ASTM E2362-22

Standard Practice for Evaluation of Pre-saturated or Impregnated Towelettes for Hard Surface Disinfection

SIGNIFICANCE AND USE
5.1 This practice may be used to determine if a pre-saturated or impregnated towelette demonstrates antimicrobial effectiveness as a disinfectant on hard surfaces. This practice provides survivor results in the form of a qualitative endpoint (growth positive versus growth negative). The results generated by following this practice do not provide for specific quantitative reductions.
SCOPE
1.1 This practice is designed to evaluate the antimicrobial activity of pre-saturated or impregnated towelettes when used as a hard surface disinfectant.  
1.2 It is the responsibility of the investigator to determine whether Good Laboratory Practices (GLP’s) are required and to follow them when appropriate.  
1.3 This practice should be performed only by those trained in microbiological techniques.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 Appropriate modifications to the practice may be required when testing organisms not specified herein.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 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.

General Information

• Status: Published
• Publication Date: 30-Sep-2022
• ICS: 71.100.35 - Chemicals for industrial and domestic disinfection purposes
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Refers: ASTM E2756-19 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Nov-2019
• Refers: ASTM E2756-18 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Apr-2018
• Refers: ASTM E2756-10 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-May-2010
• Refers: ASTM E1054-08 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 01-Apr-2008
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM E1054-02 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 10-May-2002
• Refers: ASTM D1193-99e1 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999
• Refers: ASTM D1193-99 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999

Overview

ASTM E2362-22: Standard Practice for Evaluation of Pre-saturated or Impregnated Towelettes for Hard Surface Disinfection provides an internationally recognized method to assess the antimicrobial effectiveness of disinfectant towelettes on hard, nonporous surfaces. Developed by ASTM International, this standard enables laboratories and manufacturers to determine whether pre-saturated or impregnated towelettes are effective as disinfectants using a qualitative endpoint. It is widely used for regulatory compliance, product development, and quality control within industries relying on hard surface sanitation.

Key Topics

• Antimicrobial Effectiveness: The standard outlines a practice to determine whether a disinfectant towelette can eliminate bacteria, mycobacteria, and fungi from hard surfaces. Results are provided as growth positive or growth negative, rather than quantitative reductions.
• Test Microorganisms: Specifies relevant test organisms such as Staphylococcus aureus, Salmonella enterica, Pseudomonas aeruginosa, Mycobacterium bovis, and Trichophyton mentagrophytes, with provisions for adapting the method for additional organisms.
• Laboratory Practice and Expertise: Emphasizes the need for qualified personnel trained in microbiological techniques, and for adherence to Good Laboratory Practices (GLP) where required.
• Safety and Compliance: Users must establish proper safety, health, and environmental practices, including adherence to local regulatory limitations.
• Materials, Equipment, and Reagents: Detailed specification of required laboratory apparatus, culture media, and test protocols to ensure reproducibility and accuracy.
• Qualitative Endpoint: The main outcome is a pass/fail assessment based on the presence or absence of microbial growth after treatment, supporting efficacy claims for disinfectant towelettes.

Applications

• Product Efficacy Validation: Manufacturers of disinfectant wipes use ASTM E2362-22 to validate antimicrobial claims for bleach wipes, alcohol wipes, and other pre-saturated towelettes intended for healthcare, laboratory, and consumer use.
• Regulatory Submissions: Results derived from this practice support regulatory registration and compliance for disinfectant products in various jurisdictions, including the United States Environmental Protection Agency (EPA) requirements.
• Quality Control: Routine application of this standard ensures consistent effectiveness of each product batch, aiding in the detection of manufacturing or formulation issues.
• Research and Development: R&D laboratories employ the methodology during the formulation and improvement of new disinfectant towelette products.
• Healthcare and Institutional Procurement: Purchasing departments use compliance with ASTM E2362-22 as a criterion when selecting hard surface cleaning products, particularly in environments where pathogen control is critical.

Related Standards

• ASTM D1193 - Specification for Reagent Water
• ASTM E1054 - Practices for Evaluation of Inactivators of Antimicrobial Agents
• ASTM E2756 - Terminology Relating to Antimicrobial and Antiviral Agents
• 40 CFR, Part 160 - Good Laboratory Practice Standards (US EPA)
• AOAC Official Method 965.12 - Tuberculocidal Activity of Disinfectants
• AOAC Official Method 961.02 - Germicidal Spray Products as Disinfectants

Practical Value

ASTM E2362-22 is an indispensable tool for companies producing or verifying disinfectant towelettes. It ensures standardized and reliable procedures for evaluating microbial efficacy, thereby safeguarding public health and supporting responsible product claims. By requiring clearly defined methodology, specified controls, and detailed reporting, the standard fosters transparency and trust in the effectiveness of hard surface disinfectant wipes across healthcare, institutional, and consumer markets.

Keywords: ASTM E2362-22, pre-saturated towelettes, disinfectant wipes, antimicrobial efficacy, hard surface disinfection, microbiological testing, quality control, regulatory compliance, healthcare sanitation

Standards Content (Sample)

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: E2362 − 22
Standard Practice for
Evaluation of Pre-saturated or Impregnated Towelettes for
Hard Surface Disinfection
This standard is issued under the fixed designation E2362; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope* E2756Terminology Relating toAntimicrobial andAntiviral
Agents
1.1 This practice is designed to evaluate the antimicrobial
2.2 Federal Standard
activity of pre-saturated or impregnated towelettes when used
as a hard surface disinfectant. 40 CFR, Part 160Good Laboratory Practice Standards
1.2 It is the responsibility of the investigator to determine
3. Terminology
whether Good Laboratory Practices (GLP’s) are required and
3.1 For definitions of terms used in this practice, refer to
to follow them when appropriate.
Terminology E2756.
1.3 This practice shouldbeperformedonlybythose trained
3.2 Definitions:
in microbiological techniques.
3.2.1 carrier, n—a transportable surface onto which a test
1.4 The values stated in SI units are to be regarded as
organism will be inoculated and dried.
standard. No other units of measurement are included in this
3.2.1.1 Discussion—The carrier will be treated with the test
standard.
substance and subcultured for survivors.
1.5 Appropriate modifications to the practice may be re-
3.2.2 CFU, n—colony forming units
quired when testing organisms not specified herein.
3.2.3 disinfectant, n—a physical or chemical agent or pro-
1.6 This standard does not purport to address all of the
cess that destroys pathogenic or potentially pathogenic micro-
safety concerns, if any, associated with its use. It is the
organisms in/on surfaces or objects.
responsibility of the user of this standard to establish appro-
3.2.4 impregnated, adj—saturated with test substance.
priate safety, health, and environmental practices and deter-
3.2.5 neutralizer, n—a component used to render an active
mine the applicability of regulatory limitations prior to use.
1.7 This international standard was developed in accor- agent incapable of destroying organisms by chemical or
dance with internationally recognized principles on standard- physical means.
ization established in the Decision on Principles for the
3.2.6 pre-saturated, adj—to be filled or impregnated with
Development of International Standards, Guides and Recom-
test substance prior to the time of its intended use.
mendations issued by the World Trade Organization Technical
3.2.7 towelette, n—A paper, cloth or non-woven blend
Barriers to Trade (TBT) Committee.
materialusedasatransporterforacleaningand/ordisinfection
agent.
2. Referenced Documents
2.1 ASTM Standards:
4. Summary of Practice
D1193Specification for Reagent Water
4.1 A towelette impregnated or pre-saturated with a test
E1054PracticesforEvaluationofInactivatorsofAntimicro-
substance is used to treat a carrier which has been inoculated
bial Agents
withatestorganismafteranaliquotofatestorganismhasbeen
inoculated, evenly distributed to an inoculation area of ap-
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides, proximately one square inch (approximately 625 mm), and
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Oct. 1, 2022 Published November 2022. Originally
approved in 2004. Last previous edition approved in 2015 as E2362–15. DOI: Available from the Superintendent of Documents, U.S. Government Printing
10.1520/E2362-22. Office, Washington D.C. 20402
2 4
For referenced ASTM standards, visit the ASTM website, www.astm.org, or UnitedStatesEnvironmentalProtectionAgency,StandardOperatingProcedure
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM for Disinfectant Towelette Test Against Staphylococcus aureus, Pseudomonas
Standards volume information, refer to the standard’s Document Summary page on aeruginosa, and Salmonella enterica, EPA/OPP Microbiology Laboratory, Ft.
the ASTM website. Meade, MD. SOP# MB09-05, Revised 1/30/13.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2362 − 22
dried onto the carrier. The carrier is wiped using the pre- 6.19 Pipette—sterile volumetric pipettes.
saturated or impregnated towelette simulating the application
6.20 Glass Jars—100 mL or other appropriate vessel.
ofthetestsubstanceandthenheldforapre-determinedcontact
6.21 Filter Paper—9 cm (Whatman No. 2, or equivalent)
time. After the specified contact time, the test substance
sterilized prior to use.
remaining on the carrier is neutralized and the carrier is
subcultured to recover surviving test organism. 6.22 Thermometer—calibrated thermometer.
6.23 Glass Beads—3 –5 mm sterile beads.
5. Significance and Use
6.24 Gauze—sterile cotton gauze.
5.1 Thispracticemaybeusedtodetermineifapre-saturated
6.25 Hemacytometer—calibrated hemacytometer.
or impregnated towelette demonstrates antimicrobial effective-
ness as a disinfectant on hard surfaces. This practice provides 6.26 Glass Wool—sterile grease free glass wool.
survivor results in the form of a qualitative endpoint (growth
6.27 Hot air oven—ability to maintain ≥180°C.
positive versus growth negative). The results generated by
6.28 Refrigerator—calibrated to maintain 5°C 6 3°C.
following this practice do not provide for specific quantitative
reductions.
6.29 Ultra-Cold Freezer, Calibrated to maintain ≤ -70°C
6.30 Glass Tissue Grinder or Macerator, sterile.
6. Apparatus
6.31 Sterile cryovials,(forexample,1.5mLwithscrewcap)
6.1 Incubator—any calibrated incubator that maintains a
6.32 Centrifuge, calibrated.
temperature specific for propagation of organisms. (for
example,bacteriaandmycobacteriaat36°C 61°Candfungi
7. Reagents
at 27.5°C 62.5°C).
7.1 Culture Media—Bacteria
6.2 Sterilizer—any suitable, calibrated steam sterilizer that
7.1.1 Nutrient Broth or Synthetic Broth—Pseudomonas
produces the conditions of sterilization is acceptable.
aeruginosa,
6.3 Test Towelettes—with instructions for use.
7.1.2 Cystine Trypticase Agar—Pseudomonas aeruginosa,
7.1.3 Synthetic Broth—Salmonella enterica and Staphylo-
6.4 Timer (Stop-clock)—acalibratedtimerthatdisplaysmin
coccus aureus.
and s.
7.1.4 Fluid Thioglycollate Broth.
6.5 Spectrophotometer—calibrated to 650 nm.
7.1.5 Tryptic Soy Broth (TSB)
6.6 Mixer—a vortex mixer is recommended. 7.1.6 Tryptic Soy Broth with 15% v/v glycerol (Cyropro-
tectant solution)
6.7 pH meter—acalibratedpHmetertodeterminethepHof
media. 7.2 Culture Media—Mycobacteria
7.2.1 Middlebrook 7H11 or 7H9 Agar Slants.
6.8 Nonporous Test Carriers—borosilicate glass slides, 25
7.2.2 Modified Proskauer-Beck Broth.
mm × 75 mm slides, pre-cleaned (or other hard surfaces and
sizes as appropriate). 7.3 Culture Media—Fungi
7.3.1 Sabouraud DextroseAgar plates/GlucoseAgar plates.
6.9 Glass Culture Tubes—20 mm × 150 mm, 25 mm × 150
7.3.2 Sabouraud Dextrose Agar slants/Glucose Agar slants.
mm, and 38 mm × 100 mm or 38 mm × 200 mm without lip,
7.4 Neutralizing Subculture Media—A neutralizing growth
or equivalent, sterile.
medium capable of supporting the growth of the test organism
6.10 Culture Tube Closures—appropriate size nontoxic clo-
following exposure to the test material in accordance with
sures.
PracticesE1054.ForMycobacterium,horseserum(whichmay
6.11 Petri Dishes—100 mm × 15 mm, glass and plastic,
besupplementedwithadditionalneutralizers)isrecommended.
sterile.
7.5 Subculture Agar
6.12 Balance—a calibrated balance sensitive to 0.1 g.
7.5.1 Tryptic Soy Agar with or without sheep blood—
Bacteria.
6.13 Micropipettor—calibrated for dispensing 10 µL.
7.5.2 Middlebrook 7H11 Agar—Mycobacteria.
6.14 Forceps—sterilizable or pre-sterilized.
7.5.3 Sabouraud Dextrose Agar or Glucose Agar—Fungi.
6.15 Sterilizer Apparatus—a bunsen burner or other appro-
7.6 Subculture Media—Mycobacteria
priate heat sterilizer.
7.6.1 Modified Proskauer-Beck Broth
6.16 Bacteriological Culture Loop— 4 mm inside diameter
7.6.2 Kirchner’s Medium
loop of platinum or platinum alloy wire or sterile disposable
7.6.3 Middlebrook 7H9 Broth or TB broth
plastic loops of appropriate size.
7.7 Other subculture agars, broths and neutralizers may be
6.17 Colony Counter—any one of several types may be
used where appropriate.
used, for example Quebec.
6.18 Gloves—sterile gloves not possessing antimicrobial
AOAC Official Method 965.12 Tubcerculocidal Activity of Disinfectants.
properties. AOAC International, Chapter 6.
E2362 − 22
7.8 Soil—Blood Serum, such as heat inactivated fetal bo- 9.1.1 Bacteria Inoculum Preparation—For S. aureus and S.
vine serum or other appropriate alternative soil. enterica, defrost a single cryovial at room temperature and
briefly vortex to mix.Add 10 µLof the thawed frozen stock to
7.9 Dilution Fluid—sterile phosphate buffered water
atubecontaining10mLsyntheticbrothandthenvortextomix.
(PBDW), sterile saline or Butterfield’s Buffer. (See Specifica-
Incubate at 36°C 6 1°C for 24h 6 2 h. Briefly vortex the 24
tion D1193.)
h culture prior to transfer. For this final subculture step,
7.10 Sterile saline + 0.05% v/v Triton X-100
inoculate a sufficient number of 20mm × 150 mm tubes
7.11 Sterile 0.1% v/v Polysorbate (Tween) 80 containing10mLsyntheticbrothwith10µLpertubeofthe24
h synthetic broth culture; incubate 48h to 54 h at 36°C 6
7.12 Carrier Preparation Solutions—70% to 95% isopro-
1°C. Using a Vortex-style mixer, mix synthetic broth test
pyl alcohol, deionized or distilled water.
cultures 3s to 4 s and let stand 10 min at room temperature
before continuing. Remove the upper portion of each culture,
8. Test Organisms
leaving behind any debris or clumps, and transfer to a sterile
8.1 Bacterial Test Organisms:
flask or tube; pool cultures in the flask and swirl to mix.
8.1.1 Staphylococcus aureus (ATCC 6538), Salmonella en-
Aliquot a sufficient volume of culture into a sterile test tube.
terica (ATCC 10708), and Pseudomonas aeruginosa (ATCC
9.1.1.1 For each bacterium, one daily transfer is required
15442)-received lyophilized.
prior to the inoculation of a final test culture. Daily cultures
8.1.2 Other bacterial organisms may be tested using appro-
may be subcultured for up to 5 d; each daily transfer may be
priate culture and subculture procedures.
usedtogenerateatestculture.Forthepurposeofachievingthe
8.2 Mycobacterial Test Organism:
carrier count range, final cultures may be adjusted by dilution
8.2.1 Mycobacterium bovis—(BCG) (Organon teknika or
ingrowthmediumorbyconcentrationusingcentrifugation(for
ATCC 35743)
example, 5000 g for 20 min) resuspending the pellet in the
8.2.2 Other mycobacterial strains may be tested using ap-
appropriate volume of sterile test culture medium.
propriate culture and subculture procedures.
9.1.2 For P. aeruginosa, defrost a single cryovial at room
8.3 Fungal Test Organisms:
temperatureandbrieflyvortextomix.Eachcryovialshouldbe
8.3.1 Trichophyton mentagrophytes (ATCC 9533)
singleuseonly.Add10µLofthethawedfrozenstocktoatube
8.3.2 Other fungi may be tested using appropriate culture
containing 10 mL broth (synthetic or nutrient broth) and then
and subculture procedures.
vortextomix.Incubateat36°C 61°Cfor24h 62h.Donot
vortex the 24 h culture prior to transfer. For this final
9. Preparation of Organism
subculture step, inoculate a sufficient number of 20 × 150 mm
9.1 Bacteria –Preparation of frozen stock cultures for S.
tubescontaining10mLbroth(syntheticornutrient)with10µL
enterica, S. aureus, and P. aeruginosa.—Using a tube contain-
per tube of the 24 h broth culture; incubate 48 to 54 h at 36°C
ing 5mL–6 mL TSB, aseptically withdraw 0.5mL to 1.0 mL
6 1°C. Do not shake 48h to 54 h test culture. The pellicle
and rehydrate the lyophilized culture. Aseptically transfer the
from the 48h to 54 h cultures must be removed from the broth
entire rehydrated pellet back into the original tube of broth.
either by decanting the liquid aseptically into a sterile tube, by
Mix well. Incubate for 24h 62hat36°C 6 1°C. Using a
gentlyaspiratingthebrothawayfromthepellicleusingapipet,
sterile spreader, inoculate a sufficient number of TSA plates
or by removal with a vacuum. Avoid harvesting pellicle from
(forexample,5to10platesperorganism)with100µLeachof
the bottom of the tube.
the culture. Incubate plates at 36°C 6 1°C for 24h 62h.
9.1.2.1 Any disruption of the pellicle resulting in dropping,
Following incubation, add 5 mL cryoprotectant solution (TSB
or breaking up of the pellicle in culture before or during its
with 15% v/v glycerol) to the surface of each agar plate.
removal renders that culture unusable in the test. This is
Resuspend the cells in this solution using a sterile spreader or
extremelycriticalbecauseanypelliclefragmentremainingwill
asterileswabandaspiratethecellsuspensionfromthesurface
result in uneven clumping and layering of organism, allowing
oftheagar.Transfersuspensionintoasterilevessel.Repeatby
for biased exposure to disinfectant and causing false-positive
adding another 5 mL cryoprotectant to the agar plates, resus-
results. Pool the test culture from each tube and visually
pendthecells,aspiratesuspensionandpoolwiththeinitialcell
inspect culture for pellicle fragments. Presence of pellicle in
suspension.Alternately, 10 mLcryoprotectant solution may be
thefinalculturemakesitunusablefortest.UsingaVortex-style
added per plate for resuspending with subsequent aspiration.
mixer, mix test cultures 3s to 4 s and let stand 10 min at room
Mix the pooled contents of the vessel thoroughly. Immediately
temperature before continuing. Remove the upper portion of
after mixing, pipet approximately 1.0 mL quantities of the
eachculture,leavingbehindanydebrisorclumps,andtransfer
diluted suspension into cryovials. Place and store cryovials in
to a sterile flask or tube; pool cultures from tubes in the flask
–70°C or below freezer; these are the frozen stock cultures.
and swirl to mix.Aliquot a sufficient volume of culture into a
Each cryovial is considered as single use only. Store stock
sterile test tube.
cultures up to 18 months. Reinitiate stocks using a new
9.1.2.2 Onedailytransferisrequiredpriortotheinoculation
lyophilized culture.
ofafinaltestculture.Dailyculturesmaybesubculturedforup
to 5 days; each daily transfer may be used to generate a test
culture. For the purpose of achieving the carrier count range,
AOAC Official Method 961.02 Germicidal Spray Products as Disinfectants.
AOAC International, Chapter 6. finalculturesmaybeadjustedbydilutioningrowthmediumor
E2362 − 22
by concentration using centrifugation (for example, 5000 g for ofserumtoatubeofappropriaterecoverybroth,(forexample,
20 min) resuspending the pellet in the appropriate volume of Fluid thioglycollate medium) and incubate with the test.
sterile test culture medium.
9.6 Organism Purity—Subculture each test organism to the
9.2 Mycobacteria—Maintain a stock culture of Mycobacte- appropriateagar,incubatewiththetest,andexamineforpurity.
rium organisms on Middlebrook 7H11 or 7H9 agar slants by
monthly transfer and incubation for 15 days to 20 days at
10. Procedure
36°C 61°C.Slantsmaybestoredat5°C 63°Cforuptosix
10.1 Preparation of Carriers:
weeks.
10.1.1 Test carriers should be submerged in 70% to 95%
9.2.1 Mycobacteria Inoculum Preparation—From stock
ethyl or isopropyl alcohol, then rinsed with deionized or
culture, inoculate Modified Proskauer-Beck (MPB) Broth
distilled water.
tubesandincubate21to2daysat36°C 61°C.Usingasterile
10.1.2 Place the test carriers into a large glass dish and
transfer loop, transfer culture to a sterile glass tissue grinder.
sterilize in a hot air oven for ≥2hat ≥180°C.
Add 1.0 mL of 0.1% polysorbate (Tween) 80. Grind to break
10.1.3 After sterilization, place each carrier horizontally
up large clumps or aggregates. Dilute the culture with 9 mLof
intoseparateglassorplasticPetridishescontaining2piecesof
Modified Proskauer-Beck Broth. Transfer the suspension to a
sterile filter paper.Transfer at a minimum the required number
sterile test tube and allow to settle for 10min to 15 min.
ofcarriersfortestingincludingaminimumof6carriersforthe
Remove the upper portion to a sterile tube or flask, leaving
populationcontrol,1to3carriersforeachviabilitycontrol,and
behind any debris or clumps. Pool cultures, as applicable, and
1 carrier for the carrier sterility control.
swirltomix.Dilutetheculturetoachieve20% 61%Tat650
nm using
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E2362 − 15 E2362 − 22
Standard Practice for
Evaluation of Pre-saturated or Impregnated Towelettes for
Hard Surface Disinfection
This standard is issued under the fixed designation E2362; 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 Scope*
1.1 This practice is designed to evaluate the antimicrobial activity of pre-saturated or impregnated towelettes when used as a hard
surface disinfectant.
1.2 It is the responsibility of the investigator to determine whether Good Laboratory Practices (GLP’s) are required and to follow
them when appropriate.
1.3 This practice should be performed only by those trained in microbiological techniques.
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 Appropriate modifications to the practice may be required when testing organisms not specified herein.
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and to determine the
applicability of regulatory limitations prior to use.
1.7 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.
2. Referenced Documents
2.1 ASTM Standards:
D1193 Specification for Reagent Water
E1054 Practices for Evaluation of Inactivators of Antimicrobial Agents
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 Federal Standard
40 CFR, Part 160 Good Laboratory Practice Standards
This practice is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Oct. 1, 2015Oct. 1, 2022 Published October 2015November 2022. Originally approved in 2004. Last previous edition approved in 20092015
as E2362 – 09.E2362 –15. DOI: 10.1520/E2362-15.10.1520/E2362-22.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Available from the Superintendent of Documents, U.S. Government Printing Office, Washington D.C. 20402
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2362 − 22
3. Terminology
3.1 carrier, n—a transportable surface onto which a test organism will be inoculated and dried. The carrier will be treated with
the test substance and subcultured for survivors.
3.1 For definitions of terms used in this practice, refer to Terminology E2756.
3.2 CFU, n—colony forming units
3.2 Definitions:
3.2.1 carrier, n—a transportable surface onto which a test organism will be inoculated and dried.
3.2.1.1 Discussion—
The carrier will be treated with the test substance and subcultured for survivors.
3.2.2 CFU, n—colony forming units
3.2.3 disinfectant, n—a physical or chemical agent or process that destroys pathogenic or potentially pathogenic microorganisms
in/on surfaces or objects.
3.2.4 impregnated, adj—saturated with test substance.
3.2.5 neutralizer, n—a component used to render an active agent incapable of destroying organisms by chemical or physical
means.
3.2.6 pre-saturated, adj—to be filled or impregnated with test substance prior to the time of its intended use.
3.2.7 towelette, n—A paper, cloth or non-woven blend material used as a transporter for a cleaning and/or disinfection agent.
3.3 disinfectant, n—a physical or chemical agent or process that destroys pathogenic or potentially pathogenic microorganisms
in/on surfaces or objects.
3.4 impregnated, adj—saturated with test substance.
3.5 neutralizer, n—a component used to render an active agent incapable of destroying organisms by chemical or physical means.
3.6 pre-saturated, adj—to be filled or impregnated with test substance prior to the time of its intended use.
3.7 towelette, n—A paper, cloth or non-woven blend material used as a transporter for a cleaning and/or disinfection agent.
4. Summary of Practice
4.1 A towelette impregnated or pre-saturated with a test substance is used to treat a carrier which has been inoculated with a test
organism after an aliquot of a test organism has been inoculated, evenly distributed to an inoculation area of approximately one
square inch (approximately 625 mm), and dried onto the carrier. The carrier is wiped using the pre-saturated or impregnated
towelette simulating the application of the test substance and then held for a pre-determined contact time. After the specified
contact time, the test substance remaining on the carrier is neutralized and the carrier is subcultured to recover surviving test
organism.
5. Significance and Use
5.1 This practice may be used to determine if a pre-saturated or impregnated towelette demonstrates antimicrobial effectiveness
United States Environmental Protection Agency, Standard Operating Procedure for Disinfectant Towelette Test Against Staphylococcus aureus, Pseudomonas aeruginosa,
and Salmonella enterica, EPA/OPP Microbiology Laboratory, Ft. Meade, MD. SOP# MB09-05, Revised 1/30/13.
E2362 − 22
as a disinfectant on hard surfaces. This practice provides survivor results in the form of a qualitative endpoint (growth positive
versus growth negative). The results generated by following this practice do not provide for specific quantitative reductions.
6. Apparatus
6.1 Incubator—any calibrated incubator that maintains a temperature specific for propagation of organisms. (for example, bacteria
and mycobacteria at 36 6 1 °C36 °C 6 1 °C and fungi at 27.5 6 2.5 °C).27.5 °C 6 2.5 °C).
6.2 Sterilizer—any suitable, calibrated steam sterilizer that produces the conditions of sterilization is acceptable.
6.3 Test Towelettes—with instructions for use.
6.4 Timer (Stop-clock)—a calibrated timer that displays min and s.
6.5 Spectrophotometer—calibrated to 650 nm.
6.6 Mixer—a vortex mixer is recommended.
6.7 pH meter—a calibrated pH meter to determine the pH of media.
6.8 Nonporous Test Carriers—borosilicate glass slides, 25 mm × 75 mm slides, pre-cleaned (or other hard surfaces and sizes as
appropriate).
6.9 Glass Culture Tubes—20 mm × 150 mm, 25 mm × 150 mm, and 38 mm × 100 mm or 38 mm × 200 mm without lip, or
equivalent, sterile.
6.10 Culture Tube Closures—appropriate size nontoxic closures.
6.11 Petri Dishes—100 mm × 15 mm, glass and plastic, sterile.
6.12 Balance—a calibrated balance sensitive to 0.1 g.
6.13 Micropipettor—calibrated for dispensing 10 μL.
6.14 Forceps—sterilizable or pre-sterilized.
6.15 Sterilizer Apparatus—a bunsen burner or other appropriate heat sterilizer.
6.16 Bacteriological Culture Loop— 4 mm inside diameter loop of platinum or platinum alloy wire or sterile disposable plastic
loops of appropriate size.
6.17 Colony Counter—any one of several types may be used, for example Quebec.
6.18 Gloves—sterile gloves not possessing antimicrobial properties.
6.19 Pipette—sterile volumetric pipettes.
6.20 Glass Jars—100 mL or other appropriate vessel.
6.21 Filter Paper—9 cm (Whatman No. 2, or equivalent) sterilized prior to use.
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6.22 Thermometer—calibrated thermometer.
6.23 Glass Beads—3 –5 mm sterile beads.
6.24 Gauze—sterile cotton gauze.
6.25 Hemacytometer—calibrated hemacytometer.
6.26 Glass Wool—sterile grease free glass wool.
6.27 Hot air oven—ability to maintain ≥180°C.
6.28 Refrigerator—calibrated to maintain 55 °C 6 3°C.
6.29 Ultra-Cold Freezer, Calibrated to maintain ≤ -70°C-70 °C
6.30 Glass Tissue Grinder or Macerator, sterile.
6.31 Sterile cryovials, (for example, 1.5 mL with screw cap)
6.32 Centrifuge, calibrated.
7. Reagents
7.1 Culture Media—Bacteria
7.1.1 Nutrient Broth or Synthetic Broth—Pseudomonas aeruginosa,
7.1.2 Cystine Trypticase Agar—Pseudomonas aeruginosa,
7.1.3 Synthetic Broth—Salmonella enterica and Staphylococcus aureus.
7.1.4 Fluid Thioglycollate Broth.
7.1.5 Tryptic Soy Broth (TSB)
7.1.6 Tryptic Soy Broth with 15% v/v glycerol (Cyroprotectant solution)
7.2 Culture Media—Mycobacteria
7.2.1 Middlebrook 7H11 or 7H9 Agar Slants.
7.2.2 Modified Proskauer-Beck Broth.
7.3 Culture Media—Fungi
7.3.1 Sabouraud Dextrose Agar plates/Glucose Agar plates.
7.3.2 Sabouraud Dextrose Agar slants/Glucose Agar slants.
7.4 Neutralizing Subculture Media—A neutralizing growth medium capable of supporting the growth of the test organism
following exposure to the test material in accordance with Practices E1054. For Mycobacterium, horse serum (which may be
supplemented with additional neutralizers) is recommended.
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7.5 Subculture Agar
7.5.1 Tryptic Soy Agar with or without sheep blood—Bacteria.
7.5.2 Middlebrook 7H11 Agar—Mycobacteria.
7.5.3 Sabouraud Dextrose Agar or Glucose Agar—Fungi.
7.6 Subculture Media—Mycobacteria
7.6.1 Modified Proskauer-Beck Broth
7.6.2 Kirchner’s Medium
7.6.3 Middlebrook 7H9 Broth or TB broth
7.7 Other subculture agars, broths and neutralizers may be used where appropriate.
7.8 Soil—Blood Serum, such as heat inactivated fetal bovine serum or other appropriate alternative soil.
7.9 Dilution Fluid—sterile phosphate buffered water (PBDW), sterile saline or Butterfield’s Buffer. (See Specification D1193.)
7.10 Sterile saline + 0.05%0.05 % v/v Triton X-100
7.11 Sterile 0.1%0.1 % v/v Polysorbate (Tween) 80
7.12 Carrier Preparation Solutions—7070 % to 95 % isopropyl alcohol, deionized or distilled water.
8. Test Organisms
8.1 Bacterial Test Organisms:
8.1.1 Staphylococcus aureus (ATCC 6538), Salmonella enterica (ATCC 10708), and Pseudomonas aeruginosa (ATCC
15442)-received lyophilized.
8.1.2 Other bacterial organisms may be tested using appropriate culture and subculture procedures.
8.2 Mycobacterial Test Organism:
8.2.1 Mycobacterium bovis—(BCG) (Organon teknika or ATCC 35743)
8.2.2 Other mycobacterial strains may be tested using appropriate culture and subculture procedures.
8.3 Fungal Test Organisms:
8.3.1 Trichophyton mentagrophytes (ATCC 9533)
8.3.2 Other fungi may be tested using appropriate culture and subculture procedures.
AOAC Official Method 965.12 Tubcerculocidal Activity of Disinfectants. AOAC International, Chapter 6.
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9. Preparation of Organism
9.1 Bacteria –Preparation of frozen stock cultures for S. enterica, S. aureus, and P. aeruginosa.—Using a tube containing
5–65 mL–6 mL TSB, aseptically withdraw 0.50.5 mL to 1.0 mL and rehydrate the lyophilized culture. Aseptically transfer the
entire rehydrated pellet back into the original tube of broth. Mix well. Incubate for 2424 h 6 2 h at 3636 °C 6 1°C.1 °C. Using
a sterile spreader, inoculate a sufficient number of TSA plates (for example, 5 to 10 plates per organism) with 100 μL each of the
culture. Incubate plates at 3636 °C 6 1ºC1 °C for 2424 h 6 2 h. Following incubation, add 5 mL cryoprotectant solution (TSB
with 15% v/v glycerol) to the surface of each agar plate. Resuspend the cells in this solution using a sterile spreader or a sterile
swab and aspirate the cell suspension from the surface of the agar. Transfer suspension into a sterile vessel. Repeat by adding
another 5 mL cryoprotectant to the agar plates, resuspend the cells, aspirate suspension and pool with the initial cell suspension.
Alternately, 10 mL cryoprotectant solution may be added per plate for resuspending with subsequent aspiration. Mix the pooled
contents of the vessel thoroughly. Immediately after mixing, pipet approximately 1.0 mL quantities of the diluted suspension into
cryovials. Place and store cryovials in –70°C or below freezer; these are the frozen stock cultures. Each cryovial is considered as
single use only. Store stock cultures up to 18 months. Reinitiate stocks using a new lyophilized culture.
9.1.1 Bacteria Inoculum Preparation—For S. aureus and S. enterica, defrost a single cryovial at room temperature and briefly
vortex to mix. Add 10 μL of the thawed frozen stock to a tube containing 10 mL synthetic broth and then vortex to mix. Incubate
at 3636 °C 6 1°C1 °C for 2424 h 6 2 h. Briefly vortex the 24 h culture prior to transfer. For this final subculture step, inoculate
a sufficient number of 2020 mm × 150 mm tubes containing 10 mL synthetic broth with 10 μL per tube of the 24 h synthetic broth
culture; incubate 4848 h to 54 h at 3636 °C 6 1°C.1 °C. Using a Vortex-style mixer, mix synthetic broth test cultures 33 s to 4 s
and let stand 10 min at room temperature before continuing. Remove the upper portion of each culture, leaving behind any debris
or clumps, and transfer to a sterile flask or tube; pool cultures in the flask and swirl to mix. Aliquot a sufficient volume of culture
into a sterile test tube.
9.1.1.1 For each bacterium, one daily transfer is required prior to the inoculation of a final test culture. Daily cultures may be
subcultured for up to 5 d; each daily transfer may be used to generate a test culture. For the purpose of achieving the carrier count
range, final cultures may be adjusted by dilution in growth medium or by concentration using centrifugation (for example, 5000
g for 20 min) resuspending the pellet in the appropriate volume of sterile test culture medium.
9.1.2 For P. aeruginosa, defrost a single cryovial at room temperature and briefly vortex to mix. Each cryovial should be single
use only. Add 10 μL of the thawed frozen stock to a tube containing 10 mL broth (synthetic or nutrient broth) and then vortex to
mix. Incubate at 3636 °C 6 1ºC1 °C for 2424 h 6 2 h. Do not vortex the 24 h culture prior to transfer. For this final subculture
step, inoculate a sufficient number of 20 × 150 mm tubes containing 10 mL broth (synthetic or nutrient) with 10 μL per tube of
the 24 h broth culture; incubate 48 to 54 h at 3636 °C 6 1°C.1 °C. Do not shake 4848 h to 54 h test culture. The pellicle from
the 4848 h to 54 h cultures must be removed from the broth either by decanting the liquid aseptically into a sterile tube, by gently
aspirating the broth away from the pellicle using a pipet, or by removal with a vacuum. Avoid harvesting pellicle from the bottom
of the tube.
9.1.2.1 Any disruption of the pellicle resulting in dropping, or breaking up of the pellicle in culture before or during its removal
renders that culture unusable in the test. This is extremely critical because any pellicle fragment remaining will result in uneven
clumping and layering of organism, allowing for biased exposure to disinfectant and causing false-positive results. Pool the test
culture from each tube and visually inspect culture for pellicle fragments. Presence of pellicle in the final culture makes it unusable
for test. Using a Vortex-style mixer, mix test cultures 33 s to 4 s and let stand 10 min at room temperature before continuing.
Remove the upper portion of each culture, leaving behind any debris or clumps, and transfer to a sterile flask or tube; pool cultures
from tubes in the flask and swirl to mix. Aliquot a sufficient volume of culture into a sterile test tube.
9.1.2.2 One daily transfer is required prior to the inoculation of a final test culture. Daily cultures may be subcultured for up to
5 days; each daily transfer may be used to generate a test culture. For the purpose of achieving the carrier count range, final cultures
may be adjusted by dilution in growth medium or by concentration using centrifugation (for example, 5000 g for 20 min)
resuspending the pellet in the appropriate volume of sterile test culture medium.
9.2 Mycobacteria—Maintain a stock culture of Mycobacterium organisms on Middlebrook 7H11 or 7H9 agar slants by monthly
transfer and incubation for 15 days to 20 days at 36 6 1 °C.36 °C 6 1 °C. Slants may be stored at 55 °C 6 3 °C for up to six
weeks.
9.2.1 Mycobacteria Inoculum Preparation—From stock culture, inoculate Modified Proskauer-Beck (MPB) Broth tubes and
AOAC Official Method 961.02 Germicidal Spray Products as Disinfectants. AOAC International, Chapter 6.
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incubate 21 to 2 days at 36 6 1 °C.36 °C 6 1 °C. Using a sterile transfer loop, transfer culture to a sterile glass tissue grinder. Add
1.0 mL of 0.1%0.1 % polysorbate (Tween) 80. Grind to break up large clumps or aggregates. Dilute the culture with 9 mL of
Modified Proskauer-Beck Broth. Transfer the suspension to a sterile test tube and allow to settle for 1010 min to 15 min. Remove
the upper portion to a sterile tube or flask, leaving behind any debris or clumps. Pool cultures, as applicable, and swirl to mix.
Dilute the culture to achieve 2061% 20 % 61 % T at 650 nm using Modified Proskauer-Beck Broth.
9.3 Fungi—Maintain a stock culture of Trichophyton mentagrophytes on Sabouraud Dextrose Agar (SDA) or Glucose agar slants
by transferring at less than or equal to 3 month intervals and incubate 10 d at 27.56 2.5°C,27.5 °C6 2.5 °C, followed by storage
at 563°C.5 °C63 °C.
9.3.1 Conidial Suspension
...