ASTM D5588-97(2008)
(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
SIGNIFICANCE AND USE
Spoilage of paint in the container is often related to the use of contaminated raw materials, water (particularly recycled washwater), vessels, piping, and equipment in the manufacturing plant. There is a need for a simple method to determine the presence or absence of microorganisms in plants that manufacture paints and coatings. Such a determination enables the 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.
Note 1—Some contamination in plant areas is to be expected, since microorganisms are ubiquitous and cannot generally be eliminated practically (it is what an in-can preservative is supposed to control). Excessive levels of contamination or contaminated raw materials can exceed the capability of the preservative. If you have excessive contamination in the plant, there are methods for decontamination including steam, preservatives, bleach, etc. These should be discussed with your biocide supplier and used with care. Recovery of spoiled or contaminated products is often not feasible, so an adequate level of the appropriate biocide in conjunction with good plant housekeeping practices are essential. Your biocide supplier can also help here.
This test method may be used by persons without basic microbiological training, but some training on aseptic techniques would be recommended.
Note 2—The reliability of the results obtained from this test method is extremely dependent on the techniques employed. Improper techniques can result in a sterile sample appearing to be contaminated, and even worse, a contaminated sample appearing to be sterile (see also 5.1). It is recommended that you consult with your biocide supplier, raw material supplier, or an independent testing laboratory to confirm questionable results.
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.
General Information
Relations
Standards Content (Sample)
Designation: D5588 − 97 (Reapproved 2008)
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 D5588; 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.
NOTE 1—Some contamination in plant areas is to be expected, since
1. Scope
microorganisms are ubiquitous and cannot generally be eliminated prac-
1.1 This test method covers a procedure for the determina-
tically (it is what an in-can preservative is supposed to control). Excessive
tion of the microbial condition (contamination or sterility) of levels of contamination or contaminated raw materials can exceed the
capability of the preservative. If you have excessive contamination in the
raw materials used in the manufacture of paint, and the
plant, there are methods for decontamination including steam, preserva-
microbial condition of paint and paint manufacturing areas.
tives, bleach, etc. These should be discussed with your biocide supplier
1.2 The values in SI units are to be regarded as the standard. and used with care. Recovery of spoiled or contaminated products is often
not feasible, so an adequate level of the appropriate biocide in conjunction
The values given in parentheses are for information only.
with good plant housekeeping practices are essential. Your biocide
1.3 This standard does not purport to address all of the
supplier can also help here.
safety concerns, if any, associated with its use. It is the
3.2 This test method may be used by persons without basic
responsibility of the user of this standard to establish appro-
microbiological training, but some training on aseptic tech-
priate safety and health practices and determine the applica-
niques would be recommended.
bility of regulatory limitations prior to use.
NOTE 2—The reliability of the results obtained from this test method is
extremely dependent on the techniques employed. Improper techniques
2. Summary of Test Method
can result in a sterile sample appearing to be contaminated, and even
2.1 This test method outlines procedures to (1) obtain
worse, a contaminated sample appearing to be sterile (see also 5.1). It is
samples for sterility testing from wet or dry materials and plant
recommended that you consult with your biocide supplier, raw material
supplier, or an independent testing laboratory to confirm questionable
sites, (2) conduct the sterility testing on those samples to see if
results.
they are contaminated, (3) evaluate the degree of contamina-
tion, if any, and (4) provide a guide for some indication of the
4. Apparatus and Materials
type of contamination present (bacterial, fungal, yeast, etc.).
4.1 Balance, capable of weighing to 0.10 g.
This test method is not designed to include all the necessary
precautionstomaintainthelevelofsterilityrequiredtoprovide
4.2 Incubator, or other device capable of maintaining a
the most accurate results. Some familiarity with microbiologi-
constant temperature between 28 and 32°C.
cal techniques is recommended.
4.3 Refrigerator.
2 3
3. Significance and Use
4.4 TrypticSoyAgar(TSA)Plates, pre-prepared. (SeeNote
3).
3.1 Spoilage of paint in the container is often related to the
use of contaminated raw materials, water (particularly recycled
4.5 Potato Dextrose Agar (PDA) Plates, or Malt Agar
washwater), vessels, piping, and equipment in the manufactur-
Plates, acidified to pH 3.5 with lactic acid, pre-prepared.
ing plant. There is a need for a simple method to determine the
presence or absence of microorganisms in plants that manu-
facture paints and coatings. Such a determination enables the
Please note that Tryptic Soy and Trypticase Soy are names used interchange-
manufacturer to establish the point of contamination (that is, ably. Pre-prepared TSA plates, BBL# 21185, are available from various microbio-
logical supply companies.
raw materials or problem housekeeping areas in the plant) to
Agar plates (media) may be purchased pre-prepared using the indicated Difco
help in solving the spoilage problem.
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
This test method is under the jurisdiction of ASTM Committee D01 on Paint techniques.
and Related Coatings, Materials, andApplications and is the direct responsibility of Pre-prepared plates available are Difco # 4360-22-0, or BBL # 96272. These
Subcommittee D01.28 on Biodeterioration. pre-prepared plates are not acidified to pH 3.5, but may be used (see also Footnote
Current edition approved June 1, 2008. Published June 2008. Originally 3).
approved in 199494. Last previous edition approved in 2002 as D5588 – 97 (2002). Pre-prepared plates available are Difco # 4265-22-6. These are not acidified,
DOI: 10.1520/D5588-97R08. but may be used (see also Footnote 3).
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5588 − 97 (Reapproved 2008)
NOTE 3—If preparing plates, Tryptic Soy Agar media with TTC
5.5 Dry materials may be sampled as in 6.3 or 9.1.To
(triphenyltetrazolium chloride) indicator dye may also be used. In general,
sample unopened, dry raw materials in bags, wipe a large area
the TTC helps visualize contamination, but it has been reported on
of the outside of the bag clean with a clean rag or paper towel.
occasion to inhibit the growth of some bacteria. Interferences from
Using a clean knife, cut open the bag within the cleaned area.
pigments in materials being tested may make the color change difficult to
Sample as in 9.1, or using a sterile tongue depressor or sterile
see. If self-prepared plates are used with the TTC indicator, 0.01 % TTC
indicator should be used and it must be added after autoclaving.
spatula, scoop 10 to 15 g into a sterile plastic bag, close and
seal bag for transport to sterile testing area.
4.6 Lactic Acid.
4.7 Sterile Swabs in tubes, pre-prepared. NOTE 4—To decrease the chances of inadvertent contamination, a
suggestion would be to carefully wipe the area of the bag to be cut, and
4.8 Swabtubes,CulturetteTubes, or a similar system (swab
the knife used for cutting it, with isopropyl alcohol. Warning: Exercise
in a test tube with a transport medium) , all sterile, pre-
care to avoid skin contact, since the isopropyl alcohol could carry
hazardous materials through the skin. Also, avoid excess alcohol that
prepared can be used if transport of collected samples to the
could affect test results.
laboratory testing area is required.
5.6 When testing open containers of raw materials, vats,
4.9 Sterile Diluent (9 mL) in tubes, pre-prepared (0.85 %
drums, etc., there is no need to sterile equipment surfaces (see
saline or other suitable diluent). These can be prepared from
Section 6). However, be aware that any contamination ob-
sterile tubes and sterile saline solution then stored in a
served may have been introduced after opening. Samples taken
refrigerator.
from equipment surfaces that show contamination do not
4.10 Laminar Flow Hood, Sterile Room, or at least a
necessarily mean that the material contained or being manu-
laboratory testing area that is relatively clean, free of blowing
factured inside is also contaminated.
dust and dirt, etc., which can be used for streaking plates.
6. Sampling Procedure for Plant Areas
4.11 Antiseptic Solution, to help maintain sterility of testing
area surfaces (4.10) (For example, 70 % ethanol solution.).
6.1 Establish a protocol for surveying probable areas of
contamination. Make sure that such areas include pipes and
4.12 Plastic or Rubber Laboratory Gloves, optional, steril-
hoses, especially if left with water standing, any storage and
ized.
mixing vessels, pumps, drains, sumps, etc. Because recycled
4.13 Facial Mask, optional.
washwater is particularly susceptible to contamination, be sure
4.14 Sterile Spatulas or Sterile Tongue Depressors, 150-
to include it.
mm, (6-in.) individually wrapped.
6.2 Sampling is best carried out when the area to be tested
4.15 Plastic Bag, sterile.
is wet. In wet areas, the swab is dipped into or wiped on the
area (see Note 3), and then returned to a sterile tube (with or
5. General Sampling Guidelines
without transport media). This swab is then used for testing as
5.1 Take all reasonable precautions to avoid microbial described in Section 8 (see also Section 7).
contamination while obtaining samples. You may choose to
6.3 Sampling dry areas provides information that is less
wear a facial mask and sterilized gloves. (Warning—Do not
conclusive, but can be carried out by swabbing the dry area
touch the swab anywhere near the cotton tip, or near parts of
with a sterile swab that has previously been dipped into sterile
the swab which could be immersed in the test sample.
diluent. This swab is then used as described in Section 8.
Microorganisms from the skin, clothing, and even air if
exposed too long, can contaminate the sample. If the swab has
7. Testing Transported Samples
a cap, do not touch any part of the swab except that cap.
7.1 Iftransportofcollectedsamplestothelaboratorytesting
Confirm suspicious results with additional testing.)
area is required, then use the swab contained in the swab tubes,
5.2 Use a new sterile swab, tongue depressor or spatula for
culturette tubes, or similar system (swab in a test tube with a
each sample. Do not reuse any sampling devices. If using
transportmedium),inplaceofthedryswabasdescribedin4.7.
gloves, dispose after use.
Any transport medium transferred to the agar or broth should
5.3 When taking samples, be sure to minimize the time
not adversely affect the results.
sterile items are exposed to the air to avoid false contamination
7.2 Test swabs in tubes without media as soon as possible to
results.
avoid drying of swab and possibly killing any contaminating
5.4 Liquid materials may be sampled as outlined in Section microorganisms. Test swabs in tubes with media within the
6. Alternately, a sterilized container may be used to transport
time specified by the manufacturer (generally 48 to 72 h).
the liquid sample to the sterile testing area. Be sure that no
non-sterile items contact the liquid sample during sampling, 8. Testing Procedure for Liquid Samples or Swabs, or
handling, and movement to the testing area (for example, use Both
sterile pipet, etc. for transfer of material to container, etc.).
8.1 Grasping the opposite end, dip the cotton end of a dry
sterile swab into the liquid (or mixture from Procedure 9),
remove the cover from a sterile tryptic soy agar (TSA) plate,
Available from microbiological supply companies. Swab tubes or culturette
and streak the agar surface with the wet swab. Make sure that
tubes 9345 with Amies medium were used.
Sterile plastic packs are available from microbiological supply companies. this is done so that the streaks are in a set pattern (for example,
D5588 − 97 (Reapproved 2008)
they are not acidified. Fungi can also grow on the TSA plates, and yeast
three streaks from left to right with 12.7-mm, ( ⁄2-in.) spacing,
in particular can look like a bacterial contamination. Differentiation
criss crossed by three streaks from top to bottom, also with
between bacterial and fungal growth can require more sophisticated
12.7-mm ( ⁄2-in.) spacing). Replace the cover. Do this as
techniques than are covered in this test method. Assistance can be
quickly as possible to avoid introducing airborne contamina-
obtained from your biocide supplier.
tion to the plates.
10.3 If there are no spots appearing on the agar surface by
NOTE5—Optimally,theseproceduresshouldbecarriedoutinalaminar the end of the incubation period, then the test sample or area
flow hood or other sterile environment. Minimally, a relatively clean area
was most likely sterile (free of contamination).
as specified in 4.10 must be used. The use of antiseptic solution (see 4.11)
toregularlysanitizecountertopsandotherworksurfacesisrecommended. NOTE 10—Very low levels of contamination or inhomogeneity of a
Unfiltered air, hands, unsanitized surfaces and equipment may introduce sample may lead to false indications of sterility. Be certain samples are as
contamination during the transfer and give a false indication of contami- homogeneous as possible prior to sampling or streaking, or both.
nation. The use of aseptic technique during transfer is very important in
10.4 If spots are observed on or just against the streaks at
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
8.2 Dip the swab again into the mixture and repeat the
Section 11) for the degree of contamination.
streaking as in 8.1 using an acidified potato dextrose agar
10.5 If there are several colonies that are not on, or do not
(PDA) plate or malt agar plate.
touch the streaks, this indicates that contamination may have
8.3 Turn the streakedTSAplates upside down, and the PDA
occurred from the air during the streaking process, and a new
or malt agar plates right side up. Place all streaked plates in an
sample should be obtained and retested for confirmation of any
incubator, and incubate at 30°C for the specified time. Make
contamination.
sure that the incubation time for fungi (PDA or malt agar
plates) is 3 to 7 days, and for bacteria (TSAplates), 24 to 48 h.
11. Rating System
NOTE 6—The 30°C temperature is generally appropriate for detecting
11.1 A rating system helps in evaluation of the relative
environmental contaminants. If two incubators are available, use 28°C for
degree of contamination of areas and materials. The streaked
the fungi and 32°C for the bacteria. If humidity control is available, use
plates can be evaluated based on the number of colonies
95 % relative humidity (rh) for the fungi and 50 % rh for the bacteria.
NOTE 7—To achieve some degree of humidity control in a non-
(spots):
humidity controlled incubator or oven, place a container (such as a
0 = no contamination,
borosilicate baking dish) filled with distilled water at the bottom of the
1 = trace of contamination (1 to 9 colonies),
incubator. This helps to prevent the drying out of the plates (which could
2 = light contamination (10 to 99 colonies),
inhibit the growth of any microorganisms and give a false indication of
sterility). Change this water regularly to avoid growth of bacteria, etc. (or 3 = moderate contamination (>100 disti
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