1、Designation: D5588 97 (Reapproved 2012)Standard Test Method forDetermination of the Microbial Condition of Paint, Paint RawMaterials, and Plant Areas1This standard is issued under the fixed designation D5588; the number immediately following the designation indicates the year oforiginal adoption or,
2、 in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers a procedure for the determina-tion of the microbial cond
3、ition (contamination or sterility) ofraw materials used in the manufacture of paint, and themicrobial 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 pur
4、port to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Summary of Test Method2.1 This test method
5、 outlines procedures to (1) obtainsamples for sterility testing from wet or dry materials and plantsites, (2) conduct the sterility testing on those samples to see ifthey are contaminated, (3) evaluate the degree of contamina-tion, if any, and (4) provide a guide for some indication of thetype of co
6、ntamination present (bacterial, fungal, yeast, etc.).This test method is not designed to include all the necessaryprecautions to maintain the level of sterility required to providethe most accurate results. Some familiarity with microbiologi-cal techniques is recommended.3. Significance and Use3.1 S
7、poilage of paint in the container is often related to theuse of contaminated raw materials, water (particularly recycledwashwater), vessels, piping, and equipment in the manufactur-ing plant. There is a need for a simple method to determine thepresence or absence of microorganisms in plants that man
8、u-facture paints and coatings. Such a determination enables themanufacturer to establish the point of contamination (that is,raw materials or problem housekeeping areas in the plant) tohelp in solving the spoilage problem.NOTE 1Some contamination in plant areas is to be expected, sincemicroorganisms
9、 are ubiquitous and cannot generally be eliminated prac-tically (it is what an in-can preservative is supposed to control). Excessivelevels of contamination or contaminated raw materials can exceed thecapability of the preservative. If you have excessive contamination in theplant, there are methods
10、for decontamination including steam, preserva-tives, bleach, etc. These should be discussed with your biocide supplierand used with care. Recovery of spoiled or contaminated products is oftennot feasible, so an adequate level of the appropriate biocide in conjunctionwith good plant housekeeping prac
11、tices are essential. Your biocidesupplier can also help here.3.2 This test method may be used by persons without basicmicrobiological training, but some training on aseptic tech-niques would be recommended.NOTE 2The reliability of the results obtained from this test method isextremely dependent on t
12、he techniques employed. Improper techniquescan result in a sterile sample appearing to be contaminated, and evenworse, a contaminated sample appearing to be sterile (see also 5.1). It isrecommended that you consult with your biocide supplier, raw materialsupplier, or an independent testing laborator
13、y to confirm questionableresults.4. Apparatus and Materials4.1 Balance, capable of weighing to 0.10 g.4.2 Incubator, or other device capable of maintaining aconstant temperature between 28 and 32C.4.3 Refrigerator.4.4 Tryptic Soy Agar (TSA) Plates,2pre-prepared.3(See Note3).4.5 Potato Dextrose Agar
14、(PDA) Plates,4or Malt AgarPlates,5acidified to pH 3.5 with lactic acid, pre-prepared.1This test method is under the jurisdiction of ASTM Committee D01 on Paintand Related Coatings, Materials, and Applications and is the direct responsibility ofSubcommittee D01.28 on Biodeterioration.Current edition
15、approved June 1, 2012. Published July 2012. Originally approvedin 1994. Last previous edition approved in 2008 as D5588 97 (2008). DOI:10.1520/D5588-97R12.2Please note that Tryptic Soy and Trypticase Soy are names used interchange-ably. Pre-prepared TSA plates, BBL# 21185, are available from various
16、 microbio-logical supply companies.3Agar plates (media) may be purchased pre-prepared using the indicated Difcoor BBL number from microbiological supply companies, or both. Media may alsobe prepared from the formulations given in the Difco Manual (Difco Laboratories,Detroit, MI) or from appropriate
17、dehydrated media using standard microbiologicaltechniques.4Pre-prepared plates available are Difco # 4360-22-0, or BBL # 96272. Thesepre-prepared plates are not acidified to pH 3.5, but may be used (see also Footnote3).5Pre-prepared plates available are Difco # 4265-22-6. These are not acidified,but
18、 may be used (see also Footnote 3).Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1NOTE 3If preparing plates, Tryptic Soy Agar media with TTC(triphenyltetrazolium chloride) indicator dye may also be used. In general,the TTC helps visu
19、alize contamination, but it has been reported onoccasion to inhibit the growth of some bacteria. Interferences frompigments in materials being tested may make the color change difficult tosee. If self-prepared plates are used with the TTC indicator, 0.01 % TTCindicator should be used and it must be
20、added after autoclaving.4.6 Lactic Acid.4.7 Sterile Swabs in tubes, pre-prepared.4.8 Swab tubes, Culturette Tubes, or a similar system (swabin a test tube with a transport medium)6, all sterile, pre-prepared can be used if transport of collected samples to thelaboratory testing area is required.4.9
21、Sterile Diluent (9 mL) in tubes, pre-prepared (0.85 %saline or other suitable diluent). These can be prepared fromsterile tubes and sterile saline solution then stored in arefrigerator.4.10 Laminar Flow Hood, Sterile Room, or at least alaboratory testing area that is relatively clean, free of blowin
22、gdust and dirt, etc., which can be used for streaking plates.4.11 Antiseptic Solution, to help maintain sterility of testingarea surfaces (4.10) (For example, 70 % ethanol solution.).4.12 Plastic or Rubber Laboratory Gloves, optional, steril-ized.4.13 Facial Mask, optional.4.14 Sterile Spatulas or S
23、terile Tongue Depressors, 150-mm, (6-in.) individually wrapped.4.15 Plastic Bag,7sterile.5. General Sampling Guidelines5.1 Take all reasonable precautions to avoid microbialcontamination while obtaining samples. You may choose towear a facial mask and sterilized gloves. (WarningDo nottouch the swab
24、anywhere near the cotton tip, or near parts ofthe swab which could be immersed in the test sample.Microorganisms from the skin, clothing, and even air ifexposed too long, can contaminate the sample. If the swab hasa cap, do not touch any part of the swab except that cap.Confirm suspicious results wi
25、th additional testing.)5.2 Use a new sterile swab, tongue depressor or spatula foreach sample. Do not reuse any sampling devices. If usinggloves, dispose after use.5.3 When taking samples, be sure to minimize the timesterile items are exposed to the air to avoid false contaminationresults.5.4 Liquid
26、 materials may be sampled as outlined in Section6. Alternately, a sterilized container may be used to transportthe liquid sample to the sterile testing area. Be sure that nonon-sterile items contact the liquid sample during sampling,handling, and movement to the testing area (for example, usesterile
27、 pipet, etc. for transfer of material to container, etc.).5.5 Dry materials may be sampled as in 6.3 or 9.1.Tosample unopened, dry raw materials in bags, wipe a large areaof 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.Sam
28、ple as in 9.1, or using a sterile tongue depressor or sterilespatula, scoop 10 to 15 g into a sterile plastic bag,7close andseal bag for transport to sterile testing area.NOTE 4To decrease the chances of inadvertent contamination, asuggestion would be to carefully wipe the area of the bag to be cut,
29、 andthe knife used for cutting it, with isopropyl alcohol. Warning: Exercisecare to avoid skin contact, since the isopropyl alcohol could carryhazardous materials through the skin. Also, avoid excess alcohol thatcould affect test results.5.6 When testing open containers of raw materials, vats,drums,
30、 etc., there is no need to sterile equipment surfaces (seeSection 6). However, be aware that any contamination ob-served may have been introduced after opening. Samples takenfrom equipment surfaces that show contamination do notnecessarily mean that the material contained or being manu-factured insi
31、de is also contaminated.6. Sampling Procedure for Plant Areas6.1 Establish a protocol for surveying probable areas ofcontamination. Make sure that such areas include pipes andhoses, especially if left with water standing, any storage andmixing vessels, pumps, drains, sumps, etc. Because recycledwash
32、water is particularly susceptible to contamination, be sureto include it.6.2 Sampling is best carried out when the area to be testedis wet. In wet areas, the swab is dipped into or wiped on thearea (see Note 3), and then returned to a sterile tube (with orwithout transport media). This swab is then
33、used for testing asdescribed in Section 8 (see also Section 7).6.3 Sampling dry areas provides information that is lessconclusive, but can be carried out by swabbing the dry areawith a sterile swab that has previously been dipped into sterilediluent. This swab is then used as described in Section 8.
34、7. Testing Transported Samples7.1 If transport of collected samples to the laboratory testingarea is required, then use the swab contained in the swab tubes,culturette tubes, or similar system (swab in a test tube with atransport medium), in place of the dry swab as described in 4.7.Any transport me
35、dium transferred to the agar or broth shouldnot adversely affect the results.7.2 Test swabs in tubes without media as soon as possible toavoid drying of swab and possibly killing any contaminatingmicroorganisms. Test swabs in tubes with media within thetime specified by the manufacturer (generally 4
36、8 to 72 h).8. Testing Procedure for Liquid Samples or Swabs, orBoth8.1 Grasping the opposite end, dip the cotton end of a drysterile swab into the liquid (or mixture from Procedure 9),remove the cover from a sterile tryptic soy agar (TSA) plate,and streak the agar surface with the wet swab. Make sur
37、e thatthis is done so that the streaks are in a set pattern (for example,6Available from microbiological supply companies. Swab tubes or culturettetubes 9345 with Amies medium were used.7Sterile plastic packs are available from microbiological supply companies.D5588 97 (2012)2three streaks from left
38、 to right with 12.7-mm, (12-in.) spacing,criss crossed by three streaks from top to bottom, also with12.7-mm (12-in.) spacing). Replace the cover. Do this asquickly as possible to avoid introducing airborne contamina-tion to the plates.NOTE 5Optimally, these procedures should be carried out in a lam
39、inarflow hood or other sterile environment. Minimally, a relatively clean areaas 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 introduceco
40、ntamination during the transfer and give a false indication of contami-nation. The use of aseptic technique during transfer is very important inensuring the reliability of these tests (see also 10.5 and the appendix todetect anaerobic bacteria).8.2 Dip the swab again into the mixture and repeat thes
41、treaking as in 8.1 using an acidified potato dextrose agar(PDA) plate or malt agar plate.8.3 Turn the streaked TSAplates upside down, and the PDAor malt agar plates right side up. Place all streaked plates in anincubator, and incubate at 30C for the specified time. Makesure that the incubation time
42、for fungi (PDA or malt agarplates) is 3 to 7 days, and for bacteria (TSA plates), 24 to 48 h.NOTE 6The 30C temperature is generally appropriate for detectingenvironmental contaminants. If two incubators are available, use 28C forthe fungi and 32C for the bacteria. If humidity control is available, u
43、se95 % relative humidity (rh) for the fungi and 50 % rh for the bacteria.NOTE 7To achieve some degree of humidity control in a non-humidity controlled incubator or oven, place a container (such as aborosilicate baking dish) filled with distilled water at the bottom of theincubator. This helps to pre
44、vent the drying out of the plates (which couldinhibit the growth of any microorganisms and give a false indication ofsterility). Change this water regularly to avoid growth of bacteria, etc. (ora piece of copper wool can be used to help control microorganismgrowth).9. Testing Procedure for Dry Mater
45、ials9.1 Obtain or weigh out a suitable amount of dry material(0.1 to 0.5 g) using sterilized equipment (either a sterile spatulaor sterile wooden tongue depressor) and add this to a tube ofsterile diluent (see 4.9). Recap the tube and shake vigorously.NOTE 8If the material does not go into solution,
46、 shake or swirl thetube so that a uniform mixture is obtained just prior to the streakingprocedure (8.1) (see also 5.1, Note 2, and Note 5).9.2 Using the resulting liquid, continue as listed in 8.1 forliquid materials.9.3 For each additional dry sample use a new sterile spatulaor tongue depressor.10
47、. Evaluation of Results10.1 Bacterial contamination (aerobic) is generally charac-terized by milky spots of varying size (bacterial colonies) onthe agar surface. These are usually slimy or shiny in appear-ance.10.2 Fungal contamination is generally characterized byspots that are usually filamentous
48、and more fuzzy in appear-ance, with the exception of yeasts which normally look similarto the bacterial colonies.NOTE 9If present, bacteria should grow on the TSA plates, butbacteria can also grow on the PDA or malt extract plates, particularly ifthey are not acidified. Fungi can also grow on the TS
49、A plates, and yeastin particular can look like a bacterial contamination. Differentiationbetween bacterial and fungal growth can require more sophisticatedtechniques than are covered in this test method. Assistance can beobtained from your biocide supplier.10.3 If there are no spots appearing on the agar surface bythe end of the incubation period, then the test sample or areawas most likely sterile (free of contamination).NOTE 10Very low levels of contamination or inhomogeneity of asample may lead to false indications of sterility. Be certain samples are ashomogeneou
