ASTM D4783-01(2008)

Standard Test Methods for Resistance of Adhesive Preparations in Container to Attack by Bacteria, Yeast, and Fungi

SIGNIFICANCE AND USE
These test methods are used to demonstrate whether an adhesive preparation is sufficiently protected with biocide to resist attack by bacteria, yeast, and fungi during its storage life. They are patterned after methods used by biological laboratories serving the adhesive industry.
These test methods may also be used to determine the efficacy of different biocide systems against specific microorganisms.
These test methods are especially useful when tested against wild-type microorganisms which have been isolated from contaminated adhesives as an aid in determining the amount and type of biocide necessary to kill or inhibit the growth of the contaminants. If an isolated microorganism not generally used as a challenge organism, is chosen as the inoculum, it is important to identify the organism and determine on which medium and under what conditions it will grow, in order to demonstrate the efficacy of the biocide.
The results obtained when using the procedures given in these methods apply only to the species which are used for the testing. The test species listed in Section 9 are frequently used by laboratories to test for antimicrobial properties, but they are not the only ones which could be used. Selection of the species to use for these test methods requires informed judgment by the testing laboratory or by the party requesting the tests. It is also important that species which commonly attack adhesives be used. See 9.4.
The presence of an active biocide carried over from the adhesive specimen to the agar could have an inhibiting effect on the growth of microorganisms, resulting in no growth during the span of a normal incubation period, when in fact, viable microorganisms are present, but their growth has been slowed down or held in stasis. The use of Letheen agar and broth is recommended to neutralize the effect of this carry-over.
Note 4—Letheen agar may be used for the streak plates, or if another agar is chosen for testing, a Letheen agar plate cou...
SCOPE
1.1 These test methods cover the determination of the resistance of liquid adhesive preparations to microbial attack in the container by challenging adhesive specimens with cultures of bacteria, yeast, or fungi, and checking for their ability to return to sterility. These test methods return qualitative results.
1.2 The values stated in SI units are to be regarded as the standard. The values in parentheses are for information only.
1.3 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 technique. Specific precautionary statements are given in Section 8.

General Information

• Status: Historical
• Publication Date: 31-Mar-2008
• ICS: 83.180 - Adhesives
• Technical Committee: D14 - Adhesives
• Drafting Committee: D14.30 - Wood Adhesives

Relations

• Replaces: ASTM D4783-01e1 - Standard Test Methods for Resistance of Adhesive Preparations in Container to Attack by Bacteria, Yeast, and Fungi
• Effective Date: 01-Apr-2008
• Referred By: ASTM D7799-12a(2021) - Standard Specification for Tufted and Woven Broadloom Carpet Adhesives Without Homogenous PVC or Non-PVC Backings
• Effective Date: 01-Apr-2008
• Referred By: ASTM C1894-22 - Standard Guide for Microbially Induced Corrosion of Concrete Products
• Effective Date: 01-Apr-2008
• Referred By: ASTM D6465-99(2016) - Standard Guide for Selecting Aerospace and General Purpose Adhesives and Sealants
• Effective Date: 01-Apr-2008
• Referred By: ASTM E3152-23 - Standard Guide for Standard Test Methods and Practices Available for Determining Antifungal Activity on Natural or Synthetic Substrates Treated with Antimicrobial Agents
• Effective Date: 01-Apr-2008
• Referred By: ASTM D8397-23 - Standard Specification for Acrylic and Reactive Adhesives for Installation of Vinyl and Rubber Floor Coverings
• Effective Date: 01-Apr-2008
• Referred By: ASTM D3498-19a - Standard Specification for Adhesives for Field-Gluing Wood Structural Panels (Plywood or Oriented Strand Board) to Wood Based Floor System Framing
• Effective Date: 01-Apr-2008

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D4783 − 01 (Reapproved2008)
Standard Test Methods for
Resistance of Adhesive Preparations in Container to Attack
by Bacteria, Yeast, and Fungi
This standard is issued under the fixed designation D4783; 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.
serial dilution method of determining plate count using a pour plate
1. Scope*
technique.
1.1 These test methods cover the determination of the
2.2 TAPPI Method:
resistanceofliquidadhesivepreparationstomicrobialattackin
T487Fungus Resistance of Paper and Paperboard
the container by challenging adhesive specimens with cultures
of bacteria, yeast, or fungi, and checking for their ability to
2.3 CSMA:
return to sterility.These test methods return qualitative results.
Cosmetics Preservation, Method38
1.2 The values stated in SI units are to be regarded as the
3. Terminology
standard. The values in parentheses are for information only.
3.1 Definitions—Many terms in these test methods are
1.3 This standard does not purport to address all of the
defined in Terminology D907.
safety concerns, if any, associated with its use. It is the
3.2 Definitions of Terms Specific to This Standard:
responsibility of the user of this standard to establish appro-
3.2.1 adhesivepreparation,n—theadhesiveaspackagedfor
priate safety and health practices and determine the applica-
distribution, storage, and use.
bility of regulatory limitations prior to use.Thesetestmethods
are designed to be used by persons trained in correct micro-
3.3 Abbreviations:
biological technique. Specific precautionary statements are
3.3.1 PBS—phosphate buffered saline.
given in Section 8.
3.3.2 PDA—potato dextrose agar.
3.3.3 YMPG—yeast malt peptone glucose (agar).
2. Referenced Documents
2.1 ASTM Standards:
4. Summary of Test Methods
D907Terminology of Adhesives
4.1 The adhesive specimen is challenged by inoculation
D4299TestMethodforEffectofBacterialContaminationon
with a culture of bacteria, yeast, or fungi, which may be a
Performance of Adhesive Preparations and Adhesives
single species or a mixed culture of several species, following
Films (Withdrawn 1990)
the guidelines given in Note 6. The inoculated adhesive
D4300Test Methods for Ability of Adhesive Films to
specimenisstoredat21to27°C(70to80°F)for7days,during
Support or Resist the Growth of Fungi
whichtimecultures(streakplates)aremadeatpresetintervals.
E640Test Method for Preservatives in Water-Containing
See Note 2. At any point in the series of challenges, if the
Cosmetics
inoculated specimen shows microbial growth on the streak
NOTE 1—Test Method E640 is under the jurisdiction of ASTM
plates made during the week following the challenge (indicat-
Committee E35 on Pesticides. The procedure in this method outlines a
ing that it has not returned to sterility), the test is discontinued,
and the sample is reported as not resistant to attack in the
These test methods are under the jurisdiction of ASTM Committee D14 on container by the species or combination of species used as the
Adhesives and are the direct responsibility of Subcommittee D14.30 on Wood
inoculum. If the cultures show no growth, the test is repeated
Adhesives.
with up to four challenges. If the specimen tests out as sterile
Current edition approved April 1, 2008. Published April 2008. Originally
´1
following the fourth challenge, it is reported to be resistant to
approved in 1988. Last previous edition approved in 2001 as D4783–01 . DOI:
10.1520/D4783-01R08.
attackinthecontainerbythespeciesorcombinationofspecies
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. Available from TechnicalAssociation of the Pulp and Paper Industry (TAPPI),
The last approved version of this historical standard is referenced on 15 Technology Parkway South, Norcross, GA 30092, http://www.tappi.org.
www.astm.org. This method is the same as Test Method E640.
*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
D4783 − 01 (2008)
biocide. Letheen broth is recommended for the diluent and Letheen agar
of bacteria, fungi, or yeast used as the inoculum. At the
for the pour plate. See Note 1.
discretion of the biological laboratory, the test may be discon-
tinued after the second or third challenge. See Section 16 for
5. Significance and Use
further interpretation.
4.2 The time necessary to kill is determined by noting the 5.1 These test methods are used to demonstrate whether an
earliest streak plate to read sterile. If the 4-h plate is positive adhesive preparation is sufficiently protected with biocide to
and the 24-h plate is negative, the kill time could be narrowed
resistattackbybacteria,yeast,andfungiduringitsstoragelife.
down further by repeating the challenge and making streak
They are patterned after methods used by biological laborato-
platesatintervalsof4,8,12,and24hfollowingthechallenge.
ries serving the adhesive industry.
4.3 The testing laboratory has the option of changing the
5.2 These test methods may also be used to determine the
timing of the challenges, the sterility checks, and the incuba-
efficacy of different biocide systems against specific microor-
tion period.
ganisms.
NOTE 2—Two proposed schedules for the challenging and sterility
5.3 These test methods are especially useful when tested
checks are shown in Table 1 and Table 2, Schedule A for bacteria and
yeast,andScheduleBforfungi.Theexactformattobefollowedwillvary, against wild-type microorganisms which have been isolated
according to the convenience of the schedule to the testing laboratory and
from contaminated adhesives as an aid in determining the
special circumstances relating to the problem being addressed.
amount and type of biocide necessary to kill or inhibit the
NOTE 3—A serial-dilution plate-count method of checking for sterility
growth of the contaminants. If an isolated microorganism not
may be used when numerical information is needed on the population of
viable organisms or the reduction in population with increasing levels of generally used as a challenge organism, is chosen as the
TABLE 1 Schedule A—Proposed Bacteria and Yeast Testing, Covering 4-h, 24-h, 48-h, 72-h, and 7-Day Tests
Day of Week Day no. First Challenge Second Challenge Third Challenge Fourth Challenge
Monday (−1) inoculate fresh bacterial . . .
or yeast culture
Tuesday 0 prepare suspension . . .
Tuesday 0 inoculate specimens . . .
Tuesday (0 + 4 h) streak 4-h plate . . .
Wednesday 1 streak 24-h plate . . .
Thursday 2 streak 48-h plate . . .
Friday 3 streak 72-h plate . . .
Sat./Sun. 4–5 . . . .
Monday 6 . inoculate fresh bacterial . .
or yeast culture
Tuesday 7 . prepare suspension . .
Tuesday 7 streak 7-day plate inoculate specimens . .
Tuesday (7 + 4 h) read 4-h plate streak 4-h plate . .
Wednesday 8 read 24-h plate streak 24-h plate . .
Thursday 9 read 48-h plate streak 48-h plate . .
Friday 10 read 72-h plate streak 72-h plate . .
Sat./Sun. 11–12 . . . .
Monday 13 . . inoculate fresh bacterial .
or yeast culture
Tuesday 14 . . prepare suspension .
Tuesday 14 read 7-day plate streak 7-day plate inoculate specimens .
Tuesday (14 + 4 h) . read 4-h plate streak 4-h plate .
Wednesday 15 . read 24-h plate streak 24-h plate .
Thursday 16 . read 48-h plate streak 48-h plate .
Friday 17 . read 72-h plate streak 72-h plate .
Sat./Sun. 18–19 . . . .
Monday 20 . . . inoculate fresh
bacterial or yeast
culture
Tuesday 21 . . . prepare suspension
Tuesday 21 . read 7-day plate streak 7-day plate inoculate specimens
Tuesday (21 + 4 h) . . read 4-h plate streak 4-h plate
Wednesday 22 . . read 24-h plate streak 24-h plate
Thursday 23 . . read 48-h plate streak 48-h plate
Friday 24 . . read 72-h plate streak 72-h plate
Sat./Sun. 25–26 . . . .
Monday 27 . . . .
Tuesday 28 . . read 7-day plate streak 7-day plate
Tuesday (28 + 4 h) . . . read 4-h plate
Wednesday 29 . . . read 24-h plate
Thursday 30 . . . read 48-h plate
Friday 31 . . . read 72-h plate
Sat./Sun. 32–33 . . . .
Monday 34 . . . .
Tuesday 35 . . . read 7-day plate
D4783 − 01 (2008)
TABLE 2 Schedule B—Proposed Fungi Testing, Covering 4-h, 24-h, 48-h, 72-h, and 7-Day Tests
Day of Week Day no. First Challenge Second Challenge Third Challenge Fourth Challenge
Friday (−10) inoculate fresh fungal . . .
culture
Friday (−3) . inoculate fresh . .
fungal culture
Monday 0 prepare spore . . .
suspension
Monday 0 inoculate specimens . . .
Monday (0 + 4 h) streak 4-h plate . . .
Tuesday 1 streak 24-h plate . . .
Wednesday 2 streak 48-h plate . . .
Thursday 3 streak 72-h plate . . .
Friday 4 . . inoculate fresh fungal .
culture
Sat./Sun. 5, 6 . . . .
Monday 7 . prepare spore . .
suspension
Monday 7 streak 7-day plate inoculate specimens . .
Monday (7 + 4 h) read 4-h plate streak 4-h plate . .
Tuesday 8 read 24-h plate streak 24-h plate . .
Wednesday 9 read 48-h plate streak 48-h plate . .
Thursday 10 read 72-h plate streak 72-h plate . inoculate fresh
fungal culture
Fri./Sat./Sun. 11, 12, 13 . . . .
Monday 14 . . prepare spore .
suspension
Monday 14 read 7-day plate streak 7-day plate inoculate specimens .
Monday (14 + 4 h) . read 4-h plate streak 4-h plate .
Tuesday 15 . read 24-h plate streak 24-h plate .
Wednesday 16 . read 48-h plate streak 48-h plate .
Thursday 17 . read 72-h plate streak 72-h plate .
Fri./Sat./Sun. 18, 19, 20 . . . .
Monday 21 . . . prepare spore
suspension
Monday 21 . read 7-day plate streak 7-day plate inoculate specimens
Monday (21 + 4 h) . . read 4-h plate streak 4-h plate
Tuesday 22 . . read 24-h plate streak 24-h plate
Wednesday 23 . . read 48-h plate streak 48-h plate
Thursday 24 . . read 72-h plate streak 72-h plate
Fri./Sat./Sun. 25, 26, 27 . . . .
Monday 28 . . read 7-day plate streak 7-day plate
Monday (28 + 4 h) . . . read 4-h plate
Tuesday 29 . . . read 24-h plate
Wednesday 30 . . . read 48-h plate
Thursday 31 . . . read 72-h plate
Fri./Sat./Sun. 32, 33, 34 . . . .
Monday 35 . . . read 7-day plate
NOTE 4—Letheen agar may be used for the streak plates, or if another
inoculum, it is important to identify the organism and deter-
agar is chosen for testing, a Letheen agar plate could be streaked as a
mineonwhichmediumandunderwhatconditionsitwillgrow,
control to test against the neutralizing effect. Even more effective would
in order to demonstrate the efficacy of the biocide.
be diluting the challenged adhesive specimen with Letheen broth and
running Letheen agar pour plates. See Note 1 and Note 3. Extending the
5.4 Theresultsobtainedwhenusingtheproceduresgivenin
incubation period of negative plates would be another safeguard. To
these methods apply only to the species which are used for the
neutralize thiazoline-based preservatives, 10 to 50 ppm of sodium
testing. The test species listed in Section 9 are frequently used
thioglycolate can be added to the medium.
by laboratories to test for antimicrobial properties, but they are
5.6 These test methods are dependent upon the physiologi-
nottheonlyoneswhichcouldbeused.Selectionofthespecies
cal action of living microorganisms under a reported set of
touseforthesetestmethodsrequiresinformedjudgmentbythe
conditions. Conclusions about the resistance of the test adhe-
testing laboratory or by the party requesting the tests. It is also
sive to microbiological attack can be drawn by comparing the
important that species which commonly attack adhesives be
resultstosimultaneouslyruncontrolsofknownresistance.See
used. See 9.4.
X5.2 for statements regarding test repeatability.
5.5 The presence of an active biocide carried over from the
6. Apparatus
adhesive specimen to the agar could have an inhibiting effect
on the growth of microorganisms, resulting in no growth 6.1 In addition to the standard equipment found in any fully
during the span of a normal incubation period, when in fact, equipped microbiological laboratory, the following items are
viable microorganisms are present, but their growth has been sometimes needed:
slowed down or held in stasis. The use of Letheen agar and 6.1.1 Autoclave, capable of producing 103 kPa of steam
broth is recommended to neutralize the effect of this carry- pressureat121°C(250°F)andmaintainingitforaminimumof
over. 15 min.
D4783 − 01 (2008)
6.1.2 Cell Counting Chamber, Petroff-Hausser, cell depth— 8. Precautions
0.02 mm (or equivalent).
8.1 These test methods employ live cultures of bacteria,
6.1.3 Bottles, Screwcap, approximately 375 mL, Boston
fungi,andyeast,someofwhicharecapableofcausingdisease,
Rounds of flint glass. Mold-A-7232-D, Finish 28-400, and
and others allergic reaction in some humans. In addition to
Black Artmold Caps BM-8041, Size 28-400, with rubber ring
other precautions, assign laboratory personnel trained in cor-
liners fastened to caps with steamproof adhesive.
rect microbiological techniques to run these tests. Use proper
6.1.4 Constant Temperature Chamber, capable of being
microbiological procedures in order to prevent contamination
maintained at 35 6 0.5°C (95 6 1°F) for bacteria, or 30 6
of the cultures or of the work area. Clean and sterilize in an
0.5°C (86 6 1°F) for fungi, or two chambers if needed
approved manner all spills and all equipment coming into
simultaneously.
contact with the cultures and the inoculated adhesive speci-
6.1.5 Glass Rods, 305 mm in length having a diameter of
mens. Also sterilize in an approved manner all cultures and
6.3 mm.
contaminateddisposableequipmentbeforediscarding.See1.3.
6.1.6 Hemacytometer,LevyCountingChamber,celldepth—
0.1 mm, Newbauer rulings.
9. Test Organisms
6.1.7 Hood, laminar flow type.
9.1 Cultures of one or more of the following bacterial
6.1.8 Jar, Screw Cap,round,approximately1L(1-qtmason
species are suggested for use:
type) for sampl
...

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:D4783–98a Designation:D4783–01(Reapproved2008)
Standard Test Methods for
Resistance of Adhesive Preparations in Container to Attack
by Bacteria, Yeast, and Fungi
This standard is issued under the fixed designation D 4783; 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
1.1These test methods cover the determination of the resistance of liquid adhesive preparations to microbial attack in the
container by challenging adhesive specimens with cultures of bacteria, yeast, or fungi, and checking for their ability to return to
sterility. *
1.1 These test methods cover the determination of the resistance of liquid adhesive preparations to microbial attack in the
container by challenging adhesive specimens with cultures of bacteria, yeast, or fungi, and checking for their ability to return to
sterility. These test methods return qualitative results.
1.2 The values stated in SI units are to be regarded as the standard. The values in parentheses are for information only.
1.3 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 technique.
Specific precautionary statements are given in Section 8.
2. Referenced Documents
2.1 ASTM Standards:
D 907Terminology of Adhesives Test Methods for Chemical Analysis of Nickel, Cobalt, and High-Temperature Alloys
D 4299 Test Methods for Effect of Bacterial Contamination on Performance of Adhesive Preparations and Adhesive Films
D4300Test Methods forAbility ofAdhesive Films to Support or Resist the Growth of Fungi 4300 Test Methods for Chemical
Analysis of Nickel, Cobalt, and High-Temperature Alloys
E 640TestMethodforPreservativesinWater-ContainingCosmetics TestMethodsforChemicalAnalysisofNickel,Cobalt,and
High-Temperature Alloys
NOTE 1—Test Method E 640 is under the jurisdiction of ASTM Committee E-35E35 on Pesticides. The procedure in this method outlines a serial
dilution method of determining plate count using a pour plate technique.
2.2 TAPPI Method:
T 487 Fungus Resistance of Paper and Paperboard
2.3 CSMA:
Cosmetics Preservation, Method 38
3. Terminology
3.1 Definitions:
3.1.1resistance, n—as related to bacteria, yeast, or fungi, the power or capacity to ward off growth. Definitions—Many terms
in these test methods are defined in Terminology D 907.
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 D4783–88. Last previous edition D4783–98.
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 .
e1
Current edition approved April 1, 2008. Published April 2008. Originally approved in 1988. Last previous edition approved in 2001 as D 4783 – 01 .
For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at [e-mail]. 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 11.05.
Available from Technical Association of the Pulp and Paper Industry (TAPPI), 15 Technology Parkway South, Norcross, GA 30092, http://www.tappi.org.
Available from TAPPI, P.O. Box 105113, Atlanta, GA30348.
This method is the same as Test Method E 640.
*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.
D4783–01 (2008)
3.2 Definitions of Terms Specific to This Standard:
3.2.1 adhesive preparationadhesive preparation, n—the adhesive as packaged for distribution, storage, and use.
3.3 Abbreviations:Abbreviations: Abbreviations:
3.3.1 PBS—phosphate buffered saline.
3.3.2 PDA—potato dextrose agar.
3.3.3 YMPG—yeast malt peptone glucose (agar).
3.4For definitions of other terms, see Terminology D907.
4. Summary of Test Methods
4.1 Theadhesivespecimenischallengedbyinoculationwithacultureofbacteria,yeast,orfungi,whichmaybeasinglespecies
or a mixed culture of several species, following the guidelines given in Note 6. The inoculated adhesive specimen is stored at 21
to 27°C (70 to 80°F) for 7 days, during which time cultures (streak plates) are made at preset intervals. See Note 2. At any point
intheseriesofchallenges,iftheinoculatedspecimenshowsmicrobialgrowthonthestreakplatesmadeduringtheweekfollowing
the challenge (indicating that it has not returned to sterility), the test is discontinued, and the sample is reported as not resistant
to attack in the container by the species or combination of species used as the inoculum. If the cultures show no growth, the test
isrepeatedwithuptofourchallenges.Ifthespecimentestsoutassterilefollowingthefourthchallenge,itisreportedtoberesistant
to attack in the container by the species or combination of species of bacteria, fungi, or yeast used as the inoculum. At the
discretion of the biological laboratory, the test may be discontinued after the second or third challenge. See Section 16 for further
interpretation.
4.2 The time necessary to kill is determined by noting the earliest streak plate to read sterile. If the 4-h plate is positive and the
24-hplateisnegative,thekilltimecouldbenarroweddownfurtherbyrepeatingthechallengeandmakingstreakplatesatintervals
of 4, 8, 12, and 24 h following the challenge.
4.3 Thetestinglaboratoryhastheoptionofchangingthetimingofthechallenges,thesterilitychecks,andtheincubationperiod.
NOTE 2—Two proposed schedules for the challenging and sterility checks are shown in Table 1 and Table 2, Schedule A for bacteria and yeast, and
Schedule B for fungi. The exact format to be followed will vary, according to the convenience of the schedule to the testing laboratory and special
circumstances relating to the problem being addressed.
NOTE 3—Aserial-dilution plate-count method of checking for sterility may be used when numerical information is needed on the population of viable
organisms or the reduction in population with increasing levels of biocide. Letheen broth is recommended for the diluent and Letheen agar for the pour
plate. See Note 1.
5. Significance and Use
5.1 These test methods are used to demonstrate whether an adhesive preparation is sufficiently protected with biocide to resist
attack by bacteria, yeast, and fungi during its storage life.They are patterned after methods used by biological laboratories serving
the adhesive industry.
5.2 These test methods may also be used to determine the efficacy of different biocide systems against specific microorganisms.
5.3 These test methods are especially useful when tested against wild-type microorganisms which have been isolated from
contaminated adhesives as an aid in determining the amount and type of biocide necessary to kill or inhibit the growth of the
contaminants. If an isolated microorganism not generally used as a challenge organism, is chosen as the inoculum, it is important
to identify the organism and determine on which medium and under what conditions it will grow, in order to demonstrate the
efficacy of the biocide.
5.4 The results obtained when using the procedures given in these methods apply only to the species which are used for the
testing. The test species listed in Section 9 are frequently used by laboratories to test for antimicrobial properties, but they are not
the only ones which could be used. Selection of the species to use for these test methods requires informed judgment by the testing
laboratory or by the party requesting the tests. It is also important that species which commonly attack adhesives be used. See 9.4.
5.5 The presence of an active biocide carried over from the adhesive specimen to the agar could have an inhibiting effect on
the growth of microorganisms, resulting in no growth during the span of a normal incubation period, when in fact, viable
microorganisms are present, but their growth has been slowed down or held in stasis. The use of Letheen agar and broth is
recommended to neutralize the effect of this carry-over.
NOTE 4—Letheen agar may be used for the streak plates, or if another agar is chosen for testing, a Letheen agar plate could be streaked as a control
to test against the neutralizing effect. Even more effective would be diluting the challenged adhesive specimen with Letheen broth and running Letheen
agar pour plates. See Note 1 and Note 3. Extending the incubation period of negative plates would be another safeguard. To neutralize thiazoline-based
preservatives, 10 to 50 ppm of sodium thioglycolate can be added to the medium.
5.6 Thesetestmethodsaredependentuponthephysiologicalactionoflivingmicroorganismsunderareportedsetofconditions.
Conclusions about the resistance of the test adhesive to microbiological attack can be drawn by comparing the results to
simultaneously run controls of known resistance. See X5.2 for statements regarding test repeatability.
6. Apparatus
6.1 In addition to the standard equipment found in any fully equipped microbiological laboratory, the following items are
sometimes needed:
D4783–01 (2008)
TABLE 1 Schedule A—Proposed Bacteria and Yeast Testing, Covering 4-h, 24-h, 48-h, 72-h, and 7-Day Tests
Day of Week Day no. First Challenge Second Challenge Third Challenge Fourth Challenge
Monday (−1) inoculate fresh bacterial . . .
or yeast culture
Tuesday 0 inoculate specimens . . .
Tuesday 0 prepare suspension . . .
Tuesday 0 prepare suspension . . .
Tuesday 0 inoculate specimens . . .
Tuesday (0 + 4 h) streak 4-h plate . . .
Wednesday 1 streak 24-h plate . . .
Thursday 2 streak 48-h plate . . .
Friday 3 streak 72-h plate . . .
Sat./Sun. 4–5 . . . .
Monday 6 . inoculate fresh bacterial . .
or yeast culture
Tuesday 7 . prepare suspension . .
Tuesday 7 streak 7-day plate inoculate specimens . .
Tuesday (7 + 4 h) read 4-h plate streak 4-h plate . .
Wednesday 8 read 24-h plate streak 24-h plate . .
Thursday 9 read 48-h plate streak 48-h plate . .
Friday 10 read 72-h plate streak 72-h plate . .
Sat./Sun. 11–12 . . . .
Monday 13 . . inoculate fresh bacterial .
or yeast culture
Tuesday 14 . . prepare suspension .
Tuesday 14 read 7-day plate streak 7-day plate inoculate specimens .
Tuesday (14 + 4 h) . read 4-h plate streak 4-h plate .
Wednesday 15 . read 24-h plate streak 24-h plate .
Thursday 16 . read 48-h plate streak 48-h plate .
Friday 17 . read 72-h plate streak 72-h plate .
Sat./Sun. 18–19 . . . .
Monday 20 . . . inoculate fresh
bacterial or yeast
culture
Tuesday 21 . . . prepare suspension
Tuesday 21 . read 7-day plate streak 7-day plate inoculate specimens
Tuesday (21 + 4 h) . . read 4-h plate streak 4-h plate
Wednesday 22 . . read 24-h plate streak 24-h plate
Thursday 23 . . read 48-h plate streak 48-h plate
Friday 24 . . read 72-h plate streak 72-h plate
Sat./Sun. 25–26 . . . .
Monday 27 . . . .
Tuesday 28 . . read 7-day plate streak 7-day plate
Tuesday (28 + 4 h) . . . read 4-h plate
Wednesday 29 . . . read 24-h plate
Thursday 30 . . . read 48-h plate
Friday 31 . . . read 72-h plate
Sat./Sun. 32–33 . . . .
Monday 34 . . . .
Tuesday 35 . . . read 7-day plate
6.1.1 Autoclave,capableofproducing103kPaofsteampressureat121°C(250°F)andmaintainingitforaminimumof15min.
6.1.2 Cell Counting Chamber, Petroff-Hausser, cell depth—0.02 mm (or equivalent).
6.1.3 Bottles, Screwcap, approximately 375 mL, Boston Rounds of flint glass. Mold-A-7232-D, Finish 28-400, and Black
Artmold Caps BM-8041, Size 28-400, with rubber ring liners fastened to caps with steamproof adhesive.
6.1.4 Constant Temperature Chamber , capable of being maintained at 35 6 0.5°C (95 6 1°F) for bacteria, or 30 6 0.5°C (86
6 1°F) for fungi, or two chambers if needed simultaneously.
6.1.5 Glass Rods, 305 mm in length having a diameter of 6.3 mm.
6.1.6 Hemacytometer, Levy Counting Chamber, cell depth—0.1 mm, Newbauer rulings.
6.1.7 Hood, laminar flow type.
6.1.8 Jar, Screw Cap, round, approximately 1 L (1-qt mason type) for samples.
6.1.9 Pasteur Pipets.
6.1.10 Pipet, 1 mL, disposable, sterile.
This method is the same as Test Method E640.
Available from most laboratory supply houses.
Available from most laboratory supply houses.
Screw cap bottles are available from Owens-Illinois Glass Co., OH Building, Toledo, OH, or equivalent bottles and caps have been found suitable for this purpose.
Screw cap bottles are available from Owens-Illinois Glass Co., OH Building, Toledo, OH, or equivalent bottles and caps have been found suitable for this purpose.
The Biogard Hood or similar equipment, The Baker Co., Sanford, ME.
D4783–01 (2008)
TABLE 2 Schedule B—Proposed Fungi Testing, Covering 4-h, 24-h, 48-h, 72-h, and 7-Day Tests
Day of Week Day no. First Challenge Second Challenge Third Challenge Fourth Challenge
Friday (−10) inoculate fresh fungal . . .
culture
Friday (−3) . inoculate fresh . .
fungal culture
Monday 0 prepare spore . . .
suspension
Monday 0 inoculate specimens . . .
Monday (0 + 4 h) streak 4-h plate . . .
Tuesday 1 streak 24-h plate . . .
Wednesday 2 streak 48-h plate . . .
Thursday 3 streak 72-h plate . . .
Friday 4 . . inoculate fresh fungal .
culture
Sat./Sun. 5, 6 . . . .
Monday 7 . prepare spore . .
suspension
Monday 7 streak 7-day plate inoculate specimens . .
Monday (7 + 4 h) read 4-h plate streak 4-h plate . .
Tuesday 8 read 24-h plate streak 24-h plate . .
Wedne
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