1、Designation: D 2574 06Standard Test Method forResistance of Emulsion Paints in the Container to Attack byMicroorganisms1This standard is issued under the fixed designation D 2574; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the
2、 year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope*1.1 This test method covers the determination of the relativeresistance of emulsion paints to attack in the con
3、tainer bymicroorganisms.1.2 The values stated in SI units are to be regarded as thestandard. The values given in parentheses are for informationonly.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this s
4、tandard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 5588 Test Method for Determination of the MicrobialCondition of Paint, Paint Raw Materials, and Plant Areas3. Summary of
5、Test Method3.1 This test method is designed to challenge samples of oneor more paints containing various levels of one or morebiocides with a known amount of bacteria and rate the abilityof the test paint(s) to control the “contamination.”4. Significance and Use4.1 Spoilage of paint in the container
6、 can result in putrefac-tion, lowered pH, gas formation, and decrease in viscosity. Thistest method provides a standard procedure for the evaluation ofthe resistance of emulsion paints to microbial deterioration.The results should enable: (1) the paint manufacturer to selectan effective preservative
7、 and (2) the supplier of preservatives toevaluate the performance in emulsion paints of competitive anddevelopmental preservatives.4.2 This test method should be used preferably by personswho have had basic microbiological training.NOTE 1The reliability of the results obtained from this test method
8、isextremely dependent on the techniques employed. Improper techniquescan result in a sterile sample appearing to be contaminated, and evenworse, a contaminated sample appearing to be sterile (see also Note 2). Itis recommended that you consult with your biocide supplier, raw materialsupplier, or an
9、independent testing laboratory to confirm questionableresults. Formulation and raw materials quality may also vary and therebyaffect the test results.5. Apparatus and Materials5.1 Balance, capable of weighing to 0.10 g.5.2 Incubator, or other device capable of maintaining aconstant temperature betwe
10、en 28 and 32C.5.3 Refrigerator, maintained at 10 to 13C.5.4 Screwcap Borosilicate Test Tubes, 125 by 15-mm.5.5 Borosilicate Flasks, 1-L.5.6 Screwcap Bottles, 150-mL.5.7 Autoclave, capable of producing 103 kPa (15 psi) ofsteam pressure at 121C and maintaining it for a minimum of15 min. An autoclave i
11、s not necessary if prepared agar slantsare used.5.8 Pipettes or an Automatic Pipettor, sterile, 1-mL, withsterile disposable pipette tips for 1 mL.5.9 Petri Dishes, sterile.5.10 Dehydrated Tryptic Soy Agar (TSA), medium, or pre-prepared slants, plates, and broth tubes.35.11 Swabs, sterile cotton.5.1
12、2 Laminar Flow Hood, Sterile Room, or at Least aLaboratory Testing Area, relatively clean, free of blowing dustand dirt, etc., which can be used for streaking plates.5.13 Antiseptic Solution, to help maintain sterility of testingarea surfaces (4.12) (for example, 70% ethanol solution).5.14 A minimum
13、 of 235 mL (12 pt) of each paint sampleunder test (pre-loaded with biocide).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 approved
14、June 1, 2006. Published June 2006. Originallyapproved in 1967. Last previous edition approved in 2000 as D 2574 00.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to t
15、he standards Document Summary page onthe ASTM website.3Available from microbiological supply companies. Media with TTC indicatordye may be used. In general, the TTC helps visualize contamination, but it has beenreported on occasion to inhibit the growth of some bacteria. Interferences frompigments i
16、n materials being tested may make the color change difficult to see. Ifself-prepared plates are used with the TTC indicator, 0.01 % TTC indicator shouldbe used and it must be added after autoclaving.1*A Summary of Changes section appears at the end of this standard.Copyright ASTM International, 100
17、Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.15 A minimum of 475 mL (1 pt) of paint identical to 5.14,but containing no biocide.5.16 Twenty-four Hour Cultures of a Pseudomonas sp. (forexample, Pseudomonas aeruginosa ATCC #10145) and anEnterobacter sp. (for exampl
18、e, Enterobacter aerogenes, ATCC#13048)These should be grown separately in tryptic soybroth. If a spoiled paint of a similar type as that under test isavailable, organisms cultured from this material can be used.NOTE 2See Appendix X1 for a method to spoil paint for use as aninoculum. Organisms isolat
19、ed following the procedures in Test MethodD 5588 may be used as challenge organisms in Test Method D 2574. AlsoBacillus sp. for example, Bacillus subtilis, ATCC #27328 or otherorganisms as agreed upon between the parties involved may be employed.When using spore-forming bacteria, care must be taken
20、to ensure onlyvegetative cells are used in the inoculation (early log phase of growth).6. Preparation of MaterialsNOTE 3Observe conventional microbiological techniques in makingthese tests. Handle all materials so as to avoid contamination from the air,fingers, or work surfaces.6.1 Preparation of Tr
21、yptic Soy Agar Plates and Slants:6.1.1 Follow the instructions on the container for prepara-tion, or purchase prepared plates and slants.6.1.2 Distribute 10 mL of the dissolved medium into each of50 test tubes and 100-mL medium in 250-mL conical flasks.6.1.3 Autoclave tubes (with caps loose) and the
22、 flask for 15min at 103 kPa (15 psi) and a temperature of 121C.6.1.4 Upon removal from the autoclave, tighten caps andplace the tubes at an approximate 30 angle position to preparethe slants with a slope of about 50 mm (2 in.) long.6.1.5 For preparing TSA plates, pour 30 mL of the agarmedium from th
23、e flask into sterile petri dishes and allow to set.6.1.6 Store the prepared TSA slants and plates in a refrig-erator at 10 to 13C until needed.6.2 Preparation of Tryptic Soy Broth Tubes (TSB):6.2.1 Follow the instructions on the container for prepara-tion, or purchase prepared tubes.6.2.2 Distribute
24、 10 mL of the dissolved medium into each of50 test tubes.6.2.3 Autoclave tubes (with caps loose) for 15 min at 103kPa (15 psi) and a temperature of 121C.6.2.4 Upon removal from the autoclave, allow the tubes tocool to room temperature, tighten the caps, and store untilneeded.6.3 Inoculation of Trypt
25、ic Soy Broth Tubes with thePseudomonas sp. and the Enterobacter sp:6.3.1 Above organisms are stored on tryptic soy agar slantsin a refrigerator. To prepare a 24-h culture of each of the aboveorganisms, the surface of a slant of each organism is scrapedoff with a sterile inoculating loop. This materi
26、al is inoculatedinto a tube of TSB each and incubated in a 30 6 2C incubatorovernight.6.3.2 The overnight cultures are used to reinoculate freshTSB tubes using a sterile inoculating loop.6.3.3 Incubate the cultures to their log phase of growth aspreviously determined by standard microbiological tech
27、niqueand growth curves using a plate count usually 16 to 24 h.6.3.4 Soak a sterile cotton swab or a loop in the inoculatedbroth culture following the incubation period described in6.3.3.6.3.5 Remove the swab or loop and prepare a second brothculture by repeating 6.3.2 and 6.3.3.6.3.6 Following the i
28、ncubation period, use the broth cultureprepared in 6.3.5 to proceed as in Section 7 to inoculate thepaint.NOTE 4Maintenance of cultures for future use: The purity of thebacterial inoculum prepared in 6.3.2 is verified by streaking a loopful fromthe growth onto a prepared TSA plate. A single isolated
29、 colony from theplate is then transferred to a previously prepared TSA slant using aninoculating loop. Incubate the slant for 24 h at 30 6 2C or until aluxuriant growth occurs on the slant surface. The slant is then stored in therefrigerator as a working stock culture until further use.NOTE 5The ino
30、culum preparation for Bacillus substilis differs fromthe other cultures. Bacillus subtilis, ATCC 27328 has been shown toproduce extracellular cellulase enzymes in the TSB medium.4Hence, it isadvised that for Bacillus inoculum, the broth culture from 6.3.5 should becentrifuged at 4000 r/m for 10 min,
31、 the supernatant containing thecellulase enzymes is discarded and the bacterial pellet is re-suspended inequal volume of sterile water and then used as the inoculum in Section 7.6.4 Preparation of Paints for Test:6.4.1 Paints may be previously loaded with biocide asprovided, or ladders of levels of
32、biocide may be added asagreed upon by the parties involved. In all testing, a negativecontrol (sample containing no biocide) should be included andappropriately identified.6.4.2 Weigh 100 g of each paint sample to be tested into asuitable container (screwcap glass jars have been found suit-able).7.
33、Procedure7.1 Inoculation of Paint Samples:7.1.1 Remove 0.1 mL from each of the individual bacterialinocula at ;109colony forming units/mL CFU/mL and inocu-late into 100 g of the test paint (provides ;106CFU/g of thepaint).7.1.2 Incubate the paint at 30 6 2C for one week, andcheck for bacterial recov
34、ery or paint sterility after 1, 2, or 3, 5,and 7 days as described in 7.3.7.1.3 For those samples which were sterile after the seventhday of the first week, repeat the inoculation using 1 mL of a;109inoculum and repeat incubation in accordance with 7.1.2.7.2 Preliminary Examination of Paint Under Te
35、st:7.2.1 Examine the container for evidence of swelling. If thecontainer/lid is swollen, exercise caution in removing the lid.7.2.2 Remove the lid and carefully smell the contents of thecontainer. Deterioration of paint by microorganisms is oftencharacterized by distinct odors. Such odors may be eit
36、herputrefactive or fermentative.7.2.3 Observe the contents of the container for the presenceof stringy structures characteristic of the presence of certainmicroorganisms.4Sadasivan, L. and Hinkle, J., “Extracellular Production of Cellulase by BacillusIsolates from Spoiled Paints,” Biodeterioration a
37、nd Biodegradation 9, pp 602608,Institution of Chemical Engineers, Rugby, UK, 1995.D25740627.2.4 Observe the contents for noticeable losses in viscosity.This physical change frequently occurs as the result of micro-biological deterioration.7.3 Determination of Recovery Microorganisms from thePaint Un
38、der Test:7.3.1 Soak a sterile cotton swab in the paint under test.Remove excess paint by pressing gently against inside ofcontainer (approximately, 200-mg quantity of paint is retainedon the cotton swab).7.3.2 Evenly spread the paint from the cotton tip onto thesurface of a TSA plate (out of 200-mg
39、quantity of paintretained on the swab, only about 50 mg of paint gets spread onthe plates).NOTE 6If desired, swabs dipped in paint may be placed in the TSBbroth and incubated for 24 h at 30 6 2C for the enrichment of low countsof bacteria. Subsequently, a loopful from the enriched TSB broth may best
40、reaked on a TSA plate or slant to check for the sterility. Caution: Thisprocedure is primarily for the qualitative assessment of the presence orabsence of bacteria in the paint. Do not use broth enrichment results forthe rating system described in 8.1.7.3.3 In order to obtain duplicate agar slants o
41、r plates, repeat7.3.1 and 7.3.2.7.3.4 Incubate the inoculated agar slants or plates at 30 62C for a minimum of 1 week.7.3.5 If colonial growth of bacteria is observed on the agarsurface at the end of the incubation period, the test is completeand may be reported in accordance with Section 8.7.3.6 If
42、 colonial growth of bacteria is not observed on theagar surface at the end of the incubation period, continue thetest as described in 7.1.3 until failure is obtained, or until aspecified number of challenges (at least two) have been madeas agreed upon between the parties involved.NOTE 7Optimally, th
43、ese procedures should be carried out in a laminarflow hood or other sterile environment. The use of antiseptic solutions toregularly sterilize countertops and other work surfaces is recommended.Unfiltered air, hands, and unsterilized surfaces and equipment mayintroduce contamination during the trans
44、fer and give a false indication ofcontamination. The sterility of the transfer is very important in ensuringthe reliability of these tests.8. Rating System8.1 A rating system helps in the evaluation of the relativedegree of contamination of areas and materials. The streakedplates can be evaluated ba
45、sed on a log scale of the number ofbacterial colonies recovered as follows:0 = No bacterial recovery.1 = Trace of contamination (1 to 9 colonies).2 = Light contamination (10 to 99 colonies).3 = Moderate contamination (100 distinct colonies).4 = Heavy contamination (continuous smear of growth,colonie
46、s have grown together and are indistinguishable).NOTE 8The observation of any growth (a rating of 1 to 4) indicatesthat the sample may not be adequately preserved against the testorganisms.9. Report9.1 Report the following information or as otherwise agreedupon between the parties involved in the te
47、sting:9.1.1 Time, date, location, lot number, and other means ofidentification from each sample.9.1.2 Notation of sterility or contamination in the paintsamples when received.9.1.3 Corresponding results of daily observations, includ-ing: rating of degree of contamination (0 to 4); notation ofpossibl
48、e contamination during streaking (off-streak spots); andany other observations noted while testing the samples (forexample, those examined in accordance with 7.2).9.1.4 If living microorganisms are found in the paint asreceived (if previously loaded with biocide), or after inocula-tion, the paint sh
49、all be reported as “not resistant in thecontainer to attack by microorganisms” (see Note 7).9.1.5 If living organisms are not found, the paint shall bereported as “resistant in the container to attack by the micro-organisms employed in the test.”NOTE 9If living organisms are not recovered from any given sample,this is not a guarantee that the sample will be resistant to all possiblecontamination organisms or sources. Appropriate housekeeping measuresshould always be employed, along with the appropriate biocide in anyoperation to avoid contamination problems.10
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