1、Designation: D6329 98 (Reapproved 2015)Standard Guide forDeveloping Methodology for Evaluating the Ability of IndoorMaterials to Support Microbial Growth Using StaticEnvironmental Chambers1This standard is issued under the fixed designation D6329; the number immediately following the designation ind
2、icates the year oforiginal adoption or, 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 Many different types of microorganisms (f
3、or example,bacteria, fungi, viruses, algae) can occupy indoor spaces.Materials that support microbial growth are potential indoorsources of biocontaminants (for example, spores and toxins)that can become airborne indoor biopollutants. This guidedescribes a simple, relatively cost effective approach
4、to evalu-ating the ability of a variety of materials to support microbialgrowth using a small chamber method.1.2 This guide is intended to assist groups in the develop-ment of specific test methods for a definite material or groupsof materials.1.3 Static chambers have certain limitations. Usually, o
5、nlysmall samples of indoor materials can be evaluated. Care mustbe taken that these samples are representative of the materialsbeing tested so that a true evaluation of the material isperformed.1.4 Static chambers provide controlled laboratory microen-vironment conditions. These chambers are not int
6、ended toduplicate room conditions, and care must be taken wheninterpreting the results. Static chambers are not a substitute fordynamic chambers or field studies.1.5 A variety of microorganisms, specifically bacteria andfungi, can be evaluated using these chambers. This guide is notintended to provi
7、de human health effect data. However, organ-isms of clinical interest, such as those described as potentiallyallergenic, may be studied using this approach.1.6 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.7 This standard do
8、es not purport 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. Referenced Documents2.1 ASTM Sta
9、ndards:2D1193 Specification for Reagent WaterD1356 Terminology Relating to Sampling and Analysis ofAtmospheresE104 Practice for Maintaining Constant Relative Humidityby Means of Aqueous Solutions2.2 APHA Standards:3Standard Methods for the Examination of Water and Waste-water3. Terminology3.1 Defini
10、tionsFor definitions of terms used in this guide,refer to Terminology D1356.3.2 Definitions of Terms Specific to This Standard:3.2.1 amplificationthe act or result of increasing thequantity of microorganisms.3.2.2 CFUcolony forming unit, which may arise from asingle organism or multiple units, such
11、as spores, in the case ofthe fungi.3.2.3 colonymacroscopically visible growth.3.2.4 inoculationthe act of introducing a microorganism(inoculum) into the test material.3.2.5 inoculumviable test microorganism introduced ontoa material by implanting a small amount on the surface orsubstrate.3.2.6 plate
12、petri dish containing microbiological agar me-dia on which microorganism are grown.1This guide is under the jurisdiction of ASTM Committee D22 on AirQuality and is the direct responsibility of Subcommittee D22.08 on Sampling andAnalysis of Mold.Current edition approved July 1, 2015. Published July 2
13、015. Originally approvedin 1998. Last previous edition approved in 2008 as D6329 98 (2008). DOI:10.1520/D6329-98R15.2For 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
14、the standards Document Summary page onthe ASTM website.3Available from American Public Health Association (APHA), 800 I St., NW,Washington, DC 20001, http:/www.apha.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.2.7 static cham
15、bera small chamber (enclosed space)with no internal forced air motion.3.2.8 susceptibilitythe vulnerability of a material or sur-face to colonization by microorganisms.4. Significance and Use4.1 The static chambers have several different applications:4.1.1 The static chambers can be used to compare
16、thesusceptibility of different materials to the colonization andamplification of various microorganisms under defined condi-tions.4.1.2 Chambers operated at high relative humidities may beused to perform worst case scenario screening tests on mate-rials by providing an atmosphere where environmental
17、 condi-tions may be favorable for microbial growth.4.1.3 Use of multiple chambers with different environmen-tal parameters, such as a range of relative humidities, permitsthe evaluation of multiple microenvironments and allowsinvestigation of materials under differing environmental con-ditions.4.1.4
18、 Drying requirements for wetted materials may also beinvestigated. This information may be relevant for determiningmaterial resistance to microbial growth after becoming wet.These conditions may simulate those where materials aresubjected to water incursion through leaks as well as duringremediation
19、 of a building after a fire.4.1.5 Growth rates of microorganisms on the material mayalso be investigated. Once it has been established that organ-isms are able to grow on a particular material under definedconditions, investigations into the rate of organism growth maybe performed. These evaluations
20、 provide base line informationand can be used to evaluate methods to limit or containamplification of microorganisms.4.2 These techniques should be performed by personnelwith training in microbiology. The individual must be compe-tent in the use of sterile technique, which is critical to excludeexte
21、rnal contamination of materials.5. Apparatus5.1 Static ChamberChambers should be relatively smalland portable, contain three or four shelves, and be easilydecontaminated. In addition, transparent walls are desirablebecause visual inspection of the test material and monitoring ofinstruments (that is,
22、 hygrometers) without opening the cham-ber is preferred. Fig. 1 is a schematic diagram of a possiblestatic chamber. Acrylic desiccators are readily available, easilyadaptable, and relatively inexpensive. Other options, such asglass, are also acceptable. Glass has the advantage of beingautoclavable;
23、however, it is frequently much less portable. Thechamber door must provide ready access to the materials butshould be airtight when closed.5.1.1 Relative HumidityMaintain humidities through theuse of saturated salt solutions contained in trays on the bottomof the chambers (see Practice E104). It is
24、essential that thechambers be tightly sealed so that the desired humidity will bemaintained. Place hygrometers in the chambers for confirma-tion that humidities are being maintained, although saturatedsalt solutions are themselves standards. Exercise care that thesalts selected for use in the chambe
25、r are not inhibitory to thetest organisms.5.1.2 TemperatureControl the temperature of the cham-bers. The chambers may be externally controlled through theuse of constant temperature environments, such as a room orincubator. Chart recorders or other data logging devices arerecommended to confirm main
26、tenance of temperature. Con-trolled temperature is critical for two reasons. First, it can havea profound effect on the growth of microorganisms. Second,relative humidity is dependent upon temperature. The controllimits may be defined by consulting a psychometric chart anddetermining the impact of t
27、emperature on a specific test RH.5.1.3 Characterize instrumentation for evaluating other pa-rameters if the instruments are to be employed during materialtesting. Conditions such as light need to be noted and con-trolled during the course of an experiment as these conditionsmay have an effect on the
28、 growth of the test organism. Lightmay be controlled externally by placing the chambers in adarkened room to remove light or in a continuously lightedroom for a constant light source.5.2 Provide ports, where needed, for the insertion of probesto monitor and record temperature and relative humidity,
29、usingexternally located instrumentation as long as it is well sealedand contamination is avoided.5.3 DecontaminationDecontaminate the chamber beforeinitiating any analysis. Surface disinfection or vapor phasedisinfection may be appropriate. Glass may be autoclaved.Follow the manufacturers instructio
30、ns, especially any safetyprecautions. If a chemical disinfectant is employed, clear thechambers of any residual disinfectant to prevent interferencewith the growth of the microorganisms on the material beingevaluated. Thoroughly ventilate the chambers in a cleanenvironment. Decontaminate the salt so
31、lutions. The methodused is dependent upon the composition of the salts selected.Any instrumentation to be used during the evaluations, such ashygrometers, may be removed from the chambers during thedecontamination procedure of the chamber surfaces and de-contaminated separately; however, it is gener
32、ally more effec-tive for them to remain in the chambers. Verify the efficacy ofthe decontamination procedure as part of the QualityAssurance/Quality Control (QA/QC) plan.5.4 Decontaminate the work area around the chambersroutinely, especially before opening the chamber door. Thechambers should be ke
33、pt in a clean room, functionally Class100 000 (M 6.5 or ISO 8) or better.FIG. 1 Schematic of Example Static ChamberD6329 98 (2015)26. Reagents6.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specificat
34、ions of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.4Other grades may be used,provided it is first ascertained that the reagent is of sufficientlyhigh purity to permit its use without lessening the accuracy ofthe determination.6.2 Purit
35、y of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water as certifiedby Type II of Specification D1193. It should conform to theType A specifications for microbial classification.6.3 Microbiological MediaChoose appropriate media de-pending upon the test micr
36、oorganism selected. Commerciallyprepared media may be acceptable, but it may be necessary toprepare organism specific media. References should be con-sulted to determine the proper media for optimal growth of thetest organism.7. Characterization of Static Chamber7.1 Characterize static chambers for
37、all environmental pa-rameters being measured before any material evaluations areperformed. Chambers should be characterized for at leastrelative humidity and temperature. Take sufficient readings toensure that the conditions will be maintained throughout thecourse of the experiment and will meet the
38、 QA/QC standardsdeveloped for a specific test.7.1.1 EquilibrationEquilibrate disinfected chambers con-taining hygrometers before taking the first relative humidityreading. Place the hygrometers on a shelf for ease of readingthrough the walls of the chamber without opening the door.Take multiple sequ
39、ential readings at appropriate intervals thatwere determined experimentally. The variation of the instru-mentation must be determined and taken into consideration.For example, a minimum of four similar readings (65 %) overan 8 h period may be determined to demonstrate equilibrium.7.1.2 RecoveryDeter
40、mine the amount of time required forthe chamber relative humidity to recover to test levels afteropening the door for 1 to 2 min. This determination may becrucial, especially at the higher relative humidities. Exercisecare to utilize hygrometers that have a rapid response time.7.2 Check chamber rela
41、tive humidity daily, and recordreadings depending on test length.8. Sample Preparation8.1 Specific details on the preparation of the samples willdepend upon the characteristics of the material to be tested.Generally, replicate small pieces of the test material should beused. Depending upon the mater
42、ial, pieces as small as 4 by 4cm may be used. Pieces should be placed on sterile petri dishesor other appropriate holders on the shelves in the chamber.Include controls and blanks within the QA/QC framework.8.2 Common microbiological practice is to sterilize a sur-face or material before inoculation
43、 to ensure that the testorganism is the only source being evaluated. Autoclaving is anextremely effective method if such a procedure does not alterthe test material. Other methods, such as ionizing and non-ionizing irradiation, ultraviolet, dry heat, and surface or vaporphase disinfection, are also
44、acceptable if these methods do notharm the material and do not have residue effects or if all tracesof the disinfectant can be removed prior to testing. Consult thetest material manufacturer or conduct tests with the testmaterial to determine the best method of sterilization. Specificdetails for dec
45、ontamination depend upon the method selectedand should be worked out before actual testing begins withinthe QA/QC framework.8.3 Equilibrate or bring to near equilibrium samples in thechamber before inoculation with the test organism. Equilibra-tion time will depend upon both the material to be teste
46、d andthe chamber relative humidity selected for the test. Determineequilibration times for each material prior to testing.8.3.1 Ascertain equilibration by determining when the bulkmoisture content of the material reaches a constant value. Theuse of a calibrated analytical balance is recommended.8.3.
47、2 Compute the bulk moisture content of the test materialas follows:MC 5 Mb2 Md!/Md# 3100 (1)where:MC = bulk moisture content (%),Mb= mass of the small piece (g), andMd= mass of the small piece after drying (g).Mdmay be determined either by oven drying at 105 to 110Cor desiccation to constant weight
48、depending upon the testmaterial. Time required for drying is determined experimen-tally. A sample can be considered dry when no significantweight change is detected in two consecutive weighings at least1 h apart.9. Selection of Test Organism9.1 Selection of the appropriate test organisms is extremel
49、yimportant. Since growth requirements vary for differentorganisms, the selection process should include a justificationfor the particular organism or organisms chosen. Testing ofmaterials with many different organisms from diverse groups isoptimal. At a minimum, representative bacteria and fungishould both be tested. Initial tests should be performed withonly one species of microorganism.9.1.1 Criteria for organism selection are based on a numberof factors.Appropriateness of the organism for the test materialand the environment where the material is used are k
