ASTM D4300-01(2008)
(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
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.
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 G 21, 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.
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.
The efficacy of some biocides may change in storage due to the chemical and thermal environment to which they are subjected as comp...
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 and health 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.
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Designation: D4300 − 01 (Reapproved2008)
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.Anumber 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 Department of Defense.
1. Scope* D1286Method ofTest for Effect of Mold Contamination on
Permanence of Adhesives Preparations and Adhesives
1.1 These test methods test the ability of adhesive films to
Bonds (Withdrawn 1983)
inhibit or support the growth of selected fungal species
G21Practice for Determining Resistance of Synthetic Poly-
growing on agar plates by providing means of testing the films
meric Materials to Fungi
on two agar substrates, one which promotes microbial growth,
2.2 TAPPI Method:
and one which does not.
T487Fungus Resistance for Paper and Paperboard
1.2 Thesetestmethodsarenotappropriateforalladhesives.
The activity of certain biocides may not be demonstrated by
3. Terminology
thesetestmethodsasaresultofirreversiblereactionwithsome
3.1 Definitions—Manytermsinthistestmethodaredefined
of the medium constituents.
in Terminology D907.
NOTE 1—As an example, quaternary ammonium compounds are
3.2 Definitions of Terms Specific to This Standard:
inactivated by agar.
3.2.1 adhesive preparation, n—theadhesiveaspackagedfor
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
3.2.2 adhesive film, n—the small portion of the adhesive
mastic-type adhesives. Also, a method approved by the gov-
preparation, as prepared for use by the consumer, either with
ernment is given.
additives or as received, which is cast on a substrate, cured 24
h, and represents the glue line.
1.4 The values stated in SI units are to be regarded as the
standard. The values given in parentheses are for information 3.2.2.1 Discussion—For purposes of these test methods the
adhesive film is the thin layer of adhesive spread on either the
only.
21-mm fiberglass disk as described in 14.2, or the adhesive
1.5 This standard does not purport to address all of the
layer 3 mm thick which is cast on the tile squares as described
safety concerns, if any, associated with its use. It is the
in 15.1.
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica- 3.2.3 zone of inhibition, n—the area on an inoculated agar
plate surrounding the adhesive-coated disk or tile, showing a
bility of regulatory limitations prior to use.Thesetestmethods
are designed to be used by persons trained in correct micro- reduced fungal growth or an absence thereof.
biological techniques. Specific precautionary statements are
3.3 Abbreviations:
given in Section 7 and in 14.3.2.
3.3.1 PDA—potato dextrose agar.
2. Referenced Documents 3.3.2 MSA—mineral salts agar.
2.1 ASTM Standards: 3.3.3 ZI—zone of inhibition.
D907Terminology of Adhesives
4. Significance and Use
These test methods are under the jurisdiction of ASTM Committee D14 on
4.1 Thesetestmethodsaredesignedtobeusedtodetermine
Adhesives and are the direct responsibility of Subcommittee D14.30 on Wood
the susceptibility of the adhesive film to biodegradation and
Adhesives.
whether the adhesive will carry into the bond line sufficient
Current edition approved April 1, 2008. Published April 2008. Originally
approved in 1983. Last previous edition approved in 2001 as D4300–01. DOI:
10.1520/D4300-01R08.
2 3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or The last approved version of this historical standard is referenced on
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM www.astm.org.
Standards volume information, refer to the standard’s Document Summary page on Available from TechnicalAssociation of the Pulp and Paper Industry (TAPPI),
the ASTM website. 15 Technology Parkway South, Norcross, GA 30092, 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 − 01 (2008)
anti-fungal properties to prevent growth of fungi frequently 5.1.5 Filter Paper Assay Disk, 1.5 cm diameter, sterile.
present on the gluing equipment, on adherends, or in the Schleicher and Schnell, Inc., or the equivalent, has been found
adhesive as applied. satisfactory for this purpose.
5.1.6 Glass Rods, 305 mm in length having a diameter of
4.2 Potato dextrose agar (PDA) provides a complete me-
6.3 mm.
dium for the growth of fungi, while mineral salts agar (MSA)
5.1.7 Glove Bag, 68 cm in length and width, 38 cm in
lacks a carbohydrate source and provides a less favorable
height.
medium. Use of PDA tests the adhesive film for its ability to
5.1.8 Hemacytometer Levy Counting Chamber, cell depth-
resist the growth of fungi on its surface as well as its ability to
0.1 mm, Newbauer rulings.
repel a copious growth of fungi on the adjacent agar surface.
5.1.9 Hood, Laminar-Flow Type, Class II Type I.
Use of MSAtests the adhesive film primarily for its ability to
5.1.10 Jar, Screw Cap, round, approximately 1 L (1 qt,
resist the growth of fungi on its surface.When it is used, there
mason type).
is a reduced possibility that the growth from the agar will be
5.1.11 Pipet, Pasteur.
mis-read as coming from the adhesive film, since fungal
5.1.12 Petri Dishes, sterile, disposable, top-diameter of
growth on the adjacent agar will be scant.
150-mm, bottom-height of 15-mm.
NOTE 2—The method given here using the MSA is based on Practice
5.1.13 Refrigerator, capable of maintaining 4 6 1°C (39 6
G21, adapted to be used with adhesives. Requirements to meet the
2°F).
approval of government specifications are the use of the MSA described
5.1.14 Teflon Paper or Grid, pressure sensitive overlay,
in 10.2, and a mixed species of fungi described in 8.2 for the inoculum.
coated with TFE-fluorocarbon (PTFE), vinyl sheet backing, to
4.3 Theresultsobtainedwhenusingtheproceduresgivenin
be used at up to 93°C (200°F).
this method apply only to the species used for the testing. The
test species listed in Section 8 are frequently used by labora-
6. Materials
toriestotestforantifungalproperties,buttheyarenottheonly
6.1 Potato Dextrose Agar, Difco or equivalent.
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-
tory or by the party requesting the tests. These methods are
6.3 Disinfectant Solution—Amphyll,Alcide, or comparable
especially useful when species that have been isolated from
product.
contaminated adhesives are used as the test species (see
6.4 Materials for Mineral Salts Agar. (See list in 10.2.1.)
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
duetothechemicalandthermalenvironmenttowhichtheyare
7. Precautions
subjected as components of certain adhesives. These test
7.1 Assignlaboratorypersonneltrainedincorrectmicrobio-
methods are not appropriate for determining the effect of
logical techniques to run these tests. These test methods
fungal contamination on adhesives under water-soaking
employ live cultures of fungi, some of which are capable of
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.
propermicrobiologicalproceduresinordertopreventcontami-
4.5 These test methods are dependent upon the physiologi-
nation of the cultures or of the work area. Disinfect and
cal action of living microorganisms under a reported set of
sterilize in an approved manner all spills and all equipment
conditions. Conclusions about the resistance of the test adhe-
coming into contact with the cultures. Also sterilize in an
sive to fungal attack can be drawn by comparing the results to
approved manner all cultures and contaminated disposable
simultaneously run controls of known resistance. See X5.2 for
equipment before discarding. See 1.5 and 14.3.2.
statements regarding test repeatability.
7.2 In addition to other precautions, the use of a Class II,
Type I containment hood is highly recommended for all
5. Apparatus
proceduresthatwouldcauseformationoffungalaerosols.This
5.1 Inadditiontothestandardequipmentfoundinanyfully
type of laminar flow hood prevents the spread of fungal spores
equipped microbiological laboratory, items from the following
throughout the laboratory and inhalation of spores by the
list are needed for various tests. Not all items are needed for
operator.Thehoodsshouldbemonitoredbyabiologicalsafety
each test.
officer or a health physicist if they are to be used with
5.1.1 Chromist Laboratory Spray Unit.
hazardous agents. Refer to the operating manual supplied by
5.1.2 Constant Temperature Chamber, capable of being
the manufacturer for detailed information. This warning ap-
maintained at 35 6 0.5°C (95 6 1°F) or 25 6 0.5°C (77 6
plies specifically to the use of the Chromist laboratory spray
1°F), or two chambers if needed simultaneously.
unit listed in 5.1.1 and in the instructions in 14.3.2.
5.1.3 Filter Disk, Glass Microfibre, 934-AM, diameter-21
mm.
Available from Instruments for Research and Industry, 108 Franklin Ave.,
5.1.4 Filter Disk, Sterile Whatman No. 1.
Cheltenham, PA, or most laboratory supply houses.
The Biogard Hood or similar equipment is available from laboratory supply
houses.
5 9
Available from Gelman Sciences, Ann Arbor, MI. Gelman Sciences, or most laboratory supply houses.
6 10
Available from laboratory supply houses. Available commercially as Tween 80.
D4300 − 01 (2008)
8. Test Species of Fungi 10.2.1 Dissolve in 1 L of water the designated amounts of
the following reagents:
8.1 Cultures of one or more of the following species are
Grams
suggested for use when PDA is the medium:
Potassium phosphate (KH PO)0.7
2 4
ATCC No.
Magnesium sulfate (MgSO ·7H O) 0.7
4 2
Ammonium nitrate (NH NO)1.0
4 3
8.1.1 Aspergillus niger 9642
Sodium chloride (NaCl) 0.005
8.1.2 Aspergillus flavus 9643
Ferrous sulfate (FeSO ·7H O) 0.002
4 2
8.1.3 Penicillium pinophilum 9644 (See X1.1.6)
Zinc sulfate (ZnSO ·7H O) 0.002
4 2
8.1.4 Phanerochaete chrysosporium 24725
Manganous sulfate (MnSO·4H O) 0.001
4 2
8.1.5 Aureobasidium pullulans
Agar 15.0
Var.—melanigenum 15233
10.2.2 Adjust the pH of the medium by the addition of
NOTE 3—The choice of test organisms is often made from the fungal
0.01N NaOH solution so that after sterilization the pH is
species listed above. Information on these and other species is given in
between 6.0 and 6.5, and sterilize by autoclaving at 103 kPa,
Appendix X1.
and 121°C (250°F) for 15 min.
8.2 Cultures of the following species are used for the
10.2.3 Prepareplatesasdescribedin10.1.2,andstoreinthe
governmentrequirementsdescribedinSection16,usingMSA:
refrigerator until needed.
ATCC No.
11. Fungal Cultures
8.2.1 Aspergillus niger 9642
8.2.2 Aureobasidium pullulans
11.1 Propagation of Fungal Cultures:
Var.—melanigenum 15233
11.1.1 Prepare a fresh culture for each species on PDAand
8.2.3 Chaetomium globosum 6205
label by species and ATCC Number. Incubate at 25 6 0.5°C
8.2.4 Gliocladium virens 9645
8.2.5 Penicillium pinophilum 9644 (See X1.1.6)
(77 6 1°F) for a minimum of 10 days or until full sporulation
is achieved.
NOTE 4—The species listed in 8.2 are used in Practice G21. The
following optional species are also sometimes used: Aspergillus flavus, 11.1.2 Refrigerate the cultures. Prepare new cultures each
(ATCCNo.9643)and Aspergillus versicolor(ATCCNo.11730).See13.2
month. If contamination occurs, discard the cultures and
and Appendix X1.
prepare new ones.
8.3 Other pure cultures or mixed cultures of fungal species
11.2 Preparation of Fungal Inoculum:
may be used, if agreed upon between the interested parties and
11.2.1 Follow the procedure in 11.1.1 to prepare fresh
upon the recommendation of the testing laboratories.
cultures on PDAslants for each species to be used to conduct
the tests.
9. Sterilization of Equipment and Media
11.2.2 Harvesting Fungal Cultures and Dislodging
9.1 Followacceptedmicrobiologicalpracticesforsterilizing
Spores—To one tube of each species of fungi, add 15 mL of
equipment and media.
sterile distilled or deionized water, containing 0.05% sorbitan
mono-oleate polyoxyethylene. Harvest fungal cultures and
NOTE5—TworeferencesforsterilizationmethodsareTAPPIT487(see
2.2) and Ref (1).
dislodge spores by rubbing the growth gently with a sterile
inoculating loop or by removing it with a sterile glass rod.
10. Preparation of Media
Transfer the washings into a sterilized container containing
10.1 Potato Dextrose Agar:
glassbeadsandshakethoroughlytobreakuptheclumps.Filter
10.1.1 Prepare sufficient agar slants and plates for culture
through sterile layered cheese cloth or sterile nonabsorbent
propagation and conducting the tests.
cotton. Adjust the spore level to 1.0 × 10 per mL, using a
10.1.2 Follow the instructions given for preparation of the
hemacytometer and the procedure in Annex. Use this spore
commercial product. Dissolve using heat and agitation. Trans-
suspension of a single species of fungi as the inoculum for the
fer an appropriate amount of the agar solution to each flask
tests described in Sections 14 and 15 when using the option
used for pouring plates, and 10 mL per test tube. Plug flask
given in 13.1.1.1.
with appropriate closures. Cap tubes loosely with metal,
11.2.3 For a mixed culture, obtain a spore count on each
plastic, or foam caps. Autoclave for 15 min at 103 kPa and a
fungal species, and adjust each suspension to the level of 1 ×
temperature of 121°C (250°F).Allow the agar to cool to 48 to 0 per mL. Combine equal portions of the spore suspensions
50°C (118 to 122°F) before pouring the plates, filling to an
from each of the species in a common sterilized container. Use
approximate depth of 3 mm. Allow plates to solidify. Tighten this mixed spore suspension for the tests described in Sections
the caps on the tubes and place them in a slanted position to
14, 15, and 16 when using the option given in 13.1.1.2.
solidify,makingaslantofabout51mm.Storeslantsandplates
12. Adhesive Sample
in refrigerator until needed.
12.1 For ready-to-use liquid adhesives, obtain an approxi-
10.2 Mineral S
...
This document is not anASTM standard and is intended only to provide the user of anASTM 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.
Designation:D4300–98a Designation:D4300–01(Reapproved2008)
Standard Test Methods for
Ability of Adhesive Films to Support or Resist the Growth of
Fungi
This standard is issued under the fixed designation D 4300; 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 (e) indicates an editorial change since the last revision or reapproval.
This standard has been approved for use by agencies of the Department of Defense.
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 and health 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 Note 9. and in 14.3.2.
2. Referenced Documents
2.1 ASTM Standards:
D907Terminology of Adhesives
D 907 Terminology of Adhesives
D1286Test Method 1286 Methods of Test for Effect of Mold Contamination on Permanence of Adhesive Preparations and
Adhesive Bonds
G 21 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:
3.1.1resist, vt, as related to fungi—to fend off or withstand the growth.
3.1.2support, vt, as related to fungi—to submit to the growth. Definitions—Many terms in this test method are defined in
Terminology D 907.
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.
These test methods are under the jurisdiction of ASTM Committee D-14 on Adhesives and are the direct responsibility of Subcommittee D14.30 on Wood Adhesives.
Current edition approved Sept. 10, 1998. Published March 1999. Originally published as D4300 – 83. Last previous edition D4300 – 98. Replaces D1286 – 57 (1979).
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, 2008. Published April 2008. Originally approved in 1983. Last previous edition approved in 2001 as D 4300 – 01.
For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
, Vol 15.06.volume information, refer to the standard’s Document Summary page on the ASTM website.
Withdrawn.
Annual Book of ASTM Standards, Vol 14.02.
Available from Technical Association of the Pulp and Paper Industry (TAPPI), 15 Technology Parkway South, Norcross, GA 30092, 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.
D4300–01 (2008)
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—theareaonaninoculatedagarplatesurroundingtheadhesive-coateddiskortile,showingareduced
fungal growth or an absence thereof.
3.3 Abbreviations:Abbreviations: 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
whethertheadhesivewillcarryintothegluebondlinesufficientanti-fungalpropertiestopreventgrowthoffungifrequentlypresent
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 PDAtests 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 MSAtests 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 MSAis based on Practice G 21, 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 as components of certain adhesives. These test methods are not appropriate for determining the effect of fungal
contaminationonadhesivesunderwater-soakingconditions,becausetheyarenotdesignedtocoverthepossibilityofwater-soluble
biocides leaching out of the gluebond line.
4.5 Thesetestmethodsaredependentuponthephysiologicalactionoflivingmicroorganismsunderareportedsetofconditions.
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 reapeatability.
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.
Available from TAPPI, P. O. Box 105113, Atlanta, GA30348.
Available from Gelman Sciences, Ann Arbor, MI.
Available from Gelman Sciences, Ann Arbor, MI.
Available from laboratory supply houses.
Available from laboratory supply houses.
Available from Instruments for Research and Industry, 108 Franklin Ave., Cheltenham, PA, or most 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.
D4300–01 (2008)
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.
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 Note 9and 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 , the instructions in
and in the instructions in 14.3.2, and Note 9.
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 9642
8.1.2 Aspergillus flavus 9643
8.1.3 Penicillium pinophilum 9644 (See X1.6)
8.1.3 Penicillium pinophilum 9644 (See X1.1.6)
8.1.4 Phanerochaete chrysosporium 24725
8.1.5 Aureobasidium pullulans
Var.—melanigenum 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 inAppendix
X1.
8.2 Cultures of the following species are used for the government requirements described in Section 16, using MSA:
ATCC No.
8.2.1 Aspergillus niger 9642
8.2.2 Aureobasidium pullulans
Var.—melanigenum 15233
8.2.3 Chaetomium globosum 6205
8.2.4 Gliocladium virens 9645
8.2.5 Penicillium pinophilum 9644 (See X1.6)
8.2.5 Penicillium pinophilum 9644 (See X1.1.6)
NOTE 4—The species listed in 8.2 are used in Practice G 21. 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.
The Biogard Hood or similar equipment is available from laboratory supply houses.
Gelman Sciences, or most laboratory supply houses.
Gelman Sciences, or most laboratory supply houses.
Available commercially as Tween 80.
Available commercially as Tween 80.
Cultures may be purchased from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852.
D4300–01 (2008)
NOTE 5—Two references for sterilization methods are TAPPIT487 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 mLper test tube. Plug flask with appropriate
closures.Captubeslooselywithmetal,plastic,orfoamcaps.Autoclavefor15minat103kPaandatemperatureof121°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 6.5,6.5, 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.
11. Fungal Cultures
11.1 Propagation of Fungal Cultures:
11.1.1
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