US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Use of Petrifilm and PetriScan
SOP Number: EQ-09-02
Date Revised: 08-13-13
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SOP No. EQ-09-02
Date Revised 08-13-13
Page 1 of 11
SOP Number
EQ-09-02
Title
Use of Petrifilm and PetriScan®
Scope
This SOP describes the use of 3M Petrifilm™ Aerobic Count (AC)
Plate and PetriScan with Bacillus subtilis, Pseudomonas aeruginosa,
Staphylococcus aureus, and Salmonella enterica.
Application
To enumerate microorganisms used in disinfectant and sporicidal
efficacy testing.
Approval Date
SOP Developer:
Print Name:
SOP Reviewer
Print Name:
Quality Assurance Unit
Print Name:
Branch Chief
Print Name:
Date SOP issued:
Controlled copy number:
Date SOP withdrawn:
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SOP No. EQ-09-02
Date Revised 08-13-13
Page 2 of 11
TABLE OF CONTENTS
Contents Page Number
1.
DEFINITIONS
3
2.
HEALTH AND SAFETY
3
3.
PERSONNEL QUALIFICATIONS AND TRAINING
3
4.
INSTRUMENT CALIBRATION
3
5.
SAMPLE HANDLING AND STORAGE
3
6.
QUALITY CONTROL
3
7.
INTERFERENCES
4
8. NON-CONFORMING DATA
4
9.
DATA MANAGEMENT
5
10.
CAUTIONS
5
11.
SPECIAL APPARATUS AND MATERIALS
5
12.
PROCEDURE AND ANALYSIS
5
13.
DATA ANALYSIS/CALCULATIONS
8
14.
FORMS AND DATA SHEETS
8
15.
REFERENCES
8
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SOP No. EQ-09-02
Date Revised 08-13-13
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1. Definitions
Additional abbreviations/definitions are provided in the text.
TM
1. Petrifilm = A sample-ready culture medium which contains "Standard
Methods" nutrients, a cold water soluble gelling agent, and a tetrazolium
indicator that facilitates colony enumeration.
2. CFU = Colony forming units
3. Audit View = A secure database that records and displays user, date, time,
and edited data when records are changed.
4. Flag = A code notation assigned by Petri Scan indicating plate edits or
changes in other plate characteristics.
5. Plate = Petrifilm plate
2. Health and
Safety
1. Follow procedures specified in SOP MB-01, Laboratory Biosafety. The
Study Director and/or lead analyst should consult the Material Safety Data
Sheet for specific hazards associated with products.
2. Counting the Petrifilm using the scanner will be performed on the bench-
top.
3. Personnel
Qualifications
and Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
1. The Petri Scan was validated after purchase; these data are archived.
2. Verification is conducted annually, refer to section 6.
5. Sample
Handling and
Storage
For additional information, refer to 15.5.
1. Store unopened Petrifilm plate pouches refrigerated or frozen at
temperatures <8°C.
2. Remove the required number of plates. Return unused plates to pouch.
Seal by folding the end of the pouch over and taping shut. Place the pouch
into a resealable container.
3. Opened pouches may be stored at room temperature for up to 30 days.
4. For longer term storage, store opened pouches of Petrifilm in a freezer that
does not have an automated defrost cycle until the expiration date listed on
the package.
5. Do not refrigerate resealed pouches.
6. Quality Control
1. For quality control purposes, the required information is documented on
the appropriate data sheets (see section 6.3).
2. Perform quality control procedures described in the PetriScan User
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SOP No. EQ-09-02
Date Revised 08-13-13
Page 4 of 11
Guide, pages 28 and 29, section 3.8 to verify the count accuracy and
consistency at both low and high colony counts of the PetriScan system
on an annual basis.
a. The electronic data generated during these quality control procedures
are stored in a database entitled Quality Control. Once per year,
archive the quality control data.
3. Sterility assessment:
a. On each test day, select one plate, spot it with 1 mL of sterile diluent,
and incubate at 36±1°C with the test system alongside inoculated
plates. Record results (e.g., growth or no growth).
b. The lack of red colonies indicates a sterile plate while the presence of
one or more red colonies indicates contamination.
c. If contamination is noted in the sterility assessment, investigate the
potential source.
7. Interferences
1. Do not use Petrifilm plates that show discoloration.
2. Do not use diluents containing citrate, bisulfite or thiosulfate with
Petrifilm plates; these compounds can inhibit growth.
3. Since the colonies grow suspended in a gel matrix, the application of
substantial pressure to the plate will cause the colonies to shift; thus avoid
the application of pressure to the plate after spreading.
4. High concentrations of colonies on the Petrifilm plates will cause the
entire growth area to become red or pink. Occasionally, on overcrowded
plates, the center may lack visible colonies, but many small colonies can
be seen on the edges. When any of these occurs, do not count the plates
using the scanner - record the results as too numerous to count (TNTC).
5. Dust particles may be picked up by the PetriScan and counted as colonies
- analysts should evaluate each film after the program performs its Auto
Count function.
8. Non-
conforming
Data
1. Management of non-conforming data will be specified in the study
protocol; procedures will be consistent with SOP ADM-07, Non-
Conformance Reports.
2. If any colonies are detected on the uninoculated films used for quality
control procedures, visually inspect the film for defects or particles. If no
particles are present, it may be necessary to clean the scanner (refer to the
PetriScan User Guide, page 41, section 5).
3. For further information involving problems encountered in operating the
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SOP No. EQ-09-02
Date Revised 08-13-13
Page 5 of 11
PetriScan®, refer to the PetriScan® User Guide, page 43, section 6
(Troubleshooting).
9. Data
Management
1. Data will be archived consistent with SOP ADM-03, Records and
Archives.
®
2. PetriScan software creates an FDA/GLP-compliant database, which
contains every saved plate, including its image and the settings used in its
analysis. This allows images to be recalled and re-analyzed at a later date.
®
3. PetriScan tracks changes made to the database, in compliance with Code
of Federal Regulations, Title 21, Part 11, Electronic Records; Electronic
Signatures, and Code of Federal Regulations, Title 21, Part 58, Good
Laboratory Practice for Non-Clinical Laboratory Studies. These changes
can be seen in the Audit View by clicking the Audit View button (refer to
®
the PetriScan User Guide, page 35, section 4.4 for information regarding
Audit View).
10. Cautions
1. Hold the pipette perpendicular to the plate to dispense the sample.
2. Immediately after spotting the sample on the Petrifilm, press the sample
with the spreader to distribute the sample.
3. Press the spreader straight down - do not twist or slide the spreader across
the plate.
11. Special
Apparatus and
Materials
1. Petrifilm™ Aerobic Count Plate
2. PetriScan®
®
3. Computer loaded with PetriScan software
4. Hewlett-Packard Scanjet 8200
12. Procedure and
Analysis
Allow plates to come to room temperature before use.
12.1 Use of
Petrifilm
a. Mark the plate identification information within the top % inch
portion of the film.
b. Prepare dilutions to be plated using appropriate sterile diluents (e.g.,
PBDW, Luria-Bertani broth, letheen broth, or distilled water).
c. For plating, use dilutions that will yield between 0-300 CFU per
plate.
d. Place the Petrifilm plate on a flat, level surface.
e. Lift the top film and hold the pipette perpendicular to dispense the
appropriate amount (800, 900 or 1000 [xL, depending upon the total
volume of the dilution) of sample suspension onto the center of the
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SOP No. EQ-09-02
Date Revised 08-13-13
Page 6 of 11
bottom film.
f. Drop the top film down onto the sample. Do not roll the top film
down.
g. Place the plastic spreader with the recessed side down on the center
of the plate. Press gently on the center of the spreader to distribute
the sample evenly. Spread the inoculum over the entire area of the
recessed side of the spreader on the Petrifilm plate growth area
before the gel is formed.
h. Remove the spreader and leave the plate undisturbed for at least one
minute to permit the gel to form.
i. Incubate plates in a horizontal position with the clear side up in
stacks of no more than 20 plates at 36±1°C for 24-48 hours
(depending on the organism being evaluated). An incubation time of
48 hours is recommended for Pseudomonas aeruginosa as well as for
microorganisms damaged by exposure to disinfectants or sporicides.
®
j. Petrifilm can be counted manually or using the PetriScan .
k. Count all red colonies regardless of size or intensity.
12.2 Confirmation
a. Colonies may be isolated for further identification by lifting up the
top film and picking up growth from the gel; these colonies can be
Gram stained or plated.
12.3 PetriScan
®
a. Turn on the computer and double click the PetriScan icon on the
desktop. The sign-on dialog box will be displayed. Enter the
appropriate user name and password.
®
b. PetriScan data are stored and maintained in database files. After
signing on, create a new database or open an existing database. To
®
create a new database, refer to the PetriScan User Guide, page 13,
®
section 2.2.1. To open an existing database, refer to the PetriScan
User Guide, page 14, section 2.2.2.
®
c. To count colonies using the PetriScan , lift the scanner lid and place
1-4 Petrifilms face-down within the cutouts on the alignment grid.
d. Do not count plates that contain any of the following conditions:
i. High concentrations of colonies on the Petrifilm plates that
cause the entire growth area to become red or pink.
ii. Overcrowded plates where the center lacks visible colonies,
but the edges contain colonies.
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SOP No. EQ-09-02
Date Revised 08-13-13
Page 7 of 11
iii. Excessive spreader growth (>50% of the film area).
e. If liquefied gel interferes with counting, an estimated count can be
made by counting the unaffected areas.
f. Close the lid and click Scan. When the scan is complete, the images
will be displayed in the grid view (see Attachment 1).
g. After scanning, enter in an ID and Dilution factor (if applicable) for
each film to be counted.
h. Click Select All, or select each film individually by placing a S mark
in the box in the lower left-hand corner of the film.
i. Click Auto Count. PetriScan will mark each counted colony with a
green circle and display the number of counted colonies and the
CFU/mL or CFU/gm. The CFU/mL or CFU/gm represents the
calculated colony forming units per milliliter or gram of the original
sample, taking into account the dilution factor.
j. To count film manually, place a S mark in the box in the lower left-
hand corner of the film to be counted and click Manual Count. As
necessary, add or delete colonies using the left and right mouse
buttons, respectively. Click Exit. Select Yes when asked if the data
should be saved.
k. After automatic counting, click Save Data. Films marked with V
marks will be saved as JPEG (.jpg) image files, and data for the
selected films will be saved to the open database and displayed in the
Data View. The grid will be cleared of images/data and will be ready
for the next scan.
i. NOTE: Data are not saved to the database until Save Data is
clicked. If the Scan button is clicked for the next set of
Petrifilm to be scanned before saving data, the images and
data will be lost.
1. It is recommended that after scanning and saving the data, the analyst
reviews each film to ensure it was counted correctly. To review each
scanned film, double-click on an individual entry to bring up the
image of the scanned film. Observe the film to make sure all
colonies have been counted (e.g., encircled in blue) and to make sure
that all spots that were counted are colonies (occasionally a dust
particle will be counted as a colony).
m. It is also recommended that the ID and Dilution Factor entered by
the analyst be checked against the ID and Dilution Factor noted on
the label of the Petrifilm.
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SOP No. EQ-09-02
Date Revised 08-13-13
Page 8 of 11
13. Data Analysis/
Calculations
1. To edit colonies (this option is only available after saving), double-click
on the row listed in the Data View.
a. To remove marked colonies, click with the right mouse button on the
green circles. The circles will turn red to show that they will not be
used in the count.
b. To add colonies that were not marked as counted, click with the left
mouse button. A blue circle will mark the colony, indicating that it
will be added to the count. To exclude a newly-marked circle,
simply click on it again with the right mouse button.
c. Click Exit and select Yes when asked if the data should be saved.
d. Enter the reason for editing the data and click OK. The saved data
will replace the row in the Data View, and a flag will be assigned
(refer to the PetriScan User Guide, page 27, section 3.6.2 for
information regarding flags). The previous data, flag notations, and
reason for change will be archived to Audit View.
14. Forms and Data
Sheets
1. Attachment 1: Sample Film with Growth
2. Attachment 2: Sample Data Spreadsheet
3. Attachment 3: Sample Audit View
15. References
1. PetriScan® User Guide, Spiral Biotech, Inc., Rev3 110104.
2. Hewlett-Packard Scanjet 8200 Series Scanners User's Manual, Hewlett-
Packard, 2003.
3. 3M Petrifilm™ Aerobic Count Plate Pamphlet, 3M, 2004 (38-9018-1246-
1).
4. 3M Petrifilm™ Aerobic Count Plate Interpretation Guide, 3M, 2005 (70-
2008-4572-8).
5. Storage and Shelf Life of 3M™ Petrifilm™ Plates, 3M Technical Bulletin,
October 2008 (TB.002.02).
6. Nelson, M.T., LaBudde, R.A., Tomasino, S.F., and Pines, R.M. (2013) J.
AO AC Int. 96, 717-722.
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SOP No. EQ-09-02
Date Revised 08-13-13
Page 9 of 11
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SOP No. EQ-09-02
Date Revised 08-13-13
Page 10 of 11
Attachment 2
Sample Data Spreadsheet
Nunc
Title
Reference
High Count Limit
Lew Cmmt Limit
Created l>v
QC
Quality Control
300
0
MLB
Plate a*
Place ID
Dilution
Count
CFLVml
Flag
Date
Time
I'ser ID
238
Template 1,03/12
A
30
3.00E+0!
ID ED
3 2312
2:04:21 PM
MLS
259
Template 2,88,12
A
2«
2.O0E+82
ID ED
3 li 2312
2:04:28 PM
">10
Uninoculated 1.03 '12
§
0
C.C3E-0S
ED
3 '.-1 2312
2:0429 PM
ME
-t i <
Uninoculated 2,03/12
0
0
O.CCE—03
3 I- 2312
2:0429 PM
MB
Template 1,06/12
ft
30
3.C3E-01
13 ED
S 28 2312
2:38:02 PM
ML3
2-3
Template 2.06/12
0
200
2.33E~32
ID ED
5 2S 2312
2:3S:tt3 PM
MB
2-U
Uninoculated 1,06/12
o
0
3.C3E-33
DED
6 2S 2312
2:3S:WPM
MB
Uninoculated 2.06/12
0
0
3.S3E-33
ID ED
S 2S 2312
3S
3-i ?2C
'.II
246
Template I, §9/S2
0
30
3.S3E-31
DED
c 2S 2312
i-
;i?m
MB
247
Template 2, 09/12
0
200
2.3SE-32
ID ED
5 26 2312
3
--
1: ?M
MB
248
Unmodulated 1.09/12
0
0
3.C3E-33
ED
S 25 2312
J
11
i: ?J.;
MB
249
Uninoculated 2,W/12
0
0
3.S3E-33
ED
; 2;n
3
li
i; ?2C
MLB
230
Template 1, 12/12
0
30
3.03E-3]
ID ED
12 21 2312
1:25:25 A2vl
MB
251
Template 112/12
0
200
-i
DED
12 2- 2Ci2
1:2
5:26 Ai:
MB
7^1
Uninoculated 1.12/12
0
0
ED
'1 1 ^ ir; } ~v
1:25:25 AM
MLB
253
Uninoculated 2.12/12
0
0
3.33E-33
-> ¦ 1^:1
A- -O
5:26 AM
MB
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SOP No. EQ-09-02
Date Revised 08-13-13
Page 11 of 11
Attachment 3
Sample Audit View
PetriScan Quality Control Procedure Records for 2012 {Audit View)
0?? Mi crofctolosv Laboratory
Nuse QC
Title Quality Control
Reference
High Count Limit 300
Low Count Limit 0
Created by MLB
Plsre i
• i.
Biluiion
Cou fit
ck; hi 3_
Hag
Mad Dare
Mod Ticie
Mod Bv
Reason
23 S
Template 1
0
31
E-"
3'14/2012
2:05:13 PM
MLB
Added date to ID deletedpiece of dust
239
Template 2
0
202
: 2 .:
2 55 34 PM
MLB
Added date to ID. deleted piece of dust
240
Umnoculated 1.C3 12
0
3
3 ji 2C52
2 05-15 PM
MLB
Deleted pieces of dust.
242
Template I
0
32
:¦
2 3? -5 PM
MLB
Added date to ID deleted pieces of dust
243
Template 2
0
202
_ _E- .
. 2: 2:.:
2 4? "*4 PM
mlb
Added date to ZD. deleted pieces of dust
244
0
2
: r-
5 25 2012
2 j-3 ;1 PM
MLB
Added ID. deleted pieces of dust.
245
0
2
2.0OE-KK}
S 2S 2S12
2 K 15 ?M
^XB
Added ID deleted pieces of dust
245
247
Template 1
Template!
0
32
-litr _r1.\
o 2 c 2 012
11 *
:• „ "i
Added date to ID. deleted pieces of dust
Added date to ID. deleted pieces of dust.
n
202
. _E- .
: " -
3 4-pm
MLB
24 §
L'tunoculated. J ^2
3
- E-
: T:' .1
34513PM
MLB
Deleted pieces of dust
249
Utiaiorulat-'i 2 2 2
2
2.00E-00
a 2'K2
5 4? "S ?M
MLB
Deleted pieces of dust
250
Template i
32
,2'IE-1
12 2- 2012
112" 02 AM
1 .IE
Added date to ID deleted pieces of dust
251
Template 2
0
201
_ z- .
:2:.2
112" 23 AM
I^XB
Added date to ID. deleted piece of dust
252
Urunoculated 1.1212
0
5
--
:2 -
IS 2'f.TAM
ISXB
Deleted pieces of dust
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