from both dilutions
- __________ Spreaders
- __________ Count colonies on representative portion only
when colonies are well distributed and area
covered or repressed does not exceed 25% of plate
- __________ Do not count if repressed growth
area > 25% of plate area
- __________ When spreaders must be counted, count each as a single colony
- __________ Count chains/spreaders from separate sources as separate colonies
- __________ If 5% of plates are more than ¼ covered by spreaders, take immediate steps to eliminate and resolve problem
- __________ If there is no 25–250 colony plate, use plate having
nearest to 250 colonies
- __________ If plates from all dilutions exceed 250 colonies,
estimate counts as follows
- __________ Count colonies in portions representative of
distribution and estimate total
- __________ Where there are < 10 colonies/sq
cm, count
colonies in 12 squares, selecting 6 consecutive
squares horizontally across the plate and six
consecutive squares at right angles
- __________ When there are 10 or more colonies/sq cm, count
4 representative squares
- __________ Multiply average number colonies/sq cm by area
of plate in sq cm
- __________ If plates yield < 25 colonies each,
record actual
number in lowest dilution
- __________ If all plates from a sample show no colonies, record
count as 0
- Coliform Count
- __________ After incubating plates, promptly count colonies
- __________
Where impossible to count at once, store plates at 0–4.4C for not longer than 24 hr (avoid as a routine
practice)
- __________
Dark red colonies measuring 0.5 mm or more in diameter
on agar plates are considered coliforms in plates
containing
£ 154 colonies
- __________
On crowded plates, coliform colonies may be atypical;
count and confirm presence of lactose fermentors
- __________
Confirmation of colonies
- __________ Pick 10% up to 10 representative colonies per
plate with relative percentages of each colony
type and inoculate into brilliant green lactose
bile broth; incubate 24–48 hr at 32±1C as appropriate
- __________ Presence of any gas in a BGB tube constitutes a
confirmed test
- __________ Record the number of picked colonies and the
number of colonies that produced gas (if
necessary calculate % confirmed and multiply
by total number of colonies)
- __________ If no colonies appear on plate(s), record count as 0
- __________ If there are 1–154 colonies on a plate, record number
counted
- __________ If > 154 colonies develop in highest
dilution plate, record number as > 150
- __________ When multiple plates of a dilution are used, sum counts
of plates
- __________ Personal Errors
- __________
Avoid inaccurate counting due to carelessness, fatigue,
or impaired vision
- __________
Discover cause and correct if unable to duplicate your
own counts on the same plate
- __________
Perform monthly counting
- __________ If 3 or more analysts use the RpSm method, see
current SMEDP, records maintained
- __________ If less than three analysts, comparative counts
agree £ 8% for the same analyst and £ 10% between two analysts, records maintained
REPORTS
- __________ Computing and Reporting Counts
- __________
Multiply number of colonies (or estimated number
if necessary) by the reciprocal of the dilution
- __________
If consecutive dilutions yield 25–250 colonies, compute
count using formula below (see current SMEDP)
N = åC/[(1 x n1) + (0.1 x n2)]d
Where, N = number of colonies per milliliter or gram
åC = sum of all colonies on all plates counted
n1 = number of plates in lower dilution counted
n2 = number of plates in next highest dilution
counted
d = dilution from which the first counts were
obtained
Example: 1:100 = 244 colonies 1:1,000 = 28 colonies
N = (244 + 28)/ [(1 x 1) + (0.1 x 1)]0.01
= 272/[1.1]0.01
= 272/0.011
= 24,727 [25,000 (reported)]
Note: In the NCIMS Program the denominator will always
be 0.11 for 1:10 dilutions and 0.011 for 1:100
dilutions
- __________
Report SPC and coliform counts only if inhibitors are
not detected
- __________
Report computed count as Standard Plate Count/mL or
/g (SPC/mL or SPC/g) when taken from plate(s) in the
25–250 range
- __________
Report count as Coliform Count (confirmed)/mL or
/g when taken from plate(s) in the l l54 range
- __________
If no colonies appear on SPC plates, report as < 25 times the reciprocal of
the dilution and report as estimated
- __________
If no colonies appear on coliform plates, report as < 1 times the reciprocal
of the dilution and report as estimated
- __________
Report SPC plate counts of 0 to 24 as < 25 times the reciprocal of the dilution
and report as estimated
- __________
When colonies on SPC plates exceed 100/sq cm, compute
count by multiplying 100 x dilution factor x area of
plate in sq cm and report as > computed count estimated
- __________
Computed counts from SPC plates outside the 25-250
range are reported as Estimated SPC (ESPC)
- __________
Counts from coliform plates > 154 are reported as > 150 Estimated Coliform
Count (ECC)
- __________
If for any reason, an entire plate is not counted, the
computed count is reported as Estimated
- __________
Report only first two left hand digits
- __________ If the third digit is 5 round the second number
using the following rules
- __________ When the second digit is odd round up
(odd up, 235 to 240)
- __________ When the second digit is even round down
(even down, 225 to 220)
- __________
If all plates from a sample have excessive spreader
growth, report as spreaders (SPR), or are known to
contain inhibitor(s) report as growth inhibitors (GI)
- __________
If a laboratory accident renders a plate uncountable,
report as laboratory accident (LA)
PLATE LOOP COUNT METHOD
APPARATUS
- __________ Loop 0.001 mL
- __________
True circle, welded I.D. 1.45±0.06 mm, calibrated to
contain 0.001 mL, made of appropriate wire
- __________
Loop fits over a No. 54 but not a No. 53 twist
drill bits (lab must have set), checked monthly,
records maintained
- __________
Modified by making a 30° bend 3–4 mm from loop,
compare to template before use
- __________
Opposite end of wire kinked in several places
- __________ Hypodermic Needle, Luer-Lok
- __________ 13 gauge (sawed off 24–36 mm from the point where the
barrel enters the hub)
- Kinked end of loop wire shank inserted into needle until bend is 12–14 mm from end of barrel, compare to template before use
- __________ Cornwall Continuous Pipetting Outfit
- __________ Consisting of metal holder, Cornwall Luer Lok syringe
and filling outfit
- __________
Syringe, 2 mL capacity, adjusted to deliver 1.0 mL
- __________ Calibrated by checking ten 1 mL discharges (10 mL)
using a 10 mL Class A graduate cylinder each day of
use, records maintained
- __________
With Luer-Lok of needle attached to Luer Lok fitting
of syringe
PREPARATION
- __________ Heat Treatment of Pipetting Equipment
- __________ Sterilize assembled outfit wrapped in kraft paper or
in a closed container by autoclaving at 120±1C for
15 min
- __________ Assembly of Complete Apparatus for Use
- __________
Place end of rubber supply tube (attached to syringe)
in sterile dilution buffer blank and depress syringe
plunger several times to pump buffer into syringe
- __________ Briefly flame loop and allow to cool 15 sec
- __________ Discharge several 1 mL portions to waste, then
discharge 1 mL portion of buffer into instrument
control plate
PROCEDURE
- __________ Comparative Test with SPC
- __________
Comparisons done by each analyst performing test
- __________ Comparison is valid only if done using similar
plate count methods, i.e. SPC agar with pipets (or
pipettors) to SPC agar with the PLC device or
Petrifilm with pipets (or pipettors) to Petrifilm
with the PLC device. Mixing methods is not
permissible
- __________ Results must be shown to be acceptable prior to
official use of test in laboratory
- __________ Copy of comparison and results in QC record (or easily
accessible file in laboratory)
- __________ Identifying Plates (as item 4)
- __________ Sample Agitation (as item 5)
- __________ Inoculating Plates
- __________
Dip loop into each sample (avoiding foam) to bend in
shank and withdraw vertically from surface three times
in 3 sec with uniform movement of 2.5 cm
- __________
Raise cover of petri dish (just high enough to insert
loop), insert loop and depress plunger causing sterile
dilution buffer to flow across charged loop washing
measured 0.001 mL of sample into dish
- __________
Do not depress plunger so rapidly that buffer fails
to flow across loop
- __________ Plating
- __________
As described in item 13 or 44
- __________
Pour plates with 12–15 mL agar
CONTROLS
- __________ Controls
- __________
See item 14
- __________
Initial rinse control, see item 25c and 29c
- __________
Determine if loop is free rinsing by preparing a rinse
control plate after every 20 samples plated
- __________
After all samples have been run discharge a final
rinse to a control plate
INCUBATION
- __________ Incubation (see item 15)
- __________
48±3 hr at 32±1C
COUNTING COLONIES
- __________ Counting Aids (see item 16 or 47b)
- __________ Recording Plate Loop Counts (see item 17 or 48)
- __________ Personal Errors (see item 19 or 49)
REPORTS
- __________ Reporting Counts
- __________ See item 20 or 50
- __________ If 0 to 24 colonies on plate report
as < 25,000 Estimated Plate Loop Count/mL (EPLC/mL) or if Petrifilm used,
EPPLC/mL
- __________ If count is between 25 and 250, report count as
PLC/mL or PPLC/mL
- __________ If colony count is > 250, report
as EPLC/mL or EPPLC/mL
- __________ When colonies exceed 100/sq cm,
compute count by
multiplying 100 x dilution factor x area of plate
in sq cm and report as > computed count estimated
PETRIFILM AEROBIC COUNT METHOD
APPARATUS
- __________ Petrifilm Aerobic Count (PAC) Films
- __________ Plastic Spreader
- __________ Provided with Petrifilm films, concave (ridge) side
used
- __________ Identifying Films (as item 4)
- __________ Sample Agitation (as item 5)
- __________ Sample Measurement (as item 6&7)
- __________ Dilution Agitation (as item 8)
- __________ Dilution Measurement (as item 9&10)
- __________ Procedure
- __________ Place the film on a level surface
- __________ Lift the top film and deposit 1 mL of sample or
dilution onto the center of the base film, touching
off the last drop
- __________ Deposit samples with pipet (since only 1 mL samples
can be used; 10 fold dilution will have to be made)
- __________ Or, deposit samples with pipettor (capable of making
a 1:10 dilution in the tip)
- __________ Or, deposit sample with PLC apparatus (item 29)
- __________ Carefully drop the top film onto the inoculum
- __________ Place the plastic spreader with the ridge side down
(item 38) on the top film over the sample and press
down gently on the center of the spreader to distribute
inoculum to the circular ridge of the spreader
- __________ Leave film undisturbed for 1 min for gel solidification
- __________ Incubate within 10 min of solidification
- __________ Controls
- __________ See item 14 above except for air plates
- __________ Air plates
- __________ Inoculate PAC film with dilution buffer (1 mL)
- __________ Drop film down onto the dilution buffer and spread
as described in item 44d above
- __________ Leave film undisturbed for 1 minute for
solidification of gel
- __________ The film must be the first one prepared immediately
before samples are shaken and must be located such
that it is in the area of the plating activity (not
off to the side)
- __________ Roll top film back and away from bottom film
and expose film for 15 min
- __________ After the 15 min roll top film back down and
incubate with other films as usual
- __________ Incubated, exposed films should
contain £ 10
colonies, if count > 10, take and note corrective actions
INCUBATION
- __________ Incubation
- __________ Place films in horizontal position, clear side up
- __________ Stack films no more than 20 high
- __________ Incubate 48±3 hr at 32±1C
COUNTING COLONIES
- __________ Counting PAC Films
- __________ See item 16, or
- __________ Optionally, count using an approved Petrifilm reader
- __________ Refer to manufacturer's instructions for set-up
and operation information
- __________ 3M Petrifilm Information Management System (PIMS)
- __________ Store control cards in a clean, dry and
enclosed container
- __________ Scan and record control card result prior to
the start of and at the end of each operation
period
- __________ Scan and record control card result hourly with
continuous operation
- __________ Control card result must fall in the 92 to 108
range, if outside of this range an alarm will
sound to alert the operator of a failure
- __________ If alarm sounds, inspect card for defects,
if defect(s) are observed replace control
card, scan and report result of new card
- __________ Do not proceed unless control card gives
acceptable result, seek technical assistance
- __________ 3M Petrifilm Plate Reader
- __________ Store System Verification Cards in a clean, dry
and enclosed container
- __________ Scan and record System Verification Card result
prior to the start of and at the end of each
operation period
- __________ Use Log File feature to automatically save
electronic pass/fail result
- __________ Scan and record System Verification Card result
hourly with continuous operation
- __________ Use Log File feature to automatically save
electronic pass/fail result
- __________ System Verification Cards used to check the
function of the Petrifilm Plate Reader prior
to reading test films (red, green and blue top
lights, and backlights flash)
- __________ If inserting the System Verification Card
results in an error message, remove and
re-insert card
- __________ If an error occurs a second time, inspect
card for visible dirt or defects, clean
and re-insert card
- __________ If card gives a third error replace card,
scan and report result of new card
- __________ Do not proceed unless System Verification
Card gives an acceptable result, seek
technical assistance
- __________ Advanced Instruments PetriScan Reader
- __________ Inspect scanner glass for spots and if necessary
clean using a soft, lint-free cloth with a mild
glass cleaner
- __________ Place templates 1 and 2, and two films with no
growth in the PetriScan grid and scan
- __________ Count and record all results prior to the start
of and at the end of each operation period
- __________ Scan, count and record template and no growth
film results hourly with continuous operation
- __________ Template 1 gives count between 27 and 33
- __________ Template 2 gives count between 190 and 210
- __________ No growth films give a count of zero
- __________ If any results out of range
- __________ Inspect templates and films for defects and
scanner glass for spots
- __________ If defect(s) found replace template or film
and scan, count and record new result(s)
- __________ Do not proceed until template and no growth
films give acceptable results, seek technical
assistance
- __________ Maintain records
- __________ Examine each test film visually prior to placing into
the reader
- __________ For films with no growth, assure reader count is
zero
- __________ For atypical films, spreader, confluent
growth, excessive growth around edge of film, etc., do not
count with reader, record as appropriate using
items 17 & 48
- __________ Petrifilm Count
- __________ Count all colonies stained various shades of red,
even those outside the circular indentation left
by the spreader
- __________ See item 17
- __________ Select spreader free films with 25–250 colonies and
count all red colonies
- __________ If films from all dilutions yield
< 25 colonies each, record actual number in lowest dilution
- __________ If all films from a sample show no colonies, record
count as 0
- __________ If films from all dilutions exceed 250 colonies,
estimate (as per manufacturer specification)
- __________ Personal Errors
- __________ See item 19, or
- __________ If using an approved film reader (item 47b) analysts
must perform monthly visual counts comparing to reader
results (reader = one analyst)
- __________ If one analyst, count must be £ 10% between visual and the reader result
- __________ If two or more analysts, use RpSm method (see current
SMEDP) using the reader result as an analyst count
REPORTS
- __________ Reporting Counts
- __________ See item 20
- __________ If the count is between 25 and 250, report count as
Petrifilm Aerobic count/mL (PAC/mL)
- __________ If count is 0 to 24, report as <
25x reciprocal of the
dilution as Estimated PAC/mL (EPAC/mL)
- __________ If count is > 250, report as EPAC/mL
- __________ When colonies exceed 100/sq cm,
compute count by
multiplying 100 x dilution factor x 20 sq cm and
report as > computed count estimated
PETRIFILM COLIFORM COUNT METHOD
APPARATUS
- __________ Petrifilm Coliform Count (PCC) Films
- __________ Plastic Spreader
- __________ Provided with Petrifilm films, smooth, flat side used
PROCEDURE
- __________ Selecting Dilutions
- __________ For milk samples, 1 mL direct and/or decimal
dilutions
- __________ For other milk products use 1/10 dilution, must plate
10 mL, i.e., use 10 PCC films or see 64d
- __________ Identifying Films (as item 4)
- __________ Sample Agitation (as item 5)
- __________ Sample Measurement (as item 6 & 7)
- __________ Procedure
- __________ Place films on level surface
- __________ Lift top film and deposit 1 mL of sample above the
center of the base film, touching off the last drop
- __________ Carefully roll the top film into the inoculum,
avoid trapping air bubbles
- __________ Place the plastic spreader with the flat side down
(item 52) on the top film and press down gently on
the center of the spreader to distribute inoculum
over growth area
- __________ Leave films undisturbed for 1 min for gel
solidification
- __________ Incubate films within 10 min of solidification
INCUBATION
- __________ Incubation
- __________ Place films in horizontal position, clear side up
- __________ Stack films no more than 20 high
- __________ Incubate 24±2 hr at 32±1C
COUNTING COLONIES
- __________ Counting Aids (see item 16)
- __________ Petrifilm Count
- __________ Count only red colonies having 1 or more gas bubbles
within 1 colony diameter
- __________ Colonies with gas bubbles are confirmed, no other
testing is required
REPORTS
- __________ Reporting Counts
- __________ See item 20
- __________ If the count is between 1 and 154, report count as
Petrifilm Coliform count/mL (PCC/mL)
- __________ If count is 0, report as < 1 Estimated
PCC/mL (EPCC/mL)
- __________ If count is > 154, report as > 150
EPCC/mL
PETRIFILM HIGH-SENSITIVITY COLIFORM COUNT METHOD
APPARATUS
- __________ Petrifilm High-Sensitivity Coliform Count (HSCC) Films
- __________ Plastic Spreader for HSCC Films
PROCEDURE
- __________ Selecting Dilutions
- __________ For milk samples, apply 5 mL direct and/or make
decimal dilutions
- __________ 1:5 minimum dilution required for: chocolate milk,
cottage cheese, dip, evaporated milk, frozen yogurt,
heavy and light cream, ice cream, sour cream, sweetened
condensed milk and/or decimal dilutions
- __________ 1:10 minimum dilution required for: butter, buttermilk,
cheese, dry dairy products, yogurt and/or decimal
dilutions
- __________ 1:10 dilutions of milk or milk products test 10 mL (5 mL
on two films)
- __________ Identifying Films (as item 4)
- __________ Sample Agitation (as item 5)
- __________ Sample Measurement (as item 6 & 7)
- __________ Procedure
- __________ Place film on level surface
- __________ Lift top film and deposit 5 mL of sample or
dilution just above the center of the bottom film,
touching off the last drop
- __________ Carefully roll the top film onto the sample gently to
prevent pushing the inoculum off the film and to avoid
trapping air bubbles
- __________ Place the plastic spreader (item 63) on the top film
over the inoculum
- __________ Distribute sample with a gentle downward pressure on
the handle of the spreader to distribute inoculum to
the circular ridge of the spreader .
- __________ Leave film undisturbed for 2–5 min for gel to solidify
- __________ Incubate within 10 min of solidification
INCUBATION
- __________ Incubation (see item 58)
- __________ Stack films no more than 10 high
COUNTING COLONIES
- __________ Counting Aids (see item 16)
- __________ Petrifilm Count (see items 18 & 60)
REPORTS
- __________ Reporting Counts
- __________ See items 20 and 61
- __________ On 5 mL direct films report:
- __________ 1 to 4 colonies as < 1 coliform/mL
or gm
- __________ 5 colonies as 1 coliform/mL or gm
- __________ > 5 colonies as 1 coliform for
every 5 colonies counted, rounding up to the next number if not even multiples
of 5 (ex. 11=3 coliforms/mL or gm)
- __________ 5 mL of 1:5 dilution provides a 1:1 sensitivity
- __________ 5 mL of 1:10 dilution provides a sensitivity of
2 coliforms/mL or gm, run 1:10 dilutions in duplicate
to get a sensitivity of 1 coliform/mL or gm as required
by the PMO