ASTM D5588-97(2002)
(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.
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Designation:D5588–97(Reapproved2002)
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.
tically (it is what an in-can preservative is supposed to control). Excessive
1. Scope
levels of contamination or contaminated raw materials can exceed the
1.1 This test method covers a procedure for the determina-
capability of the preservative. If you have excessive contamination in the
tion of the microbial condition (contamination or sterility) of
plant, there are methods for decontamination including steam, preserva-
raw materials used in the manufacture of paint, and the
tives, bleach, etc. These should be discussed with your biocide supplier
microbial condition of paint and paint manufacturing areas. and used with care. Recovery of spoiled or contaminated products is often
not feasible, so an adequate level of the appropriate biocide in conjunction
1.2 The values in SI units are to be regarded as the standard.
with good plant housekeeping practices are essential. Your biocide
The values given in parentheses are for information only.
supplier can also help here.
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-
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
recommended that you consult with your biocide supplier, raw material
samples for sterility testing from wet or dry materials and plant
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
4.2 Incubator, or other device capable of maintaining a
precautionstomaintainthelevelofsterilityrequiredtoprovide
constant temperature between 28 and 32°C.
the most accurate results. Some familiarity with microbiologi-
4.3 Refrigerator.
cal techniques is recommended.
2 3
4.4 Tryptic Soy Agar (TSA) Plates, pre-prepared. (See
Note 3).
3. Significance and Use
4.5 Potato Dextrose Agar (PDA) Plates, or Malt Agar
3.1 Spoilage of paint in the container is often related to the
Plates, acidified to pH 3.5 with lactic acid, pre-prepared.
use of contaminated raw materials, water (particularly recycled
washwater), vessels, piping, and equipment in the manufactur- NOTE 3—If preparing plates, Tryptic Soy Agar media with TTC
(triphenyltetrazolium chloride) indicator dye may also be used. In general,
ing plant. There is a need for a simple method to determine the
the TTC helps visualize contamination, but it has been reported on
presence or absence of microorganisms in plants that manu-
facture 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 Please note that Tryptic Soy and Trypticase Soy are names used interchange-
ably. Pre-prepared TSA plates, BBL# 21185, are available from various microbio-
help in solving the spoilage problem.
logical supply companies.
Agar plates (media) may be purchased pre-prepared using the indicated Difco
NOTE 1—Some contamination in plant areas is to be expected, since
or BBL number from microbiological supply companies, or both. Media may also
microorganisms are ubiquitous and cannot generally be eliminated prac-
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 ofASTM Committee D1 on Paint and Pre-prepared plates available are Difco # 4360-22-0, or BBL # 96272. These
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).
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D5588–97 (2002)
occasion to inhibit the growth of some bacteria. Interferences from NOTE 4—To decrease the chances of inadvertent contamination, a
pigments in materials being tested may make the color change difficult to suggestion would be to carefully wipe the area of the bag to be cut, and
see. If self-prepared plates are used with the TTC indicator, 0.01 % TTC the knife used for cutting it, with isopropyl alcohol. Warning: Exercise
indicator should be used and it must be added after autoclaving. care to avoid skin contact, since the isopropyl alcohol could carry
hazardous materials through the skin. Also, avoid excess alcohol that
4.6 Lactic Acid.
could affect test results.
4.7 Sterile Swabs in tubes, pre-prepared.
5.6 When testing open containers of raw materials, vats,
4.8 Swab tubes, Culturette Tubes, or a similar system (swab
drums, etc., there is no need to sterile equipment surfaces (see
in a test tube with a transport medium) , all sterile, pre-
Section 6). However, be aware that any contamination ob-
prepared can be used if transport of collected samples to the
served may have been introduced after opening. Samples taken
laboratory testing area is required.
from equipment surfaces that show contamination do not
4.9 Sterile Diluent (9 mL) in tubes, pre-prepared (0.85 %
necessarily mean that the material contained or being manu-
saline or other suitable diluent). These can be prepared from
factured inside is also contaminated.
sterile tubes and sterile saline solution then stored in a
refrigerator.
6. Sampling Procedure for Plant Areas
4.10 Laminar Flow Hood, Sterile Room, or at least a
6.1 Establish a protocol for surveying probable areas of
laboratory testing area that is relatively clean, free of blowing
contamination. Make sure that such areas include pipes and
dust and dirt, etc., which can be used for streaking plates.
hoses, especially if left with water standing, any storage and
4.11 Antiseptic Solution, to help maintain sterility of testing
mixing vessels, pumps, drains, sumps, etc. Because recycled
area surfaces (4.10) (For example, 70 % ethanol solution.).
washwater is particularly susceptible to contamination, be sure
4.12 Plastic or Rubber Laboratory Gloves, optional, steril-
to include it.
ized.
6.2 Sampling is best carried out when the area to be tested
4.13 Facial Mask, optional.
is wet. In wet areas, the swab is dipped into or wiped on the
4.14 Sterile Spatulas or Sterile Tongue Depressors, 150-
area (see Note 3), and then returned to a sterile tube (with or
mm, (6-in.) individually wrapped.
without transport media). This swab is then used for testing as
4.15 Plastic Bag, sterile.
described in Section 8 (see also Section 7).
5. General Sampling Guidelines
6.3 Sampling dry areas provides information that is less
conclusive, but can be carried out by swabbing the dry area
5.1 Take all reasonable precautions to avoid microbial
contamination while obtaining samples. You may choose to with a sterile swab that has previously been dipped into sterile
diluent. This swab is then used as described in Section 8.
wear a facial mask and sterilized gloves. (Warning—Do not
touch the swab anywhere near the cotton tip, or near parts of
7. Testing Transported Samples
the swab which could be immersed in the test sample.
Microorganisms from the skin, clothing, and even air if 7.1 Iftransportofcollectedsamplestothelaboratorytesting
exposed too long, can contaminate the sample. If the swab has area is required, then use the swab contained in the swab tubes,
a cap, do not touch any part of the swab except that cap. culturette tubes, or similar system (swab in a test tube with a
Confirm suspicious results with additional testing.) transportmedium),inplaceofthedryswabasdescribedin4.7.
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
handling, and movement to the testing area (for example, use
sterile swab into the liquid (or mixture from Procedure 9),
sterile pipet, etc. for transfer of material to container, etc.).
remove the cover from a sterile tryptic soy agar (TSA) plate,
5.5 Dry materials may be sampled as in 6.3 or 9.1. To
and streak the agar surface with the wet swab. Make sure that
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,
of the outside of the bag clean with a clean rag or paper towel.
three streaks from left to right with 12.7-mm, ( ⁄2-in.) spacing,
Using a clean knife, cut open the bag within the cleaned area.
criss crossed by three streaks from top to bottom, also with
Sample as in 9.1, or using a sterile tongue depressor or sterile
12.7-mm ( ⁄2-in.) spacing). Replace the cover. Do this as
spatula, scoop 10 to 15 g into a sterile plastic bag, close and
quickly as possible to avoid introducing airborne contamina-
seal bag for transport to sterile testing area.
tion to the plates.
NOTE 5—Optimally,theseproceduresshouldbecarriedoutinalaminar
flow hood or other sterile environment. Minimally, a relatively clean area
Available from microbiological supply companies. Swab tubes or culturette
as specified in 4.10 must be used. The use of antiseptic solution (see 4.11)
tubes 9345 with Amies medium were used.
Sterile plastic packs are available from microbiological supply companies. toregularlysanitizecountertopsandotherworksurfacesisrecommended.
D5588–97 (2002)
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 TurnthestreakedTSAplatesupsidedown,andthePDA
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.
(spots):
NOTE 7—To achieve some degree of humidity control in a non-
0 = no contamination,
humidity controlled incubator or oven, place a container (such as a
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
3 = moderate contamination (>100 distinct colonies), and
sterility). Change this water regularly to avoid growth of bacteria, etc. (or
4 = heavy contamination (continuous smear of growth,
a piece of copper wool can be used to help control microorganism
colonies have grown together and are indistinguishable).
growth).
11.2 Theratingsforgrowthof1to4shouldbemadeassoon
9. Testing Procedure for Dry Materials as practical after growth is observed. This avoids having the
colonies become too large for making comparisons of the
9.1 Obtain or weigh out a suitable amount
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