1 Interim Quantitative Method for Evaluating the Efficacy of Antimicrobial Test Substances
2 on Porous Surfaces Against Bacteria
3 (12/12/2022)
4
5 Scope
6 The Environmental Protection Agency (EPA) Office of Pesticide Programs (OPP) recommends
7 that applicants utilize this interim method to support efficacy criteria for the registration of
8 products bearing claims for use on soft, porous surface claims. The method provides a
9 quantitative assessment of the performance of antimicrobial substances against l'scmlomonas
10 aeruginosa and Staphylococcus aureus on soft-porous surfaces.
11 This method provides log reduction (LR) as the quantitative measure of efficacy lor disinfectants
12 against the test microbes on a soft-porous surface.
13
14 Method Overview
15 In brief, the method uses 1 cm diameter discs (carriers) of a sel of representative soft-porous
16 surface materials. Each disc receives 10 |iL of microbial inoculum (with a three-part organic and
17 inorganic soil load) deposited in the center of each carrier. The inoculum is allowed to dry and is
18 then exposed to 50 |iL of the antimicrobial treatment, control carriers receive an equivalent
19 volume of an innocuous fluid (e.g., phosphate buffered saline). The exposure time is allowed to
20 elapse; a liquid neutralizer is then added to the vial to halt the antimicrobial action. Each vial
21 with the carrier is vortexed, serially diluted, and the contents are filtered to recover viable
22 microorganisms. Based on the difference betw een the mean logio density values of the untreated
23 control and treated carriers, a mean logio reduction (LR) in viable bacteria is calculated. The LR
24 value is used as the measure of product effectiveness.
25 Appropriate safety procedures should always be used when working with laboratory test
26 systems which include human pathogenic microorganisms. Laboratory safety is discussed in the
27 current edition of "Biosafety in Microbiological and Biomedical Laboratories (BMBL)" 6th
28 edition, from the subject matter experts within the U.S. Department of Health and Human
29 Services (III IS), including experts from the Centers for Disease Control and Prevention (CDC)
30 and Tvitional Institutes of Health (NIH).
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TABLE OF CONTENTS
1) Special Apparatus and Materials 1
2) Carriers 5
3) Preparation of Test Cultures and Carrier Inoculation 7
4) Performance Assessment - Efficacy 9
5) Data Requirements 12
Attachment A: Preparation of Frozen Stock Culture 14
Attachment B: Neutralization Assay and Flowcharts 16
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1) Special Apparatus and Materials
a. Test microbes: Pseudomonas aeruginosa (ATCC #15442) and Staphylococcus aureus
(ATCC #6538).
i. Additional bacteria may be tested (for an additional label claim) per the Agency's
guidance.
b. Culture media
i. Tryptic Soy Broth (TSB). Use to rehydrate lyophilized cultures Purchase broth
from a reputable source or prepare according to manufacturer's instructions.
ii. Synthetic broth (SB). Growth medium for test cultures. Commercial media
(HIMEDIA, Synthetic Broth, AO AC, #M334-500G). Suspend 16 9 g in 1000 mL
DI water. Heat if necessary, to dissolve the medium completely l-'inal pH at 25°C
should be 7.1±02. Medium may be dispensed in 10ml. amounts in 20x150 mm
culture tubes or alternatively in 500 mL volumes in a I I. bottle; steam sterilize at
15 lbs pressure (121°C) for 15 minutes. Cool to room temperature and just before
use, aseptically add 0.1 mL of 10% sterile dextrose solution. Store prepared SB at
2-8°C.
1. Alternatively, SB made in-house per the recipe provided in AO AC
Methods 955.15, 964.02, and l->55.14 may be substituted.
iii. 10% dextrose solution. Add 5 <) u dextrose to 50 mL de-ionized water and mix by
stirring. Filter sterilize the solution using a 0.2 |im filter. Store the sterile solution
at 2-5°C for up to 30 days.
iv. TSB with 15% (v/v) glycerol. I se as a cryoprotectant. 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 steam sterilize for 15 min at 121°C.
v. Tryptic soy agar (ISA) and ISA with 5% sheep blood. Use for culturing,
isolation, and characterization of the test microbes. Purchase plates from a
reputable source or prepare according to manufacturer's instructions.
\ i Selective media, (optional) Mannitol salt agar and Cetrimide agar. Use for quality
control of test microbes listed in this procedure. Purchase plates or prepare
according to manufacturer's instructions.
c. Reagents
i. Neutralizer. A liquid reagent used to inactivate and/or dilute the antimicrobial
treatment to end the contact time.
ii. Phosphate buffered saline stock solution (e.g., 10X). To prepare IX phosphate
buffered saline. The stock solution has a pH of approximately 7.2±0.2.
iii. Phosphate buffered saline (PBS), IX. Dilution blanks and filtration. PBS with a
pH of approximately 7.0±0.5 is desirable.
iv. Soil load, 3-part. Use as the soiling agent. Add to the test suspension in the
following manner:
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1. BSA: Add 0.5 g bovine serum albumin (BSA, radio immunoassay (RIA)
grade or equivalent, CAS# 9048-46-8) to 10 mL of PBS, mix and pass
through a 0.2 |im pore diameter (polyethersulfone) membrane filter,
aliquot (e.g., a minimum of 50 |iL), and store at -20±2°C.
2. Yeast Extract: Add 0.5 g yeast extract to 10 mL of PBS, mix, and pass
through a 0.2 |im pore diameter (polyethersulfone) membrane filter,
aliquot (e.g., a minimum of 70 |iL), and store at -20±2°C.
3. Mucin: Add 0.04 g mucin (from bovine submaxillary gland. CAS #
84195-52-8) to 10 mL of PBS, stir or vortex-mix until thoroughly
dissolved, and pass through a 0.2 |im pore diameter (polyethersullbne)
membrane filter, aliquot, and store at -20±2°C.
4. The three stock solutions of the soil load arc single use only. Do not
refreeze; store up to one year at -20±2°C.
v. Antimicrobial Test substance. Ready-to-use, activated, or concentrated
antimicrobial. If the antimicrobial test substance is prepared by diluting a
concentrate, adequately mix antimicrobial test substance with the appropriate
diluent (e.g., hard water), then use prepared tesl substance within 3 hours of
preparation or as otherwise instructed by the manufacturer. Measuring error
increases as delivery volume decreases To minimize variability due to measuring
error, a minimum of 1.0 mT. or I g of concentrated antimicrobial test substance
should be used when preparing use-dilutions for testing. Use v/v dilutions for
liquids antimicrobial test substances and w/v dilutions for solid antimicrobial test
substances. The use of a positive displacement pipette is recommended for
viscous liquids.
vi. 1 NNa()// am! / A' //('/. I scd for pH adjustment of media/reagents.
vii. Water. l)e-ioni/.ed (I>1). distilled water or water with equivalent quality for
making reagent solutions and culture media.
viii 7iiwn-S() (polysorbate 80). Used to prepare PBS-T.
i\ (Irani stain. Used for diagnostic staining,
d Apparatus
i ('arriers: Discs (1 cm in diameter) cut from porous material. Carriers are single
use only. See Section 2 for carrier specifications.
ii. llule punch: If necessary, for use in the preparation of 1 cm disc from material.
Model number: SKU# HP-MEI448R or equivalent
iii. Calibrated 10 piL positive displacement pipette with corresponding 10 |iL tips, for
carrier inoculation.
iv. Filter paper. Whatman No. 2, to line glass Petri plates.
v. Calibratedmicropipettes (e.g., 200 |iL, 1 mL) with appropriate corresponding
tips, for deposition of test substance on carriers and preparing dilutions.
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vi. Bottle-top dispensers, squirt bottles, pre-measured volumes in tubes, or pipettes,
bottles, etc. For rinsing vials and filters.
vii. 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.
viii. Poly ether sulfone (PES) membranes. Use for recovery of test microbe, 47 mm
diameter and 0.2 |im pore size.
1. Use filter membranes in either a reusable or disposable filtration unit.
ix. Filter Sterilization Unit (withPES, 0.2 |im pore size). Use to filler sterilize soil
components.
x. 20 x 150 m glass culture tubes with Morton closures for lest culture preparation.
xi. Spectrophotometer. For culture standardization (if deemed necessary)
xii. Vials with lids (plastic or comparable). Sterile, flat-bollomed, wide-mouthed (at
least 25 mm diameter), approximately 20 mL capacity. Ibr holding inoculated
carriers to be exposed to the test chemical and for accommodating neutralizer.
1. Transparent vials are more desirable to facilitate application of 50 |iL test
substance or control substance to inoculated carrier.
xiii. Certified timer. Readable in mi miles and seconds, for tracking of timed events and
intervals.
xiv. Desiccation unit (with gauge to measure \ acuum level) with fresh desiccant (e.g.,
anhydrous CaCCb). For drying inoculated carriers.
xv. Vacuum source. In-house line or suitable vacuum pump capable of achieving
0.068 to 0.085 MPa, for drying inoculated carriers in desiccation unit and to
perform membrane filtration.
xvi. Titration kit. (i.e., Hach digital titrator) Used for measuring water hardness.
xvii. Voriex-siy/e mixer Used for vortex-mixing of various solutions.
\\ iii 15 ml conical centrifuge tubes. Used for centrifugation of test cultures.
\i\ ('eiitrifugc (with rotor capable of achieving 5,000g). Used for test culture
preparation.
2) Carriers
a. Carrier Materials
i. Privacy Curtain Fabric (PCF-03): 54% Polyester, 46% Fire Resistant (FR)
Polyester. CF Stinson, LLC. Mambo MAM34 Nights.
ii. Non-PVC Fabric (NVF-01): Polyurethane Face made with Polycarbonate and
Poly ether Resins, Polyester Backing. CF Stinson, LLC. Kid BlueSky KID 17.
iii. Vinyl Seating Fabric (VF-01): Vinyl Face, Polyester Backing. CF Stinson, LLC.
Hopsack - HOP24 Fjord.
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ir^L . jjpfcSrl
Lnil
Figure 1: Examples of carrier materials cut into 1 cm discs; materials 2.a.i, 2.a.ii,
and 2.a.iii (from left to right)
b. Carrier Preparation
i. Punch, or obtain, 1 cm round carriers or use comparable cutting procedure from
fabric.
ii. Visually screen carriers to ensure consistent surface characteristics; trim any
jagged edges or loose fabric.
in. No pre-cleaning of carriers is necessary. To sterilize carriers, sterilize using a
gravity cycle, 121°C for 20 minutes; ensure carriers are diy following
sterilization. Test sterility of carriers prior to testing.
1. Carriers may not be entirely flat after autoclaving; however, minor
distortion of carriers is acceptable for testing.
2. Prior to use in testing, document the condition of the screened and sterile
earners (e.g., digital photographs).
3) Preparation of Test Culture and Carrier Inoculation
a. Refer to Attachment A 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. Within 15 minutes prior to inoculation, using a calibrated pipette to aseptically add 0.1
mL of 10% sterile dextrose (w/v) solution to each 10 mL tube of SB.
d. Using a calibrated micropipette, add 100 uL of defrosted stock culture to 10 mL SB with
dextrose, briefly vortex-mix and incubate for 24±2 h at 36±1°C.
i. Incubate without disrupting the culture.
ii. In addition, inoculate an agar plate (e.g., TSA or TSA with 5% sheep blood) from
the inoculated tube and streak for isolation. Incubate plate with the test culture.
e. Following incubation, use the SB cultures to prepare a test suspension for each organism.
i. The 24±2 h culture should exhibit a titer of at least 108 CFU/mL.
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f. For P. aeruginosa, inspect culture prior to harvest; visible pellicle on the surface of the
culture is expected to form during incubation (record its presence). Discard the culture if
pellicle has been disrupted (fragments in culture).
i. Remove visible pellicle on surface of medium and around associated interior
edges of the tube by pipetting or with vacuum suction.
ii. Using a serological pipette, withdraw the remaining broth culture (at least 5 mL)
avoiding any sediment on the bottom of the tube and transfer it into a 15 mL
conical centrifuge tube.
iii. Record approximate volume harvested and transferred to ] 5 nil. conical lube.
g. For S. aureus, briefly vortex-mix the 24±2 h culture and transfer lo a I 5 mL conical
centrifuge tube.
h. Within 15 min, centrifuge the 24±2 h harvested broth cultures at 5,000gi\ for 20±5 min.
i. Remove the supernatant without disrupting the pellet. Re-suspend the pellet in 5-10 mL
PBS. Record resuspension volume.
i. Prepare the final test suspension within 30 min of resuspending the culture.
ii. If necessary, disrupt the pellet usi ng \ ortex-mixing or repetitive tapping/striking
against a hard surface to disaggregate the pel lei completely prior to re-suspending
it in 10 mL. If necessary, add I nil. of I'liS lo the pellet to aid in the
disaggregation.
j. If needed, dilute the 5-10 mL of resuspended culture in PBS to achieve a mean control
carrier count level of 4.0-5.5 logs CFl "carrier for S. aureus and P. aeruginosa.
i. Optical density/absorbance (c g . (->50 nm) may be used as a tool to monitor/adjust
the diluted test suspension
k. Use the resuspended or diluted culture to prepare the final test suspension with the
addition of the soil load
1 To obtain 5<)0 uL of the final test suspension with the 3-part soil load, vortex-mix each
component and combine in the following order using a calibrated micropipette:
i 25 |iL BSA stock
i i 3 5 u I. yeast extract stock
iii. 11iii jliL mucin stock
iv. Vortex soil suspension for 10s prior to adding microbial test suspension.
v. 340 |iL microbial test suspension
m. Briefly vortex the final test suspension with 3-part soil load (at room temperature,
21±3°C) and use to inoculate carriers within 30 min of preparation.
i. Streak inoculate an agar plate with a loopful of the final test suspension. Incubate
plate with the treated and control carrier plates and examine for purity after
incubation at 36±1°C for 72±4 h.
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n. It is advisable to briefly rescreen each sterilized carrier for abnormalities prior to
inoculation. Place carriers screened side up inside an empty, sterile plastic Petri dish (no
more than 20 carriers/dish).
i. Privacy curtain carriers have no backing material and may be inoculated on either
side.
ii. Non-PVC and vinyl carriers are layered materials comprised of a smooth, colored
top surface and a white fabric bottom; only the top surface will be inoculated.
o. Vortex-mix the final test suspension for 10 s following the addition of ilie soil load and
immediately prior to use.
p. Inoculate the number of carriers required for the evaluation of ilie lest substance (3
controls and 5 treated) along with a few extra carriers.
q. Using a calibrated positive displacement pipette with a 10 ul. lip, 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-mix the inoculum frequently during inoculation of the
carrier set.
ii. The same pipette tip may be used lo inoculate all carriers (unless the tip is
compromised).
iii. Discard any inoculated carrier where the final test suspension has run over the
edge.
r. 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.
i. Note: do not exceed 40 inoculated carriers per desiccator to ensure carriers dry
within the prescribed time.
s Maintain and monitor the vacuum level using a gauge. Achieve and maintain consistent
le\ el of vacuum (at Z<)-25 in of mercury, 508-635 torr, 677-847 mbar, or 68000-85000
Pascal) by leaving the vacuum on during the drying period with the desiccator stopcock
opened or closed as necessary.
t. Hold the inoculated carriers in the evacuated desiccation unit at 21±3°C for 45 to 60 min.
Visually inspect inoculated carriers to verify that they have completely dried and remove
from desiccation unit. Do not use carriers that are visibly wet for testing.
i. Record the time for all timed events.
ii. Depressurize the desiccator slowly to avoid the potential for carriers to move or
flip.
u. Use dried inoculated carriers for testing within 30 min following removal from
desiccation unit; hold carriers in closed Petri dish at room temperature (21±3°C) until
use.
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4) Performance Assessment - Efficacy
a. Evaluate 3 control carriers and 5 treated carriers for each test substance tested (one test
organism and contact time /carrier type combination) unless specified otherwise.
i. One set of control carriers per carrier type may be used for evaluating multiple
test substances against one organism on one test day (assuming the carrier
material, neutralizer, and soil load are the same).
b. 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.
c. Prepare the antimicrobial test substance. Use antimicrobial test substance within 3 hours
of preparation or as specified by the manufacturer.
d. In a timed fashion with appropriate intervals, sequentially deposit 50 li I. of the test
substance (equilibrated to 21±3°C) with a calibrated micropipelte over the dried
inoculum on each test carrier, ensuring complete coverage
i. Note: Gently apply the antimicrobial test substance ill a perpendicular angle to the
inoculated carrier; do not forcefully deposit the disinfectant.
e. Use a new tip for each carrier; do not touch the carrier surface with a pipette tip during
the application of the test substance or the control substance; 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, obtain the test
substance by dispensing the product into a sterile vessel for collection. Cap the
vessel and use dispensed product within 30 min.
ii. For foaming spray formulations, allow the foam to break down for at least 5-10
minutes for the generation of a 1 -2 mL liquid sample. Cap the vessel and use
dispensed product within 30 min.
f. Do not process carriers where the test substance runs off the carrier or does not
completely co\ er the inoculum spot; replace with new carrier(s) and vial(s) if this occurs.
g Conduct the test at room temperature (21±3°C) for the selected contact time. Use a
certified timer to ensure that each carrier receives the required contact time.
h Process control carriers last. Each control carrier receives 50 |iL PBS, equilibrated to
21 3 C. instead of the test substance. Hold the control carriers for the same contact time
as used lor the test substance.
i. Within ±5 s of the end of the contact period, add 10 mL of neutralizer equilibrated to
21±3°C to each vial in the specified order according to the predetermined schedule.
Briefly vortex-mix (2-3 s) each vial following the addition of the neutralizer.
i. For calculation purposes, the solution in the neutralized vial with carrier is
considered to be 10° dilution.
ii. The neutralizer for the control carriers is the same as that for the treated carriers.
j. Immediately following the addition of the neutralizer and briefly (2-3 s) vortex, allow
carriers to sit in the vials for 5 minutes undisturbed then proceed as follows:
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i. Vortex-mix vials at high speed for 30 s (vortex-mix #1).
ii. Allow carriers to sit undisturbed in the vials for 5 minutes.
iii. Vortex-mix vials at high speed for 30 s (vortex-mix #2).
iv. Allow carriers to sit undisturbed in the vials for 5 minutes.
v. Vortex-mix vials at high speed for 30 s (vortex-mix #3).
k. Initiate dilutions within 30 min after neutralization and vortex-mixing. Initiate filtration
within 30 min of preparing the dilutions.
1. Dilute and filter samples from the treated and control carriers; process treated carriers
first.
m. Serially dilute the eluate from the 10° dilution prior to filtration by transferring 1 mL into
9 mL PBS in a dilution tube.
n. Turn on vacuum and leave on for the duration of the filtration process.
o. Prior to filtration, pre-wet each membrane filter with I <) in I. PBS.
p. Use separate membrane filters for each eluate (neutralized solution); however, the same
filtration unit may be used for processing cluates from a given carrier set starting with the
most dilute sample first.
q. Filter each sample through a separate <> 2 mil IM-S membrane filter.
r. For eluates from treated carriers remaining in the vial (10° dilution), vortex-mix the vial
for ~5 s, carefully pour the eluate i nto the filter unit.
i. If a carrier tails onto the tiller membrane, aseptically remove it using sterile
forceps.
s. Rinse the treated \ ial with 2') ml. PBS, vortex-mix for ~5 s, pour the wash into the same
filter unit. For dilution tubes, rinse tube once with -10 mL PBS, briefly vortex-mix, and
pour into filter unit.
t. Swirl the contents of the filter unit and quickly filter with limited pooling of liquid in the
lllter apparatus.
u Ri use the inside surface of the funnel unit with at least 20 mL PBS and filter the contents.
v. AsepticalK remove the membrane filter and place on the appropriate recovery medium.
Avoid trapping any air bubbles between the filter and the agar surface.
w. Sterility controls.
i. On the day of the test, filter -20 mL of neutralizer and -20 mL of the PBS used in
the test using two separate membrane filters and place on TSA.
ii. Incubate these filters along with a plate of recovery medium (e.g., TSA) for 72±4
h at 36±1°C, record sterility results.
x. Incubate plates at 36±1°C for 48±4 h for control carriers and for a minimum of 72±4 h
for treated carriers.
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i. Monitor filters daily to optimize counting of colonies. CFUs may be counted
daily. Record controls after 48±4 h and treated carriers after 72±4 h.
y. Count colonies and record results.
i. Any level of contamination which interferes with the recording and interpretation
of results will result in invalid data.
ii. For example, contamination occuring on multiple filters within one set of serial
dilutions and/or across multiple carriers is considered systemic and the test is
deemed invalid.
z. For colony counts on filters in excess of 200 record as Too Numerous to Count ( TNTC).
aa. If no colonies are present, record as zero.
bb. Report non-conforming data (e.g., systemic contamination and al\ pical serial dilution
results) and repeat tests as necessary.
i. Systemic contamination
ii. Atypical serial dilution results (e.g., higher (Tl s at more dilute levels).
cc. Inspect the growth on the filters for purity and ly pical characteristics of the test microbe,
see Attachment A, Table 1.
dd. If isolated colonies are present, assess one representative colony per 5-carrier set (treated)
or 3-carrier set (controls) using a Gram stain
i. If confluent growth is present, perform a streak isolation on TSA or TSA with 5%
sheep blood on growth taken from at least 1 carrier incubate at 36±1°C for 24-48
ee. If additional verification of the test organism is required, perform further confirmatory
analyses (e.g., Vitek and biochemical analyses) and isolation streaks on selective media.
5) Data Requirements
a Per test, use colony counts to determine log reduction.
b I-'or an acceptable test, each of the three control carriers must exhibit counts between 4.0-
5 5 logs CFU/carrier.
c. Use \ allies 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).
d. Calculate the Colony Forming Units (CFU)/carrier using the following equation:
h.
where:
Y = CFU per filter,
C = volume filtered,
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= total volume of neutralizer,
D = 10"k,
k = dilution,
n = number of dilutions, and
i = lower limit of summation (the fewest number of dilutions).
358 e. When TNTC (TooNumerous To Count) values are observed for each dilution filtered,
359 substitute 200 for the TNTC at the highest (most dilute) dilution and account lor the
360 dilution factor in the calculations.
361 f. Calculate the log density of each carrier by taking the logio of tlie density per carrier.
362 g. Calculate the mean logio density across treated carriers.
363 h. Calculate the mean logio density across control carriers.
364 i. Calculate the logio reduction (LR) for treated carriers: logio reduction = the mean logio
365 density for control carriers minus the mean logio density lor treated carriers.
366 j. For a set of treated carriers: when the 10'" dilution (the contents of the vial with the
367 carrier) is filtered either by itself or in addition to other dilutions and the data for each
368 carrier result in zeros for each dilution filtered, report the LR as greater than or equal to
369 the mean logio density for the control carriers.
370 k. Log reduction data based on estimates due to the occurrence of TNTC outcomes at each
371 dilution in a dilution series for control and treated carriers is deemed unacceptable.
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Attachment A
Preparation of Frozen Stock Culture
1) Initiate new stock cultures from lyophilized cultures of Pseudomonas aeruginosa and
Staphylococcus aureus from a reputable vender at least every 18 months.
a. New frozen stock culture may be initiated one time using an existing,
unexpired frozen stock culture as the source. Begin process at step 3 below,
by streaking a loopful of the frozen stock culture onto 2 TSA plates.
2) Open ampule of freeze-dried organism per manufacturer's instructions. Using a lube
containing 5-6 mL of TSB, aseptically withdraw 0.5 to 1.0 mL and ichydiate the
lyophilized culture. Aseptically transfer the entire rehydrated pel let back into the
original tube of broth. Mix thoroughly. Incubate broth culture al 3ot 1 °C lor 24±2 h.
3) 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 lor 24±2 h at 36±1°C.
a. Record results at the end of the incubation limelYame. Refer to Table 1 for results on
selective media and diagnostic characteristics of the test microbes.
4) From the TSA plates, select 3-5 isolated colonies of the lest 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 6-1 n 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).
a. 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).
b. Incubate all plates for 24 2 h at 36±1°C. Record results. Refer to Table 1 for results on
selective media and diagnostic characteristics of the test microbes.
5) 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.
6) 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.
7) Immediately after mixing, dispense 0.5-1.0 mL aliquots of the harvested suspension into
cryovials; these represent the frozen stock cultures.
13
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411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
a. 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).
b. Incubate all plates for 24±2 h at 36±1°C.
c. Record results. Refer to Table 1 for results on selective media and diagnostic
characteristics of the test microbes.
d. After incubation, perform a Gram stain on growth from the BAP; observe the
Gram reaction by using brightfield microscopy at 1000X magnification (oil
immersion).
e. Conduct confirmation using an automated identification system (i.e., Vitek) or
biochemical and antigenic analyses from growth taken from the IJAP
according to the manufacturer's instructions.
8) Store the cryovials at approximately -80°C for a maximum of IS months. These cultures are
single use only.
9) 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.
Table 1. Sckvti\c media and diagnostic characteristics lor /'. aeruginosa and S. aureus
Aspect
P. iicni'fiiniisii"
S. aureus
(inun slum reaction
\euali\ e
I'osiiin e
Maumiol Sail \uar
(Sclccli\ c mediumi
\ \
Circular, small, \ ellou colonies, agar
turning fluorescent yellow
Cclriniidc \iiar
(Sclccli\c mediumi
Circular, small. imiialK opaque,
luriiiim riikireseeui ureeu overtime;
auar lliiorcsecui \cllou isli green
N/A
1 >lood auar (13 \l'i
1 lal. opaque lo off-u line, round
spreadiuu 111. metallic sheen, slightly
hela liemoK lie
Small, circular, yellow or white,
glistening, beta hemolytic
lApiciil Microscopic ( haraclcristics
Cell appearance
Si might or slightly curved rods, single
polar flagella, rods formed in chains;
ii 5-1.0 nm in diameter x 1.5-5.0 |im
in length
Spherical, occurring singly, in pairs
and tetrads, sometimes forming
irregular clusters; 0.5-1.0 |im in
diameter
\ I kT J" 4 "" li (I j /' aeruginosa may display two phenotypes.
14
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430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
Attachment B
Neutralization Assay
The purpose of this section is to assess the effectiveness of the neutralization processes
associated with this method. Perform the neutralization assay with both microbes for each
carrier type prior to testing to demonstrate the neutralizer's ability to inactivate the chemical.
Select a neutralizing medium that is not inhibitory to the test microbe. The acceptance criteria for
acceptable neutralization is < 50% difference in colony counts between the neutralization
effectiveness, neutralization toxicity control, titer control, and carrier control
1) Refer to Section 3 in the preceding method for preparation of the test cultures. Conduct
preliminary tests as necessary to determine appropriate dilution(s) of Test Suspension A (used
to prepare Test Suspension B) to achieve the target challenge of 2<~>-2<)i) CI I' per 10 |iL or per
carrier.
a. Prepare Test Suspension A (without soil load). Serially dilute the microbial test
suspension with PBS (e.g., through 10"4 or 10"'). Select appropriate dilutions of Test
Suspension A so that after the addition of the soil load. I lie Test Suspension B will achieve
an average challenge of 20-200 CFU per 10 |iL. Use I est Suspension A within 30 min of
preparation.
b. Prepare Test Suspension B (with soil load) Prepare the soil load: using a vortex, mix each
component and combine 25 |iL bovine serum albumin (BSA), 35 |iL yeast extract, and
100 |iL of mucin; then vortex-mix the solution. Combine 340 |iL of diluted Test
Suspension A and the 160 |iL of the soil load (SL) and vortex-mix for 10 seconds.
c. Ensure Test Suspension B provides an average challenge of 20-200 CFU per 10 |iL.
d. Two separate serial dilutions of lest Suspension A may be used to prepare two different
concentrations of Test Suspension R to ensure at least one dilution with an average
challenge of 20-200 CFU per in liL.
e. A calibration curve (Ol) (III 650nm) may be used to estimate the number of viable
organisms in Test Suspension A.
2) Neutral i/.ation Treatments (see Attachment B, Figure 2)
a Treatment 1: Neutralizer Effectiveness. Add 50 |iL of the test substance to each
ol" three reaction vessels. At timed intervals, add 10 mL neutralizer to each vessel
and briefly swirl (by hand). After 10 s, gently add 10 |iL of neutralizer test
suspension using a micropipette to each vessel and briefly vortex. Proceed with
section 4).
b. Treatment 2: Neutralizer Toxicity Control Add 10 mL neutralizer to each of three
reaction vessels. At timed intervals, add 10 |j.L of Test Suspension B using a micropipette
to each vessel and briefly vortex. Proceed with section 4).
c. Treatment 3: Titer Control. Add 10 mL PBS to each of three reaction vessels. At timed
intervals, add 10 |j.L of Test Suspension B using a micropipette to each vessel and briefly
vortex. Proceed with section 4).
15
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469
470
471
472
473
474
475
476
All
478
479
480
481
482
d. Treatment 4: Carrier Interference Control. Add one carrier to each of three reaction
vessels. At timed intervals, add 10 mL neutralizer to each vessel and briefly swirl (by
hand). After 10 s. gently add 10 |j.L of Test Suspension B using a micropipette to each
vessel and briefly vortex. Proceed with section 4).
3) Hold the neutralization treatments for 10±1 at room temperature (21±3°C).
4) At the conclusion of the holding period, vortex each reaction and filter each mixture through a
separate, pre-wetted 0.2 |jm PES membrane filter.
5) Wash each reaction vessel with ~20 mL PBS and vortex; filter the wash through ihe same
filter membrane. Finish the filtering process by rinsing the inside of the funnel unil w ith ~20
mL PBS and filter the rinsing liquid through the same filter membrane
a. Initiate filtration as soon as possible (e.g., within 30 min).
6) Remove the membrane aseptically with sterile forceps and place ii carefully over the surface
of the recovery medium. Avoid trapping air bubbles belw een the filler and the agar surface.
7) Count and record CFUs daily, up to 72±4 h.
16
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483
484
Treatment
Add 50 |iL of test
substance to each
vessel. At timed
intervals add 10 mL
neutralizer and swirl by
hand.
Treatment
Add 10 of Test
Suspension B to each
vessel containing 10 mL
neutralizer.
Neutralizer
Effectiveness
Neutralizer Toxicity
Control
Figure 2: Neutralization Schematic
Add 10 |iL of Test
^ v Suspension B to c. icli
Wait 10 s vessel containing 5< > u I.
test substance and In
50 mL test substance mL ncutrali/cr
Vortex and hold for
, 10 min at 21±3°C.
Proceed to
vortexing/filtering.
Vortex and hold fur
10 minat2lJ ' ( '.
I'roceed lo
\ oi'Icmiiu lilk'iiim
Treatment 3
Add 10 |iL of Test
Suspension B to each
vessel containing 10
mL PBS.
Titer Control
Vortex and hold for
10 min at 21±3°C.
Proceed lo
vo rlc \ i ng/filtering.
Treatment 4
Add 1 sterile carrier to
each vessel. At timed
intervals, add 10 mL
neutralizer and swirl by
hand.
V
Tiler Interference
Control
Wait 10 s
Add 10 ^L of Test
Suspension B to each
vessel containing the
carrier and 10 mL
neutralizer.
Vortex and hold for
. 10 minat21±3°C.
Proceed to
vortexing/filtering.
17
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