Standard Operating Procedure for Neutralization of Microbicidal Activity using the OECD Quantitative Method for Evaluating Bactericidal Activity of Microbicides Used on Hard, Non-Porous Surfaces SOP Number: MB-26-01 Date Revised: 08-26-14

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 of Microbicidal Activity using the OECD
Quantitative Method for Evaluating Bactericidal Activity of
Microbicides Used on Hard, Non-Porous Surfaces
SOP Number: MB-26-01
Date Revised: 08-26-14

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SOP No. MB-26-01
Date Revised 08-26-14
Page 1 of 8
SOP Number
MB-26-01
Title
Neutralization of Microbicidal Activity using the OECD
Quantitative Method for Evaluating Bactericidal Activity of
Microbicides Used on Hard, Non-Porous Surfaces
Scope
To verify the neutralization efficacy of a test substance (refer to
reference 15.1).
Application
Identify a suitable neutralizer in advance of or concurrently with
testing. Verify neutralization using the highest concentration of test
substance if there are multiple concentrations being evaluated.


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Date SOP withdrawn: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. MB-26-01
Date Revised 08-26-14
Page 2 of 8
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
4
12.
PROCEDURE AND ANALYSIS
4
13.
DATA ANALYSIS/CALCULATIONS
6
14.
FORMS AND DATA SHEETS
7
15.
REFERENCES
7

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SOP No. MB-26-01
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1. Definitions
Additional abbreviations/definitions are provided in the text.
1.	Reaction vessel = vessel used to conduct the assay (vial or test tube).
2.	Eluent = any liquid that is harmless to the test organism(s) and that is
added to the reaction vessel to recover the test organism.
3.	Eluate = recovered eluent that contains the test organism.
4.	Test suspension = suspension of the test microbe prior to the addition of
the soil load ( Test Suspension A)
5.	Final test suspension= test suspension with soil load (Test Suspension B)
6.	Stock culture = frozen culture used to prepare the test culture
7.	Test substance = a product or formulation that is under evaluation for its
microbicidal activity
2. Health and
Safety
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.
3. Personnel
Qualifications
and Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
Refer to SOP EQ-01 (pH meters), EQ-02 (thermometers), EQ-03 (weigh
balances), EQ-04 (spectrophotometers) and EQ-05 (timers) 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
Prolonged exposure of cells to the neutralizer agent in excess of 30 minutes
may result in erroneous values due to bacterial replication; timely filtration
will mitigate this potential interference.
8. Non-
conforming
Data
Management of non-conforming data will be specified in the study protocol;
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
Avoid extended soaking of the carriers in water or detergent and prolonged
rinsing to reduce risk of corrosion or rusting.

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SOP No. MB-26-01
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11. Special
Apparatus and
Materials
Refer to SOP MB-25, OECD Quantitative Method, section 11.
12. Procedure and
Analysis
1. General description of the assay:
a. The test substance is first mixed with a candidate neutralizer. A
diluted suspension of the test organism is then added to the reaction
mixture; if desired, additional evaluations may be conducted using
the test organism as dried inoculum on a carrier. The neutralization
process is deemed acceptable if the criteria outlined in section 13 are
met.
12.1 Preparation/
sterilization of
carriers
a. Refer to SOP MB-25, OECD Quantitative Method, section 12.1.
12.2 Preparation of
test organisms
a.	Refer to SOP MB-25, OECD Quantitative Method; section 12.2a
through 12.2i fori5, aeruginosa and S. aureus, Attachment 2 section
A4.a through A4.e for M. terrae.
b.	Prepare Test Suspension A (without soil load): Dilute the test
microbial suspension with PBS to achieve an average challenge of
20-200 CFU per 10 |j.L (e.g., serially dilute cultures through 10"4 or
10"5; refer to the Neutralization Test Suspension Preparation Sheet
in section 14.0). Test Suspension A should be used within 4 hours
of preparation.
i. If performing the assay with dried carriers, prior testing may
be required to account for differences in the loss of viability
of the different test organisms upon drying.
c.	Prepare Final Test Suspension B (with soil load) : Prepare the soil
load: vortex each component and combine 25 |j.L bovine serum
albumin (BSA), 35 |j.L yeast extract, and 100 |j.L of mucin, mix
well. Combine 340 |j.L of Test Suspension A and 160 |j.L of the soil
load (SL). The test microbial suspension with soil load should
provide an average challenge of 20-200 CFU/tube.
i. If performing the assay with dried carriers, ensure an average
challenge of 20-200 CFU/carrier after drying. For example,
serially dilute 10 mL cultures of P. aeruginosa through 10"3,
serially dilute 10 mL cultures of S. aureus through 10"4,
serially dilute 5 mL cultures of M. terrae through 10"2.
Note: Two separate serial dilutions of Test Suspension A may be
used to prepare two different concentrations of Final Test

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SOP No. MB-26-01
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Suspension B to ensure at least one dilution with an average
challenge of 20-200 CFU. If performing the assay with dried
carriers, the use of two separate dilutions results in a total of 20
carriers to be processed; however, the dilutions may be evaluated
separately.
d. It is recommended that a calibration curve, using optical density
(OD @ 650nm), be created to estimate the number of viable
organisms in Test Suspension A.
12.3 Carrier
inoculation
(for alternative
dried carrier-
based assay)
a.	Inoculate at least 13 carriers with Final Test Suspension B (per
concentration of Final Test Suspension B) by adding 10 |j.L using a
positive displacement pipette to each carrier. Refer to SOP MB-25,
OECD Quantitative Method, sections 12.4c through 12.4d for carrier
drying instructions.
b.	After drying, evaluate the dried carriers per section 12.5.
12.4 Suspension-
based assay
a.	Treatment 1: Neutralizer Effectiveness. Add 50 |iL of the test
substance to each of three vessels. At timed intervals, add 10 mL
neutralizer to each vessel and briefly swirl. After 10 s, gently add 10
|iL Final Test Suspension B to each vessel and briefly vortex.
Proceed with section 12.6.
b.	Treatment 2: Neutralizer Toxicity Control Add 10 mL neutralizer to
each of three reaction vessels. At timed intervals, add 10 |j.L of Final
Test Suspension B gently to each vessel and briefly vortex. Proceed
with section 12.6.
c.	Treatment 3: Titer Control. Add 10 mL PBS to each of three
reaction vessels. At timed intervals, add 10 |j.L of Final Test
Suspension B gently to each vessel and briefly vortex. Proceed with
section 12.6.
12.5 Alternative
dried carrier-
based assay
a.	Treatment 1: Neutralizer Effectiveness. Add 50 |iL of the test
substance to each of three vials. At timed intervals, add 10 mL
neutralizer to each vial and briefly swirl. After 10 s, gently add one
dried carrier inoculated with Final Test Suspension B to each vessel
and vortex for 30±2 s. Proceed with section 12.6.
b.	Treatment 2: Neutralizer Toxicity Control Add 10 mL neutralizer to
each of three reaction vessels. At timed intervals, add one dried carrier
inoculated with Final Test Suspension B gently to each vessel and
vortex for 30±2 s. Proceed with section 12.6.
c.	Treatment 3: Titer Control. Add 10 mL PBS to each of three
reaction vessels. At timed intervals, add one dried carrier inoculated

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SOP No. MB-26-01
Date Revised 08-26-14
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with Final Test Suspension B gently to each vessel and vortex for
30±2 s. Proceed with section 12.6.
12.6 Processing and
recovery
a.	Hold the mixtures from 12.4 and 12.5 for 10±1 min at room
temperature (22±2°C). Steps (e.g., addition of organism, neutralizer)
should be conducted at timed intervals (e.g., 30 s. intervals for
suspension-based assay, 1 min. intervals for dried carrier-based assay)
to ensure consistent time of contact.
b.	At the conclusion of the holding period, briefly vortex each vessel (for
the suspension-based assay) and pass each mixture through a separate,
pre-wetted 0.2 or 0.45 |j.m polyethersulfone (PES) membrane filter.
i. If performing the assay with dried carriers, vortex each vessel
for 30±2 s at the conclusion of the holding period. Use a
magnet to prevent carriers from falling onto the filter
membrane.
c.	Wash each vessel with approximately 20 mL PBS and briefly vortex;
filter the washes through the same filter membrane. Repeat once.
Finish the filtering process by rinsing the inside of the funnel unit with
about 40 mL of PBS and filtering the rinsing liquid through the same
filter membrane.
Note: Initiate filtration as soon as possible (e.g., within 30 minutes).
Two analysts are recommended to perform vortexing and filtration
steps to reduce holding time after vortexing.
d.	Remove the membrane aseptically with sterile forceps and place it
carefully over the surface of the recovery medium (trypticase soy agar
for P. aeruginosa and S. aureus, Middlebrook 7H11 agar for M
terrae). Avoid trapping air bubbles between the filter and the agar
surface. Incubate the plates for 24-48 hours at 36±1°C for P.
aeruginosa and S. aureus, 17-21 days at 36±1°C forM terrae.
e.	Examine the plates after incubation and count colonies. Calculate the
average CFU for each set of test conditions.
13. Data Analysis/
Calculations
1.	For the assay to be considered valid, ensure that:
a. The recovered number of CFU in the Titer Control (see section
12.4c) using Final Test Suspension B yields 20-200 CFU per tube.
The same must also be true when using the dried carrier-based assay
(see section 12.5c).
2.	For determining and verifying the effectiveness of the neutralizer, ensure
that:
a. The recovered number of CFU in the Neutralizer Toxicity Control

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SOP No. MB-26-01
Date Revised 08-26-14
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(see section 12.4b) is at least 50% of the Titer Control (see section
12.4c). A count lower than 50% indicates that the neutralizer is
harmful to the test organism. Note: counts higher than the Titer
Control (e.g., 120% of the Titer Control) are also deemed valid.
b. The recovered number of CFU in the Neutralizer Effectiveness
treatment (see section 12.4a) is at least 50% of the Titer Control;
this verifies effective neutralization. Note: counts higher than the
Titer Control (e.g., 120% of the Titer Control) are also deemed
valid.
3.	For both the suspension-based assay and the carrier-based assay, the
criteria in sections 13.1 and 13.2 must be met. If the criteria are not met,
another neutralizer or mixture of neutralizers must be identified and
verified.
4.	For the suspension-based assay (see section 12.4), compare the average
CFU of the Titer Control with the average CFU of the Neutralizer
Toxicity Control and Neutralizer Effectiveness treatment. Compare
results from the dried carrier-based assay (see section 12.5) in the same
manner.
14. Forms and Data
Sheets
1.	Attachment 1: OECD Neutralization Assay Flow Chart
2.	Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
OECD Method for Bactericidal Activity: , „ m p| ,
Neutralization Test Information Sheet - ' °CX
OECD Method for Bactericidal Activity: 26 01 F2 docx
Neutralization Test Suspension Preparation Sheet -
OECD Method for Bactericidal Activity: , „ _, m „ ,
Neutralization Time Recording and Results Sheet - ' °CX
15. References
1. OECD Guidance Document: Quantitative Method for Evaluating
Bactericidal Activity of Microbicides Used on Hard Non-Porous Surfaces
(January 29, 2013).

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Attachment 1
OECD Neutralization Assav Flow ("hart
Trenltiit-ut 1
SOP No. MB-26-01
Date Revised 08-26-14
Page 8 of 8
Add 50 |il of test
subsranee to each vessel,
add 10 ml neutrali.zer
and briefly swirl
U
"J | j—> w
'an 10 sec.
Add 10 fiL of Ten
Sitspensw. B to
each vessel
containing 50 ui,
test substance and
10 iilL neutralize!'

Briefly vortex and
( hold for 10 mm. at
20-25=C. Proceed to
vortexing filtering.
"sent! alizrr Effectiveness
Tresitiiteul ;
Add 10 uL of Tat
Suspension B to each
tube containing lu mL
neutralize!'
i
v \/
Neutralize!' Toxicity
Control
Briefly vertex and
• hold for 10 min at
20-25:C. Proceed to
vortexing filtering
Trcntmeut 3
Add 10 [iL of list 					Briefly vortex and
Suspixshv! B to each	| 1 I IT I	hold for 10 mm. at
tube containing 10 mL	j i I 11 I	^ 20-25'C-. Proceed to
PBS	V J \ / I	vortexine filteiins.
Titer Control

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