ASTM E3160-18

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: E3160 − 18
Standard Test Method for
Quantitative Evaluation of the Antibacterial Properties of
Porous Antibacterial Treated Articles
This standard is issued under the fixed designation E3160; 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 Todeterminethebactericidalorbacteriostaticproperties
E691Practice for Conducting an Interlaboratory Study to
ofporousarticlestreatedwithanactivebiocidalagent,samples
Determine the Precision of a Test Method
of porous treated materials, such as textiles or paper, are
E1054Test Methods for Evaluation of Inactivators of Anti-
inoculated with a defined suspension of microorganisms and
microbial Agents
then incubated. The changes in numbers of the bacterial
E2180Test Method for Determining the Activity of Incor-
populations on the treated article are compared with untreated
porated Antimicrobial Agent(s) In Polymeric or Hydro-
articleseitheroverdesignatedtimeortheyarecomparedtothe
phobic Materials
initial bacterial population at “zero time” for the treated article
E2149Test Method for Determining the Antimicrobial Ac-
to measure antibacterial properties.
tivity of Antimicrobial Agents Under Dynamic Contact
Conditions
1.2 This test method is used for measuring the quantitative
E2756Terminology Relating toAntimicrobial andAntiviral
antibacterialactivityofporousmaterialsthathavebeentreated
Agents
with a biocide to inhibit the growth of bacteria on the treated
E2922Guide for The Use of Standard Test Methods and
materials.Thismethodmayalsobeusedtomeasuretheability
Practices for EvaluatingAntibacterialActivity on Textiles
of the treated material to inhibit the growth of a microorgan-
2.2 AATCC (American Association of Textile Chemists and
ism.Itcanmeasurebothbactericidalandbacteriostaticactivity.
Colorists) Documents:
1.3 This test method shall be performed by individuals
AATCC TM100: Antibacterial Finishes on Textile Materi-
experienced and adept in microbiological procedures and in
als: Assessment of:
facilitiessuitableforthehandlingofthemicroorganismsunder
2.3 ISO (International Organization for Standardization)
test.
Documents:
ISO 22196Plastics – Measurement of Antibacterial Action
1.4 This test method may involve hazardous materials,
on Plastic Surfaces.
operations, and equipment. This standard does not purport to
ISO 20743Textiles – Determination of Antibacterial Activ-
address all of the safety concerns, if any, associated with its
ity of Antibacterial Finished Products
use. It is the responsibility of the user of this standard to
2.4 IBRG (International Biodeterioration Research Group)
establish appropriate safety, health, and environmental prac-
Documents
tices and determine the applicability of regulatory limitations
IBRG TEX13/005/1.0Quantitative Method for Evaluating
prior to use.
Bacterial Activity of Textiles and Porous Material and
1.5 This international standard was developed in accor-
Articles
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Development of International Standards, Guides and Recom-
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
mendations issued by the World Trade Organization Technical
Standards volume information, refer to the standard’s Document Summary page on
Barriers to Trade (TBT) Committee.
the ASTM website.
Available from American Association of Textile Chemists and Colorists
(AATCC), P.O. Box 12215, Research Triangle Park, NC 27709-2215, http://
www.aatcc.org.
1 4
This test method is under the jurisdiction of ASTM Committee E35 on Available from International Organization for Standardization (ISO), ISO
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier,
responsibility of Subcommittee E35.15 on Antimicrobial Agents. Geneva, Switzerland, http://www.iso.org.
Current edition approved April 1, 2018. Published July 2018. DOI: 10.1520/ Available from IBRG, Pale Lane, Hartley Wintney, Hants, UK RG27 8DH,
E3160–18 http://www.ibrg.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3160 − 18
3. Terminology 5.4 Very specific parameters are identified within this
method to limit any variability that may be seen between
3.1 Defintions—For definition of terms used in this test
laboratories. Identifying and clarifying potential variables
method, refer to, E2756 Standard Terminology Relating to
found in other guides or methods used in the industry will
Antimicrobial and Antiviral Agents.
allow for better reproducibility and repeatability between and
4. Summary of Test Method within laboratories.
5.5 Thistestmethodprovidesthefoundationforconducting
4.1 A liquid suspension of bacteria is applied to porous
materials both untreated and treated with antimicrobial fin- tests on porous antibacterial treated articles. Modifications of
this method that simulate intended use, durability and compat-
ishes.
ibility of the treated article should be outlined to ensure an
4.2 Samples of each treated or untreated material are inocu-
accurate assessment of antimicrobial activity with each par-
lated with a specified concentration of bacteria suspended in a
ticular biocide that substantiates end use claims made for the
solution containing a defined concentration of nutrients.
article. A list of these typical modifications and current test
4.3 The inoculated materials are then incubated under con-
methods for textiles can be found in Guide E2922.
ditions of controlled temperature and humidity for a specified
5.6 This test method is appropriate for porous materials
period of time.
such as textiles, paper, or similar porous materials. It is
4.4 After incubation, the samples are immersed in a neu-
intended to measure the antibacterial properties of such mate-
tralizersuitablefordeactivatingtheactivesubstance(s)usedto
rials. In most instances, further studies will be required to
produce the intended antimicrobial effect, and agitated to
support and substantiate actual claims being made for the
remove surviving organisms.
performance of treated materials in practice or as part of a
regulatory process.
4.5 The number of colony forming units present in the
resulting suspension is then determined using standard plate
5.7 This test method or indicated modifications may be
counting techniques.
used to determine antimicrobial activity as indicated in 5.6 or
4.6 Changes in the number of the test organism are then may be used as a routine bioassay in standard quality control
programs.
calculated in relation to the numbers present on the untreated
materials after a specific contact time or in relation to the
6. Apparatus
numbers applied (inoculum count), or both.
6.1 analytical balance—with capacity and precision of 0.01
5. Significance and Use
to 10 g 60.001, respectively, to weigh chemicals and to
5.1 Porous articles (often textiles) are often treated with
calibrate inoculum delivery volumes by pipettes.
antimicrobial agents to reduce the growth of microorganisms
6.2 biological safety cabinet—suitable for the containment
during use, in storage, or while waiting to be laundered, or
of the test organisms used.
both.Additionally, antimicrobial agents are added to reduce or
control the overall microbial growth on porous articles that
6.3 Bunsen burner—with a gas source and flame igniter.
may affect the material’s odor, visual, chemical or physical
6.4 colony counter—(optional.)
integrity, or both.
6.5 dispensers—for dispensing sterile 10-ml aliquots of
5.2 Antimicrobial textile test methods that measure the
diluent/neutralizer.
antimicrobial behavior of treated textiles do exist but they are
6.6 forceps—sterile for handling treated articles.
often specific for one type of antimicrobial agent or are
designed to or may artificially (not expected in real life)
6.7 freezers—afreezerat-20 62°Cforthestorageofmedia
promote the release of some specific antibacterial agents over
and additives.Asecond freezer at -70°C or lower to store the
others. This test method is designed to be able to measure the
stocks of test organisms (optional).
antimicrobial activity from all common antimicrobial agents
6.8 glass rods—sterile.foruseinholdingporoussamplesin
used to treat porous articles, including textiles, without giving
place. Rods should be no more than 40 mm in length.
either positive or negative bias to one type of chemistry or
6.9 gloves—sterile, disposable, for handling test items.
product over another.
6.10 hot air oven—an oven at 60 6 2°C to dry clean and
5.3 In an effort to avoid excessive use or abuse of
wrapped sterile glassware.
antimicrobial agents in the environment, it is important to
understand if untreated porous articles are susceptible to
6.11 incubator—an incubator to maintain a temperature of
microbial contamination and growth. In this test method, a
35 6 2°C and maintain a relative humidity of not less than
small amount of nutrients is added to each test sample in order
80%.
to promote some microbial growth on susceptible test samples
6.12 inoculating loops—Sterile plastic or sterilizable metal
but not enough to overwhelm potential antimicrobial agents
inoculating loops (10-µl).
that may be effective in real life situations. Furthermore, low
levelsofnutrientsallowinvestigatorstoaddsoilingagentsthat 6.13 magnetic stir plate and stir bars—large enough for a
may be more reflective of a specific treated product’s end use 5-L beaker or Erlenmeyer flask for preparing culture media or
or expected performance. other solutions.
E3160 − 18
6.14 sterile (plastic) or sterilizable petri dishes—100×15 7.3 Neutralizing Broth, appropriate for the antimicrobial
mm for microbial growth and recovery media. compound tested (See Practice E1054).
6.15 pH meter—having an accuracy of 60.1 pH units to
7.4 Suspension Medium, 1/500 TSB plus wetting agent
measurepHofmediaandsuspensions.Apunctureelectrodeor
(1/500 TSB). Dilute the TSB (7.1) 1:500 with distilled or
aflatmembraneelectrodeshouldbeusedformeasuringthepH
deionized water plus 0.05% v⁄v Triton X-100 and then adjust
of agar media. the pH to a value between 6.8 and 7.2 with either sodium
hydroxideorhydrochloricacid.Sterilizebyautoclavingat121
6.16 sterile or sterilizable pipette and tips (electronic or
62 ºC. If it is not used immediately after preparation, store it
non-electronic positive displacement)—100 to 1000-µl pipette
at 4 6 2°C.
and appropriate pipette tips fitted with “plungers” that can
dispense accurately 200-µl.
7.5 Sterile Distilled or Deionized Water.
6.17 refrigerator—4 6 2°C; for storage of media, culture
7.6 Ethyl Alcohol, for forcep sterilization.
plates and reagents.
8. Test Organism
6.18 sterile or sterilizable serological pipettes—reusable or
single-use pipettes of 1.0, 5.0 and 10.0-ml capacity (optional).
8.1 Escherichia coli, American Type Culture Collection
6.19 sterilizer (autoclave)—any steam sterilizer suitable for (ATCC) No. 25922.
processing culture media, reagents and labware; the steam
8.1.1 Cultures of the test organism should be maintained
supplied to the sterilizer should be free from additives toxic to
according to good microbiological practice and checked for
the test organisms.
purity on a routine basis. Consistent and accurate testing
requires maintenance of a pure, uncontaminated test culture.
6.20 sterile or sterilizable vials or tubes for dilution—
Avoidcontaminationbyuseofgoodsteriletechniqueinplating
suitable to hold 30-ml easily.
and transferring. Avoid mutation or reversion by strict adher-
6.21 sterile containers for sample inoculation and
ence to monthly stock transfers. Check culture purity by
incubation—4 oz, screw-on lid, sterile, disposable and indi-
making streak plates periodically, observing for colonies char-
vidually sealed specimen cups with a bottom surface diameter
acteristic of Escherichia coli, Gram-staining or performing
of 45 mm have been shown to be suitable.
other forms of microbial identification.
6.22 vortex mixer—to mix the cell suspensions and neutral-
NOTE 2—This method was developed and validated using ATCC No.
izer suspensions to ensure efficient recovery of the test organ-
25922 as the test organism. If an alternative culture is used, the results
ism(s).
mustbereportedashavingbeenobtainedusinga modifiedtestmethod. E.
coli was chosen for this method due to its proven reproducibility in initial
6.23 water bath—capable of reaching and maintaining a
laboratory ring tests. If the test method is modified in any way, the report
temperature of 45 6 2ºC to keep agar media from solidifying
must also include a detailed description of all modifications made,
when making culture plates.
including, but not limited to: test organisms, media, buffer, bacterial
concentration, etc.
6.24 incubator shaker—an orbital incubator shaker capable
of agitating broth cultures of bacteria and able to maintain a
9. Preparation of Bacterial Inoculum
temperature of 35 6 2°C.
9.1 Grow a fresh overnight (18-24 h) culture of E. coli in
6.25 test validity control substrate—a suitable control ma-
sterile 100% TSB at 35 62°C prior to performing the test on
terial has been found to be a cellulosic filter paper such as
an orbital incubator shaker allowing for maximum aeration of
Whatman #4 or any textile substrate shown to promote at least
culture. This culture should originate from an 18-24 h growth
1 log CFU/g of bacterial growth under the parameters outlined
coming from stock culture plates or growth on agar slants.
below.
NOTE 1—Sterilize all laboratory ware and equipment as appropriate.
9.2 The fresh overnight culture is then diluted with suspen-
Sterilization can be achieved by moist heat in an autoclave, by dry heat in
sion medium (7.4), as appropriate to obtain a bacterial concen-
a hot-air oven or other appropriate, validated sterilization process. Many
tration that is between 2.5 × 10 colony forming units
of the consumable items used in this guideline can be purchased
(CFUs)/mland1.0×10 CFUs/mL,withatargetconcent
...