US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Mic ro bio lo gy La bo ra to ry
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Neutralization Confirmation Procedure for Products Evaluated
with the AO AC Use Dilution Method (UDM), the AO AC
Germicidal Spray Products as Disinfectants Test (GSPT),
and the Disinfectant Towelette Test (DTT)
SOP Number: MB-17-03
Date Revised: 9-29-14

-------
SOP No. MB-17-03
Date Revised 09-29-14
Page 1 of 10
SOP Number
MB-17-03
Title
Neutralization Confirmation Procedure for Products Evaluated with
the AO AC Use Dilution Method (UDM), the AO AC Germicidal
Spray Products as Disinfectants Test (GSPT), and the Disinfectant
Towelette Test (DTT)
Scope
This SOP describes methodology used to determine the effectiveness
of neutralizers specified for disinfectant testing (UDM, GSPT, and
DTT). A quantitative approach is used to assess the effectiveness of
the neutralizer and any bacteriostatic action resulting from the
neutralizer itself or neutralizer/disinfectant interactions across a
range of microbe concentrations. This SOP can be modified to
accommodate other test methods.
Application
This assay is designed to simulate the conditions of the UDM,
GSPT, and DTT; however, sterile carriers are used instead of
inoculated carriers. The test conditions specified for product testing
(e.g., water hardness, use-dilution, pH, organic soil, neutralizer,
contact time, temperature) are used.


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Date SOP withdrawn:

-------
SOP No. MB-17-03
Date Revised 09-29-14
Page 2 of 10
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
3
8. NON-CONFORMING DATA
3
9.
DATA MANAGEMENT
3
10.
CAUTIONS
3
11.
SPECIAL APPARATUS AND MATERIALS
3
12.
PROCEDURE AND ANALYSIS
4
13.
DATA ANALYSIS/CALCULATIONS
10
14.
FORMS AND DATA SHEETS
10
15.
REFERENCES
10

-------
SOP No. MB-17-03
Date Revised 09-29-14
Page 3 of 10
1. Definitions
Additional abbreviations/definitions are provided in the text.
1.	Bacteriostatic = Capable of inhibiting or controlling the growth or
reproduction of bacteria without killing the cells
2.	CFU = Colony Forming Unit
2. Health and
Safety
Follow procedures specified in SOP MB-01, Laboratory Biosafety. The
Study Director and/or lead analyst should consult the Safety Data Sheet for
specific hazards associated with products.
3. Personnel
Qualifications
and Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
Refer to SOPs EQ-02 (thermometers), EQ-03 (weigh balances), EQ-05
(timers), and QC-19 (pipettes) for details on method and frequency of
calibration.
5. Sample
Handling and
Storage
Refer to SOP MB-22, Disinfectant Sample Preparation, and SOP COC-01,
Chain of Custody Procedures.
6. Quality
Control
For quality control purposes, the required information is documented on the
appropriate form(s) (see section 14).
7. Interferences
For each neutralizer and subculture medium tested per study, use one batch
(preparation) of neutralizer and medium for all treatment and control
groups. Differences in performance (quality) between batches of media
may lead to misleading neutralization results.
8. Non-
conforming
Data
Management of non-conforming data will be specified; procedures will be
consistent with SOP ADM-07, Non-Conformance Reports.
9. Data
Management
Data will be archived consistent with SOP ADM-03, Records and
Archives.
10. Cautions
1.	To ensure the stability of the test disinfectant solution, perform testing
within 3 hours of preparation.
2.	Strict adherence to the procedure is necessary for validity of test results.
3.	Use appropriate aseptic techniques for all test procedures involving the
manipulation of test organisms and associated test components.
11. Special
Apparatus
and Materials
1. Gram stain kit. Purchased from Becton Dickinson (BD).

-------
SOP No. MB-17-03
Date Revised 09-29-14
Page 4 of 10
12. Procedure and
Analysis
a.	Sterile carriers are used for this assay.
b.	The neutralization assay is performed in advance of product
testing to verify that the prescribed neutralizer is suitable for the
efficacy evaluation. Two test scenarios are conducted
concurrently to determine an appropriate approach for performing
the product efficacy evaluation:
i.	The First Scenario involves exposing carries to the
disinfectant and transferring the carriers into the
neutralizer subculture medium (primary tube). No
secondary subculture medium transfers are conducted.
The neutralizer tubes with the carrier are inoculated with a
test organism suspension to deliver 5-100 CFU/mL.
ii.	The Second Scenario is conducted by exposing carriers to
the disinfectant and transferring the carriers into the
neutralizer subculture medium (primary tube); in addition,
the carriers are subsequently transferred to a secondary
subculture medium (secondary tube). Tubes are inoculated
with a test organism suspension to deliver 5-100 CFU/mL.
c.	The purpose of the two scenario approach is to determine if the
prescribed neutralizer for the disinfectant is sufficient to support
growth.
12.1 Inoculum
Preparation
a. Prepare the inoculum according to SOP MB-05, AO AC UDM,
sections 12.1 through 12.2b.
12.2 Inoculum
Enumeration
a.	Prepare serial ten-fold dilutions of the inoculum by pipetting 1 mL
of the final test culture into 9 mL of PBDW. Use four dilutions,
(e.g., 10"4, 10"5, 10"6, and 10"7) to inoculate the neutralizer (primary
tubes) and subculture medium (secondary tubes). The target
number of cells is 5-100 CFU/mL; this level should be seen in one
of the two highest dilutions.
b.	To estimate CFU/mL, plate 0.1 mL of each of the four dilutions in
duplicate on TSA or blood agar plates (BAP). Briefly vortex each
dilution tube prior to plating. Plates must be dry prior to
incubation.
c.	Record the dilution and plating information on the Neutralization
Confirmation Assay: Enumeration Form (see section 14).
d.	Incubate plates (inverted) at 36±1°C for up to 48±2 hours and
record colony counts. Plates that have colony counts over 300 are
labeled as too numerous to count (TNTC). Record the counts on

-------
SOP No. MB-17-03
Date Revised 09-29-14
Page 5 of 10

the Neutralization Confirmation Assay: Enumeration Form (see
section 14).
12.3 Product
Sample
Preparation
a. Prepare the product according to the test parameters; follow
guidelines for disinfectant sample preparation provided in SOP
MB-22, Disinfectant Sample Preparation, and SOP COC-01,
Chain of Custody Procedures.
12.4 Carrier
Preparation
a. Prepare carriers according to the applicable SOP: for the UDM,
SOP MB-05 (stainless steel penicylinders), for the GSPT, SOP
MB-06 (25 x 25 mm glass slide carriers), and for the DTT, SOP
MB-09 (25 x 75 mm glass slide carriers).
i.	For UDM: Follow carrier inoculation (SOP MB-05,
section 12.2) except use sterile broth. Add organic soil to
the sterile broth as necessary per the test parameters.
ii.	For GSPT and DTT: Follow carrier inoculation (SOP MB-
06 and SOP MB-09, section 12.2) except use sterile broth.
Add organic soil to the sterile broth as necessary per the
test parameters.
12.5 First
Scenario:
Neutralizer -
Primary
Subculture
Treatment
only
a.	Requires four dried carriers (with broth culture added) per
organism. Use the carrier type required for the specific test.
b.	Apply the product to the carriers according to specific instruction
provided in the test parameters (e.g., use dilution, spray distance,
spray period, wipe pattern, and contact time).
c.	Per test, per one test organism, expose four of the carriers to the
disinfectant for the specified contact time in the same manner as
product efficacy testing. Record the carrier transfer information
on the Neutralization Confirmation Assay: Time Recording Sheet
for Carrier Transfers (see section 14).
d.	After the last carrier of a set (4 total carriers) has been treated with
the disinfectant, and the contact time is complete, aseptically
transfer carriers in order in a timed fashion into tubes containing
the specified neutralizer, in the same manner as product efficacy
testing. Drain excess liquid from the carrier prior to the transfer.
This set of neutralizer tubes (4 total tubes) will represent the
Neutralizer-Primary Subculture Treatment. Refer to section
12.8 for treatment inoculation (Table 1).
Note: For GSPT and DTT, the amount of neutralizer is 20 mL per
tube (38 x 100 mm tubes) compared to 10 mL (20 x 150 mm
tubes) used in the UDM.

-------
SOP No. MB-17-03
Date Revised 09-29-14
Page 6 of 10

e. Proceed immediately with the Second Scenario.
12.6 Second
Scenario:
Neutralizer
Subculture
Treatment
plus
Secondary
Subculture
Treatment
a.	Requires four dried carriers (with broth culture added) per
organism. Use the carrier type required for the specific test.
b.	Apply the product to the carriers according to specific instruction
provided in the test parameters (e.g., use dilution, spray distance,
spray period, wipe pattern, and contact time).
c.	Per test, per one test organism, expose four of the carriers to the
disinfectant for the specified contact time in the same manner as
product efficacy testing. Record the carrier transfer information
on the Neutralization Confirmation Assay: Time Recording Sheet
for Carrier Transfers.
d.	After the last carrier of a set (4 total carriers) has been treated with
the disinfectant, and the contact time is complete, aseptically
transfer carriers in order in a timed fashion into tubes containing
the specified neutralizer, in the same manner as product efficacy
testing. Drain excess liquid from the carrier prior to the transfer.
This set of neutralizer tubes (4 total tubes) will represent the
Neutralizer-Primary Subculture Treatment.
Note: For GSPT and DTT, the amount of neutralizer is 20 mL per
tube (38 x 100 mm tubes) compared to 10 mL (20 x 150 mm
tubes) used in the test method for liquid products.
e.	Following the last carrier transfer into the neutralizer tube,
incubate both First and Second Scenario neutralizer tubes at room
temperature for 30-45 min. Then, for the Second Scenario,
transfer each carrier in order into a culture tube containing the
secondary subculture medium. This portion of the assay is not
timed. This set of tubes (4 total tubes) will represent the
Secondary Subculture Treatment. Refer to section 12.8 for
treatment inoculation (Table 2).
f.	Repeat the assay for the second test organism, if required.
12.7 Controls
a. Inoculated controls
i.	The Neutralizer-Primary Inoculated Control contains
four tubes of fresh, unexposed (to disinfectant) neutralizer-
primary media.
ii.	The Secondary Subculture Inoculated Control contains
four tubes of secondary subculture media.
iii.	It is highly desirable that the preparation (media
preparation number) of each medium be the same as used

-------
SOP No. MB-I7-U5
Date Revised (W-29-1 I
Page 7 of 10
in the treatments. Refer to section 12,8 for treatment
inoculation (Table 3),
Uninoculated controls
i Ntuti ali/er-Priiuary and Secondary Subculture
I'ninoculateri Controls. One tube eaeli of uninoeulnted
neutralizer and secondary subculture media will be
included in the test and incubated with the other tubes.
Confirm sterility of earners in advance or concurrently with
testing by adding an uninoculated carrier to a tube of 10-20 ml.
fluid thioglyeollate medium or letheen broth and incubating at
36±1°C for 3-10 days.
12.8 Treatment
Inoculation
a.	After step 12.6e. inoculate all tubes concurrently using Tables 1.
2. and 3.
b.	First Scenario: Inoculation of Treatment Group with Dilutions of
the Test Organism*
Table I
Treatment
Dilutions Added
10"4
io-5
10'
io-7
Neutralizer-Primary Subculture
Treatment
0,1 hiL
0.1 mL
0.1 mL
0.1 mL
*1 '• 1(H through I -10"': based on an approx. starting suspension of 10s to 10® CFUmiL
c. Second Scenario: Inoculation of Treatment Groups with Dilutions
of the Test Organism*
Table 2




Treatments
Dilutions Added
10"4
10"5
io~6
10~7
Neutra 1 izer-Priniary Subculture
Treatment
0.1 nil
0.1 nil.
0,1 mL
0.1 mL
Secondary Subculture Treatment
0.1 uiL
0.1 ml
0.1 mL
0.1 mL
* 1 ¦* 10"4 tlirough 1 -10~?: based on an approx. starting suspension of 10s to 10p CFUaiiL
d. Controls: Inoculation of Control Groups with Dilutions of the Test
Organism*

-------
SOP No. MB-17-03
Date Revised 09-29-14
Page 8 of 10
Table 3
C ontrois
Dilutions Added
10-4
If)-5
lCr6
10-7
Inoculated
Controls
(media
performance)
Neutralizer-
Primarv
0.1 tuL
0.1 ml.
0.1 ml.
0.1 aiL
Secondary
Subculture
0.1 mL
0.1 mL
0.1 mL
0.1 mL
Sterility
Controls
Neutralizer-
Primary
HA
N/A
N< A
N'/A
Secondary
Subculture
N.'A
N.'A
MA
N.A
2.9 Reeoiding
Results and
Confirmation
Testing
*110"4 through 1 10"7: based on an approx. starting suspension of 10s to 10® CPU, mL
e. Shake tubes thoroughly. Incubate all tubes for up to 4S±2 h at
36±I°C.
a.	Record results as + (growth turbidity) or 0 (no growth) on the
Neutralization Confirmation Assay Results Form (see section 14).
b.	For each treatment and control group. Gram stain a minimum of
one positive lube per Treatment. Select the tube with the highest
dilution showing growth (inoculated with the dilution with fewest
CFL' niL delivered),
e Record confirmation results on the Neutralization Confirmation
Assay: Microbe Confirmation Sheet (see section 14).
12.10
Interpretation
of Results
Plate count data. One of the four dilutions plated should provide
counts within the approximate target range. 5-100 CFU/mL.
i. Note: The lack of complete neutralization of the
disinfectant or bacteriostatic activity of the neutralize!'
itself may be masked when a high level of inoculum is
added to the subculture tubes.
C ontrois. Growth in the Secondary Subculture Inoculated
Control verifies the presence of the test microbe, performance of
the media, and piuvides a basis for eompan.son of growth in the
neutralize!- and subculture treatment tubes. No growth or only
growth in tabes which received high levels of inoculum (e.g., a
dilution with plate counts which are too numerous to count)
indicates poor media performance, Growth in the Neutralizer-
Primary Inoculated Control should be comparable to the
Secondary Subculture Inoculated Control if the neutralize] is
the same as the seeondarv subculture media.
i. There may be cases when the neutrahzer (primary tubes) is
	significantly different from the secondary subculture	

-------
SOP No. MB-17-03
Date Revised 09-29-14
Page 9 of 10

media. In these cases, growth may not be comparable to
the Secondary Subculture inoculated Control.
ii. The Neutralizer-Primary Uninoculated Control and
Secondary Subculture Uninoculated Control tubes are
used to determine sterility and must show no growth for
the test to be valid.
c. Treatments. The occurrence of growth in the Neutralizer-
Primary Subculture and Secondary Subculture Treatment
tubes are used to assess the effectiveness of the neutralizer.
i.	First Scenario: The neutralizer itself mav exhibit
bacteriostatic activity against the test microbe. No growth
or growth only in tubes which received a high titer of
inoculum (e.g., the dilution with plate counts which are too
numerous to count) indicates poor neutralization and/or
presence of bacteriostatic properties of the neutralizer or
neutralizer-disinfectant interactions. For the neutralizer
to be deemed effective, growth must occur in the
Neutralizer Primary Subculture Treatment tubes which
received lower titer of inoculum (e.g., 5-100 CFU/mL).
ii.	Second Scenario: The neutralizer itself or in combination
with the recovery (subculture) medium may exhibit
bacteriostatic activity against the test microbe. No growth
or growth only in tubes which received a high titer of
inoculum (e.g., the dilution with plate counts which are too
numerous to count) indicates poor neutralization and/or
presence of bacteriostatic properties of the neutralizer or
neutralizer-disinfectant interactions. For the neutralizer
to be deemed effective, growth must occur in the
Secondary Subculture Treatment tubes which received
lower titer of inoculum (e.g., 5-100 CFU/mL).
12.11 Efficacy
Evaluation
based on
Neutral-
ization
Results
a.	If results from the First Scenario indicate effective neutralization,
the efficacy evaluation will be conducted using only the
neutralizer subculture tubes (i.e., primary tubes).
b.	If results from the First Scenario (Neutralizer-Primary Subculture
Treatment only) are inconclusive and/or indicate that a
bacteriostatic effect from the neutralizer or neutralizer-disinfectant
interaction is present, results from the Second Scenario are
evaluated to determine if the Secondary Subculture tube provide
appropriate neutralization.

-------
SOP No. MB-17-03
Date Revised 09-29-14
Page 10 of 10

c.	If the Second Scenario is deemed effective, the efficacy evaluation
is conducted using both subculture media tubes (i.e., primary and
secondary tubes).
d.	If results from the Second Scenario (Neutralizer-Primary
Subculture Treatment tubes and Secondary Subculture Treatment
tubes) are inconclusive and/or indicate that a bacteriostatic effect
from the neutralizer or neutralizer-disinfectant interaction is
present, an alternative neutralizer will be assayed prior to
conducting the efficacy evaluation. The alternative neutralizer
may not be specified in the test parameters.
13. Data Analysis/
Calculations
1.	Plate counts are enumerated and CFU/mL added to each tube is
calculated based on the average of countable plates. Apply TNTC for
counts above 300 CFUs.
2.	To calculate the average CFU/mL per dilution added to each tube, add
the plate counts for each plate within the dilution and divide by two.
3.	Counts from 0 through 300 are used in the calculations.
14. Forms and
Data Sheets
Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
Neutralization Confirmation Assay: Time a/tr 17 m f 1 a
Recording Sheet for Carrier Transfers - ' °CX
Neutralization Confirmation Assay: Test , _ 1
T J MB-17-03 F2.docx
Information Sheet -
Neutralization Confirmation Assay: Results , _ 1
„ J MB-17-03 F3.docx
Form -
Neutralization Confirmation Assay: Test , _ ^ 1
Microbe Confirmation Sheet MB-17-03_F4 docx
Neutralization Confirmation Assay: , _ 1
_ . „ J MB-17-03 F5.docx
Enumerati on F orm -
Neutralization Confirmation Assay: Processing - „ ^ ,
jo MB-17-03 F6.docx
Sheet -
15. References
1.	Official Methods of Analysis. Methods 955.15 and 964.02. Posted
September 2013. AO AC INTERNATIONAL, Gaithersburg, MD.
2.	Official Methods of Analysis. Method 961.02. Posted March 2013.
AO AC INTERNATIONAL, Gaithersburg, MD.
3.	Package Insert - Gram Stain Kit and Reagents. Becton, Dickinson and
Company. Part no. 8820201JAA. Revision 03/2011.

-------