US Environmental Protection Agency
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Determining the Presence of
Microbial Contamination in Disinfectant Products
Standard Operating Procedures (SOPs)
Office of Pesticide Programs
SOP Number: QC-21-01
Date Revised: 03-05-09

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SOP No. QC-21-01
Date Revised 03-05-09
Page 1 of 18
EPA/OPP MICROBIOLOGY LABORATORY
ESC, Ft. Meade, MD
Standard Operating Procedure
for
Determining the Presence of Microbial Contamination in Disinfectant Products
SOP Number: QC-21-01
Date Revised: 03-05-09
Initiated By:
Technical Review:
Print Name:
Print Name:
Technical Staff
QA Review:
Print Name:
QA Officer
Approved By:
Print Name:
Branch Chief
Date: / /
Date: / /
Date: / /
Date: / /
Effective Date:
/ /
Controlled Copy No.:
Withdrawn By:
Date: / /

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SOP No. QC-21-01
Date Revised 03-05-09
Page 2 of 18
TABLE OF CONTENTS
Contents	Page Number
1.0	SCOPE AND APPLICATION	 2
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	 4
8.0	INSTRUMENT OR METHOD CALIBRATION	 5
9.0	SAMPLE HANDLING AND STORAGE	 5
10.0	PROCEDURE AND ANALYSIS	 6
11.0	DATA ANALYSIS/CALCULATIONS	 8
12.0	DATA MANAGEMENT/RECORDS MANAGEMENT	 8
13.0	QUALITY CONTROL	 8
14.0	NONCONFORMANCE AND CORRECTIVE ACTION	8
15.0	REFERENCES	 9
16.0	FORMS AND DATA SHEETS	 9
1.0
SCOPE AND APPLICATION:

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SOP No. QC-21-01
Date Revised 03-05-09
Page 3 of 18
1.1	The assay provides data on the occurrence of microbial contamination of liquid
and spray antimicrobial products. This assay is consistent with methodology for
conducting the AO AC Use Dilution Method or the AO AC Germicidal Spray
Products as Disinfectants Test, but with sterile carriers.
1.2	The assay is qualitative and designed to recover and culture bacterial
contamination (i.e., spores of Bacillus sp.) from a hospital disinfectant sample. In
most instances, the assay will be deemed necessary due to historical evidence of
bacterial contamination associated with the sample itself, a previously-collected
sample of the same product, or a registrant's product line.
1.3	Products that show no evidence of product-borne contamination when tested by
this procedure are considered adequate for efficacy testing; however, samples
with confirmed contamination will be deemed unsuitable for conducting efficacy
tests.
2.0 DEFINITIONS:
2.1	AO AC = AO AC INTERNATIONAL
2.2	UDM = AO AC Use Dilution Method
2.3	GSPT = AOAC Germicidal Spray Products as Disinfectants Test
3.0 HEALTH AND SAFETY:
3.1 Disinfectants may contain a number of different active ingredients, such as heavy
metals, aldehydes, peroxides, phenol, etc. Personal protective clothing or devices
are recommended during the handling of these items for the purpose of activation,
dilution, or efficacy testing. A chemical fume hood or other containment
equipment is employed when performing tasks with concentrated products. The
study analyst may wish to consult the Material Safety Data Sheet for the specific
product/active ingredient to determine the best course of action.
4.0 CAUTIONS:
4.1	Use aseptic techniques to prevent contamination.
4.2	The volume of neutralizer tubes and subculture tubes will be verified in advance
and adjusted accordingly.

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SOP No. QC-21-01
Date Revised 03-05-09
Page 4 of 18
4.3	Strict adherence to the protocol is necessary for the validity of the test results.
4.4	Follow appropriate chain-of-custody guidelines during testing as stipulated in
SOP-COC-Ol, Sample Log-in and Tracking. Sample handling and custody
procedures are considered the same as for efficacy testing.
4.5	To avoid introduction of contaminating organisms into the test system and cross-
contamination from tube to tube during the assay, it is recommended that hooks
be autoclaved prior to use, that one hook be used for a predetermined number of
tubes and then be replaced with an autoclaved, unused hook, that there is
sufficient time between transfers (from disinfectant to neutralizer and neutralizer
to subculture medium) to thoroughly flame (to red-hot) the wire hook, and that
the use of a water bath be avoided (see section 10.1.3). In addition, the ultraviolet
light (UV) in the biological safety cabinet should be left on overnight prior to
performing the assay (see section 10.1.4).
5.0 INTERFERENCES:
5.1	Exposing the product to external contaminants during opening and dispensing,
and the use of non-sterile laboratory supplies may interfere with the outcome of
this analysis.
5.2	Quality control measures for media and reagents used in this evaluation must be
followed as outlined in SOP QC-11, Performance and Sterility of Media and
Reagents.
6.0 PERSONNEL QUALIFICATIONS:
6.1 Personnel are required to be knowledgeable about the procedures in this SOP.
Documentation of training and familiarization with this SOP can be found in the
training file for each employee.
7.0 SPECIAL APPARATUS AND MATERIALS:
7.1	Carriers. Polished stainless steel cylinders, 8  1 mm OD, 6  mm ID, 10  1mm
length; type 304 stainless steel, SS 18-8 (S& L Aerospace Metals, Maspeth, NY
or Fisher Scientific, catalog number 07-907-5Q).
7.2	Glass Slide Carriers. Bellco 25 mm x 25 mm (or comparable size) borosilicate
glass cover slips with number 4 thickness (Bellco Glass, Inc., item number: 1916-
S0131).

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SOP No. QC-21-01
Date Revised 03-05-09
Page 5 of 18
7.3 Appropriate Spray Disinfectant Apparatus.
1A Glassware. For disinfectant (UDM), use autoclavable 25 x 100 mm tubes (Bellco
Glass Inc., Vineland, NJ). For neutralizer and subculture media, use autoclavable
reusable or disposable 20 x 150 mm tubes. For the GSPT, use 38 mm x 100 mm
tubes (Bellco Glass Inc.) for neutralizer and subculture media. Sterilize all
glassware in hot air oven at 180C or steam sterilize for a minimum of 20 minutes
at 121C with drying cycle.
7.5	Test tube racks. Any convenient style.
7.6	Wire hook. For carrier transfer. Make 3 mm right angle bend at end of 50-75 mm
nichrome wire No. 18 B&S gage. Place other end in suitable holder.
7.7	Positive displacementpipet (to deliver 10|il to glass slides) and sterile tips.
7.8	Timer. For managing timed activities, any certified timer that can display time in
seconds.
7.9	Gram stain kit.
7.10	VITEK 2 Compact. For the automated identification of microorganisms.
7.11	VITEK 2 Compact Identification Cards.
8.0 INSTRUMENT OR METHOD CALIBRATION:
8.1 Refer to the laboratory equipment calibration and maintenance SOPs (SOP EQ
series) for details on method and frequency of calibration.
9.0 SAMPLE HANDLING AND STORAGE:
9.1	Follow appropriate chain-of-custody (COC) guidelines during testing as
stipulated in SOP COC-01, Sample Log-in and Tracking.
9.2	Disinfectants are stored according to manufacturers' recommendations or at room
temperature if the product label or testing parameters do not identify a storage
temperature. Those disinfectants requiring activation or dilution prior to use will
only be activated or diluted within three hours of testing unless test parameters
specify otherwise.
9.3	Prepare disinfectant sample per SOP MB-22, Disinfectant Sample Preparation.

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SOP No. QC-21-01
Date Revised 03-05-09
Page 6 of 18
10.0 PROCEDURE AND ANALYSIS:
10.1 To conduct the assay, follow OPP Microbiology Laboratory SOP MB-05, AO AC
Use Dilution Method for Testing Disinfectants for liquid products, and SOP MB-
06, Germicidal Spray Products as Disinfectants Test for spray products, including
liquid products to be applied as a spray. The following exceptions/comments will
apply:
10.1.1	No inoculum will be generated as test microbes are not used in this
assay.
10.1.2	If test parameters call for an organic soil load, disregard this
requirement. However, see section 10.1.7 of this SOP for a
recommendation to apply an organic substance to glass slides in the
event that the spray product beads up and flows off of a glass slide in
the AO AC Germicidal Spray Products as Disinfectants Test. The
addition of an organic substance (e.g., 5% horse serum in sterile
deionized water, letheen broth) in this case is to increase dispersion of
the spray product across the entire slide.
10.1.3	For liquid products, the recirculating chiller and water bath specified
in SOP MB-05 to maintain the temperature of the disinfectant will not
be used. Elimination of the water bath will reduce any potential for
introducing water-borne contaminants into the test system. Place
racks of disinfectant-containing tubes directly onto the surface of the
biological safety cabinet. Exposure of carriers to disinfectant will be
performed at room temperature.
10.1.4	The ultraviolet light (UV) in the biological safety cabinet should be
left on overnight prior to performing the test. Necessary test
equipment (i.e., vortex, hooks, timer) will be placed into the biological
safety cabinet prior to turning on the UV light.
10.1.5	A single assay involves the evaluation of 60 sterile carriers for one
product sample. Approximately 65-70 sterile carriers (60 plus extras)
are prepared.
10.1.6	For liquid products: Sterile stainless steel carriers are removed from
the water, placed onto filter paper in sterile glass Petri dishes, and
dried for 40 minutes per SOP MB-05. Once drying is complete, the
AO AC Use Dilution Method is conducted using the dried,

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SOP No. QC-21-01
Date Revised 03-05-09
Page 7 of 18
uninoculated carriers according to the test parameters (i.e., product
dilution, neutralizer, subculture media, contact time). Alternately, the
drying step may be eliminated, and sterile stainless steel carriers may
be transferred from the tube of sterile water directly to tubes of
disinfectant according to the test timing scheme (e.g., 30 second drop
interval).
10.1.7	For spray products: Conduct the AO AC Germicidal Spray Products as
Disinfectants Test method using the sterile glass carriers according to
the test parameters (i.e., product dilution, neutralizer, subculture
media, contact time).
Recommendation:
	Before conducting the spray test, the analyst must practice
applying the spray product to sterile glass slides to determine
the product's level of dispersion.
	If the product beads up and flows off of the slide rather than
completely covering the glass slide as it would in a typical
efficacy evaluation (with inoculated slides), the analyst should
apply 10 |iL of an organic material (e.g., 5% horse serum in
sterile deionized water, letheen broth) onto the surface of each
sterile glass slide and spread it with a sterile loop.
	Dry the slides for 40 minutes per SOP MB-06.
	Once drying is complete, apply the spray product again to
determine if product dispersion is improved.
	Based on previous observations in our laboratory, the addition
of an organic substance to the surface of the slide increases
product dispersion on glass slides.
	If the application of an organic substance is deemed necessary
to perform the test, apply the substance, dry the slides for 40
minutes as per SOP MB-06, and conduct the modified AO AC
Germicidal Spray Products as Disinfectants Test method using
the dried, sterile carriers according to the test parameters (i.e.,
product dilution, neutralizer, subculture media, contact time).
10.1.8	Carrier counts are not to be performed.
10.1.9	Report results as (+) for microbial growth or (0) for no growth on the
results sheet. In the "Comments" section of the results form, describe
the growth in positive tubes (e.g., uniformly turbid, string-like, etc.).
Note: Shaking tubes prior to recording results may disrupt the unique

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SOP No. QC-21-01
Date Revised 03-05-09
Page 8 of 18
physical appearance of typical Bacillus sp. growing in liquid media
(i.e., string-like, fibrous, not producing uniformly turbid media).
10.1.10	A minimum of three positive (showing microbial growth) carrier sets,
if available, should be confirmed using Gram staining, growth on
trypticase soy agar (TSA; for initial identification and isolation), and
VITEK. If there are less than three positive carrier sets, then each
carrier set will be confirmed. If both tubes are positive in a carrier set,
only one tube is selected for confirmatory testing.
10.1.11	For a test with greater than 20 positive carrier sets, confirm at least
20% by Gram staining, TSA, and VITEK analysis. Again, if both
tubes are positive in a carrier set, only one tube is selected for
confirmatory testing.
11.0 DATA ANALYSIS/CALCULATIONS: None
12.0 DATA MANAGEMENT/RECORDS MANAGEMENT:
12.1	Data will be recorded promptly, legibly, and in indelible ink on the appropriate
forms. Completed forms are archived in notebooks kept in secured file cabinets
in the file room D217. Only authorized personnel have access to the secured
files. Archived data is subject to OPP's official retention schedule contained in
SOP ADM-03, Records and Archives.
12.2	A performance report will be compiled as stipulated in SOP ADM-01,
Performance Reports.
13.0 QUALITY CONTROL:
13.1 For quality control purposes, the required information is documented on the
appropriate form(s) (see 16.0).
14.0 NONCONFORMANCE AND CORRECTIVE ACTION:
14.1	If a product is confirmed to be contaminated, no further efficacy testing will be
initiated.
14.2	Strict adherence to the protocol is necessary for the validity of the test results.
Any deviation from the standard protocol must be brought to the Study Director's
attention and recorded in the raw data along with an explanation for the deviation
given. The deviation and reason for it must be documented on the GLP

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SOP No. QC-21-01
Date Revised 03-05-09
Page 9 of 18
Compliance form in the final report.
15.0 REFERENCES:
15.1	Official Methods of Analysis. 2006. 18th Ed., AO AC INTERNATIONAL,
Gaithersburg, MD, (Methods 955.14, 955.15, and 964.02).
15.2	Official Methods of Analysis. 1990. 15th Ed., AO AC INTERNATIONAL,
Gaithersburg, MD, (Method 961.02).
16.0 FORMS AND DATA SHEETS:
16.1	Determining Microbial Contamination in Liquid Disinfectants: Time Recording
Sheet for Carrier Inoculation Steps
16.2	Determining Microbial Contamination in Liquid Disinfectants: Time Recording
Sheet for Carrier Transfers
16.3	Determining Microbial Contamination in Liquid Disinfectants: Information Sheet
16.4	Determining Microbial Contamination in Liquid Disinfectants: Results Sheet
16.5	Determining Microbial Contamination in Spray Disinfectants: Time Recording
Sheet for Carrier Inoculation Steps
16.6	Determining Microbial Contamination in Spray Disinfectants: Time Recording
Sheet for Carrier Transfers
16.7	Determining Microbial Contamination in Spray Disinfectants: Information Sheet
16.8	Determining Microbial Contamination in Spray Disinfectants: Results Sheet
16.9	Test Microbe Confirmation Sheet

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SOP No. QC-21-01
Date Revised 03-05-09
Page 10 of 18
16.1
Determining Microbial Contamination in Liquid Disinfectants: Time Recording Sheet for Carrier
Inoculation Steps
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
Tcsl Dale

Product Reg. No.

Product Name

Sample No(s).

Organism
Not Applicable
Initials/Date
Test ID
Carrier Dry Time*
Start Time
End Time

UDT


















* Recorded from laboratory clock/and timer.

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SOP No. QC-21-01
Date Revised 03-05-09
Page 11 of 18
16.2
Determining Microbial Contamination in Liquid Disinfectants: Time Recording Sheet for Carrier Transfers
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
Tcsl Dale

Product Reg. No.

Product Name

Sample No(s).

Organism
Not Applicable
Initials/Dale
Set
Drop
Interval
Carrier Drop Start Time (into the
disinfectant.)
Carrier Drop End Time (into the
primary subcullurc/ncutralizcr media)
Carrier Transfer (into secondary
subculture)
Clock
Tinier
Clock
Timer
Start Time'

1-20







21-40







41-60






Comments:

Carrier transfer into secondary subculture (time elapsed after last carrier dropped in primary); taken from clock

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SOP No. QC-21-01
Date Revised 03-05-09
Page 12 of 18
16.3
Determining Microbial Contamination in Liquid Disinfectants: Information
Sheet
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
EPA Reg. No.

SOP

Name

Test Date

Sample No.

Comments: Assay will be performed without test
microbes.
Lot No.

Expiration Date

TEST PARAMETERS/Confirmed by:
H20 Hardness (CaCOi) ppm	Specified
Use Dilution
Nculralizcr
Specified
Specified
Titrated (Burct)/Datc/Inil.
HACH/Datc/Init.
As Preparcd/Date/Init.
Contact Time
Other Parameters
Specified
As Tested
Specified
REAGENT/MEDIA INFORMATION/Confirmed by:
Reagent/Media
Prep. No.
Reagent/Media
Prep. No.












Neutralizer volume verified: ~ Yes
~ No
Subculture volume verified: ~ Yes ~ No

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SOP No. QC-21-01
Date Revised 03-05-09
Page 13 of 18
16.4
Determining Microbial Contamination in Liquid Disinfectants: Results Sheet
OPP Microbiology Laboratory
PRODUCT INFORMATION/Confirmed by:
EPA Reg. No.

Test Date

Name

Test Organism
Not Applicable
Sample No.


CARRIER INFORMATION (to be completed by Analyst)
Carrier Drop Time Interval
Carrier Set
Analyst









TEST RESULTS
Dale Recorded/Initials

Primary Subculture / Secondary Subculture (carrier)
1
2
3
4
5
6
7
8
9
10
/
/
/
/
/
/
/
/
/
/
11
12
13
14
15
16
17
18
19
20
/
/
/
/
/
/
/
/
/
/
21
22
23
24
25
26
27
28
29
30
/
/
/
/
/
/
/
/
/
/
31
32
jj
34
35
36
37
38
39
40
/
/
/
/
/
/
/
/
/
/
41
42
43
44
45
46
47
48
49
50
/
/
/
/
/
/
/
/
/
/
51
52
53
54
55
56
57
58
59
60
/
/
/
/
/
/
/
/
/
/
Results Summary
Number of Carrier Sets with Growth

Number of Carrier Sets without Growth

Modifications/Comments:

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SOP No. QC-21-01
Date Revised 03-05-09
Page 14 of 18
16.5
Determining Microbial Contamination in Spray Disinfectants: Time Recording Sheet for Carrier
Inoculation Steps
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
Test Dale

Product Reg. No.

Product Name

Sample No(s).

Test Organism
Not Applicable
Initials/Dale
Test ID
Carrier Dry Time*
Start Time
End Time

GSPT


















* Recorded from laboratory clock/and timer.

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SOP No. QC-21-01
Date Revised 03-05-09
Page 15 of 18
16.6
Determining Microbial Contamination in Spray Disinfectants: Time Recording Sheet for Carrier Transfers
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
Test Dale

Product Reg. No.

Product Name

Sample No(s).

Test Organism
Not Applicable
Initials/dale
Set
Spray
Interval
Carrier Spray Start Time (into the
disinfectant)
Carrier Spray End Time (into the
neutralizcr/primarv subculture)'
Carrier Transfer (into
secondary subculture)
Clock
Timer
Clock
Timer
Start Time

1-20







21-40







41-60






Comments: Carriers sprayed by: . Carriers transferred by:


Carrier transfer into secondary subculture; taken from clock.

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SOP No. QC-21-01
Date Revised 03-05-09
Page 16 of 18
16.7
Determining Microbial Contamination in Spray Disinfectants: Information Sheet
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
EPA Reg. No.

SOP

Name

Test Date

Sample No.

Comments: Assay will be performed without test
microbes.
Lot No.

Expiration Date

TEST PARAMETERS/Confirmed by:
H20 Hardness (CaCOO ppm	Specified
Use Dilution
Ncutralizer
Specified
Specified
Titrated (Buret)/Dale/Init.
HACH/Date/Init.
As Prepa red/Da te/I nil.
Contact Time
Other Parameters
Specified
As Tested
Specified
REAGENT/MEDIA INFORMATION/Confirmed by:
Reagent/Media
Prep. No.
Reagent/Media
Prep. No.












Neutralizer volume verified: ~ Yes
~ No
Subculture volume verified: ~ Yes ~ No

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SOP No. QC-21-01
Date Revised 03-05-09
Page 17 of 18
16.8
Determining Microbial Contamination in Spray Products: Results Sheet
OPP Microbiology Laboratory
PRODUCT INFORMATION/Confirmed by:
EPA Reg. No.

Test Dale

Name

Test Organism
Not Applicable
Sample No.


TEST RESULTS
Dale Recorded/Initials

Primary Subculture / Secondary Subculture (carrier)
1
2
3
4
5
6
7
8
9
10
/
/
/
/
/
/
/
/
/
/
11
12
13
14
15
16
17
18
19
20
/
/
/
/
/
/
/
/
/
/
21
22
23
24
25
26
27
28
29
30
/
/
/
/
/
/
/
/
/
/
31
32
33
34
35
36
37
38
39
40
/
/
/
/
/
/
/
/
/
/
41
42
43
44
45
46
47
48
49
50
/
/
/
/
/
/
/
/
/
/
51
52
53
54
55
56
57
58
59
60
/
/
/
/
/
/
/
/
/
/
Results Summary
Number of Carrier Sets with Growth

Number of Carrier Sets without Growth

Modifications/Comments:


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SOP No. QC-21-01
Date Revised 03-05-09
Page 18 of 18
16.9
Test Microbe Confirmation Sheet
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
EPA Reg. No.

Test Dale

Name

Test Organism
Not Applicable
Sample No.

Comments

Source:
Tubc/Plalc ID
Dale/
Initials
Slain
Results*
Media Information
Results
Name
Prep. No.
Inc. Time/
Temp.
Date/
Initials
Colony Characteristics
VITEK ID**
(if applicable)































































* GPC=Gram positive cocci, GNR=Gram negative rods, GPR=Gram positive rods ** VITEK numerical profile number

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