US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
OECD Quantitative Method for Evaluating
Bactericidal and Mycobactericidal Activity of
Microbicides Used on Hard, Non-Porous Surfaces
SOP Number: MB-25-05
Date Revised: 04-16-19
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SOP No. MB-25-05
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SOP Number
MB-25-05
Title
OECD Quantitative Method for Evaluating Bactericidal and
Mycobactericidal Activity of Microbicides Used on Hard, Non-
Porous Surfaces
Revisions Made
• Included additional Caution (section 10).
• Attachments 3 and 4: (1) clarified frozen stock culture preparation
steps and quality control procedures, (2) provided additional
specifications for initiating frozen stock cultures from a current
unexpired frozen stock culture.
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SOP Number
MB-25-05
Title
OECD Quantitative Method for Evaluating Bactericidal and
Mycobactericidal Activity of Microbicides Used on Hard, Non-
Porous Surfaces
Scope
The method provides a quantitative assessment of the performance
of liquid antimicrobial substances against Pseudomonas aeruginosa,
Salmonella en/erica., Staphylococcus aureus, and Mycobacterium
terrae designed for use on hard, non-porous surfaces. This method
is based on an Organization for Economic Co-operation and
Development (OECD) Guidance Document, dated June 21, 2013
(see ref. 15.1); however, the SOP contains revisions based on
information and data collected by the EPA since 2013.
Application
This method provides log reduction (LR) as the quantitative measure
of efficacy for liquid disinfectants against the test microbes on a
hard, non-porous surface.
Approval Date
SOP Developer:
Print Name:
SOP Reviewer
Print Name:
Quality Assurance Unit
Print Name:
Branch Chief
Print Name:
Data SOP issued:
Controlled copy number:
Date SOP withdrawn:
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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
4
9.
DATA MANAGEMENT
4
10.
CAUTIONS
4
11.
SPECIAL APPARATUS AND MATERIALS
4
12.
PROCEDURE AND ANALYSIS
8
13.
DATA ANALYSIS/CALCULATIONS
16
14.
FORMS AND DATA SHEETS
17
15.
REFERENCES
18
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1. Definitions
Additional abbreviations/definitions are provided in the text.
1. OECD = Organization for Economic Co-operation and Development
2. LR = logio reduction
3. 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.
4. Eluate = recovered eluent that contains the test organism
5. Stock culture = frozen culture used to prepare the test culture
6. Final test suspension = the harvested test culture with the addition of the
OECD soil load
7. CFU = colony forming unit
2. Health and
Safety
1. Follow procedures specified in SOP MB-01, Laboratory Biosafety.
2. Consult the Safety Data Sheet for specific hazards associated with the test
substance or other potentially hazardous materials.
3. Personnel
Qualifications
and Training
1. Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
2. A test system control based on lab-grade sodium hypochlorite solutions may
be used to verify the suitability of the test system and analyst proficiency.
4. Instrument
Calibration
1. Refer to SOPs EQ-01 (pH meters), EQ-02 (thermometers), EQ-03 (weigh
balances), EQ-04 (spectrophotometers), EQ-05 (timers), and QC-19
(pipettes) for details on method and frequency of calibration.
5. Sample Handling
and Storage
1. Refer to SOP MB-22, Preparation and Sampling Procedures for
Antimicrobial Test Substances, and SOP COC-01, Chain of Custody
Procedures.
6. Quality Control
1. For quality control purposes, the required information is documented on the
appropriate form(s) (see section 14).
2. Refer to SOP MB-10, Media and Reagents Used in Microbiological Assays,
for QC of media and reagents.
7. Interferences
1. Inadequate neutralization may lead to errors in the measurement of test
substance efficacy. Prior to efficacy testing, verify neutralizer effectiveness
using the procedure outlined in SOP MB-26 (Neutralization of Microbicidal
Activity using the OECD Quantitative Method).
2. During testing, do not process carriers where the test substance runs off of
the carrier; replace and retest with new inoculated carrier(s) and vial(s).
3. Avoid touching the carrier surface with a pipette tip during the application of
the test substance or the control substance.
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4. Transparent vials are more desirable to facilitate the application of 50 |iL test
substance or control substance on inoculated carriers.
8. Non-conforming
Data
1. For an acceptable test, achieve mean control carrier counts of 5.0-6.0 logs
CFU/carrier for Pseudomonas aeruginosa, Staphylococcus aureus and
Mycobacterium terrae, and 4.5-5.5 logs CFU/carrier for Salmonella
enterica; up to a one log difference between the three control carriers is
permitted.
2. Any level of contamination which interferes with the recording and
interpretation of results will result in invalid data.
9. Data
Management
1. Data will be archived consistent with SOP ADM-03, Records and Archives.
10. Cautions
1. Avoid extended soaking of the carriers in water or detergent and prolonged
rinsing to reduce risk of corrosion or rusting.
2. For storage, ensure carriers are completely dry following sterilization.
3. Ensure quality of media (sterility and performance) is adequate.
11. Special
Apparatus and
Materials
1. Test microbes: Pseudomonas aeruginosa (ATCC #15442,), Salmonella
enterica (ATCC #10708), Staphylococcus aureus (ATCC #6538), and
Mycobacterium terrae (ATCC #15755).
a. Additional bacteria may be evaluated using this method per the
Agency's guidance or as identified in a research protocol. Record
detailed information on the preparation of frozen stock cultures and the
final test suspension.
2. Culture media fori5, aeruginosa, S. enterica and S. aureus.
a. Tryptic Soy Broth (TSB). Use to rehydrate lyophilized cultures.
Purchase broth from a reputable source or prepare according to
manufacturer's instructions.
b. TSB with 15% (v/v) glycerol. Used as a cryoprotectant solution.
Suspend 7.5 g tryptic soy broth in 212.5 mL de-ionized water. Add
37.5 mL glycerol and stir, warm slightly to dissolve. Dispense into
bottles and autoclave for 15 min at 121 °C.
c. Tryptic soy agar (TSA) and TSA with 5% sheep blood (BAP). Used for
culturing, isolation, and characterization of the test microbes. Purchase
plates from a reputable source or prepare according to manufacturer's
instructions.
d. Selective media. Used for quality control of test microbes listed in this
procedure. Mannitol salt agar, Cetrimide agar, and Xylose lysine
deoxycholate agar. See Table 1 for use. Purchase plates from a
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reputable source or prepare according to manufacturer's instructions.
3. Culture media forM terrae.
a. Middlebrook 7H9 broth with 10% (v/v) ADC enrichment and 15% (v/v)
glycerol (MADC). Used as the growth medium for test cultures.
Combine 4.7 g Middlebrook 7H9 broth powder, 150 mL glycerol, 750
mL water and sterilize in autoclave per the manufacturer's instructions.
After the medium has cooled to 40-45°C, aseptically add 100 mL
Middlebrook ADC enrichment and then add sterilized water up to
1,000 mL. The pH of the medium should be 6.6±0.2.
b. Middlebrook 7H11 agar. Used for recovery and enumeration of the test
microbe. Purchase prepared agar from a reputable source or prepare
according to manufacturer's instructions.
4. Reagents
a. Neutralizer. Various neutralizers may be used including PBS with
0.1% (v/v) Tween-80 (PBS-T). If necessary, other ingredients may be
added to PBS-T. Additionally, non-PBS based neutralizers may be
used.
b. Phosphate buffered saline stock solution (e.g., 10X). Use to prepare IX
phosphate buffered saline. The stock solution has a pH of
approximately 7.2±0.2.
c. Phosphate buffered saline (PBS), IX. Use for dilution blanks and
filtration. PBS with a pH of approximately 7.0±0.5 is desirable.
d. PBS-T with 0.1% (w/v) sodium thiosulfate. Neutralizer for sodium
hypochlorite-based test substances, including the test system control. A
pH of approximately 7.2±0.2 is desirable.
e. Soil load. The OECD soil load to be incorporated in the test
suspension is a mixture of the following stock solutions in PBS:
1. BSA: Add 0.5 g bovine serum albumin (BSA) to 10 mL of PBS,
mix and pass through a 0.2 |im pore diameter membrane filter,
aliquot, and store at -20±2°C.
ii. Yeast Extract: Add 0.5 g yeast extract to 10 mL of PBS, mix,
and pass through a 0.2 |im pore diameter membrane filter,
aliquot, and store at -20±2°C.
iii. Mucin: Add 0.04 g mucin (bovine or porcine) to 10 mL of PBS,
mix thoroughly until dissolved, and autoclave (15 min at
121°C), aliquot, and store at -20±2°C.
iv. The stock solutions of the soil load are single use only. Do not
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refreeze once thawed; store up to one year at -20±2°C.
v. See section 12.4 for addition of soil load to inoculum.
vi. Additional soil loads may be used per the Agency's guidance or
research protocol.
f. Test substance. Antimicrobial test solution or product. If dilution is
required, see section 11.4g for diluent.
g. Test substance diluent. Used for the preparation of dilutable products.
The OECD test substance diluent is 375 ppm hard water. Adjust the
recipe for volumes other than 1 L.
i. Prepare Solution A by dissolving 19.84 g anhydrous magnesium
chloride (or 42.36 g MgCl 6H2O) and 46.24 g anhydrous
calcium chloride (CaCh) in de-ionized water and dilute to 1,000
mL. Sterilize by membrane filtration. Store the solution in the
refrigerator (2-8°C) for up to 30 days; do not use after that time.
ii. Prepare Solution B by dissolving 35.02 g sodium bicarbonate
(NaHCCte) in water and dilute to 1,000 mL. Sterilize by
membrane filtration. Store the solution in the refrigerator (2-
8°C) for up to 30 days; do not use after that time.
iii. To prepare 1 L of 375 ppm hard water, place 600-700 mL of
de-ionized water in a 1,000 mL volumetric flask and add 6.0
mL of Solution A and then 8.0 mL of Solution B. Mix and add
water to the flask to reach 1,000 mL. The pH of the hard water
should be 7.0±0.2 at room temperature. If necessary, adjust the
pH by using 1 N NaOH or 1 N HC1. Ensure sterility of hard
water.
iv. Prepare the hard water under aseptic conditions and use within
5 days of preparation. On the day of the test, measure the
hardness of the water using a water hardness test kit or other
suitable titration method.
v. The target hardness expressed as mg/L calcium carbonate
(CaC03) is 375 mg/L +5%/-10% (338-394 ppm).
vi. Additional diluents and levels of water hardness may be used
per the Agency's guidance or research protocol.
h. Water. De-ionized (DI), distilled water or water with equivalent
quality for making reagent solutions and culture media.
i. Tween-80 (polysorbate 80). To prepare PBS-T.
j. Laboratory grade sodium hypochlorite (NaOCl) with total chlorine
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>4%. For preparation of the test substance used in the test system
control.
k. Liquinox (1% solution) or equivalent. To clean carriers.
1. Gram stain. Used for diagnostic staining of P. aeruginosa, S. enterica
and S. aureus.
m. Acidfast stain. Used for diagnostic staining of M. terrae.
5. Apparatus
a. Carriers: Disks (1 cm in diameter) made from 0.8 mm thick sheets of
brushed and magnetized stainless steel (AISI #430) (Pegen Industries,
part #430-107). The top of the disk is brushed and has rounded edges;
only the top is visually screened and inoculated. Carriers are single-use
only. See Attachment 2 for carrier specifications and photographs of
screened carriers.
i. Additional carriers (e.g., carriers composed of AISI #304
stainless steel, carriers 2 cm in diameter) may be utilized per the
Agency's guidance or as identified in a research protocol. 304
stainless steel is non-magnetized and must be managed in the
efficacy assay without the use of a magnet. Carrier
specifications for surface finishing will be retained according to
Attachment 2.
b. Calibrated 10 juL positive displacement pipette with corresponding 10
|iL tips, for carrier inoculation.
c. Filter paper. Whatman No. 2, to line glass Petri plates.
d. Calibratedmicropipettes (e.g., 200 |iL, 1 mL) with 10-100 or 20-200
|iL tips, for deposition of test substance on carriers and preparing
dilutions.
e. Bottle-top dispensers, squirt bottles, pre-measured volumes in tubes, or
pipettes, bottles etc. For rinsing vials and filters.
f. Forceps, straight or curved, non-magnetic, disposable with smooth flat
tips to handle membrane filters, appropriate to pick up the carriers for
placement in vials.
g. Magnet. To hold the carrier (430 stainless steel) in place in the vial
while the liquid is being poured out of it for membrane filtration.
h. Membranes (polyethersulfone) for recovery of test microbe, 47 mm
diameter and 0.2 jam pore size (fori5, aeruginosa, S. enterica, and S.
aureus) and or 0.45 [am pore size (forM terrae). Filtration units
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(reusable or disposable) may be used.
i. Spectrophotometer. For culture standardization.
j. Sterile vials (plastic or comparable) to hold test carriers: flat bottom
and wide-mouth to accommodate addition and removal of the carriers,
for holding inoculated carriers to be exposed to the test substance and
for accommodating neutralizer/eluent. Suitable vials should be at least
25 mm in neck diameter and capable of holding at least 20 mL of
liquid.
i. Transparent vials are more desirable to facilitate application of
50 |iL test substance or control substance to inoculated carrier.
k. Certified timer. Readable in minutes and seconds, for tracking of timed
events and intervals.
1. Desiccation unit (with gauge to measure vacuum) with fresh desiccant
(e.g., CaCCte). For drying inoculated carriers.
m. Vacuum. In-house line or suitable vacuum pump for drying inoculated
carriers in desiccation unit and to aid in filtration.
n. Hach digital titrator kit. For measuring total chlorine and water
hardness.
o. Sterile Bijou bottles (or equivalent). For M terrae test culture
preparation; glass (with aluminum caps) or plastic with capacity of
approximately 5-7 mL, with 10 glass beads (3-5 mm in diameter).
p. Tissue grinder. Any glass or plastic tissue grinder with a capacity of
approximately 15 mL for homogenization of theM terrae culture.
q. Vortex-style mixer. For vortex mixing of various solutions.
r. Centrifuge (with rotor capable of achieving 10,000g). For test culture
preparation.
12. Procedure and
Analysis
12.1 Preparation and
sterilization of
carriers
a. Without magnification, visually check the brushed top surface of the
carriers (with the rounded edge) for abnormalities (e.g., rust, chipping,
deep striations) and discard if observed.
b. Soak visually screened carriers in a suitable detergent solution for 2-4 h
to degrease and then rinse thoroughly in distilled or deionized water.
c. Record physical screening of carriers on appropriate form (see section
14).
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d. Prior to sterilization, place up to 20 clean dry carriers on filter paper
inside the bottom surface of a glass Petri dish (150 mm in diameter).
Cover the Petri dish with its lid and sterilize at 121°C for 45 min.
Ensure carriers are dry following sterilization.
e. Use sterilized carriers for up to six months. After six months, re-
sterilize any remaining unused carriers and assign a new preparation
number.
12.2 Preparation of
P. aeruginosa,
S. enterica, and
S. aureus test
cultures
a. Refer to Attachment 3 for preparation of the frozen stock cultures.
b. Defrost a cryovial; defrost rapidly to avoid loss in the viability of the
preserved cells. Each cryovial is single use only.
c. Add 100 |iL of defrosted stock culture to 10 mL TSB, briefly vortex
mix and incubate for 18-24 h at 36±1°C. In addition, inoculate an agar
plate (e.g., TSA or TSA with 5% sheep blood) with a loopful from the
inoculated tube and streak for isolation. Incubate plate with the test
culture and examine for purity.
d. Following incubation, use the broth cultures to prepare a test
suspension for each organism.
e. Fori5, aeruginosa, inspect culture prior to harvest; discard if pellicle
has been disrupted (fragments in culture). Remove visible pellicle on
surface of medium and around associated interior edges of the tube by
pipetting or with vacuum suction. Using a serological pipette, withdraw
the remaining broth culture (approx. 7-8 mL) avoiding any sediment on
the bottom of the tube and transfer it into a 15 mL centrifuge tube.
Alternatively, the culture may be removed by gently aspirating the
broth away from the pellicle material.
f. For S. enterica and S. aureus, briefly vortex the 18-24 h culture and
transfer to a 15 mL centrifuge tube.
g. Centrifuge the 18-24 h broth cultures at 5,000g\ for 20±5 min.
h. Remove the supernatant without disrupting the pellet. Re-suspend the
pellet in a maximum of 10 mL PBS. Resuspension of the pellet in a
smaller volume (5 mL) is permissible to concentrate culture.
i. For S. enterica and S. aureus, disrupt the pellet using vortexing
or repetitive tapping/striking against a hard surface to
disaggregate the pellet completely prior to re-suspending it in a
maximum of 10 mL. If necessary, add 1 mL of PBS to the pellet
to aid in the disaggregation.
ii. Further dilute the resuspended culture as necessary in PBS to
achieve a mean control carrier count level of 5.0-6.0 logs
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CFU/carrier for S. aureus and I\ aeruginosa, and 4.5-5.5 logs
CFU/carrier for S. enterica.
i. Use the diluted culture to prepare the final test suspension with the
addition of the soil load per section 12.4.
j. Optical density/absorbance (at 650 nm) may be used as a tool to
monitor/adjust the diluted test suspension.
12.3 Preparation of
M terrae test
cultures
a. Refer to Attachment 4 for preparation of the frozen stock cultures for
M terrae.
b. Defrost a cryovial; defrost rapidly to avoid loss in the viability of the
preserved cells. Each cryovial is for single use only.
c. Add 1 mL thawed culture to a flask of 100 mL MADC and incubate at
36±1°C for 7-10 days under agitation at 150 rpm. Seal the flask (e.g.,
with foil and Parafilm) to reduce evaporation and inhibit
contamination.
d. In addition, inoculate an agar plate (e.g., M7H11) with a loopful from
the inoculated flask and streak for isolation. Incubate plate for 17-21
days and examine for purity.
e. Following incubation, aliquot 25 mL portions of the 7-10 day-old
MADC broth culture into each of 2-50 mL conical screw cap tubes and
centrifuge at 10,000g\ for 20±5 min.
f. Carefully remove the supernatant and re-suspend each pellet in 25 mL
sterile water.
g. Centrifuge the tubes a second time at 10,000g\ for 20±5 min. After
centrifuging, re-suspend the pellets in a total of 5 mL sterile DI water
(1/10 of the starting volume), pool, and place in a tissue grinder.
Homogenize the culture for approximately 1 min to eliminate visible
clumps of bacteria.
h. After homogenizing, transfer the culture to a bijou bottle (or
equivalent) with 10 glass beads; vortex for 5 min.
i. For preparation of the test suspension, dilution of the culture from the
bijou bottle with sterile water may be required to achieve mean carrier
counts within the range of 5.0-6.0 logs CFU/carrier.
j. The approximate titer of test suspension may be estimated
spectrophotometrically at 650 nm, based on a standard curve specific to
the test organism.
i. Prior to inoculation of carriers, aseptically add the OECD soil
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load per the instructions in 12.4.
12.4 Preparation of
the final test
suspension with
OECD soil load
a. Vortex the test suspension for 10-30 s.
b. To obtain 500 |iL of the final test suspension with the OECD soil load,
vortex each component and combine the following (or appropriate
ratio):
i. 25 |iL BSA stock
ii. 35 |iL yeast extract stock
iii. 100 |iL mucin stock
iv. 340 |iL microbial test suspension
c. Use final test suspension with soil load (at room temperature, 22±2°C)
to inoculate carriers within 30 min of preparation.
12.5 Inoculation and
drying of
carriers
a. Vortex the final test suspension for 10 s following the addition of the
soil load and immediately prior to use.
b. Inoculate the number of carriers required for the evaluation of the test
substance (3 controls and 3 treated) along with carrier(s) to serve as
extras. If performing the test system control, refer to Attachment 1.
c. Using a calibrated positive displacement pipette with a 10 |iL tip,
withdraw 10 |iL of the final test suspension and deposit it at the center
of each carrier (clean, screened and sterile); avoid contact of pipette tip
with carrier and do not spread the final test suspension with the pipette
tip.
i. For consistency, vortex the inoculum frequently during
inoculation of the carrier set. The same pipette tip may be used
to inoculate all carriers (unless the tip is compromised). Discard
any inoculated carrier where the final test suspension has run
over the edge.
d. Transfer the Petri dish(es) with the inoculated carriers into a
desiccation unit (with desiccant) and completely remove the lid of the
Petri dish. Close the desiccation unit door (or lid) and seal the unit.
Apply vacuum to evacuate the desiccation unit.
e. Maintain and monitor the vacuum level using a gauge. Achieve and
maintain consistent level of vacuum (at 20-25 in of mercury, 508-635
torr, 677-847 mbar, or 68000-85000 Pascal).
f. Hold the inoculated carriers in the evacuated desiccation unit at 22±2°C
for 30 to 50 min. Visually inspect inoculated carriers to verify that they
have completely dried and remove from desiccation unit. Do not use
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carriers that are visibly wet for testing.
i. If the carriers are not dry within the recommended time,
continue to dry for up to an additional 10 min and record the
time to dry on the paperwork.
ii. If carriers dry very quickly (e.g., 5-10 min) or are not dry within
10 min beyond the specified time, check the desiccation unit
and the vacuum system to ensure proper function (e.g., replace
the desiccant if necessary and check the seal for leaks).
g. Following the inoculation of carriers, streak inoculate an agar plate
(e.g., TSA or TSA with 5% sheep blood fori5, aeruginosa, S. enterica
and S. aureus; M7H11 agar plate for M terrae) with a loopful of the
final test suspension (with OECD soil load). Incubate plate with the
treated and control carrier plates generated from the test day and
examine for purity at the end of the incubation period. The purity plate
should be free of contamination.
h. Use dried inoculated carriers for testing within 30 min following
removal from desiccation unit; hold carriers in closed Petri dish at room
temperature (22±2°C) until use.
12.6 Exposure of the
dried inoculum
to the test
substance or
control
substance
a. Evaluate 3 control carriers and 3 treated carriers for each test substance
tested (one test organism and contact time/temperature combination)
unless specified otherwise. Note: One set of control carriers may be
used for evaluating multiple test substances against one organism on
one test day (assuming the neutralizer is the same).
b. See Attachment 1 for conducting the test system control. Conduct the
test system control per the Agency's guidance or as identified in a
research protocol.
c. Using sterile forceps, transfer each dried carrier with the inoculated
side up to a flat-bottom vial and cap the vial. Repeat until all carriers
are transferred.
d. In a timed fashion, deposit 50 |iL of the test substance (equilibrated to
22±2°C) with a calibrated micropipette (or positive displacement
pipette) over the dried inoculum on each test carrier, ensuring complete
coverage, at predetermined staggered intervals. Use a new tip for each
carrier; do not touch the pipette tip to the carrier surface. During
testing, do not process carriers where the test substance runs off of the
carrier; replace with new carrier(s) and vial(s) if this occurs. Do not cap
the vials.
i. For non-foaming aerosols and pump/trigger spray products,
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obtain the test substance by dispensing the product into a sterile
vessel for collection. Cap the vessel. Using aseptic technique,
remove 50 |iL of the liquid from the vessel and deposit it on the
inoculated carrier.
ii. For foaming products, allow up to 5 min for the foam to break
down and generate at least 1-2 mL for sample collection.
iii. Use dispensed product within 30 min of collection.
e. Hold the test carriers at 22±2°C (or other specified temperature) for the
selected contact period. Use a certified timer to ensure that each carrier
receives the required exposure time (OECD default exposure time is 5
min±5 s).
f. Evaluate control carriers last. Each control carrier receives 50 |iL PBS,
equilibrated to 22±2°C, instead of the test substance. Hold the control
carriers at 22±2°C for the contact period.
12.7 Neutralization
of test substance
and elution of
test organisms
a. The neutralizer for the control carriers is the same as that for the treated
carriers.
b. Within ±5 s of the end of the contact period, add 10 mL of room
temperature neutralizer to each vial in the specified order according to
the predetermined schedule. Briefly vortex (2-3 s) each vial following
the addition of the neutralizer.
i. The neutralized vial with carrier is documented as the 10°
dilution.
c. Following the neutralization of the entire set of carriers, vortex each
vial for 30±5 s at high speed to recover and disaggregate the inoculum;
ensure that the liquid and carrier are fully vortexed. Do not remove the
carrier from the vial.
12.8 Dilution and
recovery
a. Initiate dilutions within 30 min after neutralization and vortexing.
Initiate filtration within 30 min of preparing the dilutions.
b. Dilute and filter samples from the treated and control carriers; process
treated carriers first.
c. Serially dilute the eluate from the 10° dilution prior to filtration by
transferring 1 mL into 9 mL PBS in a dilution tube.
d. For the treated carriers, dilute out to 10"1 and filter the entire contents of
the 10° and 10"1 dilutions; additional dilutions may be prepared and
filtered as necessary.
e. For control carriers, dilute out to 10"4 and filter the entire contents of
the 10"3 through 10"4 dilutions; additional dilutions may be prepared and
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filtered as necessary.
f. Prior to filtration, pre-wet each membrane filter with -10 mL PBS;
apply vacuum to filter contents. Leave the vacuum on for the duration
of the filtration process.
g. Use separate membrane filters for each eluate; however, the same
filtration unit may be used for processing eluates from a given carrier
set starting with the most dilute sample first.
h. Filter samples through separate 0.2 |im PES membrane filters fori5.
aeruginosa, S. enterica and S. aureus or 0.45 |im PES membrane filters
for M. terrae.
i. For eluates from treated carriers remaining in the vial (10° dilution),
vortex the vial for ~5 s and, holding a magnet at the bottom of the vial
to keep the carrier in place, pour the eluate into the filter unit.
j. Rinse the treated vial with -20 mL PBS, vortex for ~5 s and keeping
magnet in place, pour the wash into the same filter unit. For dilution
tubes, rinse tube once with -10 mL PBS, briefly vortex, and pour into
filter unit.
k. Swirl the contents of the filter unit and quickly filter with limited
pooling of liquid in the filter apparatus.
1. Rinse the inside surface of the funnel unit with -40 mL PBS and filter
the contents.
m. Aseptically remove the membrane filter and place on the appropriate
recovery medium. Avoid trapping any air bubbles between the filter
and the agar surface.
i. Use TSA for recovery of P. aeruginosa, S. enterica and S.
aureus.
ii. Use M7H11 agar for recovery of M terrae.
n. Fori5, aeruginosa, S. enterica and S. aureus, incubate plates at 36±1°C
for 48±4 h for control carriers and for a minimum of 72±4 h for treated
carriers. Following incubation, count the number of colonies.
i. Monitor filters after 24 h of incubation to facilitate appropriate
timing for counting the colonies.
ii. If no growth appears on filters for the treated carrier after 72±4
h, continue to incubate for up to five days (total) and record the
results.
o. ForM terrae, incubate all plates from treated and control carriers at
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SOP No. MB-25-05
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Page 15 of 26
36±1°C for 17-21 days; however, monitor filters for growth and count
the number of colonies beginning at 10-14 days.
12.9 Recording
results
a. Count colonies and record results.
b. For colony counts on filters in excess of 200 record as Too Numerous
to Count (TNTC).
c. If no colonies are present, record as zero.
d. Inspect the growth on the filters for purity and typical characteristics of
the test microbe
i. See Table 1 fori5, aeruginosa, S. enterica and S. aureus.
ii. Colony morphology for M terrae includes irregular margins,
rough appearance, buff color, opaque and umbonate; M. terrae
is acid fast positive.
e. If isolated colonies are present, use the appropriate stain to assess one
representative colony per 3-carrier set (treated and controls). Use Gram
stain for P. aeruginosa, S. enterica and S. aureus and acid fast stain for
M. terrae.
f. If confluent growth is present, perform a streak isolation on the
appropriate agar on growth taken from at least 1 carrier.
i. Fori5, aeruginosa, S. enterica and S. aureus, use TSA or TSA
with 5% sheep blood and incubate at 36±1°C for 24-48 h.
ii. For M. terrae use M7H11 agar and incubate at 36±1°C for 17-
21 days
g. If additional verification of the test organism is required, perform
further confirmatory analyses (e.g., Vitek and biochemical analyses for
i5. aeruginosa, S. enterica and S. aureus) and isolation streaks on
selective media.
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Table 1. Selective media and diagnostic characteristics fori5, aeruginosa, S.
aureus, and S. enterica. (see ref. 15.7 and 15.8)
Aspccl
P. ucru'fiiiKisir
S. aureus
.V. culcricu
(nam slain reaction
\euali\ e
Positive
Negative
Manmiol Sail \uar
\ \
Circular, small, yellow
colonies, agar turning
fluorescent yellow
N/A
Ceiraniide \uar
( irenlar. small,
i n11 la 11> opaque,
liirinim fluorescent
ureen n\er lime, agar
IliKireseeni \elli>wish
ureen
N/A
N/A
\\ lose |\ sine
deowcholale i \l.1)
a nan
\ \
N/A
Round, clear red
colonies with black
centers
IJIood auar (15 \l'i
I 'lal. upaqne In off-
whilc. round
spreadmu 111. metallic
sheen, shdillv heta
hemoK lie
Small, circular, yellow
or white, glistening,
beta hemolytic
Entire, glistening,
circular, smooth,
translucent, low
convex, non-
hemolytic
l >pic;il Mici'oscopic ( hai aclci islics
Cell appearance
Siraiuhl or slmlitly
enr\ed rods, single
polar flauella. rods
lornied in chains. 0.5
1 i) miii in diameter x
1 5 5 ii miii in length
Spherical, occurring
singly, in pairs and
tetrads, sometimes
forming irregular
clusters; 0.5 - 1.0 |im
in diameter
Straight rods,
peritrichous flagella;
0.7 - 1.5 umin
diameter x 2.0 - 5.0
|im in length
*After 24±2 h (1) P. aeruginosa may display two phenotypes.
13. Data Analysis/
Calculations
1. Per test, use colony counts to determine log reductions.
2. Use values with at least three significant figures when performing
calculations (e.g., log density, mean log density). Report the final mean log
reduction value with two significant figures (e.g., round up to the nearest
tenth).
3. Calculate the Colony Forming Units (CFU)/carrier using the following
equation:
CFU for Wy + CFU for 10
-z \
xc
where 10"y and 10"z are the
(axKTO + ^xKT2)
dilutions filtered, "a" and "b" are the volumes filtered at each dilution
(typically 9 or 10 mL), and "c" is the volume of medium originally in the
vial with the carrier (10 mL). Account for the volume filtered in the
calculations. See examples below:
4. Example 1. When 10° dilution yields 130 CFU and the 10"1 dilution yields 20
CFU:
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130 CFU+ 20 CFU
k(9 mL x 10°) + (10 mL x 10"1)
^ x 10 mL {vol. in vial) = 1.50 x 102 CFU/carrier
5. When TNTC (Too Numerous To Count) values are observed for each
dilution filtered, substitute 200 for the TNTC at the highest (most dilute)
dilution and account for the dilution factor in the calculations.
6. Example 2. When 10° dilution yields TNTC (>200) CFU and thelO"1
dilution yields TNTC (>200) CFU:
( 200 CFU \
I x 10 mL (vol. in vial) = 2.00 x 103 CFU/carrier
\10 mL x 10-1/ v 1 '
7. Calculate the log density of each carrier by taking the logio of the density per
carrier.
8. Calculate the mean logio density across treated carriers.
9. Calculate the mean logio density across control carriers.
10. Calculate the logio reduction (LR) for treated carriers: logio reduction = the
mean logio density for control carriers minus the mean logio density for
treated carriers.
11. For a set of 3 treated carriers: when the 10° dilution (the contents of the vial
with the carrier) is filtered either by itself or in addition to other dilutions
and the data for each carrier result in zeros for each dilution filtered, report
the LR as greater than or equal to the mean logio density for the control
carriers.
14. Forms and Data
Sheets
1. Attachment 1: Test System Control
2. Attachment 2: Carrier Specifications
3. Attachment 3: Maintenance of Bacterial Cultures - Preparation of Frozen
Stock Cultures
4. Attachment 4: Maintenance of Mycobacterium terrae - Preparation of
Frozen Stock Culture
5. Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
Physical Screening of Carriers Record Form
OECD Method for Bactericidal Activity:
Organism Culture Tracking Form
OECD Method for Bactericidal Activity: Test
Microbe Confirmation Sheet (Quality Control)
OECD Method for Bactericidal Activity: Test
Information Sheet
MB-03_Fl.docx
MB-25-05_Fl.docx
MB-25-05_F2.docx
MB-25-05 F3.docx
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OECD Method for Bactericidal Activity: Serial MB-25-05 F4.docx
Dilution Plating/Tracking Form
OECD Method for Bactericidal Activity: Results MB-25-05 F5.docx
Sheet
OECD Method for Bactericidal Activity: Test MB-25-05 F6.docx
Microbe Confirmation Sheet
OECD Method for Bactericidal Activity: Test MB-25-05 F7.docx
Processing Sheet
OECD Method for Bactericidal Activity: Test MB-25-05 F8.docx
Processing Sheet (.Mycobacteria)
15. References
1. OECD Guidance Document on Quantitative Methods for Evaluating the
Activity of Microbicides Used on Hard Non-Porous Surfaces (June 21,
2013)
2. Krieg, Noel R. and Holt, John G. 1984. Bergey's Manual of Systematic
Bacteriology Volume 1. Williams & Wilkins, Baltimore, MD.
P. aeruginosa p. 164.
3. Sneath, P., Mair, N., Sharpe, M.E., and Holt, J. eds. 1986. Bergey's Manual
of Systematic Bacteriology Volume 2. Williams & Wilkins, Baltimore, MD.
S. aureus p. 1015.
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Attachment 1
Test System Control for the OECD Quantitative Method for Bacteria and Mycobacteria
Purpose: Conduct the test system control periodically (per Agency guidance or research protocol)
to verify the suitability of the overall test system using the test microbe to be evaluated against the
antimicrobial product. It is recommended that the analyst responsible for conducting the product
efficacy evaluation perform the test system control.
Process: Conduct the test system control per methodology in MLB SOP MB-25. Derive the final
test suspension from the same set of frozen stock cultures used for the product efficacy test.
Evaluate two concentrations of laboratory-grade sodium hypochlorite (NaOCl) using three carriers
per NaOCl concentration and three control carriers per microbe. Use a 5 min contact time, the
OECD 375 ppm hard water as the diluent, and the OECD three-part soil load included in the
inoculum. See Table 1 for the concentrations of NaOCl and recommended dilutions for recovery.
Use PBS with 0.1% (v/v) Tween-80 and 0.1% (w/v) sodium thiosulfate as the neutralizer. Verify
the water hardness and concentration of the NaOCl solutions using an appropriate titration
procedure (e.g., Hach digital titrator) prior to use. Evaluate the high NaOCl concentration (2000
ppm) first, followed by the low NaOCl concentration (e.g., 200 ppm) and then the control carriers.
The three control carriers used in the product efficacy test may be used in the LR calculations for
the test system control, assuming the OECD three-part soil load is incorporated into the inoculum.
Table 1. Test system control - concentrations of NaOCl and recommended dilutions to be
assayed for treated carriers per test microbe
P. aeruginosa
S. enterica
S. aureus
XI. terrae
2000 ppm (±5%)
(filter dilutionlO0)
2000 ppm (±5%>)
(filter dilution 10°)
2000 ppm (±5%)
(filter dilution 10°)
4000 ppm (±5%)
(filter dilution 10°)
150 ppm (±5%>)
(filter dilutions 10° to 10"2)
100 ppm (±5%)
(filter dilutions 10° to 10"2)
500 ppm (±5%>)
(filter dilutions 10"1 to 10"3)
1000 ppm (±5%)
(filter dilutions 10"1 to 10"3)
Results: Count the number of colonies and calculate the mean control counts and the mean LR
values per EPA MLB SOP MB-25, section 13.
Desired outcomes:
• Control carrier counts.
o Fori5, aeruginosa, S. aureus, andM. terrae, mean control carrier counts to be
within 5.0-6.0 logs per carrier. For S. en/erica, the mean control carrier counts to
be within 4.5-5.5 logs per carrier. Individual control carrier counts should be
within one log of each other.
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• Log reduction.
o The high concentration NaOCl results in no recovery (i.e., complete kill of the test
microbe) to very limited recovery (i.e., almost complete kill of the test microbe)
for each of the three treated carriers:
¦ Mean LR of >4.5 fori5, aeruginosa, S. aureus andM terrae
¦ Mean LR >4.0 for S. enterica.
o The low concentration of NaOCl results in a significant recovery of viable
bacteria for each of the three treated carriers:
¦ Mean LR between 0.0 to 3.5 for each microbe.
Note: Test system controls for additional microbes may be proposed per the Agency guidance or
a research protocol.
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Attachment 2
Carrier Specifications
(AISI Type 430 Stainless Steel Carriers)
General Description: 1 cm magnetic disc made of AISI Type 430 Stainless Steel (SS) with
No.4 finish on one side and rounded edges on top side.
Material: AISI Type 430 Ferretic stainless steel consisting of 16% to 18% Chromium, a maximum
of 0.5% Nickel and a maximum of 0.12% Carbon.
• European Specification X6Crl7 Number 1.4016
Japanese Specification: JIS G4305; EN10088-2
Dimensions:
• Diameter: lcm (0.39") in diameter
• Thickness: 0.8 mm (22 gauge / 0.031")
Flatness: some concavity desired at edges
Finish
No. 4 Finish is produced with short, parallel polishing lines. The final finish can be anywhere
between 120 and 320 grit.
Tumbling
To remove burrs from the edges of the discs they are tumble deburred in a vibratory tumbler using
ceramic median and cleanser. Tumbling time is dependent on the extent to which burring occurs.
Passivation
Parts are passivated according to ASTM A967 in a citric acid solution and prepared as follows:
Degrease with citrus based degreaser
Rinse with tap water
Passivate
o 7%> Citric Acid Solution
o Minimum of 20 min at 20-50°C.
Rinse with de-ionized water
• Air dry
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Attachment 2 (continued)
Examples of Physically Screened Carriers1
Fig. 1: Examples of typical acceptable 430 SS carriers.
Fig. 2: Examples of typical unacceptable 430 SS carriers.
'The same acceptance criteria used to screen the 430 SS carriers are used to screen the 304 SS carriers. Carriers are
screened without magnification.
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Attachment 3
Maintenance of Bacterial Cultures - Preparation of Frozen Stock Cultures
(Refer to SOP MB-02 for establishment of the organism control number.)
A. Initiate new stock cultures from lyophilized cultures of Pseudomonas aeruginosa,
Salmonella enterica and Staphylococcus aureus from ATCC (or other reputable
vendor) at least every 18 months. Record all microbe transfers on the Organism
Culture Tracking Form, see section 14.
i. New frozen stock culture may be initiated one time using an existing,
unexpired frozen stock culture as the source. Begin process at step C
below, by streaking a loopful of the frozen stock culture onto 2 TSA
plates.
B. Open ampule of freeze-dried organism per manufacturer's instructions. Using a
tube containing 5-6 mL of TSB, aseptically withdraw 0.5 to 1.0 mL and rehydrate
the lyophilized culture. Aseptically transfer the entire rehydrated pellet back into
the original tube of broth. Mix thoroughly. Incubate broth culture at 36±1°C for
24±2 h.
C. At the end of the incubation timeframe, streak a loopful of the broth culture onto 2
TSA plates to obtain isolated colonies. Perform a streak isolation of the broth
culture onto BAP as a purity check, and streak the broth culture onto the
appropriate selective media. Refer to appropriate selective media in Table 1.
Incubate all plates for 24±2 h at 36±1°C.
i. Record results at the end of the incubation timeframe. Refer to Table 1
(section 12.9) for results on selective media and diagnostic characteristics
of the test microbes.
D. From the TSA plates (step C), select 3-5 isolated colonies of the test organism and
re-suspend in 1 mL of TSB. For S. aureus, select only golden yellow colonies.
Fori5, aeruginosa, select colonies from each of the two possible phenotypes
present. Spread plate 0.1 mL of the suspension onto each of 6-10 TSA plates.
Incubate the plates for 24±2 h at 36±1°C. If necessary to obtain more frozen stock
cultures, a larger suspension (e.g., 2 mL) may be prepared using the same ratio of
TSB (1 mL) to number of colonies (3-5 colonies).
i. Using the TSB suspension, perform a streak isolation of the suspension
onto a BAP as a purity check, and streak on the appropriate selective
media (refer to Table 1).
ii. Incubate all plates for 24±2 h at 36±1°C. Record results. Refer to Table 1
(section 12.9) for results on selective media and diagnostic characteristics
of the test microbes.
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E. After the incubation period, harvest growth from TSA plates by adding
approximately 5 mL sterile cryoprotectant solution (TSB with 15% (v/v) glycerol)
on the surface of each plate. Re-suspend the growth in the cryoprotectant solution
using a sterile spreader without damaging the agar surface. Aspirate the
suspension from the plate with a pipette and place it in a sterile vessel large
enough to hold about 30 mL.
F. Repeat the growth harvesting procedure with the remaining plates and continue
adding the suspension to the vessel (more than 1 vessel may be used if necessary).
Mix the contents of the vessel(s) thoroughly; if more than 1 vessel is used, pool
the vessels prior to aliquoting culture.
G. Immediately after mixing, dispense 0.5-1.0 mL aliquots of the harvested
suspension into cryovials; these represent the frozen stock cultures.
i. For QC purposes, perform a streak isolation of the pooled culture onto a
BAP as a purity check and streak on appropriate selective media (refer to
Table 1).
ii. Incubate all plates for 24±2 h at 36±1°C.
iii. Record results. Refer to Table 1 for results on selective media and
diagnostic characteristics of the test microbes.
iv. After incubation, perform a Gram stain on growth from the BAP; observe
the Gram reaction by using brightfield microscopy at 1000X
magnification (oil immersion).
v. Conduct Vitek confirmation from growth taken from the BAP. Conduct
VITEK according to the manufacturer's instructions.
vi. Record all confirmation results on the Test Microbe Confirmation Sheet
(Quality Control) (see section 14).
H. Store the cryovials at -70°C or lower for a maximum of 18 months. These cultures
are single-use only.
I. If the characteristics of the organism are not consistent with the information in
Table 1 at any step in the process, or the Vitek profile is inconsistent with the
organism, discard the cultures and re-initiate the process.
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Attachment 4
Maintenance of Mycobacterium terrae - Preparation of Frozen Stock Culture
(Refer to SOP MB-02 for establishment of the organism control number.)
I. Preparation of Frozen Stock Cultures
A. Initiate new stock cultures from a lyophilized culture of Mycobacterium terrae
(ATCC #15755) from ATCC at least every 18 months.
i. New stock culture may be initiated one time using an existing, unexpired
frozen stock culture as the source. Begin process at step C below, by
spreading 0.1 mL of the frozen stock culture onto 6-10 M7H9 or M7H11
agar plates.
B. Aseptically add 1.0 mL of Middlebrook 7H9 Broth with 10% ADC enrichment
(MADC) and rehydrate the lyophilized culture. Aseptically transfer the entire
rehydrated pellet back into 5 mL of MADC. Mix thoroughly.
C. Spread 0.1 mL of the test organism suspension onto approximately 6-10 M7H9 or
M7H11 agar plates. Incubate for 20-22 days at 36±1°C. Ensure sterility of the
plates during the incubation period (e.g., place in sterile bags or wrap with
Parafilm).
i. Note: Each plate will yield 10 mL of harvested suspension and
consequently nine to ten cryovials, each containing 1-1.5 mL of frozen
stock culture.
D. At the end of the incubation period, add 5 mL MADC to the surface of each agar
plate. Re-suspend the cells in MADC using a sterile spreader without damaging
the agar surface.
E. Aspirate the suspension from the plate with a pipette and place it in a sterile
vessel large enough to hold about 30 mL.
F. Repeat by adding another 5 mL of MADC to the agar plates, re-suspend the cells,
aspirate the suspension and pool with the initial cell suspension.
G. Repeat the growth harvesting procedure with the remaining plates and continue
adding the suspension to the vessel (more than 1 vessel may be used if necessary).
H. Mix the contents of the vessel(s) thoroughly; if more than 1 vessel is used, pool
the vessels prior to aliquoting the culture.
I. While mixing continuously, dispense 1-1.5 mL aliquots of the harvested
suspension into separate cryovials; these represent the frozen stock cultures.
J. Store the cryovials at -70°C or lower for a maximum of 18 months (from the date
of harvesting/freezing). These cultures are single-use only.
J. A titer of the frozen stock culture of approximately 1 x 109 CFU/mL is appropriate.
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II. QC of Frozen Stock Cultures
A. Conduct QC of the pooled culture concurrently with freezing. Streak a loopful on
M7H11 agar and incubate at 36±1°C for 17-21 days. Following the incubation
period, record the colony morphology as observed on the plates. Typical colony
morphology forM terrae includes irregular margins, rough appearance, buff
colored, opaque. Record all confirmation results on the Test Microbe
Confirmation Sheet (Quality Control) (see section 14).
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