ASTM D5588-97
(Test Method)Standard Test Method for Determination of the Microbial Condition of Paint, Paint Raw Materials, and Plant Areas
Standard Test Method for Determination of the Microbial Condition of Paint, Paint Raw Materials, and Plant Areas
SCOPE
1.1 This test method covers a procedure for the determination of the microbial condition (contamination or sterility) of raw materials used in the manufacture of paint, and the microbial condition of paint and paint manufacturing areas.
1.2 The values 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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NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
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Designation: D 5588 – 97
AMERICAN SOCIETY FOR TESTING AND MATERIALS
100 Barr Harbor Dr., West Conshohocken, PA 19428
Reprinted from the Annual Book of ASTM Standards. Copyright ASTM
Standard Test Method for
Determination of the Microbial Condition of Paint, Paint Raw
Materials, and Plant Areas
This standard is issued under the fixed designation D 5588; 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.
levels of contamination or contaminated raw materials can exceed the
1. Scope
capability of the preservative. If you have excessive contamination in the
1.1 This test method covers a procedure for the determina-
plant, there are methods for decontamination including steam, preserva-
tion of the microbial condition (contamination or sterility) of
tives, bleach, etc. These should be discussed with your biocide supplier
raw materials used in the manufacture of paint, and the
and used with care. Recovery of spoiled or contaminated products is often
microbial condition of paint and paint manufacturing areas. not feasible, so an adequate level of the appropriate biocide in conjunction
with good plant housekeeping practices are essential. Your biocide
1.2 The values in SI units are to be regarded as the standard.
supplier can also help here.
The values given in parentheses are for information only.
1.3 This standard does not purport to address all of the 3.2 This test method may be used by persons without basic
safety concerns, if any, associated with its use. It is the microbiological training, but some training on aseptic tech-
responsibility of the user of this standard to establish appro-
niques would be recommended.
priate safety and health practices and determine the applica-
NOTE 2—The reliability of the results obtained from this test method is
bility of regulatory limitations prior to use.
extremely dependent on the techniques employed. Improper techniques
can result in a sterile sample appearing to be contaminated, and even
2. Summary of Test Method
worse, a contaminated sample appearing to be sterile (see also Note 4). It
2.1 This test method outlines procedures to (1) obtain is recommended that you consult with your biocide supplier, raw material
supplier, or an independent testing laboratory to confirm questionable
samples for sterility testing from wet or dry materials and plant
results.
sites, (2) conduct the sterility testing on those samples to see if
they are contaminated, (3) evaluate the degree of contamina-
4. Apparatus and Materials
tion, if any, and (4) provide a guide for some indication of the
4.1 Balance, capable of weighing to 0.10 g.
type of contamination present (bacterial, fungal, yeast, etc.).
4.2 Incubator, or other device capable of maintaining a
This test method is not designed to include all the necessary
constant temperature between 28 and 32°C.
precautions to maintain the level of sterility required to provide
4.3 Refrigerator.
the most accurate results. Some familiarity with microbiologi-
2 3
4.4 Tryptic Soy Agar (TSA) Plates, pre-prepared. (See
cal techniques is recommended.
Note 3).
3. Significance and Use 4.5 Potato Dextrose Agar (PDA) Plates, or Malt Agar
Plates, acidified to pH 3.5 with lactic acid, pre-prepared.
3.1 Spoilage of paint in the container is often related to the
use of contaminated raw materials, water (particularly recycled
NOTE 3—If preparing plates, Tryptic Soy Agar media with TTC
washwater), vessels, piping, and equipment in the manufactur- (triphenyltetrazolium chloride) indicator dye may also be used. In general,
the TTC helps visualize contamination, but it has been reported on
ing plant. There is a need for a simple method to determine the
occasion to inhibit the growth of some bacteria. Interferences from
presence or absence of microorganisms in plants that manu-
pigments in materials being tested may make the color change difficult to
facture paints and coatings. Such a determination enables the
see. If self-prepared plates are used with the TTC indicator, 0.01 % TTC
manufacturer to establish the point of contamination (that is,
raw materials or problem housekeeping areas in the plant) to
help in solving the spoilage problem.
Please note that Tryptic Soy and Trypticase Soy are names used interchange-
ably. Pre-prepared TSA plates, BBL# 21185, are available from various microbio-
NOTE 1—Some contamination in plant areas is to be expected, since
logical supply companies.
microorganisms are ubiquitous and cannot generally be eliminated prac-
Agar plates (media) may be purchased pre-prepared using the indicated Difco
tically (it is what an in-can preservative is supposed to control). Excessive or BBL number from microbiological supply companies, or both. Media may also
be prepared from the formulations given in the Difco Manual (Difco Laboratories,
Detroit, MI) or from appropriate dehydrated media using standard microbiological
techniques.
1 4
This test method is under the jurisdiction of ASTM Committee D-1 on Paint Pre-prepared plates available are Difco # 4360-22-0, or BBL # 96272. These
and Related Coatings, Materials, and Applications and is the direct responsibility of pre-prepared plates are not acidified to pH 3.5, but may be used (see also Footnote
Subcommittee D01.28 Biodeterioration. 3).
Current edition approved July 10, 1997. Published September 1997. Originally Pre-prepared plates available are Difco # 4265-22-6. These are not acidified,
e1
published as D 5588 – 94. Last previous edition D 5588 – 94 . but may be used (see also Footnote 3).
NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
D 5588
indicator should be used and it must be added after autoclaving. the knife used for cutting it, with isopropyl alcohol. Precaution: Exercise
care to avoid skin contact, since the isopropyl alcohol could carry
4.6 Lactic Acid.
hazardous materials through the skin. Also, avoid excess alcohol that
4.7 Sterile Swabs in tubes, pre-prepared.
could affect test results.
4.8 Swab tubes, Culturette Tubes, or a similar system (swab
5.6 When testing open containers of raw materials, vats,
in a test tube with a transport medium) , all sterile, pre-
drums, etc., there is no need to sterile equipment surfaces (see
prepared can be used if transport of collected samples to the
Section 6). However, be aware that any contamination ob-
laboratory testing area is required.
served may have been introduced after opening. Samples taken
4.9 Sterile Diluent (9 mL) in tubes, pre-prepared (0.85 %
from equipment surfaces that show contamination do not
saline or other suitable diluent). These can be prepared from
necessarily mean that the material contained or being manu-
sterile tubes and sterile saline solution then stored in a
factured inside is also contaminated.
refrigerator.
4.10 Laminar Flow Hood, Sterile Room, or at least a
6. Sampling Procedure for Plant Areas
laboratory testing area that is relatively clean, free of blowing
6.1 Establish a protocol for surveying probable areas of
dust and dirt, etc., which can be used for streaking plates.
contamination. Make sure that such areas include pipes and
4.11 Antiseptic Solution, to help maintain sterility of testing
hoses, especially if left with water standing, any storage and
area surfaces (4.10) (For example, 70 % ethanol solution.).
mixing vessels, pumps, drains, sumps, etc. Because recycled
4.12 Plastic or Rubber Laboratory Gloves, optional, steril-
washwater is particularly susceptible to contamination, be sure
ized.
to include it.
4.13 Facial Mask, optional.
6.2 Sampling is best carried out when the area to be tested
4.14 Sterile Spatulas or Sterile Tongue Depressors, 6-in.,
is wet. In wet areas, the swab is dipped into or wiped on the
(150-mm) individually wrapped.
area (see Note 3), and then returned to a sterile tube (with or
4.15 Plastic Bag, sterile.
without transport media). This swab is then used for testing as
described in Section 8 (see also Section 7).
5. General Sampling Guidelines
6.3 Sampling dry areas provides information that is less
5.1 Take all reasonable precautions to avoid microbial
conclusive, but can be carried out by swabbing the dry area
contamination while obtaining samples. You may choose to
with a sterile swab that has previously been dipped into sterile
wear a facial mask and sterilized gloves.
diluent. This swab is then used as described in Section 8.
NOTE 4—Caution: Do not touch the swab anywhere near the cotton tip,
7. Testing Transported Samples
or near parts of the swab which could be immersed in the test sample.
Microorganisms from the skin, clothing, and even air if exposed too long,
7.1 If transport of collected samples to the laboratory testing
can contaminate the sample. If the swab has a cap, do not touch any part
area is required, then use the swab contained in the swab tubes,
of the swab except that cap. Confirm suspicious results with additional
culturette tubes, or similar system (swab in a test tube with a
testing.
transport medium), in place of the dry swab as described in 4.7.
5.2 Use a new sterile swab, tongue depressor or spatula for
Any transport medium transferred to the agar or broth should
each sample. Do not reuse any sampling devices. If using
not adversely affect the results.
gloves, dispose after use.
7.2 Test swabs in tubes without media as soon as possible to
5.3 When taking samples, be sure to minimize the time
avoid drying of swab and possibly killing any contaminating
sterile items are exposed to the air to avoid false contamination
microorganisms. Test swabs in tubes with media within the
results.
time specified by the manufacturer (generally 48 to 72 h).
5.4 Liquid materials may be sampled as outlined in Section
8. Testing Procedure for Liquid Samples or Swabs, or
6. Alternately, a sterilized container may be used to transport
Both
the liquid sample to the sterile testing area. Be sure that no
non-sterile items contact the liquid sample during sampling, 8.1 Grasping the opposite end, dip the cotton end of a dry
sterile swab into the liquid (or mixture from Procedure 9),
handling, and movement to the testing area (for example, use
sterile pipet, etc. for transfer of material to container, etc.). remove the cover from a sterile tryptic soy agar (TSA) plate,
and streak the agar surface with the wet swab. Make sure that
5.5 Dry materials may be sampled as in 6.3 or 9.1. To
sample unopened, dry raw materials in bags, wipe a large area this is done so that the streaks are in a set pattern (for example,
three streaks from left to right with 12.7-mm, ( ⁄2-in.) spacing,
of the outside of the bag clean with a clean rag or paper towel.
Using a clean knife, cut open the bag within the cleaned area. criss crossed by three streaks from top to bottom, also with
12.7-mm ( ⁄2-in.) spacing). Replace the cover. Do this as
Sample as in 9.1, or using a sterile tongue depressor or sterile
spatula, scoop 10 to 15 g into a sterile plastic bag, close and quickly as possible to avoid introducing airborne contamina-
tion to the plates.
seal bag for transport to sterile testing area.
NOTE 6—Optimally, these procedures should be carried out in a laminar
NOTE 5—To decrease the chances of inadvertent contamination, a
flow hood or other sterile environment. Minimally, a relatively clean area
suggestion would be to carefully wipe the area of the bag to be cut, and
as specified in 4.10 must be used. The use of antiseptic solution (see 4.11)
to regularly sanitize countertops and other work surfaces is recommended.
Unfiltered air, hands, unsanitized surfaces and equipment may introduce
Available from microbiological supply companies. Swab tubes or culturette
contamination during the transfer and give a false indication of contami-
tubes 9345 with Amies medium were used.
Sterile plastic packs are available from microbiological supply companies. nation. The use of aseptic technique during transfer is very important in
NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
D 5588
ensuring the reliability of these tests (see also 10.5 and the appendix to
the end of the incubation period, then the tested material was
detect anaerobic bacteria).
contaminated (not sterile). A rating system is described (see
Section 11) for the degree of contamination.
8.2 Dip the swab again into the mixture and repeat the
10.5 If there are several colonies that are not on, or do not
streaking as in 8.1 using an acidified potato dextrose agar
touch the streaks, this indicates that contamination may have
(PDA) plate or malt agar plate.
occurred from the air during the streaking process, and a new
8.3 Turn the streaked TSA plates upside down, and the PDA
sample should be obtained and retested for confirmation of any
or malt agar plates right side up. Place all streaked plates in an
contamination.
incubator, and incubate at 30°C for the specified time. Make
sure that the incubation time for fungi (PDA or malt agar
11. Rating System
plates) is 3 to 7 days, and for bacteria (TSA plates), 24 to 48 h.
11.1 A rating system helps in evaluation of the relative
NOTE 7—The 30°C temperature is generally appropriate for detecting
degree of contamination of areas and materials. The streaked
environmental contaminants. If two incubators are available, use 28°C for
plates can be evaluated based on the number of colonies
the fungi and 32°C for the bacteria. If humidity control is available, use
(spots):
95 % relative humidity (rh) for the fungi and 50 % rh for the bacteria.
NOTE 8—To achieve some degree of humidity control in a non- 0 5 no contamination,
humidity controlled incubator or oven, place a container (such as a
1 5 trace of contamination (1 to 9 colonies),
borosilicate baking dish) filled with distilled water at the bottom of the
2 5 light contamination (10 to 99 colonies),
incubator. This helps to prevent the drying out of the plates (which could
3 5 moderate contamination (>100 distinct colonies), and
inhibit the growth of any microorganisms and give a false indication of
4 5 heavy contamination (continuous smear of growth,
sterility). Change this water regularly to avoid growth of bacteria, etc. (or
colonies have grown together and are indistinguishable).
a piece of copper wool can be used to help control microorganism
11.2 The ratings for growth of 1 to 4 should
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