1、Designation: D2574 16Standard Test Method forResistance of Emulsion Paints in the Container to Attack byMicroorganisms1This standard is issued under the fixed designation D2574; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the y
2、ear 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. Scope*1.1 This test method covers the determination of the relativeresistance of emulsion paints to attack in the contai
3、ner 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 stan
4、dard 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:2D5588 Test Method for Determination of the MicrobialCondition of Paint, Paint Raw Materials, and Plant Areas3. Summary of Test
5、 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 can
6、 result inputrefaction, lowered pH, gas formation, and decrease inviscosity. This test method provides a standard procedure forthe evaluation of the resistance of emulsion paints to microbialdeterioration. The results should enable: (1) the paint manu-facturer to select an effective preservative and
7、 (2) the supplierof preservatives to evaluate the performance in emulsion paintsof competitive and developmental 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 isex
8、tremely 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 inde
9、pendent 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 between 2
10、8 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 is no
11、t 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.1This t
12、est 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 June 1, 2016. Published July 2016. Originally approvedin 1967. Last previous editio
13、n approved in 2012 as D2574 06 (2012). DOI:10.1520/D2574-16.2For 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 the standards Document Summary page onthe ASTM website.3
14、Available 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 in materials being tested may make the color change diff
15、icult to see. Ifself-prepared plates are used with the TTC indicator, 0.01 % TTC indicator shouldbe used and it must be added after autoclaving.*A Summary of Changes section appears at the end of this standardCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 194
16、28-2959. United States15.12 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% ethano
17、l solution).5.14 A minimum of 235 mL (12 pt) of each paint sampleunder test (pre-loaded with biocide).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 anEntero
18、bacter sp. (for example, 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 for a method to spoil paint for use as an inoculum.Organi
19、sms isolated following the procedures in Test Method D5588 maybe used as challenge organisms in Test Method D2574. Also Bacillus sp.for example, Bacillus subtilis, ATCC #27328 or other organisms as agreedupon between the parties involved may be employed. When usingspore-forming bacteria, care must b
20、e taken to ensure only vegetative cellsare 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 Preparati
21、on of Tryptic Soy Agar Plates and Slants:6.1.1 Follow the instructions on the container forpreparation, 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) a
22、nd the 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 f
23、rom the 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 forpreparation, or purchase prepared tubes.6.2.2 Distri
24、bute 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 T
25、ryptic 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 ma
26、terial 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
27、techniqueand 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 t
28、he incubation 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 isol
29、ated 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
30、 inoculum 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
31、min, 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
32、 of biocide may be added asagreed upon by the parties involved. In all testing, a negativecontrol (sample containing no biocide) should be included andappropriately identified. If an untreated control is not available,confirm viability of each culture by streaking the broth onto afresh TSA plate.6.4
33、.2 Weigh 100 g of each paint sample to be tested into asuitable container (screwcap glass jars have been found suit-able).6.4.3 Check all samples for native bacterial contaminationby streaking each, prior to testing, as described in 7.3.7. Procedure7.1 Inoculation of Paint Samples:7.1.1 Remove 0.1 m
34、L 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 recovery or paint sterility after 1, 2, or 3, 5,and 7 days as des
35、cribed 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 7.1.2.4Sadasivan, L. and Hinkle, J., “Extracellular Production of Cellulase by BacillusIsolates from Spoiled Pa
36、ints,” Biodeterioration and Biodegradation 9, pp 602608,Institution of Chemical Engineers, Rugby, UK, 1995.D2574 1627.2 Preliminary Examination of Paint Under Test: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
37、the lid and carefully smell the contents of thecontainer. Deterioration of paint by microorganisms is oftencharacterized by distinct odors. Such odors may be eitherputrefactive or fermentative.7.2.3 Observe the contents of the container for the presenceof stringy structures characteristic of the pre
38、sence of certainmicroorganisms.7.2.4 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 Under Test:7.3.1 Soak a sterile cotton swab in the paint unde
39、r 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 quantity of paintretained on the swab, only about 50 mg of
40、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 bestreaked on a TSA plate or slant to check for the sterility.
41、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 or plates, repeat7.3.1 and 7.3.2.7.3.4 Incubate the inoculat
42、ed 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 colonial growth of bacteria is not observed on theagar sur
43、face 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, these procedures should be carried out in a laminarflow hood
44、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 transfer and give a false indication ofcontamination. The steril
45、ity 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 based on a log scale of the number ofbacterial colonies recov
46、ered 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,colonies have grown together and are indistinguishable).NOTE 8The
47、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 testing:9.1.1 Time, date, location, lot number, and other mea
48、ns 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 ofpossible contamination during streaking (off-streak spots); andany
49、 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 afterinoculation, the paint shall 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
