ASTM D4300-01(2021)e1
(Test Method)Standard Test Methods for Ability of Adhesive Films to Support or Resist the Growth of Fungi
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 to meet the approval of government specifications are the use of the MSA described in 10.2, and a mixed species of fungi described 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...
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.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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´1
Designation: D4300 − 01 (Reapproved 2021)
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 mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
1.1 These test methods test the ability of adhesive films to
inhibit or support the growth of selected fungal species
2. Referenced Documents
growing on agar plates by providing means of testing the films
on two agar substrates, one which promotes microbial growth, 2.1 ASTM Standards:
and one which does not. D907 Terminology of Adhesives
D1286 Method of Test for Effect of Mold Contamination on
1.2 These test methods are not appropriate for all adhesives.
Permanence of Adhesives Preparations and Adhesives
The activity of certain biocides may not be demonstrated by
Bonds (Withdrawn 1983)
these test methods as a result of irreversible reaction with some
G21 Practice for Determining Resistance of Synthetic Poly-
of the medium constituents.
meric Materials to Fungi
NOTE 1—As an example, quaternary ammonium compounds are
2.2 TAPPI Method:
inactivated by agar.
T487 Fungus Resistance for Paper and Paperboard
1.3 A test method is included for use with low-viscosity
adhesives along with an alternative method for use with
3. Terminology
mastic-type adhesives. Also, a method approved by the gov-
3.1 Definitions—Many terms in this test method are defined
ernment is given.
in Terminology D907.
1.4 The values stated in SI units are to be regarded as the
3.2 Definitions of Terms Specific to This Standard:
standard. The values given in parentheses are for information
3.2.1 adhesive preparation, n—the adhesive as packaged for
only.
distribution, storage, and use.
1.5 This standard does not purport to address all of the
3.2.2 adhesive film, n—the small portion of the adhesive
safety concerns, if any, associated with its use. It is the
preparation, as prepared for use by the consumer, either with
responsibility of the user of this standard to establish appro-
additives or as received, which is cast on a substrate, cured 24
priate safety, health, and environmental practices and deter-
h, and represents the glue line.
mine the applicability of regulatory limitations prior to use.
3.2.2.1 Discussion—For purposes of these test methods the
These test methods are designed to be used by persons trained
adhesive film is the thin layer of adhesive spread on either the
in correct microbiological techniques. Specific precautionary
21-mm fiberglass disk as described in 14.2, or the adhesive
statements are given in Section 7 and in 14.3.2.
layer 3 mm thick which is cast on the tile squares as described
1.6 This international standard was developed in accor-
in 15.1.
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
These test methods are under the jurisdiction of ASTM Committee D14 on the ASTM website.
Adhesives and are the direct responsibility of Subcommittee D14.30 on Wood The last approved version of this historical standard is referenced on
Adhesives. www.astm.org.
Current edition approved April 1, 2021. Published April 2021. Originally Available from Technological Association of the Pulp and Paper Industry
approved in 1983. Last previous edition approved in 2013 as D4300 – 01 (2013). (TAPPI), 15 Technology Parkway South, Suite 115, Peachtree Corners, GA 30092,
DOI: 10.1520/D4300-01R21E01. http://www.tappi.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
´1
D4300 − 01 (2021)
3.2.3 zone of inhibition, n—the area on an inoculated agar 5. Apparatus
plate surrounding the adhesive-coated disk or tile, showing a
5.1 In addition to the standard equipment found in any fully
reduced fungal growth or an absence thereof.
equipped microbiological laboratory, items from the following
list are needed for various tests. Not all items are needed for
3.3 Abbreviations:
each test.
3.3.1 PDA—potato dextrose agar.
5.1.1 Chromist Laboratory Spray Unit.
3.3.2 MSA—mineral salts agar.
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
3.3.3 ZI—zone of inhibition.
1°F), or two chambers if needed simultaneously.
5.1.3 Filter Disk, Glass Microfibre, 934-AM, diameter-21
4. Significance and Use
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
satisfactory for this purpose.
anti-fungal properties to prevent growth of fungi frequently
5.1.6 Glass Rods, 305 mm in length having a diameter of
present on the gluing equipment, on adherends, or in the
6.3 mm.
adhesive as applied.
5.1.7 Glove Bag, 68 cm in length and width, 38 cm in
4.2 Potato dextrose agar (PDA) provides a complete me- 7
height.
dium for the growth of fungi, while mineral salts agar (MSA)
5.1.8 Hemacytometer Levy Counting Chamber, cell depth-
lacks a carbohydrate source and provides a less favorable
0.1 mm, Newbauer rulings.
medium. Use of PDA tests the adhesive film for its ability to
5.1.9 Hood, Laminar-Flow Type, Class II Type I.
resist the growth of fungi on its surface as well as its ability to
5.1.10 Jar, Screw Cap, round, approximately 1 L (1 qt,
repel a copious growth of fungi on the adjacent agar surface.
mason type).
Use of MSA tests the adhesive film primarily for its ability to
5.1.11 Pipet, Pasteur.
resist the growth of fungi on its surface. When it is used, there
5.1.12 Petri Dishes, sterile, disposable, top-diameter of
is a reduced possibility that the growth from the agar will be
150-mm, bottom-height of 15-mm.
mis-read as coming from the adhesive film, since fungal
5.1.13 Refrigerator, capable of maintaining 4 6 1°C (39 6
growth on the adjacent agar will be scant. 2°F).
5.1.14 Teflon Paper or Grid, pressure sensitive overlay,
NOTE 2—The method given here using the MSA is based on Practice
coated with TFE-fluorocarbon (PTFE), vinyl sheet backing, to
G21, adapted to be used with adhesives. Requirements to meet the
be used at up to 93°C (200°F).
approval of government specifications are the use of the MSA described
in 10.2, and a mixed species of fungi described in 8.2 for the inoculum.
6. Materials
4.3 The results obtained when using the procedures given in
6.1 Potato Dextrose Agar, Difco or equivalent.
this method apply only to the species used for the testing. The
6.2 Sterile Deionized or Distilled Water.
test species listed in Section 8 are frequently used by labora-
tories to test for antifungal properties, but they are not the only 6.3 Disinfectant Solution—Amphyll, Alcide, or comparable
product.
ones which could be used. Selection of the fungal species to
test against requires informed judgment by the testing labora-
6.4 Materials for Mineral Salts Agar. (See list in 10.2.1.)
tory or by the party requesting the tests. These methods are
6.5 Sorbitan mono-oleate polyoxyethylene.
especially useful when species that have been isolated from
contaminated adhesives are used as the test species (see
7. Precautions
Section 8) to aid in the selection of more effective fungicides.
7.1 Assign laboratory personnel trained in correct microbio-
4.4 The efficacy of some biocides may change in storage logical techniques to run these tests. These test methods
employ live cultures of fungi, some of which are capable of
due to the chemical and thermal environment to which they are
causing disease or allergic reaction in some humans. Use
subjected as components of certain adhesives. These test
proper microbiological procedures in order to prevent contami-
methods are not appropriate for determining the effect of
nation of the cultures or of the work area. Disinfect and
fungal contamination on adhesives under water-soaking
sterilize in an approved manner all spills and all equipment
conditions, because they are not designed to cover the possi-
bility of water-soluble biocides leaching out of the bond line.
Available from Gelman Sciences, Ann Arbor, MI.
4.5 These test methods are dependent upon the physiologi-
Available from laboratory supply houses.
cal action of living microorganisms under a reported set of 7
Available from Instruments for Research and Industry, 108 Franklin Ave.,
conditions. Conclusions about the resistance of the test adhe- Cheltenham, PA, or most laboratory supply houses.
The Biogard Hood or similar equipment is available from laboratory supply
sive to fungal attack can be drawn by comparing the results to
houses.
simultaneously run controls of known resistance. See X5.2 for 9
Gelman Sciences, or most laboratory supply houses.
statements regarding test repeatability. Available commercially as Tween 80.
´1
D4300 − 01 (2021)
coming into contact with the cultures. Also sterilize in an 10.1.1 Prepare sufficient agar slants and plates for culture
approved manner all cultures and contaminated disposable propagation and conducting the tests.
equipment before discarding. See 1.5 and 14.3.2.
10.1.2 Follow the instructions given for preparation of the
commercial product. Dissolve using heat and agitation. Trans-
7.2 In addition to other precautions, the use of a Class II,
fer an appropriate amount of the agar solution to each flask
Type I containment hood is highly recommended for all
used for pouring plates, and 10 mL per test tube. Plug flask
procedures that would cause formation of fungal aerosols. This
with appropriate closures. Cap tubes loosely with metal,
type of laminar flow hood prevents the spread of fungal spores
plastic, or foam caps. Autoclave for 15 min at 103 kPa and a
throughout the laboratory and inhalation of spores by the
temperature of 121°C (250°F). Allow the agar to cool to 48 to
operator. The hoods should be monitored by a biological safety
50°C (118 to 122°F) before pouring the plates, filling to an
officer or a health physicist if they are to be used with
approximate depth of 3 mm. Allow plates to solidify. Tighten
hazardous agents. Refer to the operating manual supplied by
the caps on the tubes and place them in a slanted position to
the manufacturer for detailed information. This warning ap-
solidify, making a slant of about 51 mm. Store slants and plates
plies specifically to the use of the Chromist laboratory spray
in refrigerator until needed.
unit listed in 5.1.1 and in the instructions in 14.3.2.
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
2 4
Aspergillus niger 9642
Magnesium sulfate (MgSO · 7H O) 0.7
4 2
Aspergillus flavus 9643
Ammonium nitrate (NH NO ) 1.0
4 3
Penicillium pinophilum 9644 (See X1.1.6)
Sodium chloride (NaCl) 0.005
Phanerochaete chrysosporium 24725
Ferrous sulfate (FeSO · 7H O) 0.002
4 2
Aureobasidium pullulans
Zinc sulfate (ZnSO · 7H O) 0.002
4 2
Var.—melanigenum 15233
Manganous sulfate (MnSO · 4H O) 0.001
4 2
Agar 15.0
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 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
between 6.0 and 6.5, and sterilize by autoclaving at 103 kPa,
8.2 Cultures of the following species are used for the
government requirements described in Section 16, using MSA: and 121°C (250°F) for 15 min.
10.2.3 Prepare plates as described in 10.1.2, and store in the
ATCC No.
refrigerator until needed.
Aspergillus niger 9642
Aureobasidium pullulans
Var.—melanigenum 15233 11. Fungal Cultures
Chaetomium globosum 6205
Gliocladium virens 9645 11.1 Propagation of Fungal Cultures:
Penicillium pinophilum 9644 (See X1.1.6)
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
label by species and ATCC Number. Incubate at 25 6 0.5°C
following optional species are also sometimes used: Aspergillus flavus,
(77 6 1°F) for a minimum of 10 days or until full sporulation
(ATCC No. 9643) and Aspergillus versicolor (ATCC No. 11730). See 13.2
is achieved.
and Appendix X1.
11.1.2 Refrigerate the cultures. Prepare new cultures each
8.3 Other pure cultures or mixed cultures of fungal species
month. If contamination occurs, discard the cultures and
may be used, if agreed upon between the interested parties and
prepare new ones.
upon the recommendation of the testing laboratories.
11.2 Preparation of Fungal Inoculum:
9. Sterilization of Equipment and Media
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
9.1 Follow accepted microbiological practices for sterilizing
the tests.
equipment and media.
11.2.2 Harvesting Fungal Cultures and Dislodging
NOTE 5—Two references for sterilization methods are TAPPI T487 (see
Spores—To one tube of each species of fungi, add 15 mL of
2.2) and Ref (1).
sterile distilled or deionized water, containing 0.05 % sorbitan
mono-oleate polyoxyethylene. Harvest fungal cultures and
10. Preparation of Media
dislodge spores by rubbing the growth gently with a sterile
10.1 Potato Dextrose Agar:
inoculating loop or by removing it with a sterile glass rod.
Transfer the washings into a sterilized container containing
glass beads and shake thoroughly to break up the clumps. Filter
Cultures may be purchased from the American Type Culture Collection, 12301
through sterile layered cheese cloth or sterile nonabsorbent
Parkl
...
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 2013) D4300 − 01 (Reapproved 2021)
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
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 safety, health, and healthenvironmental 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
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 Oct. 1, 2013April 1, 2021. Published October 2013April 2021. Originally approved in 1983. Last previous edition approved in 20082013 as
D4300 – 01 (2008).(2013). DOI: 10.1520/D4300-01R13.10.1520/D4300-01R21E01.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
´1
D4300 − 01 (2021)
D1286 Method of Test for Effect of Mold Contamination on Permanence of Adhesives Preparations and Adhesives Bonds
(Withdrawn 1983)
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, 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-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 use of the MSA described in 10.2, and a mixed species of fungi described 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
The last approved version of this historical standard is referenced on www.astm.org.
Available from TechnicalTechnological Association of the Pulp and Paper Industry (TAPPI), 15 Technology Parkway South, Norcross, Suite 115, Peachtree Corners, GA
30092, http://www.tappi.org.
´1
D4300 − 01 (2021)
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.
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), or two
chambers if needed simultaneously.
5.1.3 Filter Disk, Glass Microfibre, 934-AM, 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, bottom-height of 15-mm.
5.1.13 Refrigerator, capable of maintaining 4 6 1°C (39 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).
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.
Available from Gelman Sciences, Ann Arbor, MI.
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 laboratory supply houses.
´1
D4300 − 01 (2021)
6.4 Materials for Mineral Salts Agar. (See list in 10.2.1.)
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 recommended for all procedures that
would cause formation of fungal aerosols. This type of laminar flow hood prevents the spread of fungal spores throughout the
laboratory and inhalation of spores by the operator. The hoods 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.
8.1.1 Aspergillus niger
Aspergillus niger 9642
8.1.2 Aspergillus flavus
Aspergillus flavus 9643
8.1.3 Penicillium pinophilum
Penicillium pinophilum 9644 (See X1.1.6)
8.1.4 Phanerochaete
chrysosporium
Phanerochaete chrysosporium 24725
8.1.5 Aureobasidium
pullulans
Aureobasidium pullulans
Var.—melanigenum
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:
Available commercially as Tween 80.
Cultures may be purchased from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852.
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ATCC No.
8.2.1 Aspergillus niger
Aspergillus niger 9642
8.2.2 Aureobasidium
pullulans
Aureobasidium pullulans
Var.—melanigenum
8.2.3 Chaetomium globosum
Chaetomium globosum 6205
8.2.4 Gliocladium virens
Gliocladium virens 9645
8.2.5 Penicillium pinophilum
Penicillium pinophilum 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.
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).
Allow the agar to cool to 48 to 50°C (118 to 122°F) before pouring the plates, filling to an approximate depth of 3 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.
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) for 15 min.
10.2.3 Prepare plates as described in 10.1.2, and store in the refrigerator until needed.
The boldface numbers in parentheses refer to the references at the end of this standard.
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11. Fungal Cultures
11.1 Propagation of Fungal Cultures:
11.1.1 Prepare a fresh culture for each species on
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