US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
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-26-00
Date Revised: 03-14-13
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SOP No. MB-26-00
Date Revised 03-14-13
Page 1 of 10
SOP Number
MB-26-00
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. This SOP is
based on Annex II of OECD test guidelines dated January 29, 2013
(seeref. 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Date SOP withdrawn: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-26-00
Date Revised 03-14-13
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
6
14.
FORMS AND DATA SHEETS
7
15.
REFERENCES
8
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1. Definitions
Additional abbreviations/definitions are provided in the text.
1. Eluent = any liquid that is harmless to the test organism(s) and that is
added to a vial containing the carrier to recover the test organism.
2. Eluate = recovered eluent that contains the test organism.
3. Test suspension = suspension of the test microbe prior to the addition of
the soil load ( Test Suspension A)
4. Final test suspension= test suspension with soil load (Test Suspension B)
5. Stock culture = frozen culture used to prepare the test culture
6. 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, EQ-02, EQ-03, EQ-04 and EQ-05 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.
11. Special
Apparatus and
Refer to SOP MB-25, section 11.
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Materials
12. Procedure and
1. General description of the assay:
Analysis
a.
The test substance is first mixed with a candidate neutralizer. The
test organism is then added to the reaction mixture as dried inoculum
on a carrier; if desired, additional evaluations may be conducted
using the test organism as a liquid. The neutralization process is
deemed acceptable if the criteria outlined in section 13 are met.
12.1 Preparation/
sterilization of
a.
Refer to SOP MB-25, section 12.1.
carriers
12.2 Preparation of
test organisms
a.
Refer to SOP MB-25; section 12.2a through 12.2i for P. aeruginosa
and S. aureus, Attachment 2 section A4.a through A4.e forM.
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/carrier after the 10 |j.L inoculum has dried (e.g.,
"2
serially dilute 10 mL cultures of P. aeruginosa through 10" , serially
dilute 10 mL cultures of S. aureus through 10"4, serially dilute 5 mL
cultures of M. terrae through 10" ; refer to the Neutralization Test
Suspension Preparation Sheet in section 14.0). Prior testing may be
required to account for differences in the loss of viability of the
different test organisms upon drying. Test Suspension A should be
used within 4 hours of preparation.
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 132 |j.L of Test Suspension A and 68 |j.L of the soil
load (SL). The test microbial suspension with soil load should also
achieve an average challenge of 20-200 CFU/carrier after drying.
Note: Two separate serial dilutions of Test Suspension A may be
used to prepare two different concentrations of Final Test
Suspension B to ensure at least one set of carriers with an average
challenge of 20-200 CFU/carrier after the inoculum has dried.
Thus, 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
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SOP No. MB-26-00
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organisms in Test Suspension A.
12.3 Carrier
inoculation
a.
Inoculate at least 13 carriers with Test Suspension A (if conducting
optional treatments) and 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, sections 12.4c through 12.4d for carrier
drying instructions.
b.
After drying, evaluate the dried carriers using the required
treatments (section 12.4). Optional treatments (section 12.5) may
be evaluated if necessary.
12.4 Required
treatments
a.
Treatment la: Titer Control (with SL). Add 10 mL PBS to each of
four vials. At timed intervals, add one dried carrier inoculated with
Final Test Suspension B gently to each vial. Vortex each vial for 30 ±
2 s. Proceed with section 12.6. If necessary, using another vial,
perform this procedure with 10 |j.L of Final Test Suspension B in
place of one of the inoculated carriers.
b.
Treatment 2: Neutralizer Toxicity Control (with SL). Add 10 mL
neutralizer to each of three vials. At timed intervals, add one dried
carrier inoculated with Final Test Suspension B gently to each vial.
Vortex each vial for 30 ± 2 s. Proceed with section 12.6. If
necessary, using another vial, perform this procedure with 10 |j.L of
Final Test Suspension B in place of one of the inoculated carriers.
c.
Treatment 3a: Neutralizer Effectiveness (with SL). 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
vial. Vortex each vial for 30 ± 2 s. Proceed with section 12.6. If
necessary, using another vial, perform this procedure with 10 |j.L of
Final Test Suspension B in place of one of the inoculated carriers.
12.5 Optional
treatments
a.
Treatment lb: Optional Titer Control (without SL). Add 10 mL
PBS to each of four vials. At timed intervals, add one dried carrier
inoculated with Test Suspension A gently to each vial. Vortex each
vial for 30 ± 2 s. Proceed with section 12.6. If necessary, using
another vial, perform this procedure with 10 |j.L of Test Suspension A
in place of one of the inoculated carriers.
b.
Treatment 3b: Optional Neutralizer Effectiveness (without SL).
Add 50 |iL of the test substance to each of three vials. Then at timed
intervals, add 10 mL neutralizer each vial and briefly swirl. After
10 s, gently add one dried carrier inoculated with Test Suspension A
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SOP No. MB-26-00
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to each vial. Vortex each vial for 30 ± 2 s. Proceed with section
12.6. If necessary, using another vial, perform this procedure with
10 |jL of Test Suspension A in place of one of the inoculated carriers.
c. Treatment 3c: Optional Neutralizer Effectiveness (independent
addition of SL). Add 50 |j.L of the test substance to each of three
vials. At timed intervals, add 10 mL of the neutralizer to each vial
followed by the addition of 10 |j.L of the 3-part soil load to each vial
and briefly swirl. After 10 s, add one dried carrier inoculated with
Test Suspension A to each vial. Vortex each vial for 30 ± 2 s.
Proceed with section 12.6. If necessary, using another vial, perform
this procedure with 10 |j.L of Test Suspension A in place of one of the
inoculated carriers.
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 PBS, neutralizer)
should be conducted at timed intervals (e.g., 1 min. intervals) to
ensure consistent time of contact.
b. At the conclusion of the holding period, vortex each vial for 30 ± 2 s
and pass each mixture through a separate, pre-wetted 0.2 or 0.45 |jm
polyethersulfone (PES) membrane filter. Use a magnet to prevent
carriers from falling onto the filter membrane. Wash each vial with
approximately 20 mL PBS and vortex for 5 ± 1 s; filter the washes
through the same filter membrane. Repeat once (altogether 40 mL of
PBS). 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.
c. Remove the membrane aseptically with sterile forceps and place it
carefully over the surface of the recovery medium (trypticase soy agar
fori5, aeruginosa and S. aureus, Middlebrook 7H11 agar forM.
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, 21-28 days at 36±1°C for M terrae.
d. Examine the plates after incubation and record as CFU per carrier and
- if necessary - per sample of the Test Suspension A or B. Calculate
the averages for each set of test conditions with carriers.
13. Data Analysis/
Calculations
1. For the assay to be considered valid, ensure that:
a. The recovered number of CFU in the Titer Control with SL (see
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SOP No. MB-26-00
Date Revised 03-14-13
Page 7 of 10
section 12.4a) using Final Test Suspension B yields 20-200 CFU per
carrier and, if necessary, when using the microbial suspension as a
liquid.
b. The recovered number of CFU in the optional Titer Control
without SL (see section 12.5a) using Test Suspension A yields 20-
200 CFU per carrier and, if necessary, when using the microbial
suspension as a liquid.
2. For determining and verifying the effectiveness of the neutralizer, ensure
that:
c. The recovered number of CFU in the Neutralizer Toxicity Control
with SL (see section 12.4b) is at least 75% of the Titer Control
with SL (see section 12.4a). A count lower than 75% indicates that
the neutralizer is harmful to the test organism. Note: counts higher
than the Titer Control with SL (e.g., 120% of the Titer Control
with SL) are also deemed valid.
d. The recovered number of CFU in the Neutralizer Effectiveness
(with SL) (see section 12.4c) treatment is within 75% of the Titer
Control with SL; this verifies effective neutralization in the
presence of SL in the inoculum. The same results are expected for
the optional Neutralizer Effectiveness (without SL) (see section
12.5b) and Neutralizer Effectiveness (independent addition of SL)
(see section 12.5c). Note: counts higher than the Titer Control with
SL (e.g., 120%) of the Titer Control with SL) are also deemed
valid.
3. The criteria in sections 13.1 and 13.2 must be met. If the criteria are not
met, another neutralizer or mixture of neutralizes must be identified and
verified.
4. Always compare the numbers of the Titer Controls determined on
carriers with the numbers of the respective Neutralizer Toxicity
Controls or Neutralizer Effectiveness assays determined on carriers. If a
liquid microbial suspension is used instead of carriers, compare the
numbers determined in the respective suspensions with each other
accordingly.
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: ,,, ,
Neutralization Test Information Sheet MB-26-00_F 1 .docx
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SOP No. MB-26-00
Date Revised 03-14-13
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OECD Method for Bactericidal Activity: , _ _, nn F9 ,
Neutralization Test Suspension Preparation Sheet - ' 0CX
OECD Method for Bactericidal Activity:
Neutralization Time Recording and Results Sheet MB-26-00_F3.docx
(Required Components)
OECD Method for Bactericidal Activity:
Neutralization Time Recording and Results Sheet MB-26-00_F4.docx
(Optional Components)
15. References
1. Draft Test Guideline: Quantitative Method for Evaluating Bactericidal
Activity of Microbicides Used on Hard Non-Porous Surfaces (January 29,
2013).
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SOP No. MB-26-00
Date Revised 03-14-13
Page 9 of 10
Attachment 1
OECD Neutralization Assay Flow Chart (required components)
Treatment la
Add 1 Lcllill' liUKllf Itcd
Ulth [l \t\f>• >!"!> It ii
to 11 v uf tont untPii
in mi PBS
Treaimvnt 2
\Jd I umia iihiuil ikn
mtn /\ o Sim/ i1? A'
In uidl \ I if UHit I'liHi'I
Hi nil iiuUtKilli/vt
*_J LiJ LlJ Lr
Titer Control (with SL)
X ortc\ lor ?!i 2
* md hnkl tur
IB nins ut 22 2 C
hocued to
\otlc\mu hltenni:
Neutraliier Toxicity
Control
Yonc\ lor J
* see .»mi hold tor
h> min as 22' ? (\
IVoi-Cvd U>
\oik-\imj illu-nrni.
Trcuhnvnt Xi
r
Vld ill or KM
vuhsl.im. i ti» eaih \ nil.
tlvil add lo Nil
nuiluh/ei u> c.Jth \ wl
jihI hiu'th suit I
Nt'iitr;iii/e«' Lffcctivtlie
(with SL)
Ov-ntK jdd ! wurict
riHKIll.lUil u |1|» jf, ¦>!
* Wait 10 seer—5^ V, V> ''Oi 'It /i1 to tMlll
\ Wil COUUllliUkJ Si| id
W ^sihst,iiuc ,md MS
ml n^utMlf/cr
\ oik'\ till ^
•a'C .Hki hold loi
|o nidi :i\ 22- 2 1
i'ilHWil no
\oite\isv_! filk-rmn.
Optional suspension lest: Using an additional vial perform each procedure using 10 fit o* tin- appmpriate test suspension in place of the inoculated carrier..
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SOP No. MB-26-00
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Page 10 of 10
Attachment 1 (cont.)
OECD Neutralization Assay Flow Chart (optional components)
Treatment lb
Add 1 Laiiic uun.uLtcd
With U f
to 11 v uf tont untr.t:
in mi PBS
Titer C ontrol
(without SL)
\ itrtcx ti>r 2
- m.v, ,iml bold tur
(0 nun .it 22 2 t
Proceed to
\ora-\Mhi lilk'im.ii
Treatment 3b __
r
Vld *»< ul »i k tl<
MV II I
• Wait 10
Neutralim J tiUhvimsN
(without i
*. K'iitl\ add 1 uurict
movuLitcd uith / >i
Sif'lii :mu ,t\ 22' 2 t
Plowed Jo
fdklini!
Treatment 3c.
r
\dd ul ol Ws\
¦•uKi mic lo each \ j.i
then .Kid 10 ml
Mc-uli.ili/cr .uul Hi uI
iho i-pun M tn c.sdi
\Uf. I»iswill
Niutj nii/i r J ffeciiveness
(iiuiepi'iidi iit addition of SL)
Gently ;idd 1 carrier
uuKuUleil uiih L'st
* Wait 10 sect—> Smj-wsffm •? to tmli
s I ^ ul
kM MlKUlfUl .lUd 10
ml iwiituii/L-i
\ <11ICX in! *-i}-2
>a sjr.d hold tut
ii< nun ,i\ 22 2 (
PtiKVCd SO
Mnic\in'2 flhci iou
Optional suspension lest: Using an additional vial perform each procedure using 10 fit *»t tin- appiopriate test suspension in place of the inoculated carrier..
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