ASTM E2276-10(2019)
(Test Method)Standard Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the Fingerpads of Adults
Standard Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the Fingerpads of Adults
SIGNIFICANCE AND USE
5.1 This in vivo procedure is designed to test the ability of hygienic handwash or handrub agents to eliminate selected types of bacteria from experimentally contaminated skin of the hands of adult subjects. Since the two thumbpads and all eight fingerpads can be used in any given test, it allows for the incorporation of an input control (two), control for viable bacteria remaining after the inoculum has been allowed to dry (two), bacteria eliminated after treatment with a control or reference solution (two), and up to four replicates to assess the bacteria-eliminating efficiency of the product under test. No more than 100 µL of the test bacterial suspension is required to complete one test. The results of testing with this test method may form the basis for confirmatory tests using a suitable whole-hand test protocol, such as Test Method E1174.
5.2 Whereas this test method relates to testing with bacteria, it can be readily adapted to work with protozoa and bacteriophages. Similar methods for work with fungi (Test Method E2613) and viruses of human origin (Test Method E1838) are already ASTM standards.
5.3 Potentially infectious microorganisms left on hands after washing can be reduced further by drying the washed hands with paper, cloth, or warm air (7). A step for the drying of fingerpads after exposure to the control or test solution, therefore, has not been included to avoid bacterial removal by the drying process itself.
5.4 This test method is not meant for use with surgical hand scrubs (Test Method E1115) or preoperative skin preps (Test Method E1173).
5.5 The level of contamination with viable bacteria on each fingerpad after the drying of the inoculum should be five- to ten-fold higher than the product performance criterion required. For example, the titer in the dried inoculum on each fingerpad should be about 105 colony forming units of the test bacterium when a >104 reduction is required under the conditions of this test method.
SCOPE
1.1 This test method is designed to determine the activity of hygienic handwash and handrub (4) agents against transient bacterial flora on hands and is not meant for use with surgical hand scrubs or preoperative skin preps.
1.2 Performance of this procedure requires the knowledge of regulations pertaining to the protection of human subjects.3
1.3 The test method should be performed by persons with training in microbiology in facilities designed and equipped for work with infectious agents at biosafety level 2 (5).
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.
1.6 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
Relations
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: E2276 − 10 (Reapproved 2019)
Standard Test Method for
Determining the Bacteria-Eliminating Effectiveness of
Hygienic Handwash and Handrub Agents Using the
Fingerpads of Adults
This standard is issued under the fixed designation E2276; 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.
INTRODUCTION
Hands can spread many types of pathogens directly (1) or by transfer of such organisms to other
surfaces and objects during casual contact (2,3). Therefore, regular and proper decontamination of
hands by caregivers and food-handlers in particular is crucial for infection control. Hygienic hand
antisepsis is meant to reduce the load of transient microflora on hands, thereby reducing the risk of
disease transmission. Such reduction in the bacterial load may be due to a combination of bacterial
inactivation and removal of viable bacteria from the skin. In this method the test bacterial suspension
is placed on the thumb- and fingerpads of adults to simulate the contamination of hands with transient
microflora, the inoculum on the fingerpads is allowed to dry and is then treated with test and control
solutions. Since in each test all ten digits on any given subject can be used, the protocol permits the
inclusion of the required controls and several replicates of the test formulation in the same trial.
1. Scope 1.6 This international standard was developed in accor-
dance with internationally recognized principles on standard-
1.1 This test method is designed to determine the activity of
ization established in the Decision on Principles for the
hygienic handwash and handrub (4) agents against transient
Development of International Standards, Guides and Recom-
bacterial flora on hands and is not meant for use with surgical
mendations issued by the World Trade Organization Technical
hand scrubs or preoperative skin preps.
Barriers to Trade (TBT) Committee.
1.2 Performance of this procedure requires the knowledge
of regulations pertaining to the protection of human subjects.
2. Referenced Documents
1.3 The test method should be performed by persons with
2.1 ASTM Standards:
traininginmicrobiologyinfacilitiesdesignedandequippedfor
D1129 Terminology Relating to Water
work with infectious agents at biosafety level 2 (5).
E1115 Test Method for Evaluation of Surgical Hand Scrub
1.4 The values stated in SI units are to be regarded as
Formulations
standard. No other units of measurement are included in this
E1173 Test Method for Evaluation of Preoperative,
standard.
Precatheterization, or Preinjection Skin Preparations
1.5 This standard does not purport to address all of the
E1174 Test Method for Evaluation of the Effectiveness of
safety concerns, if any, associated with its use. It is the
Health Care Personnel Handwash Formulations
responsibility of the user of this standard to establish appro-
E1838 Test Method for Determining the Virus-Eliminating
priate safety, health, and environmental practices and deter-
Effectiveness of Hygienic Handwash and HandrubAgents
mine the applicability of regulatory limitations prior to use.
Using the Fingerpads of Adults
E2613 Test Method for Determining Fungus-Eliminating
Effectiveness of Hygienic Handwash and HandrubAgents
This test method is under the jurisdiction of ASTM Committee E35 on
Using Fingerpads of Adults
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Jan. 1, 2019. Published January 2019. Originally
approved in 2003. Last previous edition approved in 2010 as E2276 – 10. DOI:
10.1520/E2276-10R19. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
The boldface numbers in parentheses refer to the list of references at the end of contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
this standard. Standards volume information, refer to the standard’s Document Summary page on
Federal Register, Vol 46, No. 17, Jan. 27, 1991. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2276 − 10 (2019)
3. Terminology volume of the test bacterial suspension is placed on a demar-
cated area on each fingerpad and the inoculum allowed to dry.
3.1 Definitions—Fordefinitionsofgeneraltermsusedinthis
The contaminated area then is exposed to the control (standard
test method, refer to Terminology D1129.
hard water) or test substance for the desired contact time and
3.2 Definitions of Terms Specific to This Standard:
organisms remaining on the fingerpad are eluted and the
3.2.1 active ingredient, n—a substance added to a formula-
eluates assayed for viable bacteria. Percent or log reductions
tion specifically for the inhibition or inactivation of microor-
in the numbers of viable bacteria after treatment with the
ganisms.
control and test substance are then determined. The fingerpad
3.2.2 dry control, n—a control to determine the number of method gives results that are comparable to those obtained
colony forming units of the test bacterium remaining viable
usingawhole-handprocedure (6).Iftwodifferentformulations
after the initial drying of the inoculum on the skin. are being compared in the same test, one of them may be
designated as a reference and used in place of the hard water
3.2.3 handrub, n—a liquid or gel which is applied by
control. If desired, one also may use tap water in parallel with
rubbing to decontaminate lightly soiled hands between hand-
the hard water control to determine the influence of water
washings and generally do not require a post-treatment water
hardness on the test product’s bacteria-eliminating activity.
rinse; such agents usually contain alcohol alone or with other
active ingredients.
5. Significance and Use
3.2.4 hard water, n—water with a standard hardness of
calcium carbonate. 5.1 This in vivo procedure is designed to test the ability of
hygienic handwash or handrub agents to eliminate selected
3.2.5 hygienic handwash agent, n—an agent generally used
types of bacteria from experimentally contaminated skin of the
for handwashing by personnel in hospitals, other health-care
hands of adult subjects. Since the two thumbpads and all eight
facilities, day-care centers, nursing homes, and food-handling
fingerpads can be used in any given test, it allows for the
establishments to eliminate transient microorganisms from
incorporation of an input control (two), control for viable
intact skin.
bacteria remaining after the inoculum has been allowed to dry
3.2.6 input control, n—a control to determine the number of
(two), bacteria eliminated after treatment with a control or
colonyformingunitsofthetestbacteriumplacedoneachdigit.
reference solution (two), and up to four replicates to assess the
3.2.7 neutralization, n—a process which results in quench-
bacteria-eliminating efficiency of the product under test. No
ing the antimicrobial activity of a test substance. This may be
more than 100 µLof the test bacterial suspension is required to
achieved through dilution of the test substance to reduce the
complete one test. The results of testing with this test method
antimicrobial activity, or through the use of chemical agents,
may form the basis for confirmatory tests using a suitable
called neutralizers, to eliminate antimicrobial activity.
whole-hand test protocol, such as Test Method E1174.
3.2.8 nonmedicatedsoap,n—asoapordetergentthatismild
5.2 Whereas this test method relates to testing with bacteria,
to the skin and does not contain any antimicrobial chemicals.
it can be readily adapted to work with protozoa and bacterio-
3.2.9 soil load, n—a solution of one or more organic and/or
phages. Similar methods for work with fungi (Test Method
inorganic substances added to the suspension of the test
E2613) and viruses of human origin (Test Method E1838) are
organism to simulate the presence of body secretions, excre-
already ASTM standards.
tions or other extraneous substances.
5.3 Potentially infectious microorganisms left on hands
3.2.10 test substance or test formulation, n—a formulation
after washing can be reduced further by drying the washed
which incorporates antimicrobial ingredients.
hands with paper, cloth, or warm air (7). A step for the drying
3.2.11 test organism, n—an applied inoculum of an organ-
of fingerpads after exposure to the control or test solution,
ism that has characteristics which allow it to be readily
therefore, has not been included to avoid bacterial removal by
identified. The test organism is used to simulate a transient
the drying process itself.
topical microbial contaminant. It may also be referred to as a
5.4 This test method is not meant for use with surgical hand
marker organism, bacterial simulant/surrogate or bacterial
scrubs (Test Method E1115) or preoperative skin preps (Test
contaminant.
Method E1173).
3.2.12 test vehicle, n—the test formulation without an
5.5 The level of contamination with viable bacteria on each
active ingredient.
fingerpad after the drying of the inoculum should be five- to
3.2.13 transient microbiota, n—microorganisms from the
ten-fold higher than the product performance criterion re-
environment that contaminate but do not normally colonize the
quired. For example, the titer in the dried inoculum on each
skin.
fingerpad should be about 10 colony forming units of the test
bacterium when a >10 reduction is required under the condi-
4. Summary of Test Method
tions of this test method.
4.1 This test method is conducted on a group of adults who
have provided informed consent and the skin of whose hands
6. Equipment and Apparatus
has been determined to be free from any apparent damage.
Subjects are to refrain from using any products containing 6.1 Colony Counter—Any of several types may be used, for
antimicrobial agents for one week prior to the test. A known example, Quebec Colony Counter.
E2276 − 10 (2019)
6.2 Freezers—A freezer at -20 6 2 °C is required for the 7.4.2.3 Add 0.04 g of bovine mucin to 10 mL of phosphate
storage of culture media.Asecond freezer at -70 °C or lower is buffer.
required to store bacterial stocks.
7.4.3 Prepare the stock solutions separately and sterilize by
passage through a 0.22 µm pore diameter membrane filter,
6.3 Handwashing Sink—A sink of sufficient size to permit
aliquot and store at either 4 62°Cor–20 6 2 °C; the stock
subjects to wash hands without touching hands to sink surface.
solution of bovine mucin can be autoclave-sterilized.
6.4 Incubator—Any incubator capable of maintaining a
7.4.4 To obtain a 500-µLinoculum of the test inoculum, add
temperature of 36 6 1 °C. Serratia marcescens requires
to 340 µL of the bacterial suspension, 35 µL of tryptone or
incubation at 25 6 2 °C for pigment formation.
yeastextract(7.4.2.1),25µLBSA(7.4.2.2),and100µLmucin
6.5 Laminar Flow Cabinet—A Class II biological safety
(7.4.2.3) stock solutions. This mixture contains approximately
cabinet is required for this work.The procedures for the proper 2 g of total protein/L, which is roughly equivalent to the
maintenance and use of such cabinets are given in Ref (2).
protein content of a 5 % solution of fetal bovine serum.
6.6 Magnetic Stirrer and Magnets—Large enough to hold a
7.5 Standard Hard Water—The quality and disinfectant (for
5-L beaker or Erlenmeyer flask for preparing culture media or
example, chlorine) residual in tap water can vary from site to
other solutions.
site and also at different times at the same site. The use of
standard hard water, therefore, is recommended here to avoid
6.7 Membrane Filtration System—A membrane filtration
variations in results due to differences in tap water quality.
system and membranes with a pore diameter of 0.22-µm are
Water prepared in accordance with AOAC 960.09 E and F (8)
required to sterilize heat-sensitive media/solutions and to
toastandardhardnessofatleast200ppmascalciumcarbonate
capture and culture viable test bacteria in control samples and
is used for dilution of the test substance, as the control solution
eluates.
to determine the baseline level of bacterial elimination, and to
6.8 Positive Displacement Pipette—A pipette and pipette
rinse the fingerpads after exposure to the test product. The
tips that accurately can dispense 10-µL volumes.
standard hard water and tap water (if used) must first be tested
6.9 Refrigerator—A refrigerator at 4 6 2 °C for storage of
to ensure that they do not have any activity against the test
prepared culture media and reagents.
bacterium.Ifwaterwithadifferentlevelofhardnessisusedfor
making the use-dilution of the test formulation, this change
6.10 Sterilizer—Any suitable steam sterilizer capable of
must be clearly justified and documented in the report.
producing the conditions of sterilization is acceptable.
7.6 Test Agents—At least two samples of the product shall
6.11 Timer(Stop-clock)—One that can be read for minutes
be tested.
and seconds.
6.11.1 Tap Water Temperature Regulator and Temperature
7.7 Diluent for Bacterial Titration—Normal saline (0.85 %
Monitor—to monitor and regulate water temperature at 40 6
NaCl) at pH 7.2 – 7.4, or appropriate buffer.
2 °C.
7.8 Eluent for Bacterial Recovery from Fingerpads—
6.11.2 Water Faucet(s)—to be located above the sink at a
Normal saline or another suitable eluent.
height that permits the hands to be held higher than the elbow
during the washing procedure. Faucets with electronic sensors 7.9 Plastic Vials—Sterile screw-capped 2.0-mL vials with
or those that are wrist-, elbow-, knee-, or foot-operated are an inside diameter of about 8 mm will be required for
preferred to avoid recontamination of the washed hands. demarcation of the fingerpads and to hold various test solu-
tions.
7. Materials and Reagents
7.10 Miscellaneous Laboratory Ware—Automatic pipettes,
7.1 Serological Pipettes—Sterile reusable or single-use pi- pipette tips, plastic vials for storing stock cultures.
pettes of 10.0, 5.0, and 1.0-mL capacity.
7.11 Broth—Tryptose phosphate broth (TPB) or equivalent,
7.2 Culture Plates—Petri plates of 100 mm diameter for
to prepare the inoculum of the test organisms.
culturing bacteria.
7.12 Agar—Trypticase soy agar (TSA) or equivalent, to
NOTE 1—Plastic culture ware may be purchased from most laboratory recover and count the colonies of the test organism in control
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.