ASTM E2946-21

Standard Test Method for Determining the Bacteria-Reducing Effectiveness of Food-Handler Handwash Formulations Using Hands of Adults

SIGNIFICANCE AND USE
5.1 Hand hygiene is considered one of the most important measures for preventing the spread of infectious microorganisms and is critical for reducing the incidence of food-borne disease. Food-handling settings are unique in that moderate to heavy soil load present on hands often can influence the ability of a product to remove or kill microorganisms (3, 4). Test methods are needed for assessing the efficacy of hand hygiene products under conditions representative of those encountered in a food-handling environment.  
5.2 This test method is specifically designed to evaluate the effectiveness of food-handler products to kill and remove bacteria from experimentally-contaminated hands under conditions of moderate to heavy organic soil load. The inclusion of soils typical of food service setting makes this a methodology more appropriate than Test Method E1174, which was designed to evaluate healthcare personnel hand washes and does not include an option to include soil (4).
SCOPE
1.1 This test method is designed to determine the activity of food-handler handwashes against transient bacterial flora on the hands.  
1.2 Performance of this procedure requires the knowledge of regulations pertaining to the protection of human subjects (1)2.  
1.3 This test method should be performed by persons with training in microbiology, in facilities designed and equipped for work with potentially infectious agents at biosafety level 2 (2).  
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 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. For more specific precautionary statements see 8.1.1.  
1.6 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: 31-Mar-2021
• 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 E1174-06 - Standard Test Method for Evaluation of the Effectiveness of Health Care Personnel Handwash Formulations
• 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 E1174-00 - Standard Test Method for Evaluation of the Effectiveness of Health Care Personnel or Consumer Handwash Formulations
• Effective Date: 10-Aug-2000

Overview

ASTM E2946-21 sets a standardized test method for evaluating the bacteria-reducing effectiveness of handwash formulations intended for food-handlers. Hand hygiene is a key factor in preventing the spread of infectious microorganisms and reducing the risk of foodborne illnesses in food service environments. This internationally recognized standard offers a method to assess the efficacy of hand hygiene products under conditions representative of moderate to heavy soil loads typical to food-handling scenarios.

ASTM E2946-21 specifically addresses the unique requirements of food-handling settings, offering a more relevant evaluation than existing standards aimed at healthcare, such as ASTM E1174. It provides protocols for using adult hands experimentally contaminated with transient bacterial flora, such as Escherichia coli, in the presence of organic soil.

Key Topics

• Purpose: To measure the ability of food-handler handwash products to remove or kill transient bacteria from adult hands under realistic food-service conditions.
• Test Conditions:
  • Hands are experimentally contaminated with known quantities of bacteria in moderate (beef broth) or heavy (ground beef) soil matrices.
  • The method includes both water- and non-water-based handwash formulations.
  • Only SI units are used.
• Requirements:
  • Performed by trained microbiologists in biosafety level 2 laboratories.
  • Compliance with regulations for human subject protection is mandatory, including informed consent and ethical oversight.
• Safety and Compliance:
  • Users must implement appropriate safety, health, and environmental practices.
  • The applicability of regulatory requirements must be determined prior to use.

Applications

ASTM E2946-21 is widely applicable across sectors in which food-handler hand hygiene is critical:

• Food Processing and Service: Facilities manufacturing or serving ready-to-eat foods use this standard to select and validate handwash products, ensuring they are effective under heavy contamination conditions.
• Product Development: Manufacturers of antimicrobial and non-antimicrobial handwash formulations rely on this method to demonstrate bacteria-reducing efficacy for regulatory approvals or labeling claims.
• Regulatory Compliance: Organizations seeking compliance with national and international hygiene requirements can use this test method to validate claims regarding the reduction of transient microorganisms on food-handler hands.
• Quality Assurance: Routine quality control for assessing hand hygiene protocols or products in catering, restaurants, institutional kitchens, and food plants.

By simulating real-world soil loads and foodborne bacteria, ASTM E2946-21 helps stakeholders improve the safety of food handling and protect public health.

Related Standards

• ASTM E1174: Standard Test Method for Evaluation of the Effectiveness of Health Care Personnel Handwash Formulations. Primarily applicable to healthcare settings and does not include soil in the testing protocol.
• ASTM E1054: Test Practices for Evaluation of Inactivators of Antimicrobial Agents. Reference for validating neutralizers in E2946 testing.
• ASTM E2756: Terminology Relating to Antimicrobial and Antiviral Agents.
• AATCC Test Method 147: Antibacterial Activity Assessment of Textile Materials; referenced for confirming the neutrality of test apparatus components.
• WHO and International Principles: Developed in accordance with the WTO's principles for international standards and technical barriers to trade.

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Keywords: ASTM E2946-21, food-handler handwash, bacteria reduction, hand hygiene, antimicrobial efficacy, food safety, handwash formulation testing, transient bacteria, E. coli, foodborne illness prevention, standard test method.

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: E2946 − 21
Standard Test Method for
Determining the Bacteria-Reducing Effectiveness of Food-
Handler Handwash Formulations Using Hands of Adults
This standard is issued under the fixed designation E2946; 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 E1174Test Method for Evaluation of the Effectiveness of
Health Care Personnel Handwash Formulations
1.1 Thistestmethodisdesignedtodeterminetheactivityof
E2756Terminology Relating toAntimicrobial andAntiviral
food-handler handwashes against transient bacterial flora on
Agents
the hands.
2.2 AATCC Standards:
1.2 Performance of this procedure requires the knowledge
Test Method 147Antibacterial Activity Assessment of Tex-
of regulations pertaining to the protection of human subjects
tile Materials: Parallel Streak Method
(1) .
3. Terminology
1.3 This test method should be performed by persons with
training in microbiology, in facilities designed and equipped
3.1 Definitions—See Terminology E2756.
for work with potentially infectious agents at biosafety level 2
3.2 food-handler handwash, n—a water or non-water-aided
(2).
formulationusedforhandwashingbypersonnelinfoodservice
1.4 Units—The values stated in SI units are to be regarded
settings intended to kill or remove transient bacteria, or both
asstandard.Nootherunitsofmeasurementareincludedinthis
from the hands.
standard.
3.3 non-medicated/non-antimicrobial soap, n—a soap or
1.5 This standard does not purport to address all of the
detergent that is mild to the skin and does not contain any
safety concerns, if any, associated with its use. It is the
germicidal chemicals.
responsibility of the user of this standard to establish appro-
3.4 test material, n—a product or formulation that incorpo-
priate safety, health, and environmental practices and deter-
rates one or more antimicrobial ingredients.
mine the applicability of regulatory limitations prior to use.
For more specific precautionary statements see 8.1.1.
4. Summary of Test Method
1.6 This international standard was developed in accor-
4.1 This test method is performed using adult subjects who
dance with internationally recognized principles on standard-
have provided written informed consent, and whose hands
ization established in the Decision on Principles for the
havebeendeterminedtobefreeofanyapparentdamageatthe
Development of International Standards, Guides and Recom-
time of participation in the study. Subjects are to refrain from
mendations issued by the World Trade Organization Technical
use of any antimicrobials for at least one week prior to the
Barriers to Trade (TBT) Committee.
initiation of test procedures (Section 11).
2. Referenced Documents
4.2 Subjects contaminate hands with E. coli incorporated
2.1 ASTM Standards:
into a medium or heavy organic soil (beef broth or hamburger,
E1054Test Practices for Evaluation of Inactivators ofAnti-
respectively), which then is distributed over all surfaces of the
microbial Agents
hands and fingers to result in a minimum baseline recovery
population of ≥10 colony forming units (CFU)/hand.
This test method is under the jurisdiction of ASTM Committee E35 on
4.3 Test material effectiveness is measured by comparing
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
thenumberofchallengebacteriarecoveredfromcontaminated
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved April 1, 2021. Published May 2021. Originally
hands after a single application of the test material to the
approved in 2013. Last previous edition approved in 2013 as E2946–13. DOI:
numberrecoveredfromcontaminatedhandsnotexposedtothe
10.1520/E2946–21
2 test material.
The boldface numbers in parentheses refer to a list of references at the end of
this standard.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Available from American Association of Textile Chemists and Colorists
Standardsvolume information,referto thestandard’sDocumentSummary page on (AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http://
the ASTM website. www.aatcc.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2946 − 21
5. Significance and Use 7. Reagents and Materials
5.1 Hand hygiene is considered one of the most important 7.1 Cleansing Wash—Any mild, proven non-antimicrobial
measures for preventing the spread of infectious microorgan- liquid soap. It may be purchased commercially or as an
isms and is critical for reducing the incidence of food-borne example it may be prepared according to instructions for
disease. Food-handling settings are unique in that moderate to making soft soap.
heavysoilloadpresentonhandsoftencaninfluencetheability
Soft Soap, 200 g/L
of a product to remove or kill microorganisms (3, 4). Test
Linseed oil 50 parts by weight
methods are needed for assessing the efficacy of hand hygiene
Potassium hydroxide 9.5 parts
products under conditions representative of those encountered
Ethanol 7 parts
Distilled or high purity water as needed
in a food-handling environment.
Add linseed oil to a solution of potassium hydroxide in 15
5.2 This test method is specifically designed to evaluate the
parts water and heat up to approximately 70°C while con-
effectiveness of food-handler products to kill and remove
stantly stirring. Add the ethanol and continue heating while
bacteria from experimentally-contaminated hands under con-
stirring until the saponification process is completed and a
ditionsofmoderatetoheavyorganicsoilload.Theinclusionof
sampledissolvesclearlyinwaterandalmostclearlyinalcohol.
soils typical of food service setting makes this a methodology
The weight of the soft soap is then brought up to 100 parts by
more appropriate than Test Method E1174, which was de-
addition of hot water. Take 200g of the soft soap in 1L of
signed to evaluate healthcare personnel hand washes and does
water. Dispense in to appropriate containers and sterilize in an
not include an option to include soil (4).
autoclave.
6. Apparatus
7.2 Chlorhexidine Skin Cleanser—Antiseptic skin cleanser
containing 4% chlorhexidine gluconate (w/v) for hand decon-
6.1 Colony Counter—Anyofseveraltypesmaybeused;for
tamination.
example, Quebec colony counters and similar devices.
Automated, computerized plater/counter systems may also be
7.3 Culture Media:
used.
7.3.1 Broth—Soybean-casein digest broth (tryptic soy
broth) is recommended.
6.2 Gloves, sterile, loose-fitting, unlined, powder-free
gloves possessing no antimicrobial properties. Perform a zone 7.3.2 Agar Plating Media:
7.3.2.1 MacConkey Agar is recommended. E. coli (ATCC
ofinhibitiontest,suchasAATCCTestMethod147,toevaluate
for antibacterial activity. #11229) will produce purple colonies on this agar.
7.4 Beef Broth—Anystandard,sterileliquidbeefbroth,such
6.3 Handwashing Sink, sufficient in size to permit hand-
asSwansonbrandbeefbrothorothersimilarproduct,wouldbe
washing without the touching of hands to sink surface or other
suitable, low sodium or other modified versions are not
subjects.
recommended.
6.3.1 Water Faucet(s)—Located above the sink at a height
to permit hands to be held higher than the elbow during the
7.5 Hamburger—Gamma-irradiated 90% lean ground beef
washing procedure.
to ensure that the ground beef contains no contaminating
6.3.2 Tap Water Temperature Regulator and Temperature
microorganisms prior to use in testing; ground beef can be
Monitor—To set and maintain the tap water temperature at
purchased pre-irradiated from a standard meat supplier.
40°C 6 2°C.
7.6 Dilution Fluid—Sterile Butterfield’s buffered phosphate
6.4 Incubator,capableofmaintainingtemperaturesof35°C
diluent (5) (or other suitable diluent) adjusted to pH 7.2 6 0.1
6 2°C.
and containing an effective inactivator of the antimicrobial
chemistry of the test material, if necessary.
6.5 Miscellaneous Labware—Continuously adjustable pi-
petters (1mL and 0.2mL capacity) and sterile pipette tips,
NOTE 1—Inactivator is required only if neutralization of the test
sterile serological pipettes (5.0mL capacity), sterile culture
material cannot be achieved upon dilution into the Sampling Solution
(7.8).
tubes, sterile disposable Petri dishes, sterile syringes, Erlen-
meyer flasks, and beakers.
7.7 Ethanol Solution—70% ethanol in water (v/v) for hand
decontamination.
6.6 Sampling Containers, sterile or sterilizable containers
having tight closures and sufficient capacity to hold 75 mL of
7.8 Sampling Solution—Dissolve 0.4 g KH PO , 10.1 g
2 4
sampling solution (7.7)
Na HPO , 1.0 g isooctylphenoxypolyethoxyethanol, and ap-
2 4
propriately validated neutralizers, if necessary, in distilled
6.7 Sterilizer, any steam sterilizer capable of processing
culture media and reagents.
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
6.8 Timer (Stop-Clock), type that can be read for minutes
Standard-Grade Reference Materials, American Chemical Society, Washington,
and seconds. DC.ForsuggestionsonthetestingofreagentsnotlistedbytheAmericanChemical
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
6.9 Tourniquets, child size or any style capable of securing
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
gloves to the wrist. copeial Convention, Inc. (USPC), Rockville, MD.
SoldunderseveralTradeNamesincludingTriton™X-100(TheDowChemical
6.10 Vortex Mixer—Any vortexing device that will ensure
Company, Midland, MI), Igepal® CA-630 (Rhodia, Inc., Cranbury, NJ) and
proper mixing of culture tubes. Protachem OP-9 (Protameen Chemicals, Inc., Totowa, NJ).
E2946 − 21
water.Adjust pH to 7.8 6 0.1 with 0.1 N HCl or 0.1 N NaOH exceed one half of the capacity of the Erlenmeyer flask.
and bring volume to 1L with distilled water. Sterilize in an Incubate for 24h 64hat35°C 6 2°C to yield a titer of
autoclave and aseptically dispense 75-mLaliquants into sterile approximately 1.0 × 10 CFU/mL.
sampling containers (6.6)(6). 9.2.2 Swirl or shake suspension prior to withdrawal of each
aliquotusedfortesting,andassayitforthenumberofbacteria
NOTE2—AneutralizervalidationshouldbeconductedaccordingtoTest
present at the beginning and at the end of the use period. Do
Method E1054 prior to the study. Test Method E1054 provides a list of
neutralizersappropriateforcommonlyusedantimicrobialagents.Insome not use a suspension for more than 8 h.The population should
cases neutralization can be achieved by dilution alone.
not vary by more than 6 0.5 log CFU/mL over the 8-h
period.
7.9 Test Material—Use directions provided with the test
9.2.3 Prior to testing, weigh approximately 4ounce (113 g)
material. If directions are not provided, use the directions
portions of gamma-irradiated ground beef (7.5). Assay a
provided in this method.
representative sample of ground beef for viable E. coli at the
8. Test Bacteria Species
beginning of the use period, if the E. coli was found to be
8.1 Escherichia coli ATCC 11229. contaminated it is recommended that subjects that used that
inoculum are replaced. Immediately prior to each hand
8.1.1 Warning—Applicationofmicroorganismstotheskin
may involve a health risk. Determine the antibiotic sensitivity contamination, dispense 5.0 ml of the challenge suspension
(9.2.1) into a ground beef portion and, using gloved hands,
profile of the test bacteria prior to applying to the skin. After
testing has been completed, decontaminate the subjects’hands knead the beef portion in a manner to ensure even distribution
of the suspension fluid evenly throughout the portion.
and follow proper procedures to reduce infection risk (12.1 –
12.2). If an infection occurs, provide the antibiotic susceptibil-
ity profile to the attending clinician. 10. Subjects
10.1 Recruit a sufficient number of healthy adult human
9. Preparation of Test Bacteria Suspension
subjects who have no clinical evidence of dermatoses, cuts,
9.1 Method 1( for moderate soil matrix with beef broth): :
lesions, hangnails, or other skin disorders on the hands or
9.1.1 Prepare a stock culture by transferring E. coli (ATCC
forearms.Aminimum of eight (8) subjects should be used for
11229)fromacryogenicstockorlyophilizedvialorpelletinto
each test material. The total number of subjects used will
approximately5mLofsoybean-caseindigestbroth(7.3.1)and
depend on the number of test materials, the purpose of the
incubatefor24h 62hat35°C 62°C.Inoculateavolumeof
study, and the regulatory requirements governing the study.
soybean-casein digest broth with 1 mLof the stock culture per
10.2 Itistheresponsibilityoftheuserofthistestmethodto
1000mLof broth to yield a volume of suspension sufficient to
obtain the necessary approval from an Institutional Review
complete the study. The volume of the sus
...

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: E2946 − 13 E2946 − 21
Standard Test Method for
Determining the Bacteria-Reducing Effectiveness of Food-
Handler Handwash Formulations Using Hands of Adults
This standard is issued under the fixed designation E2946; 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
1.1 This test method is designed to determine the activity of food-handler handwashes against transient bacterial flora on the
hands.
1.2 Performance of this procedure requires the knowledge of regulations pertaining to the protection of human subjects (1) .
1.3 This test method should be performed by persons with training in microbiology, in facilities designed and equipped for work
with potentially infectious agents at biosafety level 2 (2).
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this
standard.
1.5 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 and health practices and determine the applicability of regulatory
limitations prior to use. For more specific precautionary statements see 8.1.1.
1.5 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. For more specific precautionary statements see 8.1.1.
1.6 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:
E1054 Test Practices for Evaluation of Inactivators of Antimicrobial Agents
E1174 Test Method for Evaluation of the Effectiveness of Health Care Personnel Handwash Formulations
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 AATCC Standards:
Test Method 147 Antibacterial Activity Assessment of Textile Materials: Parallel Streak Method
This test method 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, 2013April 1, 2021. Published November 2013May 2021. Originally approved in 2013. Last previous edition approved in 2013 as
E2946–13. DOI: 10.1520/E2946–1310.1520/E2946–21
The boldface numbers in parentheses refer to a list of references at the end of this standard.
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 American Association of Textile Chemists and Colorists (AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http://www.aatcc.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2946 − 21
3. Terminology
3.1 Definitions—See Terminology E2756.
3.2 food-handler handwash, n—a water or non-water-aided formulation for use used for handwashing by personnel in food service
settings intended to kill or remove transient bacteria, or both from the hands.
3.3 non-medicated/non-antimicrobial soap, n—a soap or detergent that is mild to the skin and does not contain any germicidal
chemicals.
3.4 test material, n—a product or formulation that incorporates one or more antimicrobial ingredients.
4. Summary of Test Method
4.1 This test method is performed using adult subjects who have provided written informed consent, and whose hands have been
determined to be free of any apparent damage at the time of participation in the study. Subjects are to refrain from use of any
antimicrobials for at least one week prior to the initiation of test procedures (Section 11).
4.2 Subjects contaminate hands with E. coli incorporated into a medium or heavy organic soil (beef broth or hamburger,
respectively), which then is distributed over all surfaces of the hands and fingers to result in a minimum baseline recovery
population of ≥10 colony forming units (CFU)/hand.
4.3 Test material effectiveness is measured by comparing the number of challenge bacteria recovered from contaminated hands
after a single application of the test material to the number recovered from contaminated hands not exposed to the test material.
5. Significance and Use
5.1 Hand hygiene is considered one of the most important measures for preventing the spread of infectious microorganisms and
is critical for reducing the incidence of food-borne disease. Food-handling settings are unique in that moderate to heavy soil load
present on hands often can influence the ability of a product to remove or kill microorganisms (3, 4). Test methods are needed for
assessing the efficacy of hand hygiene products under conditions representative of those encountered in a food-handling
environment.
5.2 This test method is specifically designed to evaluate the effectiveness of food-handler products to kill and remove bacteria
from experimentally-contaminated hands under conditions of moderate to heavy organic soil load. The inclusion of soils typical
of food service setting makes this a methodology more appropriate than Test Method E1174, which was designed to evaluate
healthcare personnel hand washes and does not include an option to include soil (4).
6. Apparatus
6.1 Colony Counter—Any of several types may be used; for example, Quebec colony counters and similar devices. Automated,
computerized plater/counter systems may also be used.
6.2 Gloves, sterile, loose-fitting, unlined, powder-free gloves possessing no antimicrobial properties. Perform a zone of inhibition
test, such as AATCC Test Method 147, to evaluate for antibacterial activity.
6.3 Handwashing Sink, sufficient in size to permit handwashing without the touching of hands to sink surface or other subjects.
6.3.1 Water Faucet(s)—Located above the sink at a height to permit hands to be held higher than the elbow during the washing
procedure.
6.3.2 Tap Water Temperature Regulator and Temperature Monitor—To set and maintain the tap water temperature at 4040 °C 6
2°C.2 °C.
6.4 Incubator, capable of maintaining temperatures of 3535 °C 6 2°C.2 °C.
E2946 − 21
6.5 Miscellaneous Labware—Continuously adjustable pipetters (1-mL(1 mL and 0.2-mL0.2 mL capacity) and sterile pipette tips,
sterile serological pipettes (5.0-mL(5.0 mL capacity), sterile culture tubes, sterile disposable Petri dishes, sterile syringes,
Erlenmeyer flasks, and beakers.
6.6 Sampling Containers, sterile or sterilizable containers having tight closures and sufficient capacity to hold 75 mL of sampling
solution (7.7)
6.7 Sterilizer, any steam sterilizer capable of processing culture media and reagents.
6.8 Timer (Stop-Clock), type that can be read for minutes and seconds.
6.9 Tourniquets, child size or any style capable of securing gloves to the wrist.
6.10 Vortex Mixer—Any vortexing device that will ensure proper mixing of culture tubes.
7. Reagents and Materials
7.1 Cleansing Wash—Any mild, proven non-antimicrobial liquid soap. It may be purchased commercially or as an example it may
be prepared according to instructions for making soft soap.
Soft Soap, 200 g/L
Linseed oil 50 parts by weight
Potassium hydroxide 9.5 parts
Ethanol 7 parts
Distilled or high purity water as needed
Add linseed oil to a solution of potassium hydroxide in 15 parts water and heat up to approximately 70°C70 °C while constantly
stirring. Add the ethanol and continue heating while stirring until the saponification process is completed and a sample dissolves
clearly in water and almost clearly in alcohol. The weight of the soft soap is then brought up to 100 parts by addition of hot water.
Take 200 g 200 g of the soft soap in 1 L 1 L of water. Dispense in to appropriate containers and sterilize in an autoclave.
7.2 Chlorhexidine Skin Cleanser—Antiseptic skin cleanser containing 4% chlorhexidine gluconate (w/v) for hand decontamina-
tion.
7.3 Culture Media:
7.3.1 Broth—Soybean-casein digest broth (tryptic soy broth) is recommended.
7.3.2 Agar Plating Media:
7.3.2.1 MacConkey Agar is recommended. E. coli (ATCC #11229) will produce purple colonies on this agar.
7.4 Beef Broth—Any standard, sterile liquid beef broth, such as Swanson brand beef broth or other similar product, would be
suitable, low sodium or other modified versions are not recommended.
7.5 Hamburger—Gamma-irradiated 90% lean ground beef to ensure that the ground beef contains no contaminating microor-
ganisms prior to use in testing; ground beef can be purchased pre-irradiated from a standard meat supplier.
7.6 Dilution Fluid—Sterile Butterfield’s buffered phosphate diluent (5) (or other suitable diluent) adjusted to pH 7.2 6 0.1 and
containing an effective inactivator of the antimicrobial chemistry of the test material, if necessary.
ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference Materials, American Chemical Society, Washington, DC. For
suggestions on the testing of reagents not listed by the American Chemical Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and
the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.
E2946 − 21
NOTE 1—Inactivator is required only if neutralization of the test material cannot be achieved upon dilution into the Sampling Solution (7.8).
7.7 Ethanol Solution—70%70 % ethanol in water (v/v) for hand decontamination.
7.8 Sampling Solution—Dissolve 0.4 g KH PO , 10.1 g Na HPO , 1.0 g isooctylphenoxypolyethoxyethanol, and appropriately
2 4 2 4
validated neutralizers, if necessary, in distilled water. Adjust pH to 7.8 6 0.1 with 0.1 N HCl or 0.1 N NaOH and bring volume
to 1 L 1 L with distilled water. Sterilize in an autoclave and aseptically dispense 75-mL aliquants into sterile sampling containers
(6.6)(6).
NOTE 2—A neutralizer validation should be conducted according to Test Method E1054 prior to the study. Test Method E1054 provides a list of
neutralizers appropriate for commonly used antimicrobial agents. In some cases neutralization can be achieved by dilution alone.
7.9 Test Material—Use directions provided with the test material. If directions are not provided, use the directions provided in this
method.
8. Test Bacteria Species
8.1 Escherichia coli ATCC 11229.
8.1.1 Warning—Application of microorganisms to the skin may involve a health risk. Determine the antibiotic sensitivity profile
of the test bacteria prior to applying to the skin. After testing has been completed, decontaminate the subjects’ hands and follow
proper procedures to reduce infection risk (12.1 – 12.2). If an infection occurs, provide the antibiotic susceptibility profile to the
attending clinician.
9. Preparation of Test Bacteria Suspension
9.1 Method 1( for moderate soil matrix with beef broth): :
9.1.1 Prepare a stock culture by transferring E. coli (ATCC 11229) from a cryogenic stock or lyophilized vial or pellet into
approximately 5 mL 5 mL of soybean-casein digest broth (7.3.1) and incubate for 2424 h 6 2 h 2 h at 3535 °C 6 2°C.2 °C.
Inoculate a volume of soybean-casein digest broth with 1 mL of the stock culture per 1000 mL 1000 mL of broth to yield a volume
of suspension sufficient to complete the study. The volume of the suspension should not exceed one half of the capacity of the
Erlenmeyer flask. Incubate for 2424 h 6 2 h 2 h at 3535 °C 6 2°C2 °C to yield a titer of approximately 1.0 × 10 CFU/mL.
NOTE 3—The frozen or lyophilized stock should be at least two, but no more than four, 24-h24 h soybean-casein digest broth transfers from the original
ATCC culture.
9.1.2 Transfer the culture to appropriately sized sterile centrifuge tubes or bottles and centrifuge to sediment the culture
completely. Recommended conditions are 4750 rpm 6 50 rpm for 30 min. Decant the supernatant and resuspend the pellet in a
volume of beef broth equal to the original culture volume (7.4) to yield a homogeneous challenge suspension containing
approximately 1.0 × 10 CFU/mL.
9.1.3 Swirl or shake suspension prior to withdrawal of each aliquot used for testing. Assay the suspension for the number of
bacteria at the beginning and at the end of the use period. Do not use a suspension for more than 8 h. The population should not
vary by more than 6 0.5 log CFU/mL over the 8-h8 h period.
9.2 Method 2 (for heavy soil matrix with hamburger):
9.2.1 Prepare a stock culture by transferring E. coli ATCC 11229 from a cryogenic stock or lyophilized vial or pellet into
approximately 5 mL of soybean-casein digest broth (7.3.1) and incubate for 24 h 6 4 h at 3535 °C 6 2°C.2 °C. Inoculate a volume
of soybean-casein digest broth with 1 mL of the stock culture per 1000 mL of broth to yield a volume of suspension sufficient to
complete the study. The volume of the suspension should not exceed one half of the capacity of the Erlenmeyer flask. Incubate
for 2424 h 6 4 h at 3535 °C 6 2°C2 °C to yield a titer of approximat
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