ASTM D2574-00
(Test Method)Standard Test Method for Resistance of Emulsion Paints in the Container to Attack by Microorganisms
Standard Test Method for Resistance of Emulsion Paints in the Container to Attack by Microorganisms
SCOPE
1.1 This test method covers the determination of the relative resistance of emulsion paints to attack in the container by microorganisms.
1.2 The values stated in SI units are to be regarded as the standard. The values given 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.
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Designation:D2574–00
Standard Test Method for
Resistance of Emulsion Paints in the Container to Attack by
Microorganisms
This standard is issued under the fixed designation D 2574; 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.
1. Scope * 4. Apparatus and Materials
1.1 This test method covers the determination of the relative 4.1 Balance, capable of weighing to 0.10 g.
resistance of emulsion paints to attack in the container by 4.2 Incubator, or other device capable of maintaining a
microorganisms. constant temperature between 28 and 32°C.
1.2 The values stated in SI units are to be regarded as the 4.3 Refrigerator, maintained at 10 to 13°C.
standard. The values given in parentheses are for information 4.4 Screwcap Borosilicate Test Tubes, 125 by 15-mm.
only. 4.5 Borosilicate Flasks, 1-L.
1.3 This standard does not purport to address all of the 4.6 Screwcap Bottles, 150-mL.
safety concerns, if any, associated with its use. It is the 4.7 Autoclave, capable of producing 103 kPa (15 psi) of
responsibility of the user of this standard to establish appro- steam pressure at 121°C and maintaining it for a minimum of
priate safety and health practices and determine the applica- 15 min. An autoclave is not necessary if prepared agar slants
bility of regulatory limitations prior to use. are used.
4.8 Pipettes or an Automatic Pipettor, sterile, 1-mL, with
2. Summary of Test Method
sterile disposable pipette tips for 1 mL.
2.1 Thistestmethodisdesignedtochallengesamplesofone
4.9 Petri Dishes, sterile.
or more paints containing various levels of one or more 4.10 Dehydrated Tryptic Soy Agar (TSA), medium, or pre-
biocides with a known amount of bacteria and rate the ability
prepared slants, plates, and broth tubes.
of the test paint(s) to control the “contamination.” 4.11 Swabs, sterile cotton.
4.12 Laminar Flow Hood, Sterile Room, or at Least a
3. Significance and Use
Laboratory Testing Area, relatively clean, free of blowing dust
3.1 Spoilage of paint in the container can result in putrefac-
and dirt, etc., which can be used for streaking plates.
tion,loweredpH,gasformation,anddecreaseinviscosity.This
4.13 Antiseptic Solution, to help maintain sterility of testing
test method provides a standard procedure for the evaluation of
area surfaces (4.12) (for example, 70% ethanol solution).
the resistance of emulsion paints to microbial deterioration.
4.14 A minimum of 235 mL ( ⁄2 pt) of each paint sample
The results should enable: (1) the paint manufacturer to select
under test (pre-loaded with biocide).
aneffectivepreservativeand(2)thesupplierofpreservativesto
4.15 Aminimum of 475 mL(1 pt) of paint identical to 4.14,
evaluatetheperformanceinemulsionpaintsofcompetitiveand
but containing no biocide.
developmental preservatives.
4.16 Twenty-fourHourCulturesofaPseudomonassp. (for
3.2 This test method should be used preferably by persons
example, Pseudomonas aeruginosa ATCC #10145) and an
who have had basic microbiological training.
Enterobacter sp. (for example, Enterobacter aerogenes, ATCC
#13048)—These should be grown separately in tryptic soy
NOTE 1—The reliability of the results obtained from this test method is
broth. If a spoiled paint of a similar type as that under test is
extremely dependent on the techniques employed. Improper techniques
available, organisms cultured from this material can be used.
can result in a sterile sample appearing to be contaminated, and even
worse, a contaminated sample appearing to be sterile (see also Note 2). It
NOTE 2—See Appendix X1 for a method to spoil paint for use as an
is recommended that you consult with your biocide supplier, raw material
inoculum. Also Bacillus sp. for example, Bacillus subtilis, ATCC #27328
supplier, or an independent testing laboratory to confirm questionable
or other organisms as agreed upon between the parties involved may be
results. Formulation and raw materials’ quality may also vary and thereby
affect the test results.
Available from microbiological supply companies. Media with TTC indicator
This test method is under the jurisdiction of ASTM Committee D01 on Paint dye may be used. In general, the TTC helps visualize contamination, but it has been
and Related Coatings, Materials, andApplications and is the direct responsibility of reported on occasion to inhibit the growth of some bacteria. Interferences from
Subcommittee D01.28 on Biodeterioration. pigments in materials being tested may make the color change difficult to see. If
Current edition approved Nov. 10, 2000. Published January 2001. Originally self-prepared plates are used with the TTC indicator, 0.01 % TTC indicator should
published as D 2574 – 67 T. Last previous edition D 2574 – 97. be used and it must be added after autoclaving.
*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.
D2574
employed. When using spore-forming bacteria, care must be taken to luxuriant growth occurs on the slant surface.The slant is then stored in the
ensure only vegetative cells are used in the inoculation (early log phase of refrigerator as a working stock culture until further use.
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
5. Preparation of Materials
advised that for Bacillus inoculum, the broth culture from 5.3.5 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 6.
5.1 Preparation of Tryptic Soy Agar Plates and Slants:
5.4 Preparation of Paints for Test:
5.1.1 Follow the instructions on the container for prepara-
5.4.1 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
5.1.2 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
5.1.3 Autoclave tubes (with caps loose) and the flask for 15 appropriately identified.
min at 103 kPa (15 psi) and a temperature of 121°C. 5.4.2 Weigh 100 g of each paint sample to be tested into a
suitable container (screwcap glass jars have been found suit-
5.1.4 Upon removal from the autoclave, tighten caps and
able).
place the tubes at an approximate 30° angle position to prepare
the slants with a slope of about 50 mm (2 in.) long.
6. Procedure
5.1.5 For preparing TSA plates, pour 30 mL of the agar
medium from the flask into sterile petri dishes and allow to set. 6.1 Inoculation of Paint Samples:
6.1.1 Remove 0.1 mL from each of the individual bacterial
5.1.6 Store the prepared TSA slants and plates in a refrig-
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
5.2 Preparation of Tryptic Soy Broth Tubes (TSB):
paint).
5.2.1 Follow the instructions on the container for prepara-
6.1.2 Incubate the paint at 30 6 2°C for one week, and
tion, or purchase prepared tubes.
check for bacterial recovery or paint sterility after 1, 2, or 3, 5,
5.2.2 Distribute10mLofthedissolvedmediumintoeachof
and 7 days as described in 6.3.
50 test tubes.
6.1.3 For those samples which were sterile after the seventh
5.2.3 Autoclave tubes (with caps loose) for 15 min at 103
day of the first week, repeat the inoculation using 1 mL of a
kPa (15 psi) and a temperature of 121°C.
;10 inoculumandrepeatincubationinaccordancewith6.1.2.
5.2.4 Upon removal from the autoclave, allow the tubes to
6.2 Preliminary Examination of Paint Under Test:
cool to room temperature, tighten the caps, and store until
6.2.1 Examine the container for evidence of swelling. If the
needed.
container/lid is swollen, exercise caution in removing the lid.
5.3 Inoculation of Tryptic Soy Broth Tubes with the
6.2.2 Remove the lid and carefully smell the contents of the
Pseudomonas sp. and the Enterobacter sp:
container. Deterioration of paint by microorganisms is often
5.3.1 Above organisms are stored on tryptic soy agar slants
characterized by distinct odors. Such odors may be either
in a refrigerator. To prepare a 24-h culture of each of the above
putrefactive or fermentative.
organisms, the surface of a slant of each organism is scraped
6.2.3 Observe the contents of the container for the presence
off with a sterile inoculating loop. This material is inoculated
of s
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