mm s r4
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. for Research Applications
Standard Operating Procedures (SOPs)
SOP Number: EQ-09-01
Date Revised: 06-02-09

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 1 of 14
EPA/OPP MICROBIOLOGY LABORATORY
ESC, Ft. Meade, MD
Standard Operating Procedure
for
Use of Petrifilm and PetriScan® for Research Applications
SOP Number: EQ-09-01
Date Revised: 06-02-09
Initiated By:
Technical Review:
QA Review:
Approved By:
Effective Date:
Print Name:
Print Name:
Technical Staff
Print Name:
QA Officer
Print Name:
Branch Chief
/ /
Date: / /
Date:
/ /
Date: / /
Date: / /
Controlled Copy No.:
Withdrawn By:
Date: / /

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 2 of 14
TABLE OF CONTENTS
Contents	Page Number
1.0 SCOPE AND APPLICATION	3
2.0 DEFINITIONS	3
3.0 HEALTH AND SAFETY	3
4.0 CAUTIONS	3
5.0 INTERFERENCES	4
6.0 PERSONNEL QUALIFICATIONS	4
7.0 SPECIAL APPARATUS AND MATERIALS	5
8.0 INSTRUMENT OR METHOD CALIBRATION	5
9.0 SAMPLE HANDLING AND STORAGE	5
10.0 PROCEDURE AM) ANALYSIS	5
11.0 DATA ANALYSIS/CALCULATIONS	8
12.0 DATA MANAGEMENT/RECORDS MANAGEMENT	9
13.0 QUALITY CONTROL	9
14.0 NONCONFORMANCE AND CORRECTIVE ACTION	10
15.0 REFERENCES	10
16.0 FORMS AND DATA SHEETS	 10

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 3 of 14
1.0 SCOPE AND APPLICATION:
1.1 The ability to recover and quantify microorganisms used in disinfectant and
sporicidal efficacy testing is critical to evaluate the efficacy of a product. This
SOP describes the use of 3M Petrifilm™ Aerobic Count (AC) Plate and
Petri Scan® with Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus
aureus, and Salmonella enterica for research purposes.
2.0 DEFINITIONS:
2.1	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.
2.2	CFU = Colony Forming Units
2.3	TNTC = Too Numerous to Count
2.4	AO AC = AO AC International
2.5	Audit View = A secure database that records and displays user, date, time, and
edited data when records are changed.
2.6	Flag = A code notation assigned by PetriScan® indicating film edits or changes in
other plate characteristics.
2.7	Pouch = One pouch contains 50 Petrifilm.
2.8	Plate = Petrifilm plate
3.0 HEALTH AND SAFETY:
3.1	All manipulations of the test organisms are required to be performed in
accordance with biosafety practices stipulated in SOP MB-01, Lab Biosafety.
3.2	Counting the Petrifilm using the scanner will be performed on the bench-top.
4.0 CAUTIONS:
4.1 Hold the pipette perpendicular to the film to dispense the sample.
4.2 Immediately after spotting the sample on the Petrifilm, the sample must be

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 4 of 14
pressed with the spreader to distribute the sample.
4.3	The sample must be pressed straight down - do not slide the spreader across the
film.
4.4	The Petrifilm must be allowed to rest undisturbed for one minute after spreading
to allow the gel to form.
4.5	Petrifilm Aerobic Count plate components are decontaminated though not
sterilized.
5.0 INTERFERENCES:
5.1	Do not use plates that show discoloration.
5.2	Do not use diluents containing citrate, bisulfite or thiosulfate with Petrifilm plates;
these compounds can inhibit growth.
5.3	Since the colonies grow suspended in a gel matrix, the application of substantial
pressure to the film will cause the colonies to shift; thus the application of
pressure to the film after spreading should be avoided.
5.4	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 TNTC.
5.5	Some organisms can liquefy the gel, allowing them to spread out and obscure the
presence of other colonies.
5.6	Dust particles may be picked up by the Petri Scan® and counted as colonies -
analysts must be sure to evaluate each film after the program performs its Auto
Count function.
6.0 PERSONNEL QUALIFICATIONS:
6.1 Personnel are required to be knowledgeable of the procedures in this SOP.
Documentation of training and familiarization with this SOP can be found in the
training file for each employee.

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 5 of 14
7.0 SPECIAL APPARATUS AND MATERIALS:
7.1	Petrifilm™ Aerobic Count Plate
7.2	PetriScan®
7.3	Computer in B202 loaded with PetriScan® software
7.4	Hewlett-Packard Scanjet 8200
7.5	Incubator at 36±1°C or another temperature suitable for growth of the target
organism
8.0 INSTRUMENT OR METHOD CALIBRATION:
8.1 The PetriScan® has been validated (refer to the PetriScan® User Guide, page 29,
section 3.9). This data will be archived.
9.0 SAMPLE HANDLING AND STORAGE:
9.1	Store unopened Petrifilm plate pouches refrigerated or frozen at temperatures
<8°C. Just prior to use, allow unopened pouches to come to room temperature
before opening.
9.2	Return unused plates to pouch. Seal by folding the end of the pouch over and
taping shut.
9.3	To prevent exposure to moisture, close pouch tightly before placing back into the
refrigerator.
9.4	Resealed pouches may be stored for no longer than one month.
10.0 PROCEDURE AND ANALYSIS:
10.1 Petrifilm
10.1.1	Mark the plate identification information within the top % inch portion
of the film. Barcode labels may also be placed in this area.
10.1.2	Prepare dilutions to be plated using appropriate sterile diluents (e.g.,
PBDW, Luria-Bertani broth, letheen broth, or distilled water).

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 6 of 14
10.1.3	For plating, use dilutions that will yield between 0-300 CFU per plate.
10.1.4	Place the Petrifilm plate on a flat, level surface.
10.1.5	Lift the top film and hold the pipette perpendicular to dispense the
appropriate amount (800, 900 or 1000 |iL, depending upon the total
volume of the dilution) of sample suspension onto the center of the
bottom film.
10.1.6	Drop the top film down onto the sample.
10.1.7	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.
10.1.8	Remove the spreader and leave the plate undisturbed for at least one
minute to permit the gel to form.
10.1.9	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
necessary for Pseudomonas aeruginosa as well as for microorganisms
damaged by exposure to disinfectants or sporicides.
10.1.10	Petrifilm can be counted using a standard colony counter or
PetriScan®.
10.1.11	Count all red colonies regardless of size or intensity.
10.1.12	Confirmation
10.1.12.1 Colonies may be isolated for further identification by lifting
up the top film and picking up growth from the gel. These
colonies can then be Gram stained or plated.
10.2 PetriScan®
10.2.1 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.

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 7 of 14
10.2.2	PetriScan® data is stored and maintained in database files. After
signing on, the analyst must either 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.
10.2.3	To count colonies using the PetriScan®, lift the scanner lid and place
1-4 Petrifilms face-down within the cutouts on the alignment grid.
10.2.3.1 Do not count plates that contain any of the following
conditions:
•	High concentrations of colonies on the Petrifilm plates
that cause the entire growth area to become red or pink.
•	Overcrowded plates where the center lacks visible
colonies, but the edges contain colonies.
•	Excessive spreader growth (>50% of the film area).
10.2.3.2 If liquefied gel interferes with counting, an estimated count
should be made by counting the unaffected areas.
10.2.4	Close the lid and click Scan. When the scan is complete, the images
will be displayed in the grid view. (Attachment 1)
10.2.5	After scanning, enter in an ID and Dilution factor (if applicable) for
each film to be counted.
10.2.6	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.
10.2.7	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.
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.

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 8 of 14
10.2.8	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.
NOTE: Data is 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.
10.2.9	It is recommended that after scanning and saving the data, the analyst
should review each film to be sure 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).
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.
11.0 DATA ANALYSIS/CALCULATIONS:
11.1 To edit colonies (this option is only available after saving), double-click on the
row listed in the Data View.
11.1.1	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.
11.1.2	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.
11.1.3	Click Exit and select Yes when asked if the data should be saved.
11.1.4
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

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 9 of 14
information regarding flags). The previous data, flag notations, and
reason for change will be archived to Audit View.
12.0 DATA MANAGEMENT/RECORDS MANAGEMENT:
12.1	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.
12.2	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).
13.0 QUALITY CONTROL:
13.1	Quality control procedures described in the PetriScan® User Guide, pages 28 and
29, section 3.8 are used to verify the count accuracy and consistency at both low
and high colony counts of the PetriScan® system once a month while the unit is in
use.
13.1.1 The electronic data generated during these quality control procedures
is stored in a database entitled Quality Control. Once per year, the
electronically archived quality control data is saved as a Microsoft
Excel spreadsheet, printed and filed.
13.2	The sterility of each pouch is checked as follows:
13.2.1	Upon receipt, each pouch (per box) is individually numbered 1-20
(there are twenty pouches per box of 1000 Petrifilm).
13.2.2	Upon opening a new pouch of Petrifilm, one film is randomly selected
per pouch, spotted with 1 mL of sterile DI water, and incubated at
36±1°C for 2-10 days. Results (e.g., growth or no growth) are
recorded on form 16.1.
13.2.3	The lack of red colonies indicates a sterile film, while the presence of
one or more red colonies indicates contamination.
13.2.4
Discard contaminated film (e.g., the film within the pouch where the

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 10 of 14
contamination was noted).
14.0 NONCONFORMANCE AND CORRECTIVE ACTION:
14.1	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).
14.2	For further information involving problems encountered in operating the
PetriScan®, refer to the PetriScan® User Guide, page 43, section 6
(Troubleshooting).
15.0 REFERENCES:
15.1	PetriScan® User Guide, Spiral Biotech, Inc., Rev3 110104.
15.2	Hewlett-Packard Scanjet 8200 Series Scanners User's Manual, Hewlett-Packard,
2003.
15.3	3M Petrifilm™ Aerobic Count Plate Pamphlet, 3M, 2004 (38-9018-1246-1).
16.0 FORMS AND DATA SHEETS:
16.1	Sterility Assessment Log
16.2	Attachment 1: Sample Film with Growth
16.3	Attachment 2: Sample Data Spreadsheet
16.4	Attachment 3: Sample Audit View

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 11 of 14
Petrifilm Sterility Assessment Log
OPP Microbiology Laboratory
Date/Initials
Pouch #
Control #
Petrifilm Lot #
Sterile DI
Water Prep #
Incubation End
Date/Initials
Was Growth
Observed?*

1




~ Yes ~ No

2




~ Yes ~ No

3




~ Yes ~ No

4




~ Yes ~ No

5




~ Yes ~ No

6




~ Yes ~ No

7




~ Yes ~ No

8




~ Yes ~ No

9




~ Yes ~ No

10




~ Yes ~ No

11




~ Yes ~ No

12




~ Yes ~ No

13




~ Yes ~ No

14




~ Yes ~ No

15




~ Yes ~ No

16




~ Yes ~ No

17




~ Yes ~ No

18




~ Yes ~ No

19




~ Yes ~ No

20




~ Yes ~ No
*The observation of no growth indicates a passing sterility assessment. If growth is observed, discard the contaminated film (e.g., all of the film within the pouch
where the contamination was noted).

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 12 of 14

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 13 of 14
Attachment 2
Sample Data Spreadsheet
Name
Title
Reference
High Count Limit 300
Low Count Limit 0
Created by	MLB
QC
Quality Control
Plate
Plate ID
Dilution
Count
CFU/ml
nag.	
Date
Time
User ID
81
Template 1,01/09
0
30
3.00E+01
ID ED
1/22/2009
4:31:33 PM
MLB
82
Template 2,01/09
0
200
2.00E+02
ID ED
1/22/2009
4:31:33 PM
MLB
83
Uninoculated 1,01/09
0
0
O.OOE+OO

1/22/2009
4:31:33 PM
MLB
84
U nino culate d 2,01/09
0
0
O.OOE+OO
ED
1/22/2009
4:31:33 PM
MLB
85
Template 1,02/D9
0
30
3.00E+01
ID ED
2/28/2009
6:39:19 PM
MLB
86
T emplate 2,02/09
0
200
2.00E+02
ED
2/28/2009
6:39:21 PM
MLB
8?
Uninoculated 1,02/09
0
0
O.OOE+OO

2/28/2009
6:39:22 PM
MLB
88
Uninoculated 2,02/09
0
0
O.OOE+OO
ED
2/28/2009
6:39:23 PM
MLB
89
Template 1,03/09
0
30
3.00E+01
ID ED
3/19/2009
3:16:43 PM
MLB
90
T emplate 2,03/D9
0
200
2.00E+02
ID ED
3/19/2009
3:16:43 PM
MLB
91
Uninoculated 1,03/09
0
0
O.OOE+OO

3/19/2009
3:16:43 PM
MLB
92
Uninoculated 2,03/09
0
0
0.00E+00

3/19/2009
3:16:43 PM
MLB

-------
SOP No. EQ-09-01
Date Revised: 06-02-09
Page 14 of 14
Attachment 3
Sample Audit View

A
B
c
D
E
F
G
H
1
J
1
PetriScan Quality Control Procedure
Records for 2008 (Audit View)




2
OPP Microbiology Laboratory







3










4
Name
QC








5
Title
Quality Control








6
Reference









7
High Count Limit
300








8
Low Count Limit
0








9
Created by
MLB








10







11
Plate U
Plate ID
Dilution
Count
CFU/ml

Mod Date
Mod Time
Mod By
Reason
12










13
128
Unino culate d 2? 12/08
0
1
1.00E+00

12/23/2008
4:13:49 PM
MLB
Deleted piece of dust.
14
126
T emplate 2
0
202
2.02E+02

12/23/2008
4:13:35 PM
MLB
Added date to ID, deleted pieces of dust.
15
125
T emplate 1
0
31
3.10E+01

12/23/2008
4:13:08 PM
MLB
Added date to ID, deleted piece of dust.
16
123
Unino culate d 1,11/08
0
2
2.00E+00

11/14/2008
9:48:52 AM
MLB
Deleted pieces of dust.
17
122
T emplate 2
0
202
2.02E-HD2

11/14/2008
9:48:26 AM
MLB
Added date to ID, deleted pieces of dust.
18
121
T emplate 1
0
31
3.10E+01

11/14/2008
9:47:57 AM
MLB
Added date to ID, delete piece of dust.
19
119
Unino culate d 1,10/08
0
2
2.00E+00

10/28/2008
3:41:35 PM
MLB
Deleted 2 pieces of dust.
20
118
T emplate 2
0
203
2.03E+02

10/28/2008
3:41:23 PM
MLB
Added date to ID, deleted 3 pieces of dust.
21
117
T emplate 1
0
32
3.20E+01

10/28/2008
3:41:00 PM
MLB
Added date to ID, deleted 2 pieces of dust.
77
115
Unino culate d 1,09/08
0
3
3.00E+00

9/19/2008
1:29:55 PM
MLB
Pieces of dust unselected.
23
114
T emplate 2
0
202
2.02E+02

9/19/2008
1:29:41 PM
MLB
Added date to ID; pieces of dust uns elected.
24
113
T emplate 1
0
31
3.10E+01

9/19/2008
1:29:21 PM
MLB
Added date to ID; piece of dust uns elected.
25
111
Unino culate d 1,08/08
0
2
2.00E+00

8/22/2008
12:18:55 PM
MLB
Rremoved pieces of dust.
26
110
T emplate 2
0
202
2.02E+02

8/22/2008
12:17:08 PM
MLB
Added date to ID; removed pieces of dust.
27
109
T emplate 1
0
31
3.10E+01

8/22/2008
12:16:29 PM
MLB
Added date to ID; removed piece of dust.
28
108
Unino culate d 2,07/08
0
1
1.00E+00

7/24/2008
10:45:51 AM
MLB
Piece of dust on template.
29
107
Unino culate d 1,07/08
0
1
1.00E+00

7/24/2008
10:45:46 AM
MLB
Piece of dust on template.
30
106
T emplate 2
0
202
2.02E+02

7/24/2008
10:45:33 AM
MLB
Added date to ID, pieces of dust on template.
31
105
T emplate 1
0
31
3.10E+01

7/24/2008
10:45:09 AM
MLB
Added date to ID, piece of dust on template.

-------