ASTM E1327-07(2018)
(Test Method)Standard Test Method for Evaluation of Antimicrobial Handwash Formulations by Utilizing Fingernail Regions (Withdrawn 2024)
Standard Test Method for Evaluation of Antimicrobial Handwash Formulations by Utilizing Fingernail Regions (Withdrawn 2024)
SIGNIFICANCE AND USE
4.1 The procedure should be used to test the degerming effectiveness of antimicrobial hand washing products used by health care personnel that are intended for frequent use, and that are intended to reduce the level of contamination acquired through contact with contaminated objects or people.
4.2 Performance of these procedures requires the knowledge of regulations pertaining to the protection of human subjects (Ref 1).3
SCOPE
1.1 This test method can be used to determine the effectiveness of antimicrobial handwashing agents (including handrubs) in the reduction of transient bacterial flora with particular emphasis on the fingernail region.
1.2 A knowledge of microbiological techniques is required for these procedures.
1.3 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 hazard statements, see 7.5.
1.4 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.
WITHDRAWN RATIONALE
This test method was used to determine the effectiveness of antimicrobial handwashing agents (including handrubs) in the reduction of transient bacterial flora with particular emphasis on the fingernail region.
Formerly under the jurisdiction of Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents, this test method was withdrawn in April 2024. This standard is being withdrawn without replacement due to its limited use by industry.
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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: E1327 − 07 (Reapproved 2018)
Standard Test Method for
Evaluation of Antimicrobial Handwash Formulations by
Utilizing Fingernail Regions
This standard is issued under the fixed designation E1327; 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 3. Summary of Test Method
3.1 This test method, involving an improved method of
1.1 This test method can be used to determine the effective-
recovering bacteria from hands, is used to study the effects of
nessofantimicrobialhandwashingagents(includinghandrubs)
antimicrobial handwashes including health care personnel
in the reduction of transient bacterial flora with particular
handwash products. The group of volunteer panelists need not
emphasis on the fingernail region.
refrain from using topical antimicrobials (such as deodorant
1.2 A knowledge of microbiological techniques is required
soaps) before participating in the study.All subjects wash their
for these procedures.
hands with a nonantimicrobial hand soap prior to testing to
remove any residual hand lotions and to lower the numbers of
1.3 This standard does not purport to address all of the
resident skin flora. Activity of products is measured by
safety concerns, if any, associated with its use. It is the
comparing the numbers of marker bacteria recovered from
responsibility of the user of this standard to establish appro-
artificially contaminated fingernail regions after use of the
priate safety, health, and environmental practices and deter-
handwashing formulations to the numbers recovered from the
mine the applicability of regulatory limitations prior to use.
artificially contaminated but unwashed fingernail regions.
For more specific hazard statements, see 7.5.
Broth cultures of Serratia marcescens (a red pigmented bac-
1.4 This international standard was developed in accor-
terial species) and Escherichia coli (which produces fluores-
dance with internationally recognized principles on standard-
cent colonies on a special agar medium) are used as test
ization established in the Decision on Principles for the
bacteria. A spore suspension of Bacillus subtilis may be
Development of International Standards, Guides and Recom-
utilized to study (1) degree of physical removal by handwash-
mendations issued by the World Trade Organization Technical
ing techniques, and (2) the recovery and precision aspects of
Barriers to Trade (TBT) Committee.
the test method.
2. Referenced Documents
4. Significance and Use
2.1 ASTM Standards:
4.1 The procedure should be used to test the degerming
E1054 Test Methods for Evaluation of Inactivators of Anti- effectiveness of antimicrobial hand washing products used by
microbial Agents
health care personnel that are intended for frequent use, and
E2276 Test Method for Determining the Bacteria- that are intended to reduce the level of contamination acquired
Eliminating Effectiveness of Hygienic Handwash and
through contact with contaminated objects or people.
Handrub Agents Using the Fingerpads of Adults
4.2 Performance of these procedures requires the knowl-
E1838 Test Method for Determining the Virus-Eliminating
edge of regulations pertaining to the protection of human
Effectiveness of Hygienic Handwash and HandrubAgents 3
subjects (Ref 1).
Using the Fingerpads of Adults
5. Apparatus
5.1 Colony Counter—Any of several types may be used, for
This test method is under the jurisdiction of ASTM Committee E35 on
example, Quebec Colony Counter.
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
responsibility of Subcommittee E35.15 on Antimicrobial Agents. 5.2 Incubators—One incubator capable of maintaining a
Current edition approved Sept. 1, 2018. Published September 2018. Originally
temperature of 25 6 2 °C (this temperature is required to
ɛ1
approved in 1990. Last previous edition approved in 2012 as E1327 – 07(2012) .
ensure pigment production of Serratia); a second incubator
DOI: 10.1520/E1327-07R18.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on The boldface numbers in parentheses refer to a list of references at the end of
the ASTM website. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1327 − 07 (2018)
capable of maintaining 37 6 2 °C used for E. coli and B. 6.6 Agar, Tryptic soy agar or equivalent. Include the appro-
subtilis incubation is acceptable. priate inactivator if needed.
5.3 Water Bath—Capable of maintaining temperature of 80 6.7 Agar with MUG—Tryptic soy agar with 60 to 80 µg/mL
6 2 °C for heat shocking of B. subtilis spores is needed. 4-methylumbelliferyl-β-D-glucuronide (MUG) is required.
5.4 Sterilizer—Any suitable steam sterilizer capable of pro- 6.8 Test Formulations—Directions for use of test formula-
ducing the conditions of sterilization is acceptable. tion should be included if available. If these are not available,
liquid antimicrobial soap formulations are tested by same
5.5 Timer—Any stop-watch that can be read in minutes and
routine as the nonantimicrobial control (10.5); alcoholic lotion
seconds is required.
type formulations are rubbed to dryness and then sampled for
5.6 Handwashing Sink—A sink of sufficient size to permit
survivors (10.7).
panelists to wash without touching hands to sink surface or
6.9 Nonantimicrobial Control Soap, a liquid castile soap or
other panelists is needed.
other liquid soap containing no antimicrobials.
5.6.1 Water Faucet(s), to be located above the sink at a
height that permits the hands to be held higher than the elbow 6.10 Broth—Tryptic soy broth or equivalent is required.
during the washing procedure.
7. Test Organisms
5.6.2 Tap Water Temperature Regulator and Temperature
Monitor, to monitor and regulate water temperature of 40 6
7.1 Serratia marcescens AmericanType Culture Collection,
2 °C.
ATCC No. 14756 is to be used as a marker organism. This is
a strain having stable pigmentation. Grow in tryptic soy broth
5.7 Quad Petri plates, 100 by 15 mm, plastic, sterile,
at 25 6 2 °C.
disposable.
7.2 Escherichia coli, ATCC No. 11229 is used as another
5.8 Small Petri Plates, 60 by 15 mm, glass.
Gram-negative marker organism. Grow in tryptic soy broth at
5.9 Large Petri Plates, 150 by 15 mm, glass.
35 6 2 °C.
5.10 Tooth Brushes:
7.3 Bacillus subtilis, ATCC No. 19659. Grow in tryptic soy
5.10.1 Young Size.
broth at 35 6 2 °C.
5.10.2 Battery Operated.
7.4 Preparation of Spore Suspension—Inoculate each sur-
5.11 Ultraviolet Lamp, having separate short wave and long
faceoftwotrypticsoyagarplates(30mLagarin150-mmpetri
wave bulbs.
plates) with 1 mL of B. subtilis tryptic soy broth culture.
5.12 Germicidal Lamp Monitor Strips. Spread over the entire surface of the agar. Incubate for 5 to 10
days at 35 6 2 °C. Suspend the growth in 20 mL of 0.1 %
5.13 Inoculating Loops or Needles, sterile.
tryptone water by rubbing the agar surface with a sterile
5.14 Plate Spreaders or Hockey Sticks, sterile.
rubber policeman. Add ethanol to the suspension to a final
concentration of 80 % (wt/wt) and store in a refrigerator.
6. Reagents and Materials
7.5 Other bacteria containing adequate markers to enable
6.1 Bacteriological Pipettes, 10.0 mL, sterile.
distinction from normal flora and of known safety may also be
6.2 Pipettors and Pipette Tips, Eppendorf, MLA or similar used for testing purposes. (Warning—The application of
types. microorganisms to the skin may involve a health risk. Prior to
applying S. marcescens or other bacteria to the skin, the
6.3 Disposable Analyzer Cups, 2 mL, plastic, not sterile.
antibiotic susceptibility profile of the strain should be deter-
6.4 Sampling Solution—Dissolve 0.4 g KH PO , 10.1 g
2 4
mined. If the Serratia strain is not sensitive to Gentamicin, it
Na PO and 1.0 g isooctylphenoxypolyethoxyethanol in1L
2 4
should not be used. If an infection occurs, the antibiotic
distilled water. Adjust pH to 7.8 with 0.1 N HCl or 0.1 N
susceptibility profile should be made available to an attending
NaOH. Dispense in 100 mL-volumes and sterile for 20 min at
clinician. Following the panelist’s contamination and testing
121 °C.
fortheday,thepanelist’shandsshouldbedecontaminatedwith
6.5 Dilution Fluid—The sampling fluid may be used for a 70-% ethanol solution. Care should be taken to decontami-
nate around the fingernail regions.)
dilutions or use Butterfields sterile phosphate buffered water
(2) adjusted to pH 7.2 with suitable inactivator for the
7.6 Preparation of Marker Culture Suspension—Inoculate a
antimicrobial. Adjust pH with 0.1 N HCl or 0.1 N NaOH (see
10-mLtryptic soy broth tube with each of the test bacteria and
Practices E1054).
incubateeachtubeatthetemperatureindicatedtoyieldinocula
8 9
of 10 –10 CFU/mL.When studying mixed inocula, mix equal
volumes of the cultures into a sterile test tube; an equivalent
Presterilized disposable quad plastic petri plates, the two sizes of glass petri
plates and other equipment are available from most local laboratory supply houses.
5 6
The sole source of supply of the apparatus (Triton X-100) known to the The sole source of supply of the Bacto Tryptone (Difco) water known to the
committee at this time is Rohm and Haas Co., Philadelphia, PA. If you are aware of committee at this time is Difco Laboratories, Detroit, MI. If you are aware of
alternative suppliers, please provide this information to ASTM International alternative suppliers, please provide this information to ASTM International
Headquarters.Your comments will receive careful consideration at a meeting of the Headquarters.Your comments will receive careful consideration at a meeting of the
1 1
responsible technical committee, which you may attend. responsible technical committee, which you may attend.
E1327 − 07 (2018)
volume of B. subtilis spore suspension (that is prepared by analyzer cup, add 0.9 mL of sterile diluent: set up sufficient
centrifuging the alcoholic suspension and resuspending cells in cups only for each day’s testing.
water) may be added for bacterial physical removal determi-
10.2 Place 7 mL of sampling solution into each of 21 small
nations.Keepmixedsuspensiononiceduringtheday’stesting.
petri plates.
8. Panelists 10.3 Place 0.02 mL of marker culture suspension on the
region surrounding the cuticle and under the fingernails of
8.1 Recruit a sufficient number of healthy adult human
three fingers of the left hand of a volunteer. The volunteer then
volunteers who have no clinical evidence of dermatoses, open
holdsthehandinfrontofanelectricfanfor5minforcomplete
wounds, hangnails, or other skin disorders. The number of
drying of the suspension.
people needed for a trial is dependent on the number of
10.4 For unwashed hand determinations, proceed directly to
treatments within a study.
10.8.
8.2 Volunteers are asked to maintain their normal use of
10.5 When testing nonantimicrobial soap (controls), wet
soaps, shampoos, and so forth. They are asked to refrain from
bothhandsunderflowingwarmtapwater(40 62 °C).Add2.5
the use of acids, bases, solvents on the hands during the test
to 3.0 mL of the liquid soap to hands, rub hands together in
period. Gloves should be provided for use where exposure to
normal washing manner for 15 s (no additional water), then
these agents is unavoidable.
rinse under the flowing water for 15 s to remove suds. Do not
9. Experimental Design
dry hands, proceed directly to 10.8.
9.1 Each fingernail of a volunteer may be assayed sepa-
10.6 For testing liquid antimicrobial soap formulations,
rately; therefore, 10 test determinations (replicates) may be
follow the use directions on the label or follow the routine of
obtained from one volunteer. For the comparison of several
10.5. After washing, proceed to 10.8 without drying hands.
products during a single study, a design such as a Latin Square
10.7 Alcoholic formulations are tested by placing the rec-
Design may be utilized (3). For example, to compare 5
ommended volume on the hands and then rubbing the hands
antimicrobial test products, one nonantimicrobial product and
together until the alcohol has evaporated. Proceed to 10.8.
unwashed hand control (7 total variables), 7 volunteers, (or
10.8 After performing the procedure for the day designated
multiples of 7) should be recruited. Each person performs one
in the Latin Square Design, the technician scrubs with a
testing of product or other variable on each of 7 test days,
toothbrush for 1 min each fingernail into a separate petri plate
according to schedule such as the following; the num-
containing 7 mL of sampling solution.
bers = day for testing that variable (see Table 1).
9.1.1 Example: Volunteer A tests Treatment 1 on Day 1,
NOTE 1—Although manual toothbrushes may be used for this purpose,
then Treatment 2 on Day 2; Volunteer B tests Treatment 2 on
greater uniformity between scrubbings may be obtained with less operator
fatigue if an electric toothbrush such as the GE model TB-9 or another
Day 1, Treatment 3 on Day 2, and so forth.
type is used. A brush which operates parallel with the handle is preferred
9.1.2 Each product or variable is tested once on each day,
because of less splashing.
unless multiple numbers of volunteers are in the study.
10.9 After each scrubbing, the brushes are dropped into a
9.1.3 The number of fingers, which are inoculated and then
beaker containing 70 % ethyl or isopropyl alcohol and allowed
assayed after using the product, should be kept standard
to stand for at least 10 min. The brushes are then rinsed in
throughout. Although the number can be as high as 10, three
sterile distilled water and allowed to dry. The brushes are not
fingers on one hand is a more convenient and cost savings
sterilized.
approach. The ring, middle, and index fingers of the left hand
have been selected for several studies; however, an operator
10.10 Perform serial 10-fold dilutions. Place 0.1 mL
mayselectthenumberandparticularfingerstoassayaslongas
amounts of the appropriate dilutions onto the surface of agar
they are held constant throughout.
sections of quad plates. These drops of liquid are spread with
sterile inoculation loops, needle spreaders, or hockey sticks to
10. Procedure
completelycoverthequads.All
...
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E1327 − 07 (Reapproved 2018)
Standard Test Method for
Evaluation of Antimicrobial Handwash Formulations by
Utilizing Fingernail Regions
This standard is issued under the fixed designation E1327; 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 3. Summary of Test Method
3.1 This test method, involving an improved method of
1.1 This test method can be used to determine the effective-
recovering bacteria from hands, is used to study the effects of
ness of antimicrobial handwashing agents (including handrubs)
antimicrobial handwashes including health care personnel
in the reduction of transient bacterial flora with particular
handwash products. The group of volunteer panelists need not
emphasis on the fingernail region.
refrain from using topical antimicrobials (such as deodorant
1.2 A knowledge of microbiological techniques is required
soaps) before participating in the study. All subjects wash their
for these procedures.
hands with a nonantimicrobial hand soap prior to testing to
remove any residual hand lotions and to lower the numbers of
1.3 This standard does not purport to address all of the
resident skin flora. Activity of products is measured by
safety concerns, if any, associated with its use. It is the
comparing the numbers of marker bacteria recovered from
responsibility of the user of this standard to establish appro-
artificially contaminated fingernail regions after use of the
priate safety, health, and environmental practices and deter-
handwashing formulations to the numbers recovered from the
mine the applicability of regulatory limitations prior to use.
artificially contaminated but unwashed fingernail regions.
For more specific hazard statements, see 7.5.
Broth cultures of Serratia marcescens (a red pigmented bac-
1.4 This international standard was developed in accor-
terial species) and Escherichia coli (which produces fluores-
dance with internationally recognized principles on standard-
cent colonies on a special agar medium) are used as test
ization established in the Decision on Principles for the
bacteria. A spore suspension of Bacillus subtilis may be
Development of International Standards, Guides and Recom-
utilized to study (1) degree of physical removal by handwash-
mendations issued by the World Trade Organization Technical
ing techniques, and (2) the recovery and precision aspects of
Barriers to Trade (TBT) Committee.
the test method.
2. Referenced Documents
4. Significance and Use
2.1 ASTM Standards:
4.1 The procedure should be used to test the degerming
E1054 Test Methods for Evaluation of Inactivators of Anti-
effectiveness of antimicrobial hand washing products used by
microbial Agents health care personnel that are intended for frequent use, and
E2276 Test Method for Determining the Bacteria-
that are intended to reduce the level of contamination acquired
Eliminating Effectiveness of Hygienic Handwash and through contact with contaminated objects or people.
Handrub Agents Using the Fingerpads of Adults
4.2 Performance of these procedures requires the knowl-
E1838 Test Method for Determining the Virus-Eliminating
edge of regulations pertaining to the protection of human
Effectiveness of Hygienic Handwash and Handrub Agents 3
subjects (Ref 1).
Using the Fingerpads of Adults
5. Apparatus
5.1 Colony Counter—Any of several types may be used, for
This test method is under the jurisdiction of ASTM Committee E35 on
example, Quebec Colony Counter.
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
5.2 Incubators—One incubator capable of maintaining a
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Sept. 1, 2018. Published September 2018. Originally
temperature of 25 6 2 °C (this temperature is required to
ɛ1
approved in 1990. Last previous edition approved in 2012 as E1327 – 07(2012) .
ensure pigment production of Serratia); a second incubator
DOI: 10.1520/E1327-07R18.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on The boldface numbers in parentheses refer to a list of references at the end of
the ASTM website. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1327 − 07 (2018)
capable of maintaining 37 6 2 °C used for E. coli and B. 6.6 Agar, Tryptic soy agar or equivalent. Include the appro-
subtilis incubation is acceptable. priate inactivator if needed.
5.3 Water Bath—Capable of maintaining temperature of 80 6.7 Agar with MUG—Tryptic soy agar with 60 to 80 µg/mL
6 2 °C for heat shocking of B. subtilis spores is needed. 4-methylumbelliferyl-β-D-glucuronide (MUG) is required.
5.4 Sterilizer—Any suitable steam sterilizer capable of pro- 6.8 Test Formulations—Directions for use of test formula-
ducing the conditions of sterilization is acceptable. tion should be included if available. If these are not available,
liquid antimicrobial soap formulations are tested by same
5.5 Timer—Any stop-watch that can be read in minutes and
routine as the nonantimicrobial control (10.5); alcoholic lotion
seconds is required.
type formulations are rubbed to dryness and then sampled for
5.6 Handwashing Sink—A sink of sufficient size to permit
survivors (10.7).
panelists to wash without touching hands to sink surface or
6.9 Nonantimicrobial Control Soap, a liquid castile soap or
other panelists is needed.
other liquid soap containing no antimicrobials.
5.6.1 Water Faucet(s), to be located above the sink at a
height that permits the hands to be held higher than the elbow 6.10 Broth—Tryptic soy broth or equivalent is required.
during the washing procedure.
7. Test Organisms
5.6.2 Tap Water Temperature Regulator and Temperature
Monitor, to monitor and regulate water temperature of 40 6
7.1 Serratia marcescens American Type Culture Collection,
2 °C.
ATCC No. 14756 is to be used as a marker organism. This is
a strain having stable pigmentation. Grow in tryptic soy broth
5.7 Quad Petri plates, 100 by 15 mm, plastic, sterile,
at 25 6 2 °C.
disposable.
7.2 Escherichia coli, ATCC No. 11229 is used as another
5.8 Small Petri Plates, 60 by 15 mm, glass.
Gram-negative marker organism. Grow in tryptic soy broth at
5.9 Large Petri Plates, 150 by 15 mm, glass.
35 6 2 °C.
5.10 Tooth Brushes:
7.3 Bacillus subtilis, ATCC No. 19659. Grow in tryptic soy
5.10.1 Young Size.
broth at 35 6 2 °C.
5.10.2 Battery Operated.
7.4 Preparation of Spore Suspension—Inoculate each sur-
5.11 Ultraviolet Lamp, having separate short wave and long
face of two tryptic soy agar plates (30 mL agar in 150-mm petri
wave bulbs.
plates) with 1 mL of B. subtilis tryptic soy broth culture.
5.12 Germicidal Lamp Monitor Strips.
Spread over the entire surface of the agar. Incubate for 5 to 10
days at 35 6 2 °C. Suspend the growth in 20 mL of 0.1 %
5.13 Inoculating Loops or Needles, sterile.
tryptone water by rubbing the agar surface with a sterile
5.14 Plate Spreaders or Hockey Sticks, sterile.
rubber policeman. Add ethanol to the suspension to a final
concentration of 80 % (wt/wt) and store in a refrigerator.
6. Reagents and Materials
7.5 Other bacteria containing adequate markers to enable
6.1 Bacteriological Pipettes, 10.0 mL, sterile.
distinction from normal flora and of known safety may also be
6.2 Pipettors and Pipette Tips, Eppendorf, MLA or similar used for testing purposes. (Warning—The application of
types. microorganisms to the skin may involve a health risk. Prior to
applying S. marcescens or other bacteria to the skin, the
6.3 Disposable Analyzer Cups, 2 mL, plastic, not sterile.
antibiotic susceptibility profile of the strain should be deter-
6.4 Sampling Solution—Dissolve 0.4 g KH PO , 10.1 g
2 4
mined. If the Serratia strain is not sensitive to Gentamicin, it
Na PO and 1.0 g isooctylphenoxypolyethoxyethanol in 1 L
2 4
should not be used. If an infection occurs, the antibiotic
distilled water. Adjust pH to 7.8 with 0.1 N HCl or 0.1 N
susceptibility profile should be made available to an attending
NaOH. Dispense in 100 mL-volumes and sterile for 20 min at
clinician. Following the panelist’s contamination and testing
121 °C.
for the day, the panelist’s hands should be decontaminated with
a 70-% ethanol solution. Care should be taken to decontami-
6.5 Dilution Fluid—The sampling fluid may be used for
dilutions or use Butterfields sterile phosphate buffered water nate around the fingernail regions.)
(2) adjusted to pH 7.2 with suitable inactivator for the
7.6 Preparation of Marker Culture Suspension—Inoculate a
antimicrobial. Adjust pH with 0.1 N HCl or 0.1 N NaOH (see
10-mL tryptic soy broth tube with each of the test bacteria and
Practices E1054).
incubate each tube at the temperature indicated to yield inocula
8 9
of 10 –10 CFU/mL. When studying mixed inocula, mix equal
volumes of the cultures into a sterile test tube; an equivalent
Presterilized disposable quad plastic petri plates, the two sizes of glass petri
plates and other equipment are available from most local laboratory supply houses.
5 6
The sole source of supply of the apparatus (Triton X-100) known to the The sole source of supply of the Bacto Tryptone (Difco) water known to the
committee at this time is Rohm and Haas Co., Philadelphia, PA. If you are aware of committee at this time is Difco Laboratories, Detroit, MI. If you are aware of
alternative suppliers, please provide this information to ASTM International alternative suppliers, please provide this information to ASTM International
Headquarters. Your comments will receive careful consideration at a meeting of the Headquarters. Your comments will receive careful consideration at a meeting of the
1 1
responsible technical committee, which you may attend. responsible technical committee, which you may attend.
E1327 − 07 (2018)
volume of B. subtilis spore suspension (that is prepared by analyzer cup, add 0.9 mL of sterile diluent: set up sufficient
centrifuging the alcoholic suspension and resuspending cells in cups only for each day’s testing.
water) may be added for bacterial physical removal determi-
10.2 Place 7 mL of sampling solution into each of 21 small
nations. Keep mixed suspension on ice during the day’s testing.
petri plates.
10.3 Place 0.02 mL of marker culture suspension on the
8. Panelists
region surrounding the cuticle and under the fingernails of
8.1 Recruit a sufficient number of healthy adult human
three fingers of the left hand of a volunteer. The volunteer then
volunteers who have no clinical evidence of dermatoses, open
holds the hand in front of an electric fan for 5 min for complete
wounds, hangnails, or other skin disorders. The number of
drying of the suspension.
people needed for a trial is dependent on the number of
10.4 For unwashed hand determinations, proceed directly to
treatments within a study.
10.8.
8.2 Volunteers are asked to maintain their normal use of
10.5 When testing nonantimicrobial soap (controls), wet
soaps, shampoos, and so forth. They are asked to refrain from
both hands under flowing warm tap water (40 6 2 °C). Add 2.5
the use of acids, bases, solvents on the hands during the test
to 3.0 mL of the liquid soap to hands, rub hands together in
period. Gloves should be provided for use where exposure to
normal washing manner for 15 s (no additional water), then
these agents is unavoidable.
rinse under the flowing water for 15 s to remove suds. Do not
9. Experimental Design
dry hands, proceed directly to 10.8.
9.1 Each fingernail of a volunteer may be assayed sepa-
10.6 For testing liquid antimicrobial soap formulations,
rately; therefore, 10 test determinations (replicates) may be
follow the use directions on the label or follow the routine of
obtained from one volunteer. For the comparison of several
10.5. After washing, proceed to 10.8 without drying hands.
products during a single study, a design such as a Latin Square
10.7 Alcoholic formulations are tested by placing the rec-
Design may be utilized (3). For example, to compare 5
ommended volume on the hands and then rubbing the hands
antimicrobial test products, one nonantimicrobial product and
together until the alcohol has evaporated. Proceed to 10.8.
unwashed hand control (7 total variables), 7 volunteers, (or
10.8 After performing the procedure for the day designated
multiples of 7) should be recruited. Each person performs one
in the Latin Square Design, the technician scrubs with a
testing of product or other variable on each of 7 test days,
toothbrush for 1 min each fingernail into a separate petri plate
according to schedule such as the following; the num-
containing 7 mL of sampling solution.
bers = day for testing that variable (see Table 1).
9.1.1 Example: Volunteer A tests Treatment 1 on Day 1,
NOTE 1—Although manual toothbrushes may be used for this purpose,
then Treatment 2 on Day 2; Volunteer B tests Treatment 2 on greater uniformity between scrubbings may be obtained with less operator
fatigue if an electric toothbrush such as the GE model TB-9 or another
Day 1, Treatment 3 on Day 2, and so forth.
type is used. A brush which operates parallel with the handle is preferred
9.1.2 Each product or variable is tested once on each day,
because of less splashing.
unless multiple numbers of volunteers are in the study.
10.9 After each scrubbing, the brushes are dropped into a
9.1.3 The number of fingers, which are inoculated and then
beaker containing 70 % ethyl or isopropyl alcohol and allowed
assayed after using the product, should be kept standard
to stand for at least 10 min. The brushes are then rinsed in
throughout. Although the number can be as high as 10, three
sterile distilled water and allowed to dry. The brushes are not
fingers on one hand is a more convenient and cost savings
sterilized.
approach. The ring, middle, and index fingers of the left hand
have been selected for several studies; however, an operator
10.10 Perform serial 10-fold dilutions. Place 0.1 mL
may select the number and particular fingers to assay as long as
amounts of the appropriate dilutions onto the surface of agar
they are held constant throughout.
sections of quad plates. These drops of liquid are spread with
sterile inoculation loops, needle spreaders, or hockey sticks to
10. Procedure
completely cover the quads. Allow drops to completely absorb.
10.1
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