ASTM D2574-06(2012)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: D2574 − 06(Reapproved 2012)
Standard Test Method for
Resistance of Emulsion Paints in the Container to Attack by
Microorganisms
This standard is issued under the fixed designation D2574; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope* ofpreservativestoevaluatetheperformanceinemulsionpaints
of competitive and developmental preservatives.
1.1 This test method covers the determination of the relative
resistance of emulsion paints to attack in the container by
4.2 This test method should be used preferably by persons
microorganisms.
who have had basic microbiological training.
1.2 The values stated in SI units are to be regarded as the
NOTE 1—The reliability of the results obtained from this test method is
standard. The values given in parentheses are for information
extremely dependent on the techniques employed. Improper techniques
can result in a sterile sample appearing to be contaminated, and even
only.
worse, a contaminated sample appearing to be sterile (see also Note 2). It
1.3 This standard does not purport to address all of the
is recommended that you consult with your biocide supplier, raw material
safety concerns, if any, associated with its use. It is the
supplier, or an independent testing laboratory to confirm questionable
responsibility of the user of this standard to establish appro- results. Formulation and raw materials’ quality may also vary and thereby
affect the test results.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
5. Apparatus and Materials
2. Referenced Documents
5.1 Balance, capable of weighing to 0.10 g.
2.1 ASTM Standards:
5.2 Incubator, or other device capable of maintaining a
D5588 Test Method for Determination of the Microbial
constant temperature between 28 and 32°C.
Condition of Paint, Paint Raw Materials, and Plant Areas
5.3 Refrigerator, maintained at 10 to 13°C.
3. Summary of Test Method
5.4 Screwcap Borosilicate Test Tubes, 125 by 15-mm.
3.1 Thistestmethodisdesignedtochallengesamplesofone
5.5 Borosilicate Flasks, 1-L.
or more paints containing various levels of one or more
5.6 Screwcap Bottles, 150-mL.
biocides with a known amount of bacteria and rate the ability
of the test paint(s) to control the “contamination.”
5.7 Autoclave, capable of producing 103 kPa (15 psi) of
steam pressure at 121°C and maintaining it for a minimum of
4. Significance and Use
15 min. An autoclave is not necessary if prepared agar slants
4.1 Spoilage of paint in the container can result in are used.
putrefaction, lowered pH, gas formation, and decrease in
5.8 Pipettes or an Automatic Pipettor, sterile, 1-mL, with
viscosity. This test method provides a standard procedure for
sterile disposable pipette tips for 1 mL.
the evaluation of the resistance of emulsion paints to microbial
5.9 Petri Dishes, sterile.
deterioration. The results should enable: (1) the paint manu-
facturer to select an effective preservative and (2) the supplier
5.10 Dehydrated Tryptic Soy Agar (TSA), medium, or pre-
prepared slants, plates, and broth tubes.
5.11 Swabs, sterile cotton.
This test method is under the jurisdiction of ASTM Committee D01 on Paint
and Related Coatings, Materials, andApplications and is the direct responsibility of
Subcommittee D01.28 on Biodeterioration.
CurrenteditionapprovedJune1,2012.PublishedJuly2012.Originallyapproved
in 1967. Last previous edition approved in 2006 as D2574 – 06. DOI: 10.1520/ Available from microbiological supply companies. Media with TTC indicator
D2574-06R12. dye may be used. In general, the TTC helps visualize contamination, but it has been
For referenced ASTM standards, visit the ASTM website, www.astm.org, or reported on occasion to inhibit the growth of some bacteria. Interferences from
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM pigments in materials being tested may make the color change difficult to see. If
Standards volume information, refer to the standard’s Document Summary page on self-prepared plates are used with the TTC indicator, 0.01 % TTC indicator should
the ASTM website. be used and it must be added after autoclaving.
*A Summary of Changes section appears at the end of this standard
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D2574 − 06 (2012)
5.12 Laminar Flow Hood, Sterile Room, or at Least a off with a sterile inoculating loop. This material is inoculated
Laboratory Testing Area, relatively clean, free of blowing dust into a tube ofTSB each and incubated in a 30 6 2°C incubator
and dirt, etc., which can be used for streaking plates. overnight.
6.3.2 The overnight cultures are used to reinoculate fresh
5.13 Antiseptic Solution, to help maintain sterility of testing
TSB tubes using a sterile inoculating loop.
area surfaces (4.12) (for example, 70% ethanol solution).
6.3.3 Incubate the cultures to their log phase of growth as
5.14 A minimum of 235 mL ( ⁄2 pt) of each paint sample
previously determined by standard microbiological technique
under test (pre-loaded with biocide).
and growth curves using a plate count usually 16 to 24 h.
6.3.4 Soak a sterile cotton swab or a loop in the inoculated
5.15 Aminimum of 475 mL(1 pt) of paint identical to 5.14,
broth culture following the incubation period described in
but containing no biocide.
6.3.3.
5.16 Twenty-four Hour Cultures of a Pseudomonas sp. (for
6.3.5 Remove the swab or loop and prepare a second broth
example, Pseudomonas aeruginosa ATCC #10145) and an
culture by repeating 6.3.2 and 6.3.3.
Enterobacter sp. (for example, Enterobacter aerogenes, ATCC
6.3.6 Following the incubation period, use the broth culture
#13048)—These should be grown separately in tryptic soy
prepared in 6.3.5 to proceed as in Section 7 to inoculate the
broth. If a spoiled paint of a similar type as that under test is
paint.
available, organisms cultured from this material can be used.
NOTE 4—Maintenance of cultures for future use: The purity of the
NOTE 2—See X1 for a method to spoil paint for use as an inoculum.
bacterialinoculumpreparedin6.3.2isverifiedbystreakingaloopfulfrom
Organisms isolated following the procedures in Test Method D5588 may
the growth onto a prepared TSA plate. A single isolated colony from the
be used as challenge organisms in Test Method D2574. Also Bacillus sp.
plate is then transferred to a previously prepared TSA slant using an
for example,Bacillussubtilis,ATCC #27328 or other organisms as agreed
inoculating loop. Incubate the slant for 24 h at 30 6 2°C or until a
upon between the parties involved may be employed. When using
luxuriant growth occurs on the slant surface.The slant is then stored in the
spore-forming bacteria, care must be taken to ensure only vegetative cells
refrigerator as a working stock culture until further use.
are used in the inoculation (early log phase of growth).
NOTE 5—The inoculum preparation for Bacillus substilis differs from
the other cultures. Bacillus subtilis , ATCC 27328 has been shown to
produce extracellular cellulase enzymes in the TSB medium. Hence, it is
6. Preparation of Materials
advised that for Bacillus inoculum, the broth culture from 6.3.5 should be
NOTE 3—Observe conventional microbiological techniques in making
centrifuged at 4000 r/m for 10 min, the supernatant containing the
these tests. Handle all materials so as to avoid contamination from the air,
cellulase enzymes is discarded and the bacterial pellet is re-suspended in
fingers, or work surfaces.
equal volume of sterile water and then used as the inoculum in Section 7.
6.1 Preparation of Tryptic Soy Agar Plates and Slants:
6.4 Preparation of Paints for Test:
6.1.1 Follow the instructions on the container for prepara-
6.4.1 Paints may be previously loaded with biocide as
tion, or purchase prepared plates and slants.
provided, or ladders of levels of biocide may be added as
6.1.2 Distribute10mLofthedissolvedmediumintoeachof
agreed upon by the parties involved. In all testing, a negative
50 test tubes and 100-mL medium in 250-mL conical flasks.
control (sample containing no biocide) should be included and
6.1.3 Autoclave tubes (with caps loose) and the flask for 15
appropriately identified.
min at 103 kPa (15 psi) and a temperature of 121°C.
6.4.2 Weigh 100 g of each paint sample to be tested into a
6.1.4 Upon removal from the autoclave, tighten caps and
suitable container (screwcap glass jars have been found suit-
place the tubes at an approximate 30° angle position to prepare able).
the slants with a slope of about 50 mm (2 in.) long.
7. Procedure
6.1.5 For preparing TSA plates, pour 30 mL of the agar
medium from the flask into sterile petri dishes and allow to set.
7.1 Inoculation of Paint Samples:
6.1.6 Store the prepared TSA slants and plates in a refrig-
7.1.1 Remove 0.1 mL from each of the individual bacterial
erator at 10 to 13°C until needed.
inocula at ;10 colony forming units/mL CFU/mL and inocu-
late into 100 g of the test paint (provides ;10 CFU/g of the
6.2 Preparation of Tryptic Soy Broth Tubes (TSB):
paint).
6.2.1 Follow the instructions on the container for prepara-
7.1.2 Incubate the paint at 30 6 2°C for one week, and
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