ASTM E3363-23

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3363 − 23
Standard Test Method for
Quantitative Performance Evaluation of Antimicrobial
Towelettes
This standard is issued under the fixed designation E3363; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.8 This international standard was developed in accor-
dance with internationally recognized principles on standard-
1.1 This test method quantitatively determines the effective-
ization established in the Decision on Principles for the
ness of various sizes of antimicrobial towelettes in treating
Development of International Standards, Guides and Recom-
hard, non-porous surfaces against Pseudomonas aeruginosa
mendations issued by the World Trade Organization Technical
and Staphylococcus aureus.
Barriers to Trade (TBT) Committee.
1.2 This test method may be used to evaluate towelettes for
antimicrobial efficacy against additional microorganisms (with 2. Referenced Documents
necessary modifications). 2
2.1 ASTM Standards:
1.2.1 This test method does not differentiate between chemi-
D5465 Practices for Determining Microbial Colony Counts
cal inactivation of the test microbe and mechanical removal of
from Waters Analyzed by Plating Methods
inoculum from a surface; rather, product efficacy is considered
E1054 Practices for Evaluation of Inactivators of Antimicro-
a combination of both attributes of a towelette-based formula-
bial Agents
tion.
E2362 Practice for Evaluation of Pre-saturated or Impreg-
1.3 This test method involves the use of hazardous
nated Towelettes for Hard Surface Disinfection
materials, chemicals, and infectious microorganisms and there- E2756 Terminology Relating to Antimicrobial and Antiviral
fore should be performed only by those trained in microbio-
Agents
logical techniques in facilities designed and equipped for work 2.2 AOAC Standards:
with infectious agents at the appropriate biosafety level, a
Official Method 955.15 Use-Dilution Method for Testing
BSL-2 or higher laboratory; specifications provided in the Disinfectants against Staphylococcus aureus. Revised
“Biosafety for Biomedical and Microbiological Laboratories”
(BMBL), 6th edition (BMBL).
Official Method 964.02 Use-Dilution Method for Testing
Disinfectants against Pseudomonas aeruginosa. Revised
1.4 It is the responsibility of the investigator to determine
whether Good Laboratory Practices (GLP Standards—For
2.3 Centers for Disease Control:
example, 40 CFR, Part 160 of FIFRA) are required and to
Biosafety in Microbiological and Biomedical Laborato-
follow them when appropriate.
ries 6th Edition, U.S. Department of Health and Human
1.5 Strict adherence to the protocol is necessary for the
Services, Public Health Service, Centers for Disease
validity of the test results.
Control and Prevention, National Institutes of Health,
1.6 The values stated in SI units are to be regarded as HHS Publication No. (CDC) 21- 1112, Revised June
standard. No other units of measurement are included in this
2020.
standard.
2.4 U.S. Government Regulations:
40 CFR Part 160 Federal Insecticide, Fungicide and Roden-
1.7 This standard does not purport to address all of the
ticide Act (FIFRA); Good Laboratory Practice Standards
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter-
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
mine the applicability of regulatory limitations prior to use.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
1 3
This test method is under the jurisdiction of ASTM Committee E35 on For referenced AOAC standards, visit the AOAC website,
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct www.aoacofficialmethod.org/index.
responsibility of Subcommittee E35.15 on Antimicrobial Agents. Available from https://www.cdc.gov/labs/BMBL.html.
Current edition approved Sept. 1, 2023. Published September 2023. DOI: Available from U.S. Government Publishing Office (GPO), 732 N. Capitol St.,
10.1520/E3363–23. NW, Washington, DC 20401, http://www.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3363 − 23
3. Terminology 4.4 One dried soiled carrier and one dried treated carrier are
together referred to as a 2-carrier set.
3.1 Definitions:
3.1.1 For definitions of terms used in this standard refer to
4.5 The dried carriers are exposed to the test substance by
Terminology E2756.
applying a pre-saturated towelette to the inner bottom surface
3.1.2 antimicrobial towelette (wipe), n—a piece of porous
of the carriers using a prescribed pattern of wiping, and the
material pre-saturated or impregnated with an antimicrobial
treated carrier is allowed to stand for a specific contact time. At
liquid that is meant for decontamination of environmental
the end of the contact time, two aliquots of an appropriate
surfaces by wiping.
neutralizer are added to the treated carrier, one prior to the
3.1.3 carriers, n—sterile plastic (for example, polystyrene) initial scraping of the carrier surface and the second aliquot
prior to the secondary scraping. Both done with a sterile cell
Petri plates (150 mm by 15 mm).
scraper at each instance to dislodge and suspend inoculum
3.1.4 colony forming unit (CFU), n—in microbiology, a
remaining on the surface. The inoculum-neutralizer suspension
visible mass of cells (algae, bacteria, or fungi) originating from
is collected, diluted, and enumerated using membrane filtration
either an individual cell or cluster of cells that have been placed
and plated onto recovery media (Tryptic Soy Agar).
onto or dispersed into a solid or semi-solid nutrient medium
and subsequently incubated under prescribed conditions.
4.6 Control and treated carrier CFUs are enumerated using
3.1.5 diluted test suspension, n—microbial suspension with membrane filtration (Practice D5465).
no soil.
4.7 The mean log density (LD) recovered from treated
3.1.6 dilution blank, n—tubes of phosphate buffered saline
carriers is compared to the mean log density recovered from
(PBS), phosphate buffered dilution water (PBDW), or similar
the control carriers. This calculation is used to determine the
inert phosphate buffer solution.
efficacy of the product based on the mean log reduction (LR)
3.1.7 final test suspension, n—microbial suspension with the value.
3-part soil load (25 μL BSA stock, 35 μL yeast extract stock,
100 μL mucin stock, and 340 μL microbial test suspension). 5. Significance and Use
3.1.8 quality control (QC), n—the procedures, products or
5.1 The plastic Petri plate (carrier) provides a closed system
services that meet a laboratory’s specified standards of quality.
for enumeration and easy application of a pre-saturated or
3.1.9 soiled carrier, n—Petri plates (see 3.1.3) spotted with impregnated antimicrobial towelette by an analyst.
sterile 5 % non-heat inactivated fetal bovine serum.
5.2 Aliquoting of sterile 5 % non-heat-inactivated fetal
3.1.10 soil suspension, n—3-part soil load, comprised of
bovine serum (five 10 μL spots) onto soiled carriers and
25 μL BSA stock, 35 μL yeast extract stock, 100 μL mucin
inoculation of final test suspension onto treated carriers (five
stock.
10 μL spots) is conducted using a template and a positive
displacement pipette, thereby ensuring a precise inoculum
NOTE 1—Fetal bovine serum or other animal serum as desired may be
added to the bacterial suspension to achieve the desired level of soil level and uniform distribution of soil and final test suspension.
depending on the target regulatory agency and claim desired.
5.3 A single towelette is tested per 2-carrier set, eliminating
3.1.11 treated carrier, n—Petri plates (see 3.1.3) inoculated
the likelihood of cross contamination between carriers.
with final test suspension.
5.4 The corkscrew-patterned circular motion of the product
3.1.12 two carrier (2-carrier) set, n—one dried soiled car-
application (wipe outside to inside, wipe inside to outside using
rier and one dried treated carrier.
the wiping template; see Annex A3 – Annex A6) ensures
4. Summary of Test Method uniform coverage and contact of disinfectant with the inocu-
lated surface.
4.1 This test method provides detailed instructions for
performing a quantitative evaluation of antimicrobial efficacy
5.5 The addition of neutralizer to the treated carriers at the
of a towelette when challenged against Pseudomonas aerugi-
end of the contact time results in neutralization of the test
nosa and Staphylococcus aureus. The test method may be
substance. This standard test method provides a procedure for
evaluated with additional microorganisms, though modifica-
performing neutralization verification to confirm that the
tion might be necessary to accommodate recovery of surviving
microbicidal, microbistatic, or both types of activity of a test
organism.
substance has been reduced by 50 % at the end of the contact
time (see Annex A1 for neutralization verification procedure).
4.2 Petri plates, spotted with a suspension of sterile 5 %
non-heat-inactivated fetal bovine serum, are used as the soiled
5.6 The design of this standard test method minimizes any
carriers. Each soiled carrier is spotted with five spots (10 μL
loss of viable organisms through carrier wash-off.
each) of sterile 5 % non-heat-inactivated fetal bovine serum
5.7 It is optional to adjust (dilution in PBS) the inoculum to
and allowed to dry.
achieve desired control counts of 5.0 log CFU/carrier to
4.3 Petri plates, inoculated with a suspension of vegetative
6.5 log CFU/carrier.
bacteria and 3-part soil, are used as the treated carriers. Each
treated carrier is inoculated with five spots (10 μL each) of the 5.8 Include, where applicable, comparisons of the test to
final test suspension and allowed to dry. other similar procedures such as Practices E1054 and E2362.
E3363 − 23
NOTE 2—All percentages prescribed in this Test Method are volume
6. Apparatus
fraction %.
6.1 Autoclave (Steam Sterilizer)—To sterilize media and
7.3.1 10 % Dextrose Solution—For use in rehydrating
reagents.
lyophilized/frozen vegetative culture of test microorganism.
6.2 Centrifuge (with Rotor Capable of Achieving 5 000
Add 5.0 g dextrose to 50 mL de-ionized water and mix by
g)—For test culture preparation.
stirring. Filter sterilize the solution using a 0.2 μm filter. Store
the sterile solution at 5 °C 6 3 °C for up to 30 days.
6.3 Freezer—To maintain appropriate temperature of 5 %
7.3.1.1 Prior to inoculation, on the day inoculated, use a
non-heat-inactivated FBS and 3-part soil reagents.
calibrated pipette to aseptically add 100 μL of 10 % sterile
6.4 Identification System (Optional)—For test microbes to
dextrose (w/v) solution to each 10 mL tube of SB (see 10.2).
appropriately identify bacteria.
7.3.2 Synthetic Broth (SB)—For use in rehydrating
6.5 Micropipette—100 μL, calibrated.
lyophilized/frozen vegetative culture of test microorganism.
Commercial media (HIMEDIA , Synthetic Broth, AOAC,
6.6 Non-Humidified Incubator—Capable of maintaining
#M334-500G). Store prepared SB at 2 °C to 8 °C.
temperature(s) at 36 °C 6 1 °C.
7.3.2.1 Alternatively, SB made in-house per the recipe
6.7 Petri Plates—Pre-sterilized, non-coated plastic (for
provided in Annex A14 and AOAC Methods 955.15 and
example, polystyrene) Petri plates used as soiled and treated
964.02 may be substituted.
carriers (150 mm by 15 mm).
7.3.3 Trypticase Soy Agar (TSA)—For use as a recovery
6.8 Positive Displacement Pipette/ Repeater Pipette—
medium for bacterial enumeration and purity checks. Prepare
Calibrated, used to dispense 10 μL aliquots of a designated TSA according to manufacturer’s instructions. Equivalent
suspension onto a soiled or treated carrier (see Annex A8).
commercially prepared agar culture medium may be pur-
chased. TSA with 5 % sheep’s blood (BAP) may be substituted.
6.9 Refrigerator—To maintain appropriate temperature of
7.3.4 Trypticase Soy Agar with 5 % Sheep’s Blood (BAP)—
media, reagents, and test suspension.
For performing streak isolation of microbial cultures as a purity
6.10 Sterile Cell Scraper—To scrape Petri plates for re-
check (quality control purposes); may be used as a substitute
moval of bacteria during neutralizing (for example, scraper
for TSA.
blade dimensions = 1.8 cm to 3.0 cm) (see Annex A7).
7.3.5 Trypticase Soy Broth (TSB), (30 g/L)—For use in
rehydrating lyophilized/frozen vegetative culture of test micro-
6.11 Sterile Forceps—To handle membrane filters. Straight
or curved with smooth flat tips. organism. Prepare TSB according to manufacturer’s instruc-
tions.
6.12 Sterile Glass Test Tubes—Reusable or disposable boro-
7.3.6 AOAC Nutrient Broth (NB)—For use in preparation of
silicate glass 20 mm by 150 mm with Morton closures for
Nutrient Agar (NA), Commercial media (HIMEDIA, Nutrient
dilution blanks and cultures/subcultures or other appropriate
Broth, AOAC, #M1680). Store prepared NA at 2 °C to 8 °C.
size.
7.3.7 Nutrient Agar (NA)—For use in propagation. Purchase
6.13 Serological Pipettes—Sterile single-use pipettes (for
plates from a reputable source or prepare as follows: Dissolve
example, (25.0, 10.0, 5.0, 2.0, 1.0) mL capacity).
1.5 % Bacto Agar (Difco ) in AOAC Nutrient Broth; final pH
should be 7.3 6 0.1. Steam sterilize at 121 °C for 20 min. See
6.14 Sterile Polyethersulfone Membrane (PES) Filters—To
Note 3.
filter serial dilutions for cell enumeration (0.2 μm pore size).
Filtration units (utilizing 0.2 μm pore size PES membrane
NOTE 3—Commercially dehydrated media that conform to the recipes
filters), reusable or disposable, may be used.
may be substituted.
7.4 Reagents:
6.15 Test Tube Racks—Any convenient size.
7.4.1 Cryoprotectant Solution—TSB with 15 % v/v glyc-
6.16 Timer—Any certified timer that can display time in
erol. Sterile solution is used in the preparation of frozen stock
seconds.
cultures.
6.17 Vacuum Source—For filtering test solutions. In-house
7.4.2 Organic Soil—Organic burden used for both soiled
line or vacuum pump.
carriers (that is, non-heat inactivated fetal bovine serum) and
for creating final test suspension used for treated carriers (that
6.18 Vortex-Style Mixer—For vortex-mixing of various so-
is, 3-part soil (BSA, Yeast Extract, Mucin)).
lutions.
7.4.2.1 Bovine Serum Albumin (BSA)—Required for prepa-
ration of final test suspension. Add 0.5 g BSA to 10 mL of PBS,
7. Reagents and Materials
mix and pass through a 0.2 μm pore diameter membrane filter,
7.1 Conical Tubes—Sterile, 15 mL and 50 mL. To collect
aliquot, and store frozen at –20 °C 6 2 °C for up to one year.
neutralizer/product/bacterial suspensions from treated carriers
Aliquots are single use only; do not refreeze once thawed.
and neutralizer/bacterial suspensions from control carriers after
7.4.2.2 Yeast Extract—Required for preparation of final test
inoculum has been dislodged by scraping.
suspension. Add 0.5 g yeast extract to 10 mL of PBS, mix, and
7.2 Cryovial—To store frozen stock cultures (for example,
1.5 mL capacity).
HIMEDIA is a registered trademark of HiMedia Technology Limitied.
7.3 Culture Media: DIFCO is a registered trademark of Becton, Dickinson and Company.
E3363 − 23
pass through a 0.2 μm pore diameter membrane filter, aliquot, TSB (30 g/L), aseptically withdraw 0.5 mL to 1.0 mL and
and store frozen at –20 °C 6 2 °C for up to one year. Aliquots rehydrate the lyophilized culture.
are single use only; do not refreeze once thawed. 9.1.3 Aseptically transfer the entire rehydrated pellet back
7.4.2.3 Mucin—CAS# 84195-52-8; Required for prepara- into the original tube of broth. Mix thoroughly by vortexing.
tion of final test suspension. Add 0.04 g mucin (from bovine Incubate broth culture at 36 °C 6 2 °C for 24 h 6 2 h.
submaxillary gland or equivalent) to 10 mL of PBS, mix 9.1.4 After incubation, streak a loopful of the suspension on
thoroughly until dissolved (see Note 4), and pass through a
TSA to obtain isolated colonies. Incubate the plate for 24 h 6
0.2 μm pore diameter membrane filter (do not steam sterilize), 2 h at 36 °C 6 2 °C.
aliquot, and store frozen at –20 °C 6 2 °C for up to one year.
NOTE 5—Perform a streak isolation of the broth culture onto TSA with
Aliquots are single use only; do not refreeze once thawed.
5 % sheep’s blood (BAP) as a purity check.
NOTE 4—Mucin may require vigorous stirring or vortex-mixing to fully
9.1.5 Select three to five isolated colonies (from the TSA
dissolve.
plate, 9.1.4) of the test organism and re-suspend into 1 mL of
7.4.3 Deionized Water—Purified water with mineral ions
TSB (30 g/L). For S. aureus, select only golden yellow
removed through pre-treatment, deionization, and filters.
colonies. Multiple phenotypes are present for P. aeruginosa—
Alternatively, reagent grade water (ultrapure water) may be
the stock culture should be representative of all phenotypes
used.
present on the streak isolation plate (select all phenotypes when
7.4.4 Neutralizer Medium—To use as chemical neutralizer
resuspending). Spread plate 0.1 mL of the suspension on each
based on active ingredients (for example, letheen broth, letheen
of 6 to 10 TSA plates. Incubate the plates for 24 h 6 2 h at
broth with 0.1 % sodium thiosulfate).
36 °C 6 2 °C.
7.4.4.1 Non-Heat-Inactivated Fetal Bovine Serum (FBS)—
9.1.5.1 If necessary, to obtain more frozen stock cultures, a
(5 % (v/v) FBS diluted in PBS) to be used for soiled carriers
larger suspension (for example, 2 mL) may be prepared using
(not inoculated with the test microbe).
the same ratio of TSB (1 mL) to number of colonies (3 colonies
7.4.5 Phosphate-Buffered Saline Stock Solution (PBS-SS)
to 5 colonies).
(Optional)—Prepare 10× stock solution of PBS by dissolving
9.1.6 Following the incubation of the agar plates from 9.1.5,
492 g PBS powder in 5 L of deionized water.
place approximately 5 mL sterile cryoprotectant solution (TSB
7.4.6 Phosphate-Buffered Saline (PBS) 1× Solution—
with 15 % glycerol) on the surface of each plate.
Purchase from a reputable supplier or prepare as follows:
9.1.7 Re-suspend the growth in the cryoprotectant solution
Dilute 9+1 [1-part (PBS-SS) 10X solution) plus 9 parts
using a sterile spreader without damaging the agar surface.
deionized water] to obtain 1× solution, distribute into bottles
9.1.8 Aspirate the suspension from the plate with a pipette
and steam sterilize for 20 min at 121 °C.
and place it in a sterile vessel large enough to hold approxi-
7.4.7 Ethanol—volume fraction = 70 %. To spray gloves
mately 30 mL. Repeat the growth harvesting procedure with
and prepare external surface of towelette container/canister/
the remaining plates and continue adding the suspension to the
flatpack for use.
vessel (more than 1 vessel may be used if necessary). Mix the
contents of the vessel(s) thoroughly; if more than 1 vessel is
8. Test Organisms
used, pool the vessels prior to aliquoting culture.
8.1 Pseudomonas Aeruginosa—ATCC 15442. The organism
9.1.9 Immediately after mixing, dispense aliquots (0.5 mL
is a Gram-negative, rod-shaped bacterium, that produces flat,
to 1.0 mL) of the harvested suspension into cryovials; these
opaque to off-white, round, spreading colonies within 24 h at
represent the frozen stock cultures. Within 60 min after
36 °C 6 1 °C when plated onto general growth media (for
harvesting, store the cryovials at ≤–70 ºC for a maximum of 18
example, TSA).
months.
8.2 Staphylococcus Aureus—ATCC 6538. The organism is a
10. Test Organism Preparation
Gram-positive, coccus-shaped bacterium, that produces small,
circular, yellow, glistening colonies within 24 h at 36 °C 6
10.1 Defrost a single cryovial at room temperature (for
1 °C when plated onto general growth media (for example,
example, 21 °C 6 4 °C) and briefly vortex to mix. Defrosting
TSA).
should be rapid to avoid loss in the viability of the preserved
cells (for example, expose to running room temperature water
9. Generation of Frozen Stock Cultures
to thaw). Each cryovial is for single use only.
9.1 Frozen stock cultures are single use only and should be
10.2 Prior to inoculation, on the day inoculated, use a
approximately 10 CFU/mL.
calibrated pipette to aseptically add 100 μL of 10 % sterile
9.1.1 Prepare new frozen stock cultures from lyophilized
dextrose (w/v) solution to each 10 mL tube of SB.
cultures of P. aeruginosa (ATCC 15442) and S. aureus (ATCC
10.2.1 Using a calibrated micropipette (6.5), add 100 μL of
6538) at least every 18 months.
defrosted stock culture to 10 mL SB with 100 μL 10 % (v/v)
9.1.1.1 New frozen stock culture may be initiated one time
dextrose solution (see 7.3.1). Briefly vortex-mix and incubate
using an existing, unexpired frozen stock culture as the source.
for 24 h 6 2 h at 36 °C 6 1 °C.
Begin process at step 9.1.4 below, by streaking a loopful of the
frozen stock culture onto 2 TSA plates. 10.3 In addition, inoculate an agar plate (for example, TSA
9.1.2 Open ampule of freeze-dried organism per manufac- or BAP) from the inoculated tube and streak for isolation.
turer’s instructions. Using a tube containing 5 mL to 6 mL of Incubate plate with the test culture.
E3363 − 23
10.4 Following incubation, use the broth culture to prepare 11. Carrier Inoculation
a test suspension for the organism.
11.1 Use sterile plastic Petri plates (150 mm by 20 mm) for
the soiled and treated carriers. See Annex A13 for pictorial
10.5 For P. aeruginosa, inspect culture prior to harvest;
representation of test assay.
discard if no visible pellicle has formed or if pellicle has been
disrupted (fragments in culture). Do not vortex-mix or shake
11.2 Soiled Carriers:
the 24 h 6 2 h test culture. Remove visible pellicle on surface
11.2.1 Use sterile 5 % non-heat-inactivated FBS as the
of medium and around associated interior edges of the tube
organic soil.
with vacuum suction.
11.2.2 Prepare soiled carriers at room temperature (21 °C 6
4 °C).
10.5.1 Using a serological pipette, withdraw the remaining
broth culture (approximately 7 mL to 8 mL) avoiding any 11.2.3 Using the inoculation template placed beneath the
sediment on the bottom of the tube and transfer it into a 15 mL Petri plates to standardize the location of the soil placement
conical centrifuge tube. sites (see Annex A2), apply five 10 μL spots of 5 % FBS to the
inside bottom surface of each Petri plate using a positive
10.5.1.1 If necessary, the culture may be harvested from two
displacement/ repeater pipette (6.8).
10 mL 24 h 6 2 h broth cultures to harvest and centrifuge a
11.2.4 Inoculate one soiled carrier for each treated carrier
maximum of 10 mL of P. aeruginosa culture.
(2-carrier set). Prepare extra soiled carriers, as necessary.
10.6 For S. aureus, briefly vortex-mix the 24 h 6 2 h culture
11.2.5 Streak inoculate an agar plate of non-selective me-
and transfer the entire contents to a 15 mL conical centrifuge
dium (for example, TSA or BAP) with a loopful of remaining
tube.
5 % FBS. Incubate plate with the treated and control carrier
10.7 Centrifuge the 24 h 6 2 h harvested broth cultures at plates and examine for purity after incubation at 36 °C 6 1 °C
5,000 g for 20 min. for 72 h 6 4 h.
11.3 Treated Carriers:
10.8 Remove the supernatant without disrupting the pellet.
11.3.1 Use final test suspension as prepared in Section 10
Resuspend the pellet in 5 mL to 10 mL PBS to target control
within 30 min of preparation at room temperature (21 °C 6
counts that are 5.0 CFU/carrier to 6.5 log CFU/carrier. The
4 °C) or within 2 h of initial preparation if refrigerated at 5 °C
resuspended pellet is the diluted test suspension.
10.10.1.2 and 10.10.2).
6 3 °C (
10.8.1 If necessary, disrupt the pellet using vortex-mixing
11.3.2 Inoculate treated carriers at room temperature (21 °C
or repetitive tapping/striking against a hard surface to disag-
6 4 °C).
gregate the pellet completely prior to re-suspending it in 5 mL
11.3.3 Direct plating on TSA may be employed for micro-
to 10 mL PBS. If necessary, add a small portion of the total
bial enumeration of test culture.
volume (5 mL to 10 mL) of PBS to the pellet to aid in the
11.3.4 Using the inoculation template placed beneath the
disaggregation.
Petri plates to standardize the location of the inoculation sites
10.8.2 Use this diluted test suspension within 30 min to
(see Annex A2), using the final test suspension, inoculate the
prepare the final test suspension.
inside bottom surface of each Petri plate with five 10 μL spots
10.8.3 If desired, determine titer of the diluted test suspen-
using a positive displacement pipette (6.8).
sion and the corresponding Optical Density at 650 nm or
11.3.5 Inoculate enough plates with the final test suspension
another appropriate wavelength.
for at least 3 controls (per challenge organism) and a minimum
10.9 Vortex-mix the diluted test suspension for 10 s to 30 s. of 5 treated carriers per test condition. Prepare extra inoculated
treated carriers, as necessary.
10.10 To obtain the final test suspension with the 3-part soil
11.4 Dry soiled and inoculated treated carriers in a non-
load, vortex-mix each component and combine in the follow-
humidified incubator with lids ajar, as shown in Annex A9, at
ing order using a calibrated micropipette: 25 μL BSA stock,
36 °C 6 1 °C for 30 min to 45 min until soil and inoculation
35 μL yeast extract stock, 100 μL mucin stock. This is the soil
spots appear visibly dry. Visually inspect each carrier to ensure
suspension.
complete drying.
10.10.1 Vortex-mix the soil suspension for 10 s prior to
11.4.1 Do not use carriers in which soil or inoculation spots
adding the diluted test suspension.
have coalesced.
10.10.1.1 Vortex-mix the diluted test suspension for 10 s.
11.4.2 Assay (wipe) and neutralize soiled and inoculated
Combine 340 μL diluted test suspension to the soil suspension.
treated carriers for product testing within 60 min after drying.
This is the final test suspension.
11.4.2.1 If holding time (60 min) for carriers is exceeded,
10.10.1.2 Use this final test suspension to inoculate carriers
additional carrier inoculations may be conducted within the
within 30 min of preparation.
same day using the same sterile 5 % non-heat inactivated FBS
10.10.2 If necessary, the final test suspension can be refrig-
and final test suspension (if previously refrigerated at 2 °C to
erated for up to 2 h (at 2 °C to 8 °C) after initial preparation.
8 °C and used within 2 h of initial preparation of final test
10.10.3 Streak inoculate an agar plate of non-selective
suspension (10.10.2)).
medium (for example, TSA or BAP) with a loopful of the final
12. Carrier Load Enumeration (Control Carrier Counts)
test suspension. Incubate plate with the treated and control
carrier plates and examine for purity after incubation at 36 °C 12.1 One control carrier is evaluated immediately prior to
6 1 °C for 72 h 6 4 h. commencing the test and two control carriers are evaluated
E3363 − 23
immediately following the test to assess inoculated treated 12.9 Dilutions resulting in up to 200 CFUs per filter are
carrier population (carrier counts). All carriers must be pro- deemed acceptable for counting purposes; all counts up to 200
cessed (assayed and neutralized) within 60 min after drying to are used in the calculations (refer to Practice D5465). CFUs
ensure an appropriate microbial challenge at the time of testing greater than 200 are indicated as Too Numerous to Count
(see 11.4.2.1). (TNTC).
12.1.1 The two control carriers evaluated immediately fol-
12.10 Calculate the viable CFU/carrier. Account for all
lowing the test should be processed after neutralization of all
dilutions that result in up to 200 CFU and
...