ASTM D4300-23

Standard Test Methods for Ability of Adhesive Films to Support or Resist the Growth of Fungi

SIGNIFICANCE AND USE
4.1 These test methods are designed to be used to determine the susceptibility of the adhesive film to biodegradation and whether the adhesive will carry into the bond line sufficient anti-fungal properties to prevent growth of fungi frequently present on the gluing equipment, on adherends, or in the adhesive as applied.  
4.2 Potato dextrose agar (PDA) provides a complete medium for the growth of fungi, while mineral salts agar (MSA) lacks a carbohydrate source and provides a less favorable medium. Use of PDA tests the adhesive film for its ability to resist the growth of fungi on its surface as well as its ability to repel a copious growth of fungi on the adjacent agar surface. Use of MSA tests the adhesive film primarily for its ability to resist the growth of fungi on its surface. When it is used, there is a reduced possibility that the growth from the agar will be mis-read as coming from the adhesive film, since fungal growth on the adjacent agar will be scant.
Note 2: The method given here using the MSA is based on Practice G21, adapted to be used with adhesives. Requirements for the use of the MSA are described in 10.2, and a mixed species of fungi is prescribed in 8.2 for the inoculum.  
4.3 The results obtained when using the procedures given in this method apply only to the species used for the testing. The test species listed in Section 8 are frequently used by laboratories to test for antifungal properties, but they are not the only ones which could be used. Selection of the fungal species to test against requires informed judgment by the testing laboratory or by the party requesting the tests. These methods are especially useful when species that have been isolated from contaminated adhesives are used as the test species (see Section 8) to aid in the selection of more effective fungicides.  
4.4 The efficacy of some biocides may change in storage due to the chemical and thermal environment to which they are subjected as components of certain adhesi...
SCOPE
1.1 These test methods test the ability of adhesive films to inhibit or support the growth of selected fungal species growing on agar plates by providing means of testing the films on two agar substrates, one which promotes microbial growth, and one which does not.  
1.2 These test methods are not appropriate for all adhesives. The activity of certain biocides may not be demonstrated by these test methods as a result of irreversible reaction with some of the medium constituents.  
Note 1: As an example, quaternary ammonium compounds are inactivated by agar.  
1.3 A test method is included for use with low-viscosity adhesives along with an alternative method for use with mastic-type adhesives.  
1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.  
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. These test methods are designed to be used by persons trained in correct microbiological techniques. Specific precautionary statements are given in Section 7 and in 14.3.2.  
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

• Status: Published
• Publication Date: 30-Apr-2023
• ICS: 83.180 - Adhesives
• Technical Committee: D14 - Adhesives
• Drafting Committee: D14.30 - Wood Adhesives

Relations

• Refers: ASTM G21-13 - Standard Practice for Determining Resistance of Synthetic Polymeric Materials to Fungi
• Effective Date: 01-Nov-2013
• Refers: ASTM D907-12a - Standard Terminology of Adhesives
• Effective Date: 01-Jul-2012
• Refers: ASTM D907-12 - Standard Terminology of Adhesives
• Effective Date: 01-May-2012
• Refers: ASTM D907-11a - Standard Terminology of Adhesives
• Effective Date: 01-Dec-2011
• Refers: ASTM D907-11 - Standard Terminology of Adhesives
• Effective Date: 01-Jan-2011
• Refers: ASTM D907-08b - Standard Terminology of Adhesives
• Effective Date: 01-Oct-2008
• Refers: ASTM D907-08a - Standard Terminology of Adhesives
• Effective Date: 15-Aug-2008
• Refers: ASTM D907-08 - Standard Terminology of Adhesives
• Effective Date: 01-Mar-2008
• Refers: ASTM D907-06 - Standard Terminology of Adhesives
• Effective Date: 01-Dec-2006
• Refers: ASTM D907-05 - Standard Terminology of Adhesives
• Effective Date: 01-Apr-2005
• Refers: ASTM D907-05e1 - Standard Terminology of Adhesives
• Effective Date: 01-Apr-2005
• Refers: ASTM D907-04a - Standard Terminology of Adhesives
• Effective Date: 01-Dec-2004
• Refers: ASTM D907-04 - Standard Terminology of Adhesives
• Effective Date: 01-Apr-2004
• Refers: ASTM D907-03 - Standard Terminology of Adhesives
• Effective Date: 10-May-2003
• Refers: ASTM D907-00 - Standard Terminology of Adhesives
• Effective Date: 10-Oct-2000

Overview

ASTM D4300-23: Standard Test Methods for Ability of Adhesive Films to Support or Resist the Growth of Fungi is an internationally recognized ASTM standard that provides laboratory procedures for evaluating the resistance-or susceptibility-of adhesive films to fungal growth. Adhesive films are widely used in industries such as construction, packaging, electronics, and automotive manufacturing, where microbial contamination can impact product performance and integrity. ASTM D4300-23 helps manufacturers, formulators, and users of adhesives determine how well an adhesive film prevents or supports the growth of fungi, enabling the selection of products with effective anti-fungal properties, especially for challenging environments.

Key Topics

• Assessment of Biodegradation: The standard outlines test methods to assess the potential of adhesive films to undergo biodegradation due to fungal contamination. This is crucial for maintaining bond integrity in humid or biologically active environments.
• Agar Plate Testing: Two types of agar are recommended:
  • Potato Dextrose Agar (PDA): A nutrient-rich medium that promotes fungal growth, used to evaluate both the adhesive surface's resistance and its effect on adjacent areas.
  • Mineral Salts Agar (MSA): A low-nutrient medium that discourages fungal proliferation, offering a more selective assessment of resistance on the adhesive surface itself.
• Test Adaptability: Methods are provided for both low-viscosity adhesives and mastic-type (high-viscosity) adhesives, accommodating a variety of adhesive formulations.
• Species Selection: The test is performed using selected fungal species commonly encountered in industrial settings. Laboratories can choose appropriate species based on sample history or known contamination risks.
• Interpretation & Reporting: Results are interpreted based on the presence or absence of fungal growth and zones of inhibition around the adhesive sample. Reporting details include agar type, fungal species, test procedure, and observed inhibition.

Applications

ASTM D4300-23 is highly relevant for:

• Quality Control: Ensures adhesives meet anti-fungal performance criteria prior to deployment in the field.
• Product Development: Aids manufacturers in evaluating and comparing new formulations or biocide additives for improved fungal resistance.
• Compliance & Certification: Supports documentation for regulatory and customer requirements, especially in industries such as construction, packaging, electronics, furniture, and medical devices.
• Remediation & Forensics: Useful in identifying causes of adhesive failure when fungal contamination is suspected, and in the selection of more effective fungicides for remediation.

This standard is particularly valuable in environments with high humidity, frequent biological contamination, or where product safety and longevity are critical. Accurate testing according to ASTM D4300-23 can minimize the risk of adhesive failure caused by mold or mildew, extend product life, and assist organizations in meeting stringent quality and safety standards.

Related Standards

When working with ASTM D4300-23, the following related standards and resources may also be of interest:

• ASTM D907: Terminology of Adhesives
• ASTM G21: Practice for Determining Resistance of Synthetic Polymeric Materials to Fungi
• TAPPI T487: Fungus Resistance of Paper and Paperboard

Consulting these related documents can enhance understanding and cross-applicability in broader material testing and fungal resistance assessment scenarios.

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Keywords: ASTM D4300-23, adhesive films, fungal resistance, biodegradation, antifungal testing, PDA, MSA, mold contamination, quality control, biocide efficacy, adhesive testing standards.

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: D4300 − 23
Standard Test Methods for
Ability of Adhesive Films to Support or Resist the Growth of
Fungi
This standard is issued under the fixed designation D4300; 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.
This standard has been approved for use by agencies of the U.S. Department of Defense.
1. Scope* 2. Referenced Documents
1.1 These test methods test the ability of adhesive films to
2.1 ASTM Standards:
inhibit or support the growth of selected fungal species D907 Terminology of Adhesives
growing on agar plates by providing means of testing the films
G21 Practice for Determining Resistance of Synthetic Poly-
on two agar substrates, one which promotes microbial growth, meric Materials to Fungi
and one which does not.
2.2 TAPPI Method:
1.2 These test methods are not appropriate for all adhesives. T487 Fungus Resistance for Paper and Paperboard
The activity of certain biocides may not be demonstrated by
these test methods as a result of irreversible reaction with some
3. Terminology
of the medium constituents.
3.1 Definitions—Many terms in this test method are defined
in Terminology D907.
NOTE 1—As an example, quaternary ammonium compounds are
inactivated by agar. 3.2 Definitions of Terms Specific to This Standard:
3.2.1 adhesive preparation, n—the adhesive as packaged for
1.3 A test method is included for use with low-viscosity
distribution, storage, and use.
adhesives along with an alternative method for use with
mastic-type adhesives.
3.2.2 adhesive film, n—the small portion of the adhesive
preparation, as prepared for use by the consumer, either with
1.4 The values stated in SI units are to be regarded as the
additives or as received, which is cast on a substrate, cured
standard. The values given in parentheses are for information
24 h, and represents the glue line.
only.
3.2.2.1 Discussion—For purposes of these test methods the
1.5 This standard does not purport to address all of the
adhesive film is the thin layer of adhesive spread on either the
safety concerns, if any, associated with its use. It is the
21 mm fiberglass disk as described in 14.2, or the adhesive
responsibility of the user of this standard to establish appro-
layer 3 mm thick which is cast on the tile squares as described
priate safety, health, and environmental practices and deter-
in 15.1.
mine the applicability of regulatory limitations prior to use.
These test methods are designed to be used by persons trained 3.2.3 zone of inhibition, n—the area on an inoculated agar
in correct microbiological techniques. Specific precautionary plate surrounding the adhesive-coated disk or tile, showing a
statements are given in Section 7 and in 14.3.2. reduced fungal growth or an absence thereof.
1.6 This international standard was developed in accor-
3.3 Abbreviations:
dance with internationally recognized principles on standard-
3.3.1 PDA—potato dextrose agar.
ization established in the Decision on Principles for the
3.3.2 MSA—mineral salts agar.
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
3.3.3 ZI—zone of inhibition.
Barriers to Trade (TBT) Committee.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
These test methods are under the jurisdiction of ASTM Committee D14 on contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Adhesives and are the direct responsibility of Subcommittee D14.30 on Wood Standards volume information, refer to the standard’s Document Summary page on
Adhesives. the ASTM website.
Current edition approved May 1, 2023. Published May 2023. Originally Available from Technological Association of the Pulp and Paper Industry
ɛ1
approved in 1983. Last previous edition approved in 2021 as D4300 – 01 (2021) . (TAPPI), 15 Technology Parkway South, Suite 115, Peachtree Corners, GA 30092,
DOI: 10.1520/D4300-23. http://www.tappi.org.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4300 − 23
4. Significance and Use 5.1.3 Filter Disk, Glass Microfibre, 934-AM, diameter-
21 mm.
4.1 These test methods are designed to be used to determine
5.1.4 Filter Disk, Sterile Whatman No. 1.
the susceptibility of the adhesive film to biodegradation and
5.1.5 Filter Paper Assay Disk, 1.5 cm diameter, sterile.
whether the adhesive will carry into the bond line sufficient
Schleicher and Schnell, Inc., or the equivalent, has been found
anti-fungal properties to prevent growth of fungi frequently
satisfactory for this purpose.
present on the gluing equipment, on adherends, or in the
5.1.6 Glass Rods, 305 mm in length having a diameter of
adhesive as applied.
6.3 mm.
4.2 Potato dextrose agar (PDA) provides a complete me-
5.1.7 Glove Bag, 68 cm in length and width, 38 cm in
dium for the growth of fungi, while mineral salts agar (MSA)
height.
lacks a carbohydrate source and provides a less favorable
5.1.8 Hemacytometer Levy Counting Chamber, cell depth-
medium. Use of PDA tests the adhesive film for its ability to
0.1 mm, Newbauer rulings.
resist the growth of fungi on its surface as well as its ability to
5.1.9 Hood, Laminar-Flow Type, Class II Type I.
repel a copious growth of fungi on the adjacent agar surface.
5.1.10 Jar, Screw Cap, round, approximately 1 L (1 qt,
Use of MSA tests the adhesive film primarily for its ability to
mason type).
resist the growth of fungi on its surface. When it is used, there
5.1.11 Pipet, Pasteur.
is a reduced possibility that the growth from the agar will be
5.1.12 Petri Dishes, sterile, disposable, top-diameter of
mis-read as coming from the adhesive film, since fungal
150 mm, bottom-height of 15 mm.
growth on the adjacent agar will be scant.
5.1.13 Refrigerator, capable of maintaining 4 °C 6 1 °C
NOTE 2—The method given here using the MSA is based on Practice
(39 °F 6 2 °F).
G21, adapted to be used with adhesives. Requirements for the use of the
5.1.14 Teflon Paper or Grid, pressure sensitive overlay,
MSA are described in 10.2, and a mixed species of fungi is prescribed in
coated with TFE-fluorocarbon (PTFE), vinyl sheet backing, to
8.2 for the inoculum.
be used at up to 93 °C (200 °F).
4.3 The results obtained when using the procedures given in
this method apply only to the species used for the testing. The
6. Materials
test species listed in Section 8 are frequently used by labora-
6.1 Potato Dextrose Agar, Difco or equivalent.
tories to test for antifungal properties, but they are not the only
ones which could be used. Selection of the fungal species to 6.2 Sterile Deionized or Distilled Water.
test against requires informed judgment by the testing labora-
6.3 Disinfectant Solution—Amphyll, Alcide, or comparable
tory or by the party requesting the tests. These methods are
product.
especially useful when species that have been isolated from
6.4 Materials for Mineral Salts Agar. (See list in 10.2.1.)
contaminated adhesives are used as the test species (see
Section 8) to aid in the selection of more effective fungicides.
6.5 Sorbitan mono-oleate polyoxyethylene.
4.4 The efficacy of some biocides may change in storage
7. Precautions
due to the chemical and thermal environment to which they are
7.1 Assign laboratory personnel trained in correct microbio-
subjected as components of certain adhesives. These test
methods are not appropriate for determining the effect of logical techniques to run these tests. These test methods
employ live cultures of fungi, some of which are capable of
fungal contamination on adhesives under water-soaking
conditions, because they are not designed to cover the possi- causing disease or allergic reaction in some humans. Use
bility of water-soluble biocides leaching out of the bond line. proper microbiological procedures in order to prevent contami-
nation of the cultures or of the work area. Disinfect and
4.5 These test methods are dependent upon the physiologi-
sterilize in an approved manner all spills and all equipment
cal action of living microorganisms under a reported set of
coming into contact with the cultures. Also sterilize in an
conditions. Conclusions about the resistance of the test adhe-
approved manner all cultures and contaminated disposable
sive to fungal attack can be drawn by comparing the results to
equipment before discarding. See 1.5 and 14.3.2.
simultaneously run controls of known resistance. See X5.2 for
statements regarding test repeatability.
7.2 In addition to other precautions, the use of a Class II,
Type A2 containment cabinet shall be used for all procedures
5. Apparatus
that would cause formation of fungal aerosols. This type of
5.1 In addition to the standard equipment found in any fully
safety cabinet prevents the spread of fungal spores throughout
equipped microbiological laboratory, items from the following
the laboratory and inhalation of spores by the operator. The
list are needed for various tests. Not all items are needed for
cabinet should be monitored by a biological safety officer or a
each test.
health physicist if they are to be used with hazardous agents.
5.1.1 Chromist Laboratory Spray Unit.
5.1.2 Constant Temperature Chamber, capable of being
maintained at 35 °C 6 0.5 °C (95 °F 6 1 °F) or 25 °C 6
Available from Instruments for Research and Industry, 108 Franklin Ave.,
Cheltenham, PA, or most laboratory supply houses.
0.5 °C (77 °F 6 1 °F), or two chambers if needed simultane-
The Biogard Hood or similar equipment is available from laboratory supply
ously.
houses.
Most laboratory supply houses.
4 8
Available from laboratory supply houses. Available commercially as Tween 80.
D4300 − 23
Refer to the operating manual supplied by the manufacturer for solidify. Tighten the caps on the tubes and place them in a
detailed information. This warning applies specifically to the slanted position to solidify, making a slant of about 51 mm.
use of the Chromist laboratory spray unit listed in 5.1.1 and in
Store slants and plates in refrigerator until needed. Ensure
the instructions in 14.3.2. plates are dry before inoculation.
10.2 Mineral Salts Agar—Prepare sufficient medium for
8. Test Species of Fungi
tests as described below:
8.1 Cultures of one or more of the following species are
10.2.1 Dissolve in 1 L of water the designated amounts of
suggested for use when PDA is the medium:
the following reagents:
ATCC No.
Grams
Potassium phosphate (KH PO ) 0.7
Aspergillus brasilensis 9642
2 4
Magnesium sulfate (MgSO · 7H O) 0.7
Aspergillus flavus 9643 4 2
Ammonium nitrate (NH NO ) 1.0
Penicillium pinophilum 9644 (See X1.1.6)
4 3
Sodium chloride (NaCl) 0.005
Phanerochaete chrysosporium 24725
Ferrous sulfate (FeSO · 7H O) 0.002
Aureobasidium 4 2
Zinc sulfate (ZnSO · 7H O) 0.002
melanogenum 15233 4 2
Manganous sulfate (MnSO · 4H O) 0.001
4 2
NOTE 3—The choice of test organisms is often made from the fungal
Agar 15.0
species listed above. Information on these and other species is given in
10.2.2 Adjust the pH of the medium by the addition of
Appendix X1.
0.01N NaOH solution so that after sterilization the pH is
8.2 Cultures of the following species are used for the
between 6.0 and 6.5, and sterilize by autoclaving at 103 kPa,
government requirements described in Section 16, using MSA:
and 121 °C (250 °F) for 15 min.
ATCC No.
10.2.3 Prepare plates as described in 10.1.2, and store in the
Aspergillus brasiliensis 9642
refrigerator until needed.
Aureobasidium pullulans
Chaetomium globosum 6205 11. Fungal Cultures
Trichoderma virens 9645
Talaromvces pinophilus (Penicillium ninohilum) 9644 (See X1.1.6) 11.1 Propagation of Fungal Cultures:
11.1.1 Prepare a fresh culture for each species on PDA and
NOTE 4—The species listed in 8.2 are used in Practice G21. The
following optional species are also sometimes used: Aspergillus flavus,
label by species and ATCC Number. Incubate at 25 °C 6
(ATCC No. 9643) and Aspergillus versicolor (ATCC No. 11730). See 13.2
0.5 °C (77 °F 6 1 °F) for a minimum of 10 days or until full
and Appendix X1.
sporulation is achieved.
8.3 Other pure cultures or mixed cultures of fungal species
11.1.2 Refrigerate the cultures. Prepare new cultures each
may be used, if agreed upon between the interested parties and
month. If contamination occurs, discard the cultures and
upon the recommendation of the testing laboratories.
prepare new ones.
9. Sterilization of Equipment and Media
11.2 Preparation of Fungal Inoculum:
11.2.1 Follow the procedure in 11.1.1 to prepare fresh
9.1 Follow accepted microbiological practices for sterilizing
cultures on PDA slants for each species to be used to conduct
equipment and media.
the tests.
NOTE 5—Two references for sterilization methods are TAPPI T487 (see
11.2.2 Harvesting Fungal Cultures and Dislodging
2.2) and Ref (1).
Spores—To one tube of each species of fungi, add 15 mL of
10. Preparation of Media
sterile distilled or deionized water, containing 0.05 % sorbitan
mono-oleate polyoxyethylene. Harvest fungal cultures and
10.1 Potato Dextrose Agar:
dislodge spores by rubbing the growth gently with a sterile
10.1.1 Prepare sufficient agar slants and plates for culture
inoculating loop or by removing it with a sterile glass rod.
propagation and conducting the tests.
Transfer the washings into a sterilized container containing
10.1.2 Follow the instructions given for preparation of the
glass beads and shake thoroughly to break up the clumps. Filter
commercial product. Dissolve using heat and agitation. Trans-
through sterile layered cheese cloth or sterile nonabsorbent
fer an appropriate amount of the agar solution to each flask
cotton. Adjust the spore level to 1.0 × 10 per mL, using a
used for pouring plates, and 10 mL per test tube. Plug flask
hemacytometer and the procedure in Annex. Use this spore
with appropriate closures. Cap tubes loosely with metal,
suspension of a single species of fungi as the inoculum for the
plastic, or foam caps. Autoclave for 15 min at 103 kPa and a
tests described in Sections 14 and 15 when using the option
temperature of 121 °C (250 °F). Allow the agar to cool to
given in 13.1.1.1.
48 °C to 50 °C (118 °F to 122 °F) before pouring the plates,
filling to an approximate depth of 5 mm. Allow plates to
11.2.3 For a mixed culture, obtain a spore count on each
fungal species, and adjust each suspension to the level of 1 ×
0 per mL. Combine equal portions of the spore suspensions
Cultures may be purchased from the American Type Culture Collection, 10801
from each of the species in a common sterilized container. Use
University Blvd. Manassas, VA 20110.
this mixed spore suspension for the tests described in Sections
The boldface numbers in parentheses refer to the references at the end of this
standard. 14, 15, and 16 when using the option given in 13.1.1.2.
D4300 − 23
NOTE 8—The drying time may need to be reduced for some adhesives
12. Adhesive Sample
to avoid curling of the disks. See Appendix X3 for a discussion on
12.1 For ready-to-use liquid adhesives, obtain an approxi-
handling adhesives.
mate 250 mL sample. For adhesives to be mixed at the time of
14.3 Inoculation and Placement of Specimens—Seed the
use, obtain a sufficient sample of each component, mix in
duplicate test plates and the control plate, using one of the
accordance with the manufacturer’s instructions, and run the
following procedures:
tests on the prepared adhesive mix. For mastics, use the
14.3.1 Inoculate a 150 mm agar plate by placing 0.1 mL of
adhesive as packaged for the consumer, directly from the
the spore suspension prepared in 11.2.2 on the surface of the
applicator tube.
plate, dropping from a pipet. Using a sterile L-shaped rod, seed
NOTE 6—The sample size given is for convenience in handling. The test
the total surface. Prepare two plates, placing the three
may be run on only a few millilitres of material. When several components
adhesive-coated fiberglass disks equidistant and flat on the
are to be mixed to yield the sample to be tested, the amount of each
surface. Prepare the control plate by placing three uncoated
component should be sufficient for remixing should a retest be necessary.
Whatman No. 1 filter paper disks on the inoculated surface.
Using a sterile capillary dropping pipet, place three drops of
13. Selection of Conditions of Testing
the spore suspension on the surface of each disk on the test
13.1 Select one option from each of the testing conditions
plates and the control plate.
given below:
14.3.2 As an alternate method, or to comply with Practice
13.1.1 Fungal Species for Inoculum—Select the fungal
G21, prepare duplicate plates by placing three adhesive coated
species to use for the tests based on the informed decision of
fiberglass disks equidistant and flat on the surface of each of
the testing laboratory or on the requirements of specifications
two 150 mm agar plates. For the control, place three uncoated
which are to be met, using one of the options below:
Whatman No. 1 filter paper disks on the agar surface of a third
13.1.1.1 Testing with spore suspensions of pure cultures of
plate. Then inoculate the test plates and the control plate by
single species, or
spraying the fungal spore suspension over the entire surface of
13.1.1.2 Testing with a mixed spore suspension of two or
the agar and the disks. Use the apparatus in 5.1.1 for the
more species.
spraying, and conduct this operation in the confines of a Class
NOTE 7—See Section 8 and Appendix X1 for options and help in II Type A2 Biological Safety Cabinet which has been properly
selecting the species to use. See Appendix X2 for guidelines on use of
monitored by a biological safety officer. Follow the precautions
mixed cultures.
given in 7.2. (Warning—In addition to other precautions, use
13.1.2 Agar Medium—Select the medium based on the special care with the spray inoculation described in 14.3.2,
informed decision of the testing laboratory or on specifications
which has a potential for gross contamination of the work area.
to be met: Before inoculation place the open plates inside the described
13.1.2.1 Potato dextrose agar,
biological safety cabinet. Inside the hood, use a secondary
13.1.2.2 Mineral salts agar, or containment, such as an empty 10 gal aquarium, partially
13.1.2.3 Other medium of choice.
covered, or a glove bag. Use disposable surgical gloves and a
13.1.3 Procedure, Based on Viscosity or Consistency of the respirator when inoculating by the spray technique and when
Adhesive:
handling the inoculated plates. Invert the plates (if this will not
13.1.3.1 Low-viscosity adhesives, or disturb the disks) and enclose them in a sealed plastic bag to
13.1.3.2 Mastic-type adhesives.
avoid contaminating the incubator. Use of a respirator provides
an extra measure of safety when inoculating by the spray
13.2 Testing According to Practice G21—To comply with
technique.)
Practice G21, select the following options: the mixed fungal
species designated in 13.1.1.2, using the species listed in 8.2,
14.4 Incubation and Examination—Seal the plates with
and the MSA designated in 13.1.2.2. Follow the procedure
parafilm to prevent drying out during incubation. Incubate both
given in Section 16.
the test and control culture plates at 25 °C 6 0.5 °C (77 °F 6
1 °F). When PDA is the culture medium, examine at 3, 7, and
14. Film Test for Low Viscosity Adhesives
14 days for zone of inhibition (ZI) or overgrowth on the
adhesive coated disks, and record results using the grading
14.1 Number of Specimens and Plates per Test—For each
system given in 14.5. For slow growing species, extend the
species or mixed species of fungi to be tested against, run two
incubation period to 21 days. Examine the control plate and the
agar plates using three adhesive-coated test specimens de-
outer edges of the test plates for presence of a normal confluent
scribed in 14.2, per 150 mm diameter plate and one plate with
growth pattern of the fungal species being tested and an
three uncoated Whatman No. 1 filter disks as a control.
absence of contaminants. When MSA is the culture medium,
14.2 Preparation of Adhesive Specimens—On the day be-
examine at 7, 14, and 21 days.
fore the tests are to be initiated, prepare the adhesive-coated
NOTE 9—To avoid transfer of developing spores during the incubation
fiberglass disks (see 5.1.3). Coat both sides of each disk with
period, handle the plates with care for the preliminary readings.
the adhesive. Allow the disks to dry until no longer tacky by
14.5 Grading System:
resting on a sheet of TFE-fluorocarbon-coated paper or a
TFE-fluorocarbon-coated grid. Then dry for 24 h at 40 °C 6 14.5.1 When PDA is the culture medium use the following
grading s
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
´1
Designation: D4300 − 01 (Reapproved 2021) D4300 − 23
Standard Test Methods for
Ability of Adhesive Films to Support or Resist the Growth of
Fungi
This standard is issued under the fixed designation D4300; 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.
This standard has been approved for use by agencies of the U.S. Department of Defense.
ε NOTE—Editorial changes were made to 8.1 and 8.2 in April 2021.
1. Scope Scope*
1.1 These test methods test the ability of adhesive films to inhibit or support the growth of selected fungal species growing on agar
plates by providing means of testing the films on two agar substrates, one which promotes microbial growth, and one which does
not.
1.2 These test methods are not appropriate for all adhesives. The activity of certain biocides may not be demonstrated by these
test methods as a result of irreversible reaction with some of the medium constituents.
NOTE 1—As an example, quaternary ammonium compounds are inactivated by agar.
1.3 A test method is included for use with low-viscosity adhesives along with an alternative method for use with mastic-type
adhesives. Also, a method approved by the government is given.
1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.
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. These test methods are designed to be used by persons trained in correct microbiological
techniques. Specific precautionary statements are given in Section 7 and in 14.3.2.
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.
2. Referenced Documents
2.1 ASTM Standards:
D907 Terminology of Adhesives
D1286 Method of Test for Effect of Mold Contamination on Permanence of Adhesives Preparations and Adhesives Bonds
(Withdrawn 1983)
These test methods are under the jurisdiction of ASTM Committee D14 on Adhesives and are the direct responsibility of Subcommittee D14.30 on Wood Adhesives.
Current edition approved April 1, 2021May 1, 2023. Published April 2021May 2023. Originally approved in 1983. Last previous edition approved in 20132021 as
ɛ1
D4300 – 01 (2013).(2021) . DOI: 10.1520/D4300-01R21E01.10.1520/D4300-23.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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G21 Practice for Determining Resistance of Synthetic Polymeric Materials to Fungi
2.2 TAPPI Method:
T487 Fungus Resistance for Paper and Paperboard
3. Terminology
3.1 Definitions—Many terms in this test method are defined in Terminology D907.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 adhesive preparation, n—the adhesive as packaged for distribution, storage, and use.
3.2.2 adhesive film, n—the small portion of the adhesive preparation, as prepared for use by the consumer, either with additives
or as received, which is cast on a substrate, cured 24 h, 24 h, and represents the glue line.
3.2.2.1 Discussion—
For purposes of these test methods the adhesive film is the thin layer of adhesive spread on either the 21-mm21 mm fiberglass disk
as described in 14.2, or the adhesive layer 3 mm thick which is cast on the tile squares as described in 15.1.
3.2.3 zone of inhibition, n—the area on an inoculated agar plate surrounding the adhesive-coated disk or tile, showing a reduced
fungal growth or an absence thereof.
3.3 Abbreviations:
3.3.1 PDA—potato dextrose agar.
3.3.2 MSA—mineral salts agar.
3.3.3 ZI—zone of inhibition.
4. Significance and Use
4.1 These test methods are designed to be used to determine the susceptibility of the adhesive film to biodegradation and whether
the adhesive will carry into the bond line sufficient anti-fungal properties to prevent growth of fungi frequently present on the
gluing equipment, on adherends, or in the adhesive as applied.
4.2 Potato dextrose agar (PDA) provides a complete medium for the growth of fungi, while mineral salts agar (MSA) lacks a
carbohydrate source and provides a less favorable medium. Use of PDA tests the adhesive film for its ability to resist the growth
of fungi on its surface as well as its ability to repel a copious growth of fungi on the adjacent agar surface. Use of MSA tests the
adhesive film primarily for its ability to resist the growth of fungi on its surface. When it is used, there is a reduced possibility
that the growth from the agar will be mis-read as coming from the adhesive film, since fungal growth on the adjacent agar will
be scant.
NOTE 2—The method given here using the MSA is based on Practice G21, adapted to be used with adhesives. Requirements to meet the approval of
government specifications are the for the use of the MSA are described in 10.2, and a mixed species of fungi described is prescribed in 8.2 for the
inoculum.
4.3 The results obtained when using the procedures given in this method apply only to the species used for the testing. The test
species listed in Section 8 are frequently used by laboratories to test for antifungal properties, but they are not the only ones which
could be used. Selection of the fungal species to test against requires informed judgment by the testing laboratory or by the party
requesting the tests. These methods are especially useful when species that have been isolated from contaminated adhesives are
used as the test species (see Section 8) to aid in the selection of more effective fungicides.
4.4 The efficacy of some biocides may change in storage due to the chemical and thermal environment to which they are subjected
as components of certain adhesives. These test methods are not appropriate for determining the effect of fungal contamination on
adhesives under water-soaking conditions, because they are not designed to cover the possibility of water-soluble biocides leaching
out of the bond line.
Available from Technological Association of the Pulp and Paper Industry (TAPPI), 15 Technology Parkway South, Suite 115, Peachtree Corners, GA 30092,
http://www.tappi.org.
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4.5 These test methods are dependent upon the physiological action of living microorganisms under a reported set of conditions.
Conclusions about the resistance of the test adhesive to fungal attack can be drawn by comparing the results to simultaneously run
controls of known resistance. See X5.2 for statements regarding test repeatability.
5. Apparatus
5.1 In addition to the standard equipment found in any fully equipped microbiological laboratory, items from the following list
are needed for various tests. Not all items are needed for each test.
5.1.1 Chromist Laboratory Spray Unit.
5.1.2 Constant Temperature Chamber, capable of being maintained at 35 6 0.5°C (95 6 1°F) or 25 6 0.5°C (77 6 1°F),35 °C
6 0.5 °C (95 °F 6 1 °F) or 25 °C 6 0.5 °C (77 °F 6 1 °F), or two chambers if needed simultaneously.
5.1.3 Filter Disk, Glass Microfibre, 934-AM, diameter-21 mm.diameter-21 mm.
5.1.4 Filter Disk, Sterile Whatman No. 1.
5.1.5 Filter Paper Assay Disk, 1.5 cm diameter, sterile. Schleicher and Schnell, Inc., or the equivalent, has been found satisfactory
for this purpose.
5.1.6 Glass Rods, 305 mm in length having a diameter of 6.3 mm.
5.1.7 Glove Bag, 68 cm in length and width, 38 cm in height.
5.1.8 Hemacytometer Levy Counting Chamber, cell depth-0.1 mm, Newbauer rulings.
5.1.9 Hood, Laminar-Flow Type, Class II Type I.
5.1.10 Jar, Screw Cap, round, approximately 1 L (1 qt, mason type).
5.1.11 Pipet, Pasteur.
5.1.12 Petri Dishes, sterile, disposable, top-diameter of 150-mm,150 mm, bottom-height of 15-mm.15 mm.
5.1.13 Refrigerator, capable of maintaining 4 6 1°C (39 6 2°F).4 °C 6 1 °C (39 °F 6 2 °F).
5.1.14 Teflon Paper or Grid, pressure sensitive overlay, coated with TFE-fluorocarbon (PTFE), vinyl sheet backing, to be used at
up to 93°C (200°F).93 °C (200 °F).
6. Materials
6.1 Potato Dextrose Agar, Difco or equivalent.
6.2 Sterile Deionized or Distilled Water.
6.3 Disinfectant Solution—Amphyll, Alcide, or comparable product.
6.4 Materials for Mineral Salts Agar. (See list in 10.2.1.)
Available from laboratory supply houses.
Available from Instruments for Research and Industry, 108 Franklin Ave., Cheltenham, PA, or most laboratory supply houses.
The Biogard Hood or similar equipment is available from laboratory supply houses.
Gelman Sciences, or most Most laboratory supply houses.
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6.5 Sorbitan mono-oleate polyoxyethylene.
7. Precautions
7.1 Assign laboratory personnel trained in correct microbiological techniques to run these tests. These test methods employ live
cultures of fungi, some of which are capable of causing disease or allergic reaction in some humans. Use proper microbiological
procedures in order to prevent contamination of the cultures or of the work area. Disinfect and sterilize in an approved manner
all spills and all equipment coming into contact with the cultures. Also sterilize in an approved manner all cultures and
contaminated disposable equipment before discarding. See 1.5 and 14.3.2.
7.2 In addition to other precautions, the use of a Class II, Type I containment hood is highly recommendedA2 containment cabinet
shall be used for all procedures that would cause formation of fungal aerosols. This type of laminar flow hood safety cabinet
prevents the spread of fungal spores throughout the laboratory and inhalation of spores by the operator. The hoodscabinet should
be monitored by a biological safety officer or a health physicist if they are to be used with hazardous agents. Refer to the operating
manual supplied by the manufacturer for detailed information. This warning applies specifically to the use of the Chromist
laboratory spray unit listed in 5.1.1 and in the instructions in 14.3.2.
8. Test Species of Fungi
8.1 Cultures of one or more of the following species are suggested for use when PDA is the medium:
ATCC No.
Aspergillus niger 9642
Aspergillus brasilensis 9642
Aspergillus flavus 9643
Penicillium pinophilum 9644 (See X1.1.6)
Phanerochaete chrysosporium 24725
Aureobasidium pullulans
Aureobasidium
Var.—melanigenum 15233
melanogenum 15233
NOTE 3—The choice of test organisms is often made from the fungal species listed above. Information on these and other species is given in Appendix
X1.
8.2 Cultures of the following species are used for the government requirements described in Section 16, using MSA:
ATCC No.
Aspergillus niger 9642
Aspergillus brasiliensis 9642
Aureobasidium pullulans
Var.—melanigenum 15233
Chaetomium globosum 6205
Gliocladium virens 9645
Trichoderma virens 9645
Penicillium pinophilum 9644 (See X1.1.6)
Talaromvces pinophilus (Penicillium ninohilum) 9644 (See X1.1.6)
NOTE 4—The species listed in 8.2 are used in Practice G21. The following optional species are also sometimes used: Aspergillus flavus, (ATCC No. 9643)
and Aspergillus versicolor (ATCC No. 11730). See 13.2 and Appendix X1.
8.3 Other pure cultures or mixed cultures of fungal species may be used, if agreed upon between the interested parties and upon
the recommendation of the testing laboratories.
Available commercially as Tween 80.
Cultures may be purchased from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852.10801 University Blvd. Manassas, VA 20110.
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9. Sterilization of Equipment and Media
9.1 Follow accepted microbiological practices for sterilizing equipment and media.
NOTE 5—Two references for sterilization methods are TAPPI T487 (see 2.2) and Ref (1).
10. Preparation of Media
10.1 Potato Dextrose Agar:
10.1.1 Prepare sufficient agar slants and plates for culture propagation and conducting the tests.
10.1.2 Follow the instructions given for preparation of the commercial product. Dissolve using heat and agitation. Transfer an
appropriate amount of the agar solution to each flask used for pouring plates, and 10 mL per test tube. Plug flask with appropriate
closures. Cap tubes loosely with metal, plastic, or foam caps. Autoclave for 15 min at 103 kPa and a temperature of 121°C
(250°F).121 °C (250 °F). Allow the agar to cool to 48 to 50°C (118 to 122°F)48 °C to 50 °C (118 °F to 122 °F) before pouring
the plates, filling to an approximate depth of 35 mm. Allow plates to solidify. Tighten the caps on the tubes and place them in a
slanted position to solidify, making a slant of about 51 mm. Store slants and plates in refrigerator until needed. Ensure plates are
dry before inoculation.
10.2 Mineral Salts Agar—Prepare sufficient medium for tests as described below:
10.2.1 Dissolve in 1 L of water the designated amounts of the following reagents:
Grams
Potassium phosphate (KH PO ) 0.7
2 4
Magnesium sulfate (MgSO · 7H O) 0.7
4 2
Ammonium nitrate (NH NO ) 1.0
4 3
Sodium chloride (NaCl) 0.005
Ferrous sulfate (FeSO · 7H O) 0.002
4 2
Zinc sulfate (ZnSO · 7H O) 0.002
4 2
Manganous sulfate (MnSO · 4H O) 0.001
4 2
Agar 15.0
10.2.2 Adjust the pH of the medium by the addition of 0.01N NaOH solution so that after sterilization the pH is between 6.0 and
6.5, and sterilize by autoclaving at 103 kPa, and 121°C (250°F)121 °C (250 °F) for 15 min.
10.2.3 Prepare plates as described in 10.1.2, and store in the refrigerator until needed.
11. Fungal Cultures
11.1 Propagation of Fungal Cultures:
11.1.1 Prepare a fresh culture for each species on PDA and label by species and ATCC Number. Incubate at 25 6 0.5°C (77 6
1°F)25 °C 6 0.5 °C (77 °F 6 1 °F) for a minimum of 10 days or until full sporulation is achieved.
11.1.2 Refrigerate the cultures. Prepare new cultures each month. If contamination occurs, discard the cultures and prepare new
ones.
11.2 Preparation of Fungal Inoculum:
11.2.1 Follow the procedure in 11.1.1 to prepare fresh cultures on PDA slants for each species to be used to conduct the tests.
11.2.2 Harvesting Fungal Cultures and Dislodging Spores—To one tube of each species of fungi, add 15 mL of sterile distilled
or deionized water, containing 0.05 % sorbitan mono-oleate polyoxyethylene. Harvest fungal cultures and dislodge spores by
rubbing the growth gently with a sterile inoculating loop or by removing it with a sterile glass rod. Transfer the washings into a
The boldface numbers in parentheses refer to the references at the end of this standard.
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sterilized container containing glass beads and shake thoroughly to break up the clumps. Filter through sterile layered cheese cloth
or sterile nonabsorbent cotton. Adjust the spore level to 1.0 × 10 per mL, using a hemacytometer and the procedure in Annex.
Use this spore suspension of a single species of fungi as the inoculum for the tests described in Sections 14 and 15 when using
the option given in 13.1.1.1.
11.2.3 For a mixed culture, obtain a spore count on each fungal species, and adjust each suspension to the level of 1 × 0 per mL.
Combine equal portions of the spore suspensions from each of the species in a common sterilized container. Use this mixed spore
suspension for the tests described in Sections 14, 15, and 16 when using the option given in 13.1.1.2.
12. Adhesive Sample
12.1 For ready-to-use liquid adhesives, obtain an approximate 250-mL250 mL sample. For adhesives to be mixed at the time of
use, obtain a sufficient sample of each component, mix in accordance with the manufacturer’s instructions, and run the tests on
the prepared adhesive mix. For mastics, use the adhesive as packaged for the consumer, directly from the applicator tube.
NOTE 6—The sample size given is for convenience in handling. The test may be run on only a few millilitres of material. When several components are
to be mixed to yield the sample to be tested, the amount of each component should be sufficient for remixing should a retest be necessary.
13. Selection of Conditions of Testing
13.1 Select one option from each of the testing conditions given below:
13.1.1 Fungal Species for Inoculum—Select the fungal species to use for the tests based on the informed decision of the testing
laboratory or on the requirements of specifications which are to be met, using one of the options below:
13.1.1.1 Testing with spore suspensions of pure cultures of single species, or
13.1.1.2 Testing with a mixed spore suspension of two or more species.
NOTE 7—See Section 8 and Appendix X1 for options and help in selecting the species to use. See Appendix X2 for guidelines on use of mixed cultures.
13.1.2 Agar Medium—Select the medium based on the informed decision of the testing laboratory or on specifications to be met:
13.1.2.1 Potato dextrose agar,
13.1.2.2 Mineral salts agar, or
13.1.2.3 Other medium of choice.
13.1.3 Procedure, Based on Viscosity or Consistency of the Adhesive:
13.1.3.1 Low-viscosity adhesives, or
13.1.3.2 Mastic-type adhesives.
13.2 Testing According to MeetPractice G21Government Specifications——To comply with governmentPractice
G21specifications,, select the following options: the mixed fungal species designated in 13.1.1.2, using the species listed in 8.2,
and the MSA designated in 13.1.2.2. Follow the procedure given in Section 16.
14. Film Test for Low Viscosity Adhesives
14.1 Number of Specimens and Plates per Test—For each species or mixed species of fungi to be tested against, run two agar plates
using three adhesive-coated test specimens described in 14.2, per 150-mm150 mm diameter plate and one plate with three uncoated
Whatman No. 1 filter disks as a control.
14.2 Preparation of Adhesive Specimens—On the day before the tests are to be initiated, prepare the adhesive-coated fiberglass
disks (see 5.1.3). Coat both sides of each disk with the adhesive. Allow the disks to dry until no longer tacky by resting on a sheet
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of TFE-fluorocarbon-coated paper or a TFE-fluorocarbon-coated grid. Then dry for 24 h at 40 6 2°C (104 6 3.6°F)40 °C 6 2 °C
(104 °F 6 3.6 °F) to allow the volatiles to dissipate. Store in sterile petri dishes until used.
NOTE 8—The drying time may need to be reduced for some adhesives to avoid curling of the disks. See Appendix X3 for a discussion on handling
adhesives.
14.3 Inoculation and Placement of Specimens—Seed the duplicate test plates and the control plate, using one of the following
procedures:
14.3.1 Inoculate a 150-mm150 mm agar plate by placing 0.1 mL of the spore suspension prepared in 11.2.2 on the surface of the
plate, dropping from a pipet. Using a sterile L-shaped rod, seed the total surface. Prepare two plates, placing the three
adhesive-coated fiberglass disks equidistant and flat on the surface. Prepare the control plate by placing three uncoated Whatman
No. 1 filter paper disks on the inoculated surface. Using a sterile capillary dropping pipet, place three drops of the spore suspension
on the surface of each disk on the test plates and the control plate.
14.3.2 As an alternate method, or to comply with Practice G21, prepare duplicate plates by placing three adhesive coated fiberglass
disks equidistant and flat on the surface of each of two 150-mm150 mm agar plates. For the control, place three uncoated Whatman
No. 1 filter paper disks on the agar surface of a third plate. Then inoculate the test plates and the control plate by spraying the fungal
spore suspension over the entire surface of the agar and the disks. Use the apparatus in 5.1.1 for the spraying, and conduct this
operation in the confines of a Class II Type IA2 Biological Safety Cabinet or laminar-flow hood which has been properly monitored
by a biological safety officer. Follow the precautions given in 7.2. (Warning—In addition to other precautions, use special care
with the spray inoculation described in 14.3.2, which has a potential for gross contamination of the work area. Before inoculation
place the open plates inside the described biological safety cabinet. Inside the hood, use a secondary containment, such as an empty
10-gal10 gal aquarium, partially covered, or a glove bag. Use disposable surgical gloves and a respirator when inoculating by the
spray technique and when handling the inoculated plates. Invert the plates (if this will not disturb the disks) and enclose them in
a sealed plastic bag to avoid contaminating the incubator. Use of a respirator provides an extra measure of safety when inoculating
by the spray technique.)
14.4 Incubation and Examination—Seal the plates with parafilm to prevent drying out during incubation. Incubate both the test
and control culture plates at 25 6 0.5°C (77 6 1°F).25 °C 6 0.5 °C (77 °F 6 1 °F). When PDA is the culture medium, examine
at 3, 7, and 14 days for zone of inhibition (ZI) or overgrowth on the adhesive coated disks, and record results using the grading
system give
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