ASTM E3152-23

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: E3152 − 23
Standard Guide for
Standard Test Methods and Practices Available for
Determining Antifungal Activity on Natural or Synthetic
Substrates Treated with Antimicrobial Agents
This standard is issued under the fixed designation E3152; 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 D4300 Test Methods for Ability of Adhesive Films to
Support or Resist the Growth of Fungi
1.1 This guide provides information on various test methods
D4445 Test Method for Fungicides for Controlling Sapstain
currently available to assess antifungal activity on natural or
and Mold on Unseasoned Lumber (Laboratory Method)
synthetic substrates.
D4576 Test Method for Mold Growth Resistance of Wet
1.2 Knowledge of microbiological techniques is required
Blue and Wet White
for the practice of this guide.
D4783 Test Methods for Resistance of Adhesive Prepara-
1.3 This standard does not purport to address all of the
tions in Container to Attack by Bacteria, Yeast, and Fungi
safety concerns, if any, associated with its use. It is the
D5259 Test Method for Isolation and Enumeration of En-
responsibility of the user of this standard to establish appro-
terococci from Water by the Membrane Filter Procedure
priate safety, health, and environmental practices and deter-
D5590 Test Method for Determining the Resistance of Paint
mine the applicability of regulatory limitations prior to use.
Films and Related Coatings to Fungal Defacement by
1.4 This international standard was developed in accor-
Accelerated Four-Week Agar Plate Assay
dance with internationally recognized principles on standard-
D6329 Guide for Developing Methodology for Evaluating
ization established in the Decision on Principles for the
the Ability of Indoor Materials to Support Microbial
Development of International Standards, Guides and Recom-
Growth Using Static Environmental Chambers
mendations issued by the World Trade Organization Technical
D6469 Guide for Microbial Contamination in Fuels and Fuel
Barriers to Trade (TBT) Committee.
Systems
D6974 Practice for Enumeration of Viable Bacteria and
2. Referenced Documents
2 Fungi in Liquid Fuels—Filtration and Culture Procedures
2.1 ASTM Standards:
D7436 Classification System for Unfilled Polyethylene Plas-
C1338 Test Method for Determining Fungi Resistance of
tics Molding and Extrusion Materials with a Fractional
Insulation Materials and Facings
Melt Index Using ISO Protocol and Methodology
D2020 Test Methods for Mildew (Fungus) Resistance of
D7584 Test Method for Evaluating the Resistance of the
Paper and Paperboard (Withdrawn 2009)
Surface of Wet Blue and Wet White to the Growth of
D3273 Test Method for Resistance to Growth of Mold on the
Fungi in an Environmental Chamber
Surface of Interior Coatings in an Environmental Cham-
ber D7855/D7855M Test Method for Determination of Mold
D3456 Practice for Determining by Exterior Exposure Tests Growth on Coated Building Products Designed for Inte-
the Susceptibility of Paint Films to Microbiological Attack
rior Applications Using an Environmental Chamber and
Indirect Inoculation
D7910 Practice for Collection of Fungal Material From
This guide is under the jurisdiction of ASTM Committee E35 on Pesticides,
Surfaces by Tape Lift
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
E1326 Guide for Evaluating Non-culture Microbiological
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved April 1, 2023. Published April 2023. Originally
Tests
approved in 2018. Last previous edition approved in 2018 as E3152 – 18. DOI:
E2111 Quantitative Carrier Test Method to Evaluate the
10.1520/E3152-23.
2 Bactericidal, Fungicidal, Mycobactericidal, and Sporicidal
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
Potencies of Liquid Chemicals
Standards volume information, refer to the standard’s Document Summary page on
E2197 Quantitative Disk Carrier Test Method for Determin-
the ASTM website.
3 ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,
The last approved version of this historical standard is referenced on
www.astm.org. and Sporicidal Activities of Chemicals
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3152 − 23
E2471 Test Method for Using Seeded-Agar for the Screen- EPA Pesticide Assessment Guidelines – Subdivision G Prod-
ing Assessment of Antimicrobial Activity In Carpets uct Performance, Section 93-30
E2756 Terminology Relating to Antimicrobial and Antiviral
Agents 3. Terminology
E3227 Test Practice for Qualitative Assessment of Antifun-
3.1 Definitions:
gal Activity on Textiles
3.1.1 For definitions of terms used in this guide, refer to
F1094 Test Methods for Microbiological Monitoring of
Terminology E2756.
Water Used for Processing Electron and Microelectronic
Devices by Direct Pressure Tap Sampling Valve and by
4. Significance and Use
the Presterilized Plastic Bag Method
4.1 Fungi are known to produce objectionable odors, stains,
G21 Practice for Determining Resistance of Synthetic Poly-
and premature biodeterioration of various consumer products
meric Materials to Fungi
and construction substrates including textiles, carpet, ceiling
2.2 AATCC Standards:
tile, gypsum wallboard, lumber, and plasticized vinyl and other
AATCC 30 Antifungal Activity, Assessment on Textile Ma-
polymers.
terials: Mildew and Rot Resistance of Textile Materials
AATCC TM 90 2016 Antimicrobial Activity Assessment of
4.2 Antifungal activity is typically:
Textile Materials: Agar Plate Method
4.2.1 Determination of article susceptibility to fungal
AATCC 174 (Part III)-2016 Antimicrobial Activity Assess-
colonization,
ment of Carpets
4.2.2 Determination of fungistatic activity (qualitative de-
2.3 AWPA Standards:
termination of prevented or delayed fungal colonization), and
AWPA E10-11 Standard Method of Testing Wood Preserva-
4.2.3 Determination of fungicidal/sporicidal activity (quan-
tives by Laboratory Soil-Block Cultures
titative determination of spore kill).
AWPA E24-15 Standard Method of Evaluating the Resis-
4.3 The degree of required surface examination varies from
tance of Wood Product Surfaces to Mold Growth
gross visual examination to detailed microscopic assessment
2.4 BSi Standards:
among these methods.
BS 3900:Part G6:1989 British Standard Methods of test for
4.4 This guide provides an overview of established methods
paints Part G6. Assessment of resistance to fungal growth
and suggestions for their applicability, with consideration to
BS EN 113:1997 Wood preservatives – Test method for
the type of substrate treated or the type of antifungal treatment
determining the protective effectiveness against wood
being assessed.
destroying basidiomycetes – Determination of the toxic
values
5. Methods Overview
BS EN 1104:2005 Paper and Board intended to come into
contact with foodstuffs – Determination of the transfer of
ASTM Standards
antimicrobial constituents
5.1 C1338 Test Method for Determining Fungi Resistance
2.5 ISO Standards:
of Insulation Materials and Facings (Qualitative measure of
ISO 846 Evaluation of the Action of Microorganisms on
susceptibility and/or fungistatic activity)
Plastics
5.1.1 Scope—This test method covers the determination of
ISO 16000 Indoor Air Sampling Strategy for Moulds
the ability of new insulation materials and their facings to
ISO 16256 Clinical Laboratory Testing and in-vitro diagnos-
support fungal growth.
tic test systems – Reference method for testing the in vitro
5.1.2 Significance and Use:
activity of antimicrobial agents against yeast fungi in-
5.1.2.1 The type of materials used in the manufacture of
volved in infectious disease
insulation products and the type of membrane used to face
2.6 JIS Standards:
these products can sometimes affect fungi sustenance in the
JIS K 1571:2010 Test methods for determining the effective-
presence of high humidity.
ness of wood preservatives and their performance require-
5.1.2.2 This test method is used to determine the relative
ments
ability of an insulation and its facing to support or resist fungal
2.7 Other Standards:
growth under conditions favorable for their development.
Ford Motor Company Specification
5.1.2.3 This test method uses a comparative material to
MIL-STD-810G Method 508.6 Fungus
determine the relative ability of a material to support fungal
TAPPI T-487 Fungus Resistance of Paper and Paperboard
growth. In some specialized product areas, it is required that no
growth takes place. In such cases, the use of the comparative
Available from American Association of Textile Chemists and Colorists
material is omitted and the pass/fail criterion is based upon
(AATCC), P.O. Box 12215, Research Triangle Park, NC 27709-2215, http://
growth.
www.aatcc.org.
Available from American Wood Protection Association (AWPA), P.O. Box
361784, Birmingham, AL 35236-1784, http://www.awpa.com.
Available from British Standards Institution (BSI), 389 Chiswick High Rd.,
London W4 4AL, U.K., http://www.bsigroup.com. Available from United States Environmental Protection Agency (EPA), William
Available from American National Standards Institute (ANSI), 25 W. 43rd St., Jefferson Clinton Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460,
4th Floor, New York, NY 10036, http://www.ansi.org. http://www.epa.gov.
E3152 − 23
5.2 D2020 Test Methods for Mildew (Fungus) Resistance of 5.3.3 Summary of Method—This method is favored for
Paper and Paperboard creating environmental conditions that are conducive for mold
growth. Use of potting soil along with a mixed fungal spore
5.2.1 Scope—These test methods cover the qualitative de-
inoculum mimics exposures in soiled and humid environments.
termination of mildew (fungus) resistance of paper and
5.3.3.1 Typical industry modifications to this method in-
paperboard, particularly those types which have been given a
clude evaluation of a variety of substrates beyond interior
fungus resistant treatment.
coatings. These include wood, ceiling tile, gypsum wall board,
5.2.2 Significance and Use—Paper products used or stored
fabrics and carpet.
in damp warm atmospheres or in contact with damp soil are
5.3.3.2 This method is useful for identification of mold
subject to attack by fungus and other microorganisms. These
susceptible components of a product.
test methods cover procedures for evaluating the degree and
5.3.3.3 The environmental chamber creates an environment
permanency of protection to attack by paper treatments.
that poses a “worst case scenario” for an incorporated antifun-
5.2.3 Summary of the Practice—This test includes two test
gal agent. The method also may be useful in assessing
methods which can be used singly or in combination. Method
durability of such treatments.
A involves direct inoculation of pure test cultures on non-
sterile specimens. Method B involves burying test samples in
5.4 D3456 Practice for Determining by Exterior Exposure
direct contact with soil. Tests the Susceptibility of Paint Films to Microbiological
Attack (qualitative assessment of microbiological disfigure-
5.2.3.1 Method A is an accelerated screen of both suscepti-
ment of exterior paint films; mold, bacterial or algal)
bility and fungistatic activity. Samples are placed on plates of
5.4.1 Scope—This practice provides guidelines for deter-
mineral-salt agar (nutrient salts agar) and tested against Asper-
mining the susceptibility of paint films to microbiological
gillus niger, Aspergillus terreus, and Chaetomium globosum.
attack on exterior exposure. While it is recognized that various
5.2.3.2 Assessment of the samples is performed at least
organisms may occur on an exposed coating, the specific types
once prior to seven days of incubation and again after seven
of organisms are mainly of academic interest. The degree to
days of incubation. If no growth is observed on specimens after
which microbiological discoloration occurs is the primary
seven days the samples are incubated an additional week.
concern.
(1) Samples are rated as fungal resistant, not fungal
5.4.2 Summary of the Practice—Simple observation of a
resistant, or moderately fungal resistant.
coated object subjected to exterior exposure is considered a
5.2.3.3 Method B is an accelerated screen where samples
practical and reliable method for determining the degree that
are buried in soil for two weeks. Samples are rated after burial
microorganisms discolor the coating. However, this applies to
the samples are removed, cleaned, dried, and tensile breaking
a specific coated object exposed under a given set of condi-
strength determined.
tions. It should be recognized that there are critical factors that
5.3 D3273 Test Method for Resistance to Growth of Mold
influence the amount of fungal growth that may occur on the
on the Surface of Interior Coatings in an Environmental
same coated object when exposed to other conditions. These
Chamber (Qualitative measure of susceptibility and fungistatic
factors include the geographic location, local atmospheric
activity)
conditions such as the dust and pollen content of the air, angle
5.3.1 Scope—This test method describes a small environ- of exposure, degree to which the coating is subjected to
mental chamber and the conditions of operation to evaluate
weathering, effects of moisture and sunlight, the substrates on
reproducibly in a 4-week period the relative resistance of paint which the coating is applied, and the coatings in the paint
films to surface mold fungi, mildew growth in a severe interior system under test. The latter factor includes the stability of the
environment. The apparatus is designed so it can be easily built coating while packaged in the container, as well as the
of obtained by any interested party. composition of the coatings included in the total system and the
thickness of each coating applied. Thus, while microorganisms
5.3.1.1 This test method can be used to evaluate the com-
occur on the surface of the last film applied, the degree of
parative resistance of interior coating to accelerated mildew
microbiological growth that will occur is also influenced by the
growth. Performance at a certain rating does not imply any
composition of the undercoats. All the above factors should be
specific period of time for a fungal free coating. However, a
considered in the selection of a coating resistant to discolor-
better rated coating nearly always performs better in actual end
ation by microorganisms.
use.
5.3.1.2 Temperature and humidity must be effectively con-
5.5 D4300 Test Method for Ability of Adhesive Films to
trolled within the relatively narrow limits specified in order for
Support or Resist the Growth of Fungi
the chamber to function reproducibly during the short test
5.5.1 Scope—These test methods test the ability of adhesive
period. Severity and rate of mold growth on a film is a function
films to inhibit or support the growth of selected fungal species
of the moisture content of both the film and the substrate.
growing on agar plates by providing means of testing the films
on two agar substrates, one which promotes microbial growth,
5.3.2 Significance and Use—An accelerated test for deter-
mining the resistance of interior coatings to mold growth is and one which does not.
useful in estimating the performance of coatings designed for 5.5.1.1 These test methods are not appropriate for all
use in interior environments that promote mold growth and in adhesives. The activity of certain biocides may not be demon-
evaluating compounds that may inhibit such growth and the strated by these test methods as a result of irreversible reaction
aggregate levels for their use. with some of the medium constituents.
E3152 − 23
5.5.1.2 A test method is included for use with low-viscosity mine on which medium and under what conditions it will grow,
adhesives along with an alternative method for use with in order to demonstrate the efficacy of the biocide.
mastic-type adhesives. Also, a method approved by the gov- 5.8.2.3 The results obtained when using the procedures
ernment is given. given in these methods apply only to the species which are
used for the testing. The test species listed in Section 9 (of the
5.5.2 Significance and Use—These test methods are de-
signed to be used to determine the susceptibility of the method) are frequently used by laboratories to test for antimi-
crobial properties, but they are not the only ones which could
adhesive film to biodegradation and whether the adhesive will
carry into the bond line sufficient anti-fungal properties to be used. Selection of the species to use for these test methods
requires informed judgment by the testing laboratory or by the
prevent growth of fungi frequently present on the gluing
equipment, on adherents, or in the adhesive as applied. party requesting the tests. It is also important that species
which commonly attack adhesives be used.
5.6 D4445 Test Method for Fungicides for Controlling
5.9 D5590 Test Method for Determining the Resistance of
Sapstain and Mold on Unseasoned Lumber (Laboratory
Paint Films and Related Coatings to Fungal Defacement by
Method)
Accelerated Four-Week Agar Plate Assay (Qualitative measure
5.6.1 Scope—This (laboratory) method is used for determin-
of susceptibility and fungistatic activity)
ing the minimum concentration of fungicide, or formulation of
5.9.1 Scope—This test method covers an accelerated
fungicides, that is effective in preventing biodeterioration by
method for determining the relative resistance of two or more
sapstain fungi and molds in selected species of wood under
paints or coating films to fungal growth.
optimum laboratory conditions.
5.9.2 Significance and Use—Defacement of paint and coat-
5.6.2 Significance and Use—This method is useful as a
ing films by fungal growth (mold, mildew) is a common
screening procedure for selecting fungicides or formulations
phenomenon, and defacement by algal growth can also occur
for more rigorous field evaluation.
under certain conditions. It is generally known that differences
5.7 D4576 Test Method for Mold Growth Resistance of Wet
in the environment, lighting, temperature, humidity, substrate
Blue and Wet White
pH, and other factors in addition to the coating composition
5.7.1 Scope—This method covers the determination of mold
affect the susceptibility of a given painted surface. This test
growth resistance of Wet Blue and Wet White subject to storage
method attempts to provide a means to comparatively evaluate
and shipping requirements and intended for use in leather
different coating formulations for their relative performance
manufacturing.
under a given set of conditions. It does not imply that a coating
5.7.2 Significance and Use—This method provides a tech-
that resists growth under these conditions will necessarily resist
nique for evaluating mold growth resistance characteristics of
growth in the actual application.
Wet Blue and Wet White, and should assist in the prediction of
5.9.3 Summary of Method—This test is an accelerated
storage time before molding occurs.
screen of both susceptibility and fungistatic activity. Use of
5.7.3 Summary of Method—Conclusions about mold growth
potato dextrose agar provides rapid growth conditions for a
resistance are drawn from comparisons of the test materials
mixed spore challenge of Aspergillus and Penicillium as well
with previously run controls of know resistance.
as a challenge plate for the slower growing mold Aureoba-
sidium.
5.8 D4783 Test Methods Resistance of Adhesive Prepara-
5.9.3.1 Weekly assessment for four weeks provides data on
tions in Container to Attack by Bacteria, Yeast, and Fungi
the susceptibility and or fungistatic activity of a treated paint or
5.8.1 Scope—The test methods cover the determination of
coating in laboratory growth conditions.
the resistance of liquid adhesive preparations to microbial
attack in the container by challenging adhesive specimens with
5.10 D6329 Guide for Developing Methodology for Evalu-
cultures of bacteria, yeast, or fungi, and checking for their
ating the Ability of Indoor Materials to Support Microbial
ability to return to sterility. These test methods return qualita-
Growth Using Static Environmental Chamber
tive results.
5.10.1 Scope—Many different types of microorganisms (for
5.8.2 Significance and Use—These test methods are used to
example, bacteria, fungi, viruses, algae) can occupy indoor
demonstrate whether an adhesive preparation is sufficiently
spaces. Materials that support microbial growth are potential
protected with biocide to resist attack by bacteria, yeast, and
indoor sources of biocontaminants (for example, spores and
fungi during its storage life. They are patterned after methods
toxins) that can become airborne indoor biopollutants. This
used by biological laboratories serving the adhesive industry.
guide describes a simple, relatively cost effective approach to
5.8.2.1 These test methods may also be used to determine evaluating the ability of a variety of materials to support
the efficacy of different biocide systems against specific mi- microbial growth using a small chamber method.
croorganisms.
5.10.1.1 This guide is intended to assist groups in the
5.8.2.2 These test methods are especially useful when tested development of specific test methods for a definite material or
groups of material.
against wild-type microorganisms which have been isolated
from contaminated adhesives as an aid in determining the 5.10.1.2 Static chambers have certain limitations. Usually,
amount and type of biocide necessary to kill or inhibit the only small samples of indoor materials can be evaluated. Care
growth of the contaminants. If an isolated microorganism not must be taken that these samples are representative of the
generally used as a challenge organism, is chosen as the materials being tested so that a true evaluation of the material
inoculum, it is important to identify the organism and deter- is performed.
E3152 − 23
5.10.1.3 Static chambers provide controlled laboratory mi- condition monitoring effort that includes other test parameters,
croenvironment conditions. These chambers are not intended in accordance with Guide D6469.
to duplicate room conditions, and care must be taken when
5.11.1.4 This practice offers alternative options for deliver-
interpreting the results. Static chambers are not a substitute for
ing fuel sample microbes to the filter membrane, volumes or
dynamic chambers or field studies.
dilutions filtered, growth media used to cultivate fuel-borne
5.10.1.4 A variety of microorganisms, specifically bacteria
microbes, and incubation temperatures. This flexibility is
and fungi, can be evaluated using these chambers. This guide
offered to facilitate diagnostic efforts. When this practice is
is not intended to provide human health effect data. However,
used as part of a monitoring program, a single procedure
organisms of clinical interest, such as those described as
should be used consistently.
potentially allergenic, may be studied this approach.
5.11.2 Significance and Use—Biodeteriogenic microbes in-
5.10.2 Significance and Use—The static chambers have
fecting fuel systems typically are most abundant within slime
several different applications.
accumulations on system surfaces or at the fuel-water interface
5.10.2.1 The static chambers can be used to compare the
(Guide D6469). However, it is often impractical to obtain
susceptibility of different materials to the colonization and
samples from these locations within fuel systems. Although the
amplification of various microorganisms under defined condi-
numbers of viable bacteria and fungi recovered from fuel-
tions.
phase samples are likely to be several orders of magnitude
5.10.2.2 Chambers operated at high relative humidity’s may
smaller than those found in water-phase samples, fuel-phase
be used to perform worst case scenario screening tests on
organisms are often the most readily available indicators of
materials by providing an atmosphere where environmental
fuel and fuel system microbial contamination.
conditions may be favorable for microbial growth.
5.11.2.1 Growth Medium Selectivity—Guide E1326 dis-
5.10.2.3 Use of multiple chambers with different environ-
cusses the limitations of growth medium selection. Any me-
mental parameters, such as a range of relative humidity’s,
dium selected will favor colony formation by some species and
permits the evaluation of multiple microenvironments and
suppress colony formation by others. As noted, physical,
allows investigation of materials under differing environmental
chemical and physiological variables can affect viable cell
conditions.
enumeration test results. Test Method D7436-16 provides a
5.10.2.4 Drying requirements for wetted materials may also
non-culture means of quantifying microbial biomass in fuels
be investigated. This information may be relevant for deter-
and fuel associated water.
mining material resistance to microbial growth after becoming
5.11.2.2 Since a wide range of sample sizes, or dilutions
wet. These conditions may simulate those where materials are
thereof, can be analyzed by the membrane filter technique (Test
subjected to water incursion through leaks as well as during
Methods D5259 and F1094), the test sensitivity can be adjusted
remediation of a building after a fire.
for the population density expected on the sample.
5.10.2.5 Growth rates of microorganisms on the material
5.11.2.3 Enumeration data should be used as part of diag-
may also be investigated. Once it has been established that
nostic efforts or routine condition monitoring programs. Enu-
organisms are able to grow on a particular material under
meration data should not be used as fuel quality criteria.
defined conditions, investigations into the rate of organism
growth may be performed. These evaluations provide base line
5.12 D7855/D7855M Test Method for Determination of
information and can be used to evaluate methods to limit or
Mold Growth on Coated Building Products Designed for
contain amplification of microorganisms.
Interior Applications Using an Environmental Chamber and
Indirect Inoculation
5.11 D6974 Practice for Enumeration of Viable Bacteria and
Fungi in Liquid Fuels-Filtration and Culture Procedures 5.12.1 Scope—This test method covers an environmental
5.11.1 Scope—This practice covers a membrane filter (MF)
chamber and the conditions of operation to evaluate in a
procedure for the detection and enumeration of Heterotrophic 4-week period the relative resistance to mold growth and
bacteria (HPC) and fungi in liquid fuels with kinematic
microbial surface defacement on coated building products
2 -1
viscosities ≤ 24 mm s at ambient temperature. designed for interior application using an indirect inoculation
5.11.1.1 This quantitative practice is drawn largely from IP
method. The apparatus is designed so it can be easily built or
Method 385 and Test Method D5259-14. obtained by any interested party.
5.11.1.2 This test may be performed either in the field or in
5.12.1.1 This test method can be used to evaluate the
the laboratory.
comparative resistance of coated building products to acceler-
5.11.1.3 The ability of individual microbes to form colonies
ated mold growth. Ratings do not imply a specific time period
on specific growth media depends on the taxonomy and
that the coated building product will be free of fungal growth
physiological state of the microbes to be enumerated, the
during installation in an interior environment.
chemistry of the growth medium, and incubation conditions.
5.12.1.2 This test method is not intended for use in the
Consequently, test results should not be interpreted as absolute
evaluation of public health claims.
values. Rather they should be used as part of a diagnostic or
5.12.1.3 This test method is intended for the accelerated
evaluation of mold growth on a coated building product
designed for interior use. This method is not intended for
F. Passman, Ed., Fuel and Fuel System Microbiology: Fundamentals,
evaluation of surfaces designed exterior applications or un-
Diagnosis, and Contamination Control, MNL47-EB, ASTM International, West
Conshohocken, PA, 2003, https://doi.org/10.1520/MNL47-EB coated surfaces. Use of this test method for evaluating exterior
E3152 − 23
performance has not been validated, nor have the limitations 5.14.2.4 This practice may help supplement consistency in
for such use been determined. mold sample during an indoor air quality investigation.
5.12.2 Significance and Use—An accelerated test for deter-
5.15 ASTM E2111 Quantitative Carrier Test Method to
mining the resistance of interior coated building products to
Evaluate the Bactericidal Fungicidal or Mycobactericidal, and
mold growth is useful in estimating the relative performance
Sporicidal Potencies of Liquid Chemicals
for use in interior environments under conditions favorable to
5.15.1 Scope—This test method is designed for use in
fungal growth.
product development and for the generation of product potency
5.12.3 Static or environmental chambers provide controlled
data. This test method permits the loading of each carrier with
laboratory micro-environment conditions. These chambers are
a known volume of the test organism. The incorporation of
not intended to duplicate room conditions, and care must be
controls can also determine the initial load of colony forming
taken when interpreting the results. Static chambers are not a
units (CFU) of organisms on the test carriers and any loss in
substitute for dynamic chambers or field studies.
CFU after the mandatory drying of the inoculum.
5.13 D7584 Standard Test Method for Evaluating the Re-
5.15.1.1 This test method is designed to have survivors and
sistance of the Surface of Wet Blue and Wet White on the
also to be used with a performance standard. The surviving
Growth of Fungi in an Environmental Chamber
microorganisms on each test carrier are compared to the mean
5.13.1 Scope—This environmental chamber method mea-
of no less than three control carriers to determine if the
sures the resistance of the treated Wet Blue and Wet White to
performance standard has been met. To allow proper statistical
the germination of spores and subsequent vegetative growth
evaluation of results, the size of the test inoculum should be
over a period of four weeks. The test method is useful in
sufficiently large to take into account both the performance
estimating the performance of fungicides and should assist in
standard of 6-log reduction in the viability titer of the test
the prediction of storage time of Wet Blue and Wet White
organism used, and an inoculum size of 10 CFU, then
before fungal growth begins. The apparatus is designed so it
theoretically a maximum of ten survivors per carrier is permit-
can be easily built or obtained by any interested party and
ted; however, because of experimental variability, the exact
duplicate the natural environment in which Wet Blue and Wet
target may need to be higher than 10 CFU/carrier, thus fewer
White is inoculated with fungal spores. Spores that germinate
survivors would be permitted.
on untreated or treated Wet Blue and Wet White can produce
5.15.2 Significance and Use—This test method is fully
fungal growth, resulting in disfigurement or discoloration, or
both, of the Wet Blue and Wet White. quantitative and it also avoids any loss of viable organisms
through wash off. This makes it possible to produce statistically
5.14 D7910 Practice for Collection of Fungal Material from
valid data using many fewer test and control carriers than other
Surfaces by Tape Lift
quantitative methods based on most probable number (MPN).
5.14.1 Scope—This practice describes the protocols for
5.15.2.1 The design of the carriers makes it possible to place
collection of surface samples using tape lifts and their delivery
into each a precisely measured volume of the test suspension.
to the laboratory.
The use of the threaded stir bars allows for efficient recovery of
5.14.1.1 The purpose of this practice is to support the field
the inoculum even after is exposure for several hours to strong
investigator in differentiating fungal materials from non-fungal
fixatives such as glutaraldehyde.
material such as scuffs, soot deposits, stains, pigments, dust,
5.15.2.2 The membrane filtration step allows processing of
efflorescence, adhesives, and water stains.
the entire eluate from the test carriers and therefore the capture
5.14.1.2 The samples collected by this practice are appro-
and subsequent detection of even low numbers of viable
priate for either qualitative or quantitative analysis by direct
organisms that may be present.
microscopy.
5.15.2.3 This test can be performed with or without a soil
5.14.1.3 This practice does not address the development of
load to determine the effect of such loading on microbicide
a formal hypothesis or the establishment of sampling objec-
tives. performance. The soil load developed for this test is a mixture
of three types of proteins (high molecular weight proteins, low
5.14.2 Significance and Use—This practice defines a con-
molecular weight proteins, and mucous material) to represent
sistent procedure for collecting surface material using clear,
the body secretions, excretions, or other extraneous substances
transparent, single sided adhesive collection medium, typically
that chemical microbicides may encounter under field condi-
tape (also known as tape lift).
tions. It is suitable for working with the various test organisms
5.14.2.1 A tape lift sample collected according to this
included here. The components of the soil load are readily
practice is intended to be used to assess the material present at
available and subject to much less variability than animal sera.
one specific location on a surface for fungal content.
5.14.2.2 A tape lift sample collected from a point of interest 5.15.2.4 Since the quality of tap water varies considerably
can be used for qualitative analysis or to quantify fungal both geographically and temporally, this test method incorpo-
material per sample or per unit area. Note that the recovery rates the use of water with a specified and documented level of
efficiency of material from the surface sampled is unknown and hardness to prepare use-dilutions of test products. The U.S.
a likely source of uncertainty for quantitative analyses.
Environmental Protection Agency’s Scientific Advisory Panel
(SAP) on Germicide Test Methodology has recommended the
5.14.2.3 A tape lift sample collected according to this
practice can be analyzed by direct microscopy. use of water with a standard hardness of 400 ppm of CaCO .
E3152 − 23
5.16 E2197 Quantitative Disc Carrier Test Method for included here. The components of the soil load are readily
Determining Bactericidal, Virucidal, Fungicidal, available and subject to much less variability than animal sera.
Mycobactericidal, and Sporicidal Activities of Chemicals
5.16.2.6 If distilled water or other diluent is not to be
specified on the product label, the diluent for the test substance
5.16.1 Scope—This test method is designed to evaluate the
ability of test substances to inactivate vegetative bacteria, is assumed to be tap water. Since the quality of tap water varies
considerably both geographically and temporally, this test
viruses, fungi, mycobacteria, and bacterial spores on disk
carriers of brushed stainless steel that represent hard, nonpo- method incorporates the use of water with a specified and
documented level of hardness to prepare use-dilutions of test
rous environmental surfaces and medical devices. It is also
designed to have survivors that can be compared to the mean substance that require dilution in water before use. While water
with a hardness of at least 300 ppm as CaCO is recommended
of no less than three control carriers to determine if the
performance standard has been met. For proper statistical consult local regulations regarding use of hard water prior to
testing.
evaluation of the results, the number of viable organisms in the
test inoculum should be sufficiently high to take into account
5.17 E2471 Test Method Using Seeded-Agar for the Screen-
both the performance standard and the experimental variations
ing Assessment of Antimicrobial Activity in Carpets
in the results.
5.17.1 Scope—This test method is designed to evaluate
5.16.1.1 The test protocol does not include and wiping or
(qualitatively) the presence of antimicrobial activity in or on
rubbing action. It is, therefore, not designed for testing any
carpets. Use this method to qualitatively evaluate both antibac-
wipes.
terial and antifungal activity.
5.16.2 Significance and Use—The design of this test elimi-
5.17.1.1 Use half strength (nutrient and agar) tryptic soy
nates any loss of viable organisms through wash off, thus
agar as the inoculum vehicle for bacteria and half strength
making it possible to produce statistically valid data using
potato dextrose agar as the inoculum vehicle for mold conidia.
many fewer test carriers than needed for methods based on
Use of half strength agars may reduce undue neutralization of
simple MPN estimates.
an antimicrobial due to excessive organic load.
5.16.2.1 The stringency in the test is provided by the use of
5.17.1.2 This method simultaneously evaluates (both visual
soil load, the microtopography of the brushed stainless steel
and stereo-microscopic) antimicrobial activity both at the fiber
carrier surface, and the smaller ratio of test substance to surface
layer and at the primary backing layer of carpet.
area typical for many disinfectant applications. Thus, the test
5.17.1.3 Use this method to assess the durability of the
substance being assessed is presented with a reasonable chal-
antimicrobial treatments on new carpets, and on those repeat-
lenge while allowing for efficient recovery of the test organ-
edly shampooed or exposed to in-use conditions.
isms from the inoculated carriers. The metal disks in the basic
5.17.2 Significance and Use—This method provides for
test are also compatible with a wide variety of actives.
rapid screening of antimicrobial treatments located in or on the
5.16.2.2 The design of the carriers makes it possible to place
carpet face fiber or incorporated into the backing structure of
onto each a precisely measured volume of the test organism (10
the carpet (or both).
μL) as well as the control fluid or test substance (50 μL).
5.17.2.1 This method simulates actual use conditions that
5.16.2.3 The inoculum is placed at the center of each disk
may occur on carpets (for example, food and beverage spills,
whereas the volume of the test substance covers nearly the
soiling from foot traffic, prolonged moisture exposure).
entire disk surface, thus virtually eliminating the risk of any
5.17.2.2 This method provides a means to screen for activity
organisms remaining unexposed.
and durability of an antimicrobial treatment under conditions
5.16.2.4 In all tests, other than those against viruses, the
of organic loading.
addition of 10 mL of an eluent/diluent gives a 1:200 dilution of
5.17.2.3 This method provides for the simultaneous assess-
the test substance immediately at the end of the contact time.
ment of multiple carpet components for antimicrobial activity.
While this step in itself may be sufficient to arrest the
5.18 E3227 Standard Practice for Qualitative Assessment of
microbicidal activity of most actives, the test protocol permits
Antifungal Activity on Textiles
the addition of a specific neutralizer to the eluent/diluent, if
5.18.1 Scope—This test practice determines the relative
required. Except for viruses, the membrane filtration step
fungal growth inhibition properties of materials treated with an
allows processing of the entire eluate from the test carriers and,
active biocidal agent. Samples of porous materials, such as
therefore, the capture and subsequent detection of even low
textiles, are inoculated with a defined suspension of fungal
numbers of viable organisms that may be present. Subsequent
conidia or spores and then incubated. The inhibition of growth
rinsing of the membrane filters with saline also reduces the risk
or visible growth present on treated compared to identical
of carrying on inhibitory residues over to the recovery medium.
untreated materials is used to measure relative antifungal
Validation of the process of neutralization of the test substance
properties of the treated identical materials.
is required by challenge with low numbers of the test organism.
5.19 G21 Practice for Determining Resistance of Synthetic
5.16.2.5 The soil load in this test is a mixture of three types
of proteins (high molecular weight proteins, low molecular Polymeric Materials to Fungi (Qualitative measure of suscep-
tibility and/or fungistatic activity)
weight proteins, and mucous material) designed to represent
the body secretions, excretions, or other extraneous substances 5.19.1 Scope—This practice covers determination of the
that microbicidal chemicals may encounter under field condi- effect of fungi on the properties of synthetic polymeric mate-
tions. It is suitable for working with all types of test organisms rials in the form of molded and fabricated articles, tubes, rods,
E3152 − 23
sheets, and film materials. Changes in optical, mechanical, and 5.22.1 This method is designed to determine the antimicro-
electrical properties may be determined by the applicable bial activity of new carpet materials and consists of three
ASTM methods.
procedures. Part I is a qualitative antibacterial test. Part II is a
quantitative antibacterial test. Part III is a seven day qualitative
5.19.2 Significance and Use—The synthetic polymer por-
antifungal test and utilizes Aspergillus brasiliensis mold.
tion of these materials is usually fungus-resistant in that it does
not serve as a carbon source for the growth of fungi. It is
5.22.2 Discs of carpet are placed onto Sabouraud dextrose
generally the other components, such as plasticizers,
agar spread-inoculated with Aspergillus brasiliensis spores.
cellulosics, lubricants, stabilizers, and colorants that are re-
Additional inocula is placed onto the carpet fiber and backing.
sponsible for fungus attack on plastic materials. It is important
5.22.3 After the 7-day incubation period the samples are
to establish the resistance to microbial attack under conditions
inspected for the presence of mold growth and reported as
favorable for such attack, namely, a temperature of 2 to 38 °C
none, microscopic or macroscopic.
(35 to 100 °F) and a relative humidity of 60 to 100 %.
5.19.3 A mixed fungal spore inoculum is sprayed onto the AWPA Standards
test article resting on mineral salts agar. The samples are
5.23 AWPA E10-11 Standard Method of Testing Wood
visually and microscopically inspected weekly for four weeks.
Preservatives by Laboratory Soil-Block Cultures
5.19.4 Because of its wide industry acceptance and relative
5.23.1 Scope—This method is a screening test to determine
ease to perform, this assay has also been specified to assess
the minimum amount of preservative that is effective in
other articles that is, open and closed cell foams as well as
preventing decay of selected species of wood by selected fungi
adhesives and coatings.
under optimum laboratory conditions. Preservative thresholds
obtained using this procedure provide an indication of reten-
AATCC Standards
tions to be used in above-ground field tests; retentions to
5.20 AATCC 30-2017 Antifungal Activity, Assessment on
include in ground-contact field tests would be greater (for
Textile Materials: Mildew and Rot Resistance of Textile
example, 1.5-2.0 times) than those obtained in soil-block tests.
Materials
5.23.2 Summary of Method—Conditioned blocks of wood
5.20.1 The two purposes of this test method are to determine
are impregnated with solutions, emulsions, or dispersions of a
the susceptibility of textile materials to mildew and rot and to
preservative in water or suitable organic
...