ASTM E2755-10

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Designation: E2755 − 10
StandardTest Method for
Determining the Bacteria-Eliminating Effectiveness of Hand
Sanitizer 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 Handrub Agents Using the Fingerpads of Adults
2.2 Other Standards:
1.1 Thistestmethodisdesignedtodeterminetheactivityof
AATCC Test Method 1472004 Antibacterial Activity As-
hand sanitizers (also known as hand rubs, hygienic hand rubs,
sessment of Textile Materials: Parallel Streak Method
or hand antiseptics) against transient bacterial flora on the
21 CFR Parts 50 and 56Protection of Human Subjects;
hands.
Institutional Review Boards
1.2 Performance of this procedure requires the knowledge
3. Terminology
of regulations pertaining to the protection of human subjects
(see 21 CFR Parts 50 and 56).
3.1 Definitions:
3.1.1 antiseptic, n—a material for use on living tissue that
1.3 This test method should be performed by persons with
training in microbiology, in facilities designed and equipped either destroys microorganisms or suppresses their growth.
for work with potentially infectious agents at biosafety level
3.1.2 hand sanitizer, n—an antimicrobial gel, foam, liquid,
2.
spray, or wipe, used on hands that are not visibly soiled to
reduce the number of transient microorganisms, which is
1.4 Units—The values stated in SI units are to be regarded
appliedbyrubbing,anddoesnotrequireapost-treatmentwater
asstandard.Nootherunitsofmeasurementareincludedinthis
rinse. Such agents may also be referred to as hand rubs,
standard.
hygienic hand rubs, or hand antiseptics.
1.5 This standard does not purport to address all of the
3.1.3 healthcare personnel handwash, n—a cleanser or
safety concerns, if any, associated with its use. It is the
waterless agent intended to reduce transient bacteria on the
responsibility of the user of this standard to establish appro-
hands.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. For more specific 3.1.4 neutralization, n—the process for inactivating or
precautionary statements, see 8.2.
quenchingtheactivityofamicrobicide,oftenachievedthrough
physical(forexample,filtrationordilution)orchemicalmeans,
2. Referenced Documents
or both.
2.1 ASTM Standards:
3.1.5 resident microorganisms, n—microorganismsthatsur-
E1054Test Methods for Evaluation of Inactivators of Anti-
vive and multiply on the skin, forming a stable population.
microbial Agents
3.1.6 test bacteria, n—an applied inoculum of bacteria that
E1174Test Method for Evaluation of the Effectiveness of
has characteristics which allow it to be readily identified. Test
Health Care Personnel Handwash Formulations
bacteria are used to simulate a transient topical microbial
E2276 Test Method for Determining the Bacteria-
contaminant. This may also be referred to as a test organism,
Eliminating Effectiveness of Hygienic Handwash and
marker organism, simulant, or contaminant.
3.1.7 test material, n—a product or formulation which
This test method is under the jurisdiction of ASTM Committee E35 on
incorporates an antimicrobial ingredient(s).
Pesticides and Alternative Control Agents and is the direct responsibility of
3.1.8 transient microorganisms, n—microorganisms that
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved June 1, 2010. Published July 2010. DOI: 10.1520/
contaminate the skin but do not form a stable population.
E2755–10.
CDC-NIH, Biosafety in Microbiological and Biomedical Laboratories, 5th ed.,
U.S. Department of Health and Human Services, U.S. Government Printing Office, Available from American Association of Textile Chemists and Colorists
Washington, DC, 2007. (AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http://
For referenced ASTM standards, visit the ASTM website, www.astm.org, or www.aatcc.org.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM AvailablefromU.S.GovernmentPrintingOfficeSuperintendentofDocuments,
Standards volume information, refer to the standard’s Document Summary page on 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
the ASTM website. www.access.gpo.gov.
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E2755 − 10
4. Summary of Test Method bacterium (Serratia marcescens) or a gram-positive bacterium
(Staphylococcus aureus) as the test organism. S. marcescens is
4.1 This test method uses adult subjects who have provided
used as a test organism in both Test Method E1174 and Test
a written informed consent and whose hands have been
Method E2276. S. aureus is a highly relevant pathogen in
determined to be free from any apparent damage at the time of
healthcare, institutional, and community settings. Moreover,
participation in the study. Subjects are to refrain from use of
hands are an important vehicle in the transfer of S. aureus
any antimicrobials for at least one week prior to the initiation
between people and the environment, and in the transfer
of the test procedure (see Section 11).
between individuals.
4.2 Subjects’ hands are artificially contaminated with 0.2
5.5 This test method may be used as an alternative to Test
mL of a high-titer suspension of the test bacteria which is
Method E2276, which limits the test bacteria to the fingerpads
distributedoverallsurfacesofthehandsandfingerstoproduce
and does not incorporate actual use conditions such as friction
a minimum baseline recovery level of 10 cfu/hand. Because
during hand decontamination.
Serratia marcescens is relatively sensitive to drying, the high
5.6 The investigator should be aware of potential health
titer suspension is prepared by growing in broth with vigorous
risks associated with the use of these organisms and precau-
aeration, followed by a 10-fold concentration with centrifuga-
tions similar to those referenced in Section 8 should be taken.
tion. Staphylococcus aureus is more resistant to drying and is
therefore not concentrated after growth with vigorous aeration
6. Apparatus
in broth.
6.1 Centrifuge—For the sedimentation of S. marcescens for
4.3 Test material effectiveness is measured by comparing
concentration.
thenumberoftestbacteriarecoveredfromcontaminatedhands
6.2 Centrifuge Tubes—Sterile,forsedimentationof S. marc-
after use of the test material to the number recovered from
escens for concentration.
contaminated hands not exposed to the test material. Activity
of the test material is measured following a single application
6.3 Colony Counter—Anyofseveraltypesmaybeused;for
and may also be measured after multiple consecutive
example, Quebec colony counters and similar devices. Auto-
contamination/application cycles in a single day.
mated, computerized plater/counter systems may also be used.
6.4 Gloves—Sterile, loose-fitting, unlined, powder-free
5. Significance and Use
gloves possessing no antimicrobial properties. Perform a zone
5.1 Hand hygiene is considered one of the most important
ofinhibitiontest,suchasAATCCTestMethod147,toevaluate
measures for preventing the spread of infectious microorgan-
the antibacterial activity.
isms. Hand sanitizers reduce the microbial load on the hands
6.5 Handwashing Sink—Sufficient in size to permit hand-
without the use of soap and water, and are thus an important
washing without the touching of hands to sink surface or other
tool in the practice of good hand hygiene. Hand sanitizers are
subjects.
recommendedforuseonhandsthatarenotvisiblysoiled.They
6.5.1 Water Faucet(s)—Located above the sink at a height
areformulatedtobeappliedfullstrengthtodryhands,“rubbed
to permit hands to be held higher than the elbow during the
in” until dry, and are not rinsed off.
washing procedure.
5.2 This test method is specifically designed to evaluate the
6.5.2 Tap Water Temperature Regulator and Temperature
bacteria-eliminating activity of hand sanitizers from
Monitor—To set and maintain the tap water temperature at
experimentally-contaminated hands. It is intended to be an
40 62°C.
alternativetoTestMethodE1174, which was designedprimar-
6.6 Incubator—Capable of maintaining temperatures of
ily to evaluate antimicrobial handwashing agents that are
35 62°C and 25 62°C. The latter temperature ensures ad-
lathered with the aid of water and then rinsed off. When using
equate pigment production for S. marcescens on solid media.
Test Method E1174 to evaluate hand sanitizers, inadequate
6.7 Miscellaneous Labware—Continuously adjustable pi-
dryingofthehandsaftercontaminationdilutesthetestproduct
petters (1-mL and 0.2-mL capacity) and sterile pipette tips,
and can compromise activity, leading to an underestimation of
sterile serological pipettes (5.0-mL capacity), sterile culture
effectiveness. By applying a higher titer test bacteria suspen-
tubes, sterile disposable Petri dishes, sterile syringes, Erlen-
sion in a smaller volume, soil load on the hands is minimized
meyer flasks, and beakers.
and hands are completely dry prior to application of the test
material. These modifications result in a better approximation
6.8 Sampling Containers—Sterile or sterilizable containers
of the in-use conditions for hand sanitizers and thus provide a
having tight closures and sufficient capacity to hold 75 mL
more reliable indication of their performance in the field.
sampling solution (see 7.7).
5.3 This test method can be used to test any form of hand 6.9 Shaking Incubator—Rotary platform shaking incubator
sanitizer, including gels, rubs, sprays, foams, and wipes ac-
capableofmaintaining35 62°Candcapableofshakingat250
cording to label directions at typical “in-use” doses. r/min. Alternatively, use an incubator capable of maintaining
35 6 2°C and able to accommodate a portable rotary shaker,
5.4 Susceptibility to biocides can vary among different
capable of shaking at 250 r/min.
species of bacteria and major differences have been noted
between gram-negative and gram-positive organisms.This test 6.10 Sterilizer—Any steam sterilizer capable of processing
method provides the option to use either a gram-negative culture media and reagents.
E2755 − 10
cases (for example, some alcohol-based hand sanitizers) neutralization is
6.11 Timer (Stop-Clock)—Typethatcanbereadforminutes
achieved by dilution alone.
and seconds.
7.8 Test Material—Use directions provided with the test
6.12 Tourniquets—Children’s size or any style capable of
material.Ifdirectionsarenotprovided,usethedirectionsgiven
securing gloves to the wrist.
in this method.
6.13 Vortex Mixer—Any vortex that will ensure proper
8. Test Bacteria
mixing of culture tubes.
8.1 Serratia marcescens (ATCC 14756).This strain forms a
7. Reagents and Materials
stable red pigmentation at 25°C.
7.1 Antibiotic Ointment—A topical, triple-antibiotic oint-
8.2 Staphylococcus aureus (ATCC 6538 (methicillin-
ment for application to the hands after the final decontamina-
sensitive) orATCC 33591 (methicillin-resistant)) is an alterna-
tion.
tive test bacteria. S. aureus is differentiated from resident
7.2 Cleansing Wash—Amild, proven non-antimicrobial liq-
microorganisms (including Staphylococcus epidermidis) with
uid soap. May be purchased commercially or prepared accord-
chromogenic indicator medium (see 7.4.2.1). (Warning—
ing to the instructions provided in Test Method E1174.
Application of microorganisms to the skin may involve a
health risk. Determine the antibiotic sensitivity profile of the
7.3 Chlorhexidine Skin Cleanser—Antiseptic skin cleanser
test bacteria prior to applying to the skin. After the test has
containing 4% chlorhexidine gluconate (w/v) for hand decon-
been completed, decontaminate the subject’s hands and follow
tamination.
proper procedures to reduce infection risk (12.1-12.4). If an
7.4 Culture Media:
infectionoccurs,providetheantibioticsensitivityprofiletothe
7.4.1 Broth—Soybean-casein digest broth (tryptic soy
attending clinician.)
broth) is recommended.
7.4.2 Agar Plating Media: 9. Preparation of Test Bacteria Suspension
7.4.2.1 S. aureus Plating Medium—HardyCHROM (trade-
9.1 Method 1 (for S. marcescens):
mark), Staph aureus, available from Hardy Diagnostics, is
9.1.1 Ahomogeneous bacterial suspension is used to inocu-
recommended. Other indicator media for S. aureus or MRSA
late the subjects’ hands. Prepare a stock culture of S. marc-
may be appropriate but should be validated prior to use.
escens (ATCC 14756) by inoculating approximately 5 mL of
soybean-casein digest broth (see 7.4.1) from a cryogenic stock
NOTE 1—S. aureus forms smooth, deep pink to fuchsia-colored colo-
nies. The growth of most other organisms, including Staphylococcus or lyophilized vial or pellet and incubate for 25 61hat35 6
epidermidis are partially to completely inhibited.
2°C.Inoculatetheappropriatevolumeofsoybean-caseindigest
broth with 1 mLof the stock culture of S. marcescens/125 mL
7.4.2.2 S. marcescens Plating Medium—Soybean-casein di-
gest agar (tryptic soy agar) is recommended. of broth to yield the volume necessary to complete the study
(that is, 2 mL per hand contamination (see 11.3) per test
7.5 Dilution Fluid—Sterile Butterfield’s buffered phosphate
6 subject). The volume of the broth culture should not exceed
diluent (orothersuitablediluent)adjustedtopH7.2 60.1and
about one fourth of the capacity of the Erlenmeyer flask to
containing an effective inactivator for the test material, if
ensure adequate aeration. Incubate for 25 61hat35 6 2°C
necessary.
with shaking at 250 r/min to yield a titer of approximately 1.0
NOTE 2—Inactivator is only required if neutralization of the test
×10 cfu/mL.
material cannot be achieved upon dilution into the sampling solution (see
NOTE 4—The frozen or lyophilized stock should be at least two but no
7.7).
morethanfour24-hsoybean-caseindigestbroth(see7.4.1)transfersfrom
7.6 Ethanol Solution—70% ethanol in water (v/v) for hand
the original ATCC culture.
decontamination.
9.1.2 Transfer the culture to appropriate sized sterile centri-
7.7 Sampling Solution—Dissolve 0.4 g KH PO , 10.1 g
2 4
fugetubesorbottlesandcentrifugeatconditionsappropriateto
Na HPO , 1.0 g isooctylphenoxypolyethoxyethanol (for ex-
2 4
sediment the culture completely (recommended conditions are
ample,TritonX-100),andappropriatelyvalidatedneutralizers,
7000 G for 10 min). Decant the supernatant and resuspend the
ifnecessary,indistilledwater.AdjustpHto7.8 60.1with0.1
pellet to one-tenth the original volume with s
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