ASTM E2755-22

Standard Test Method for Determining the Bacteria-Eliminating Effectiveness of Healthcare Personnel Hand Rub 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. Hand rubs reduce the microbial load on the hands without the use of soap and water, and are thus an important tool in the practice of good hand hygiene. Alcohol-based hand rubs are recommended in healthcare settings for use on hands that are not visibly soiled. They are formulated to be applied full strength to dry hands, “rubbed in” until dry, and are not rinsed off.  
5.2 This test method is designed specifically to evaluate hand rubs for efficacy in eliminating bacteria from experimentally-contaminated hands. It is designed as an alternative to Test Method E1174, which was intended primarily to evaluate antimicrobial handwashing agents that are lathered with the aid of water and then rinsed off. When using Test Method E1174 to evaluate hand rubs, inadequate drying of the hands after contamination dilutes the test material and can compromise activity, to result in an underestimation of effectiveness. Additionally, because hand rubs are not rinsed after product use, activity can be further degraded by build-up of soil from the contaminating broth and inactivated challenge bacteria on the hands.  
5.2.1 In this method, application to the hands of a small volume of high-titer test bacteria suspension minimizes soil load such that the skin is completely dry prior to application of the test material. Further, by applying the bacterial suspension only prior to those test material application cycles followed by sampling, excessive buildup of killed bacteria on the hands is avoided, and the potential impact of non-volatile test product ingredients on bacteria-eliminating effectiveness after ten consecutive applications can be specifically assessed.  
5.3 A reference control is evaluated for each subject prior to evaluation of the test material. Data from the reference control helps to control for inter-subject variability, inter-experimental variabi...
SCOPE
1.1 This test method is designed to determine the activity of healthcare personnel hand rubs, (also known as hand rubs, hygienic hand rubs, hand sanitizers, or hand antiseptics) against transient microbial skin flora on the hands after a single application and after repeated applications.  
1.2 Performance of this procedure requires the knowledge of regulations pertaining to the protection of human subjects (see 21 CFR Parts 50 and 56).  
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 2.2,3  
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 8.2.  
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-2022
• ICS: 11.080.20 - Disinfectants and antiseptics
• 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 E2276-10(2019) - Standard Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the Fingerpads of Adults
• Effective Date: 01-Jan-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 E2276-10 - Standard Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the Fingerpads of Adults
• Effective Date: 01-Apr-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 E2276-03e1 - Standard Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the Fingerpads of Adult Subjects
• Effective Date: 10-May-2003
• 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 E2755-22, issued by ASTM International, specifies the standard test method for determining the bacteria-eliminating effectiveness of healthcare personnel hand rub formulations using the hands of adult subjects. Hand hygiene is crucial in minimizing the spread of infectious microorganisms, and alcohol-based hand rubs are widely used in healthcare settings to reduce microbial load when soap and water are not available. This standard provides a consistent method for evaluating whether these products effectively eliminate bacteria, thereby supporting the development and assessment of hand sanitizers, hygienic hand rubs, hand antiseptics, and related products for routine and repeated use.

Key Topics

• Hand Rub Effectiveness: Assesses the bacteria-eliminating capability of healthcare personnel hand rubs (sanitizers, gels, foams, sprays, and wipes).
• Microbial Test Subjects: Utilizes specific bacterial organisms, such as Serratia marcescens or Staphylococcus aureus, to simulate transient microbial contamination.
• Human Test Subjects: Involves adult volunteers, following strict criteria to ensure subject safety and data consistency.
• Controlled Test Environment: Emphasizes procedure execution in microbiology laboratories at biosafety level 2, under ethical review and regulatory compliance.
• Testing Procedures: Measures hygiene product performance after both a single and repeated applications to mimic real-world healthcare use.
• Data Collection and Analysis: Includes controlled baselines and reference controls for robust statistical comparison between products and across laboratories.
• Safety and Compliance: Requires adherence to institutional review board (IRB) guidelines, informed consent, and post-test care.

Applications

ASTM E2755-22 is a vital standard for various stakeholders in the healthcare and hygiene product sectors:

• Product Development: Manufacturers can use this method to demonstrate the efficacy of new hand rub formulations or compare product performance to benchmarks.
• Regulatory Submissions: Provides recognized data for product approval processes by demonstrating compliance with internationally accepted hygiene product standards.
• Quality Assurance: Ensures that marketed hand sanitizers and antiseptics meet efficacy requirements for reducing transient microbial flora.
• Clinical Environments: Offers evidence-based data to justify the selection of hand rubs for hospitals, clinics, and institutional settings where infection prevention is essential.
• Research and Comparative Studies: Enables independent laboratories and research institutions to compare the performance of different hand hygiene products under controlled, repeatable conditions.

Related Standards

Organizations and laboratories applying ASTM E2755-22 may also reference these related standards:

• ASTM E1054: Practices for Evaluation of Inactivators of Antimicrobial Agents.
• ASTM E1174: Test Method for Evaluation of the Effectiveness of Healthcare Personnel Handwash Formulations.
• ASTM E2276: Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the Fingerpads of Adults.
• ASTM E2756: Terminology Relating to Antimicrobial and Antiviral Agents.
• AATCC Test Method 147: Antibacterial Activity Assessment of Textile Materials.
• 21 CFR Parts 50 and 56: U.S. regulations concerning the protection of human subjects and institutional review boards.

Practical Value

By standardizing the assessment of hand rub efficacy, ASTM E2755-22 provides reliable, repeatable methods essential to product claims, regulatory compliance, and public health protection. Adherence to this method helps organizations ensure that hand hygiene products meet the stringent demands of healthcare environments, ultimately contributing to safer patient care and improved infection control.

Keywords: ASTM E2755-22, hand rub, hand hygiene, hand sanitizer, antimicrobial, bacteria elimination, healthcare, infection 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: E2755 − 22
Standard Test Method for
Determining the Bacteria-Eliminating Effectiveness of
Healthcare Personnel Hand Rub Formulations Using Hands
of Adults
This standard is issued under the fixed designation E2755; 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 2. Referenced Documents
2.1 ASTM Standards:
1.1 Thistestmethodisdesignedtodeterminetheactivityof
E1054PracticesforEvaluationofInactivatorsofAntimicro-
healthcare personnel hand rubs, (also known as hand rubs,
bial Agents
hygienic hand rubs, hand sanitizers, or hand antiseptics)
E1174Test Method for Evaluation of the Effectiveness of
againsttransientmicrobialskinfloraonthehandsafterasingle
Healthcare Personnel Handwash Formulations
application and after repeated applications.
E2276 Test Method for Determining the Bacteria-
1.2 Performance of this procedure requires the knowledge
Eliminating Effectiveness of Hygienic Handwash and
of regulations pertaining to the protection of human subjects
Handrub Agents Using the Fingerpads of Adults
(see 21 CFR Parts 50 and 56).
E2756Terminology Relating toAntimicrobial andAntiviral
Agents
1.3 This test method should be performed by persons with
2.2 Other Standards:
training in microbiology, in facilities designed and equipped
AATCC Test Method 1472004 Antibacterial Activity As-
for work with potentially infectious agents at biosafety level
2,3
sessment of Textile Materials: Parallel Streak Method
2.
21 CFR Parts 50 and 56Protection of Human Subjects;
1.4 Units—The values stated in SI units are to be regarded
Institutional Review Boards
asstandard.Nootherunitsofmeasurementareincludedinthis
standard.
3. Terminology
1.5 This standard does not purport to address all of the 3.1 Definitions: For definitions of terms used in this
safety concerns, if any, associated with its use. It is the
document, see Terminology E2756.
responsibility of the user of this standard to establish appro-
3.2 Definitions of Terms Specific to This Standard:
priate safety, health, and environmental practices and deter-
3.2.1 healthcare personnel handrub, n—an antimicrobial
mine the applicability of regulatory limitations prior to use.
gel, foam, liquid, spray, or wipe, applied by rubbing to reduce
For more specific precautionary statements, see 8.2.
the transient microbial skin flora on hands that are not visibly
1.6 This international standard was developed in accor-
soiled,andwhichdoesnotrequireapost-treatmentwaterrinse.
dance with internationally recognized principles on standard-
Such agents may also be referred to as hand rubs, hygienic
ization established in the Decision on Principles for the
hand rubs, or hand antiseptics.
Development of International Standards, Guides and Recom-
3.2.2 healthcare personnel handwash, n—a cleanser or
mendations issued by the World Trade Organization Technical
waterless agent intended to reduce transient microbial skin
Barriers to Trade (TBT) Committee.
flora on the hands.
1 4
This test method is under the jurisdiction of ASTM Committee E35 on For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
responsibility of Subcommittee E35.15 on Antimicrobial Agents. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved April 1, 2022. Published July 2022. Originally the ASTM website.
approved in 2010. Last previous edition approved in 2015 as E2755–15. DOI: Available from American Association of Textile Chemists and Colorists
10.1520/E2755–22 (AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http://
CDC-NIH, Biosafety in Microbiological and Biomedical Laboratories, 5th ed., www.aatcc.org.
U.S. Department of Health and Human Services, U.S. Government Printing Office, AvailablefromU.S.GovernmentPrintingOfficeSuperintendentofDocuments,
Washington, DC, 2007. 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
45 CFR Part 46 Protection of Human Subjects (Effective July 19, 2018). www.access.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2755 − 22
3.2.3 test bacteria, n—an applied inoculum of bacteria that Method E1174 to evaluate hand rubs, inadequate drying of the
has characteristics which allow it to be readily identified. Test hands after contamination dilutes the test material and can
bacteria are used to simulate a topical transient microbial compromise activity, to result in an underestimation of effec-
contaminant. This may also be referred to as a test organism, tiveness. Additionally, because hand rubs are not rinsed after
marker organism, simulant, or contaminant. product use, activity can be further degraded by build-up of
soil from the contaminating broth and inactivated challenge
3.2.4 test material, n—a product or formulation which
bacteria on the hands.
incorporates an antimicrobial ingredient(s).
5.2.1 In this method, application to the hands of a small
4. Summary of Test Method volume of high-titer test bacteria suspension minimizes soil
loadsuchthattheskiniscompletelydrypriortoapplicationof
4.1 This test method uses adult subjects who have provided
the test material. Further, by applying the bacterial suspension
a written informed consent and whose hands have been
only prior to those test material application cycles followed by
determined to be free from any apparent damage at the time of
sampling, excessive buildup of killed bacteria on the hands is
participation in the study. Subjects are to refrain from use of
avoided, and the potential impact of non-volatile test product
any antimicrobials for at least one week prior to the initiation
ingredients on bacteria-eliminating effectiveness after ten con-
of the test procedure (see Section 11).
secutive applications can be specifically assessed.
4.2 Subjects’ hands are artificially contaminated with 0.2
5.3 Areferencecontrolisevaluatedforeachsubjectpriorto
mL of a high-titer suspension of the test bacteria which is
evaluation of the test material. Data from the reference control
distributedoverallsurfacesofthehandsandfingerstoproduce
8 helps to control for inter-subject variability, inter-experimental
a minimum baseline recovery level of 10 cfu/hand. Because
variability, and inter-laboratory variability; and enables im-
Serratia marcescens is relatively sensitive to drying, the high
proved statistical comparison of test materials evaluated in the
titer suspension is prepared by growing in broth with vigorous
same experiment.
aeration, followed by a 10-fold concentration with centrifuga-
tion. 5.4 This test method can be used to test any form of hand
rubs, including gels, rinses, sprays, foams, and wipes when
4.3 An alternate test organism that can be used is Staphy-
used according to label directions at typical “in-use” doses.
lococcus aureus if deemed appropriate. This organism is more
resistant to drying and is therefore not concentrated after 5.5 Susceptibility to biocides can vary among different
growth with vigorous aeration in broth. species of bacteria and major differences have been noted
between gram-negative and gram-positive organisms.This test
4.4 Test material effectiveness is measured by comparing
method provides the option to use either a gram-negative
thenumberoftestbacteriarecoveredfromcontaminatedhands
bacterium (Serratia marcescens) or a gram-positive bacterium
after use of the test material to the number recovered from
(Staphylococcus aureus) as the test organism. S. marcescens is
contaminated hands not exposed to the test material. Activity
used as a test organism in both Test Method E1174 and Test
of the test material is measured following a single application
Method E2276. S. aureus is a highly relevant pathogen in
and after multiple consecutive contamination/application
healthcare, institutional, and community settings. Moreover,
cycles in a single day. Evaluating effectiveness after multiple
hands are an important vehicle in the transfer of S. aureus
applications simulates repeated use of hand rubs in clinical
between people and the environment, and in the transfer
settings and determines whether progressive build-up of non-
between individuals.
volatile ingredients from the test material inhibits the antimi-
crobial action. An abbreviated test measuring activity of the 5.6 This test method may be used as an alternative to Test
test material following a single application may be used to Method E2276, which limits the test bacteria to the fingerpads
simulate situations where high frequency use is not expected. and does not incorporate actual use conditions such as friction
during hand rubbing.
5. Significance and Use
5.7 The investigator should be aware of potential health
5.1 Hand hygiene is considered one of the most important
risks associated with the use of these organisms and precau-
measures for preventing the spread of infectious microorgan-
tions similar to those referenced in Section 8 should be taken.
isms. Hand rubs reduce the microbial load on the hands
6. Apparatus
without the use of soap and water, and are thus an important
tool in the practice of good hand hygiene.Alcohol-based hand
6.1 Centrifuge—For the sedimentation of S. marcescens for
rubs are recommended in healthcare settings for use on hands
concentration.
that are not visibly soiled. They are formulated to be applied
6.2 Centrifuge Tubes—Sterile,forsedimentationof S. marc-
full strength to dry hands, “rubbed in” until dry, and are not
escens for concentration.
rinsed off.
6.3 Colony Counter—Anyofseveraltypesmaybeused;for
5.2 This test method is designed specifically to evaluate
example, Quebec colony counters and similar devices.
hand rubs for efficacy in eliminating bacteria from
Automated, computerized plater/counter systems may also be
experimentally-contaminated hands. It is designed as an alter-
used.
native toTest Method E1174, which was intended primarily to
evaluate antimicrobial handwashing agents that are lathered 6.4 Gloves—Sterile, loose-fitting, unlined, powder-free
with the aid of water and then rinsed off. When using Test gloves possessing no antimicrobial properties. Perform a zone
E2755 − 22
ofinhibitiontest,suchasAATCCTestMethod147,toevaluate 7.4.1 Broth—Soybean-casein digest broth (tryptic soy
the antibacterial activity. broth) is recommended.
7.4.2 Agar Plating Media:
6.5 Handwashing Sink—Sufficient in size to permit hand-
7.4.2.1 S. aureus Plating Medium—HardyCHROM
washing without the touching of hands to sink surface or other
(trademark), Staph aureus, available from Hardy Diagnostics,
subjects.
isrecommended.Otherindicatormediafor S. aureusorMRSA
6.5.1 Water Faucet(s)—Located above the sink at a height
may be appropriate but should be validated prior to use.
to permit hands to be held higher than the elbow during the
washing procedure. NOTE 1—S. aureus forms smooth, deep pink to fuchsia-colored colo-
nies. The growth of most other organisms, including Staphylococcus
6.5.2 Tap Water Temperature Regulator and Temperature
epidermidis are partially to completely inhibited.
Monitor—To set and maintain the tap water temperature at
7.4.2.2 S. marcescens Plating Medium—Soybean-casein di-
40°C 62°C.
gest agar (tryptic soy agar) is recommended.
6.6 Incubator—Capable of maintaining temperatures of
7.5 Dilution Fluid—Sterile Butterfield’s buffered phosphate
35°C 62°C and 25°C 62°C. The latter temperature ensures
diluent (orothersuitablediluent)adjustedtopH7.2 60.1and
adequatepigmentproductionfor S. marcescensonsolidmedia.
containing an effective inactivator for the test material, if
6.7 Miscellaneous Labware—Continuously adjustable pi-
necessary.
petters (1mL and 0.2mL capacity) and sterile pipette tips,
NOTE 2—Inactivator is only required if neutralization of the test
sterile serological pipettes (5.0mL capacity), sterile culture
material cannot be achieved upon dilution into the sampling solution (see
tubes, sterile disposable Petri dishes, sterile syringes, Erlen-
7.7).
meyer flasks, and beakers.
7.6 Ethanol Solution—70% ethanol in water (v/v) for hand
6.8 Plastic Bags—May be used in place of gloves (6.4).
decontamination.
Bags should be approximately 29cm by 31 cm, possess no
7.7 Sampling Solution—Dissolve 0.4 g KH PO , 10.1 g
2 4
antimicrobial properties, and have a low bioburden. Perform a
Na HPO , 1.0 g isooctylphenoxypolyethoxyethanol (for
2 4
zone of inhibition test, such as AATCC Test Method 147, to
example, Triton X-100), and appropriately validated
evaluate the antibacterial activity
neutralizers,ifnecessary,indistilledwater.AdjustpHto7.8 6
6.9 Sampling Containers—Sterile or sterilizable containers
0.1 with 0.1 N HCl or 0.1 N NaOH and bring volume to 1 L
having tight closures and sufficient capacity to hold 75 mL
with distilled water. Sterilize in an autoclave and aseptically
sampling solution (see 7.7).
dispense 75-mL portions into sterile sampling containers (see
6.9).
6.10 Shaking Incubator—Rotaryplatformshakingincubator
capableofmaintaining35 62°Candcapableofshakingat250
NOTE3—AneutralizervalidationshouldbeconductedaccordingtoTest
r/min. Alternatively, use an incubator capable of maintaining
Methods prior to the study. Test Methods E1054 provides a list of
neutralizersappropriateforcommonlyusedantimicrobialagents.Insome
35 6 2°C and able to accommodate a portable rotary shaker,
cases (for example, some alcohol-based hand rubs) neutralization is
capable of shaking at 250 r/min.
achieved by dilution alone.
6.11 Sterilizer—Any steam sterilizer capable of processing
7.8 Test Material—Use directions provided with the test
culture media and reagents.
material.Ifdirectionsarenotprovided,usethedirectionsgiven
6.12 Timer (Stop-Clock)—Typethatcanbereadforminutes in this method.
and seconds.
7.9 Reference Control—60% isopropanol in water (v/v).
6.13 Tourniquets—Children’s size or any style capable of
8. Test Bacteria
securing gloves to the wrist.
8.1 Serratia marcescens (ATCC 14756).This strain forms a
6.14 Vortex Mixer—Any vortex that will ensure proper
stable red pigmentation at 25°C.
mixing of culture tubes.
8.2 Staphylococcus aureus (ATCC 6538 (methicillin-
7. Reagents and Materials sensitive) orATCC 33591 (methicillin-resistant)) is an alterna-
tive test bacteria. S. aureus is differentiated from resident
7.1 Antibiotic Ointment—A topical, triple-antibiotic oint-
microbial skin flora (including Staphylococcus epidermidis)
ment for application to the hands and forearms after the final
with chromogenic indicator medium (see 7.4.2.1).
decontamination.Ifnecessary,consultwithaphysicianpriorto
(Warning—Application of microorganisms to the skin may
use.
involve a health risk. Determine the antibiotic sensitivity
7.2 Cleansing Wash—Amild, proven non-antimicrobial liq-
profile of the test bacteria prior to applying to the skin. After
uid soap. May be purchased commercially or prepared accord-
ing to the instructions provided in Test Method E1174.
Horowitz, W., (Ed.), Offıcial Methods of Analysis of the AOAC International,
7.3 Chlorhexidine Skin Cleanser—Antiseptic skin cleanser
18th Ed., Sec. 6.3.03 A.(f), Chapter 6, p. 10. AOAC International, Gaithersburg,
containing 4% chlorhexidine gluconate (w/v) for hand decon-
MD, 2000.
Peterson,A. F., “The Microbiology of the Hands: Evaluating the Effects of the
tamination.
Surgical Scrubs,” Developments in Industrial Microbiology, Vol. 14, 1973, pp.
7.4 Culture Media: 125–130.
E2755 − 22
thetesthasbeencompleted,decontaminatethesubject’shands testmaterial.Thetotalnumberofsubjectsusedwilldependon
and follow proper procedures to reduce infection risk (12.1 – the number of test materials, the purpose of the study, and the
12.4). If an infection occurs, provide the antibiotic susceptibil- regulatory requirements governing the study.
ity profile to the attending clinician.)
10.2 It is theresponsibilityof theuserof thistest methodto
obtain the necessary approval from an Institutional Review
9. Preparation of Test Bacteria Suspension
Board (IRB) or Independent Ethics Commission (IEC) for the
9.1 Method 1 (for S. marcescens):
use of adult human subjects for testing and to obtain informed
9.1.1 Ahomogeneous bacterial suspension is used to inocu-
and written consent from those selected for the study before
late the subjects’ hands. Prepare a stock culture of S. marc-
starting the tests.
escens (ATCC 14756) by inoculating approximately 5 mL of
10.3 Instruct subjects to avoid contact with antimicrobial
soybean-casein digest broth (see 7.4.1) from a cryogenic stock
products for the duration of the test and for at least one week
or lyophilized vial or pellet and incubate for 25 61hat35 6
prior to the test. This restriction includes antimicrobial-
2°C.Inoculatetheappropriatevolumeofsoybean-caseindigest
containing antiperspirants, deodorants, shampoos, lotions, and
broth with 1 mLof the stock culture of S. marcescens/125 mL
soaps. Bathing in biocide-treated pools, hot tubs, or spas
of broth to yield the volume necessary to complete the study
should be avoided. Harsh chemicals such as acids, bases, and
(that is, 0.2 mL per hand contamination (see 11.3) per test
solvents should also be avoided. Subjects may not have or
subject). The volume of the broth culture should not exceed
applynailpolish,artificialnails,ornailpolishremover,orhave
about one fourth of the capacity of the Erlenmeyer flask to
undergonenailtreatmentduringthe7-daypre-testconditioning
ensure adequate aeration. Incubate for 25h 61hat35°C 6
periodoronthesingletestday.Subjectsmaynotusetopicalor
2°Cwithshakingat250r/mintoyieldatiterofapproximately
systemic antimicrobials, antibiotics, or steroids other than f
...

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: E2755 − 15 E2755 − 22
Standard Test Method for
Determining the Bacteria-Eliminating Effectiveness of
Healthcare Personnel Hand Rub Formulations Using Hands
of Adults
This standard is issued under the fixed designation E2755; 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 healthcare personnel hand rubs, (also known as hand rubs, hygienic
hand rubs, hand sanitizers, or hand antiseptics) against transient microbial skin flora on the hands after a single application and
after repeated applications.
1.2 Performance of this procedure requires the knowledge of regulations pertaining to the protection of human subjects (see 21
CFR Parts 50 and 56).
1.3 This test method should be performed by persons with training in microbiology, in facilities designed and equipped for work
2,3
with potentially infectious agents at biosafety level 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 8.2.
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 8.2.
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.
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 May 15, 2015April 1, 2022. Published June 2015July 2022. Originally approved in 2010. Last previous edition approved in 20102015 as
E2755–10.–15. DOI: 10.1520/E2755–1510.1520/E2755–22
CDC-NIH, Biosafety in Microbiological and Biomedical Laboratories, 5th ed., U.S. Department of Health and Human Services, U.S. Government Printing Office,
Washington, DC, 2007.
45 CFR Part 46 Protection of Human Subjects (Effective July 19, 2018).
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2755 − 22
2. Referenced Documents
2.1 ASTM Standards:
E1054 Practices for Evaluation of Inactivators of Antimicrobial Agents
E1174 Test Method for Evaluation of the Effectiveness of Healthcare Personnel Handwash Formulations
E2276 Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using
the Fingerpads of Adults
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 Other Standards:
AATCC Test Method 147 2004 Antibacterial Activity Assessment of Textile Materials: Parallel Streak Method
21 CFR Parts 50 and 56 Protection of Human Subjects; Institutional Review Boards
3. Terminology
3.1 Definitions: For definitions of terms used in this document, see Terminology E2756.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 healthcare personnel handrub, n—an antimicrobial gel, foam, liquid, spray, or wipe, applied by rubbing to reduce the
transient microbial skin flora on hands that are not visibly soiled, and which does not require a post-treatment water rinse. Such
agents may also be referred to as hand rubs, hygienic hand rubs, or hand antiseptics.
3.2.2 healthcare personnel handwash, n—a cleanser or waterless agent intended to reduce transient microbial skin flora on the
hands.
3.2.3 test bacteria, n—an applied inoculum of bacteria that has characteristics which allow it to be readily identified. Test bacteria
are used to simulate a topical transient microbial contaminant. This may also be referred to as a test organism, marker organism,
simulant, or contaminant.
3.2.4 test material, n—a product or formulation which incorporates an antimicrobial ingredient(s).
4. Summary of Test Method
4.1 This test method uses adult subjects who have provided a written informed consent and whose hands have been determined
to be free from 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 the test procedure (see Section 11).
4.2 Subjects’ hands are artificially contaminated with 0.2 mL of a high-titer suspension of the test bacteria which is distributed
over all surfaces of the hands and fingers to produce a minimum baseline recovery level of 10 cfu/hand. Because Serratia
marcescens is relatively sensitive to drying, the high titer suspension is prepared by growing in broth with vigorous aeration,
followed by a 10-fold concentration with centrifugation. Staphylococcus aureus is more resistant to drying and is therefore not
concentrated after growth with vigorous aeration in broth.
4.3 An alternate test organism that can be used is Staphylococcus aureus if deemed appropriate. This organism is more resistant
to drying and is therefore not concentrated after growth with vigorous aeration in broth.
4.4 Test material effectiveness is measured by comparing the number of test bacteria recovered from contaminated hands after use
of the test material to the number recovered from contaminated hands not exposed to the test material. Activity of the test material
is measured following a single application and after multiple consecutive contamination/application cycles in a single day.
Evaluating effectiveness after multiple applications simulates repeated use of hand rubs in clinical settings and determines whether
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.
Available from U.S. Government Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
www.access.gpo.gov.
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progressive build-up of non-volatile ingredients from the test material inhibits the antimicrobial action. An abbreviated test
measuring activity of the test material following a single application may be used to simulate situations where high frequency use
is not expected.
5. Significance and Use
5.1 Hand hygiene is considered one of the most important measures for preventing the spread of infectious microorganisms. Hand
rubs reduce the microbial load on the hands without the use of soap and water, and are thus an important tool in the practice of
good hand hygiene. Alcohol-based hand rubs are recommended in healthcare settings for use on hands that are not visibly soiled.
They are formulated to be applied full strength to dry hands, “rubbed in” until dry, and are not rinsed off.
5.2 This test method is designed specifically to evaluate hand rubs for efficacy in eliminating bacteria from experimentally-
contaminated hands. It is designed as an alternative to Test Method E1174, which was intended primarily to evaluate antimicrobial
handwashing agents that are lathered with the aid of water and then rinsed off. When using Test Method E1174 to evaluate hand
rubs, inadequate drying of the hands after contamination dilutes the test material and can compromise activity, to result in an
underestimation of effectiveness. Additionally, because hand rubs are not rinsed after product use, activity can be further degraded
by build-up of soil from the contaminating broth and inactivated challenge bacteria on the hands.
5.2.1 In this method, application to the hands of a small volume of high-titer test bacteria suspension minimizes soil load such
that the skin is completely dry prior to application of the test material. Further, by applying the bacterial suspension only prior to
those test material application cycles followed by sampling, excessive buildup of killed bacteria on the hands is avoided, and the
potential impact of non-volatile test product ingredients on bacteria-eliminating effectiveness after ten consecutive applications can
be specifically assessed.
5.3 A reference control is evaluated for each subject prior to evaluation of the test material. Data from the reference control helps
to control for inter-subject variability, inter-experimental variability, and inter-laboratory variability; and enables improved
statistical comparison of test materials evaluated in the same experiment.
5.4 This test method can be used to test any form of hand rubs, including gels, rinses, sprays, foams, and wipes when used
according to label directions at typical “in-use” doses.
5.5 Susceptibility to biocides can vary among different species of bacteria and major differences have been noted between
gram-negative and gram-positive organisms. This test method provides the option to use either a gram-negative bacterium
(Serratia marcescens) or a gram-positive bacterium (Staphylococcus aureus) as the test organism. S. marcescens is used as a test
organism in both Test Method E1174 and Test Method E2276. S. aureus is a highly relevant pathogen in healthcare, institutional,
and community settings. Moreover, hands are an important vehicle in the transfer of S. aureus between people and the environment,
and in the transfer between individuals.
5.6 This test method may be used as an alternative to Test Method E2276, which limits the test bacteria to the fingerpads and does
not incorporate actual use conditions such as friction during hand rubbing.
5.7 The investigator should be aware of potential health risks associated with the use of these organisms and precautions similar
to those referenced in Section 8 should be taken.
6. Apparatus
6.1 Centrifuge—For the sedimentation of S. marcescens for concentration.
6.2 Centrifuge Tubes—Sterile, for sedimentation of S. marcescens for concentration.
6.3 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.
6.4 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 the antibacterial activity.
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6.5 Handwashing Sink—Sufficient in size to permit handwashing without the touching of hands to sink surface or other subjects.
6.5.1 Water Faucet(s)—Located above the sink at a height to permit hands to be held higher than the elbow during the washing
procedure.
6.5.2 Tap Water Temperature Regulator and Temperature Monitor—To set and maintain the tap water temperature at
40 6 2°C.40 °C 6 2 °C.
6.6 Incubator—Capable of maintaining temperatures of 35 6 2°C35 °C 6 2°C and 25 6 2°C.25 °C 6 2°C. The latter tempera-
ture ensures adequate pigment production for S. marcescens on solid media.
6.7 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.
6.8 Plastic Bags—May be used in place of gloves (6.4). Bags should be approximately 2929 cm by 31 cm, possess no
antimicrobial properties, and have a low bioburden. Perform a zone of inhibition test, such as AATCC Test Method 147, to evaluate
the antibacterial activity
6.9 Sampling Containers—Sterile or sterilizable containers having tight closures and sufficient capacity to hold 75 mL sampling
solution (see 7.7).
6.10 Shaking Incubator—Rotary platform shaking incubator capable of maintaining 35 6 2°C and capable of shaking at 250
r/min. Alternatively, use an incubator capable of maintaining 35 6 2°C and able to accommodate a portable rotary shaker, capable
of shaking at 250 r/min.
6.11 Sterilizer—Any steam sterilizer capable of processing culture media and reagents.
6.12 Timer (Stop-Clock)—Type that can be read for minutes and seconds.
6.13 Tourniquets—Children’s size or any style capable of securing gloves to the wrist.
6.14 Vortex Mixer—Any vortex that will ensure proper mixing of culture tubes.
7. Reagents and Materials
7.1 Antibiotic Ointment—A topical, triple-antibiotic ointment for application to the hands and forearms after the final
decontamination. If necessary, consult with a physician prior to use.
7.2 Cleansing Wash—A mild, proven non-antimicrobial liquid soap. May be purchased commercially or prepared according to the
instructions provided in Test Method E1174.
7.3 Chlorhexidine Skin Cleanser—Antiseptic skin cleanser containing 4 % chlorhexidine gluconate (w/v) for hand decontamina-
tion.
7.4 Culture Media:
7.4.1 Broth—Soybean-casein digest broth (tryptic soy broth) is recommended.
7.4.2 Agar Plating Media:
7.4.2.1 S. aureus Plating Medium—HardyCHROM (trademark), Staph aureus, available from Hardy Diagnostics, is recom-
mended. Other indicator media for S. aureus or MRSA may be appropriate but should be validated prior to use.
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NOTE 1—S. aureus forms smooth, deep pink to fuchsia-colored colonies. The growth of most other organisms, including Staphylococcus epidermidis are
partially to completely inhibited.
7.4.2.2 S. marcescens Plating Medium—Soybean-casein digest agar (tryptic soy agar) is recommended.
7.5 Dilution Fluid—Sterile Butterfield’s buffered phosphate diluent (or other suitable diluent) adjusted to pH 7.2 6 0.1 and
containing an effective inactivator for the test material, if necessary.
NOTE 2—Inactivator is only required if neutralization of the test material cannot be achieved upon dilution into the sampling solution (see 7.7).
7.6 Ethanol Solution—70 % ethanol in water (v/v) for hand decontamination.
7.7 Sampling Solution—Dissolve 0.4 g KH PO , 10.1 g Na HPO , 1.0 g isooctylphenoxypolyethoxyethanol (for example, Triton
2 4 2 4
X-100), and appropriately 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 with distilled water. Sterilize in an autoclave and aseptically dispense 75-mL portions into sterile
sampling containers (see 6.9).
NOTE 3—A neutralizer validation should be conducted according to Test Methods prior to the study. Test Methods E1054 provides a list of neutralizers
appropriate for commonly used antimicrobial agents. In some cases (for example, some alcohol-based hand rubs) neutralization is achieved by dilution
alone.
7.8 Test Material—Use directions provided with the test material. If directions are not provided, use the directions given in this
method.
7.9 Reference Control—60% isopropanol in water (v/v).
8. Test Bacteria
8.1 Serratia marcescens (ATCC 14756). This strain forms a stable red pigmentation at 25°C.
8.2 Staphylococcus aureus (ATCC 6538 (methicillin-sensitive) or ATCC 33591 (methicillin-resistant)) is an alternative test
bacteria. S. aureus is differentiated from resident microbial skin flora (including Staphylococcus epidermidis) with chromogenic
indicator medium (see 7.4.2.1). (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 the test has been completed, decontaminate the
subject’s hands and follow proper procedures to reduce infection risk (12.1 – 12.4). If an infection occurs, provide the antibiotic
susceptibility profile to the attending clinician.)
9. Preparation of Test Bacteria Suspension
9.1 Method 1 (for S. marcescens):
9.1.1 A homogeneous bacterial suspension is used to inoculate the subjects’ hands. Prepare a stock culture of S. marcescens
(ATCC 14756) by inoculating approximately 5 mL of soybean-casein digest broth (see 7.4.1) from a cryogenic stock or lyophilized
vial or pellet and incubate for 25 6 1 h at 35 6 2°C. Inoculate the appropriate volume of soybean-casein digest broth with 1 mL
of the stock culture of S. marcescens/125 mL of broth to yield the volume necessary to complete the study (that is, 0.2 mL per
hand contamination (see 11.3) per test subject). The volume of the broth culture should not exceed about one fourth of the capacity
of the Erlenmeyer flask to ensure adequate aeration. Incubate for 2525 h 6 1 h at 3535 °C 6 2°C2 °C with shaking at 250 r/min
to yield a titer of approximately 1.0 cfu/mL × 10 cfu/mL.
NOTE 4—The frozen or lyophilized stock should be at least two but no more than four 24-h24 h soybean-casein digest broth (see 7.4.1) transfers from
the original ATCC culture.
Horowitz, W., (Ed.), Offıcial Methods of Analysis of the AOAC International, 18th Ed., Sec. 6.3.03 A.(f), Chapter 6, p. 10. AOAC International, Gaithersburg, MD, 2000.
Peterson, A. F., “The Microbiology of the Hands: Evaluating the Effects of the Surgical Scrubs,” Developments in Industrial Microbiology, Vol. 14, 1973, pp. 125–130.
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9.1.2 Transfer the culture to appropriate sized sterile centrifuge tubes or bottles and centrifuge at conditions appropriate to
sediment the culture completely (recommended conditions are 7000 G for 10 min). Decant the supernatant and resuspend the pellet
to one-tenth the original volume with soybean-casein digest broth (see 7.4.1) to yield a homogeneous suspension containing
10 11
between 5.0 × 10 and 1.0 × 10 cfu/mL.
9.2 Method 2 (for S. aureus):
9.2.1 Use a homogeneous bacterial suspension to inoculate the subjects’ hands. Prepare a stock culture of S. aureus (AATCC 6538
or ATCC 33591) by inoculating approximately 5 mL of soybean-casein digest broth (see 7.4.1) from a frozen stock or lyophilized
vial and incubate for 2525 h 6 1 h at 3535 °C 6 2°C2 °C (see Note 4). Inoculate the appropriate volume of soybean-casein digest
broth with 1 mL of stock culture of S. aureus/125
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