1、Designation: D7391 171Standard Test Method forCategorization and Quantification of Airborne FungalStructures in an Inertial Impaction Sample by OpticalMicroscopy1This standard is issued under the fixed designation D7391; the number immediately following the designation indicates the year oforiginal
2、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.1NOTEResearch report information added editorially in May 2017.1. Scope1.1 This
3、test method is a procedure that uses direct micros-copy to analyze the deposit on an inertial impaction sample.1.2 This test method describes procedures for categorizingand enumerating fungal structures by morphological type.Typically, categories may be as small as genus (for example,Cladosporium) o
4、r as large as phylum (for example, basidi-ospores).1.3 This test method contains two procedures for enumer-ating fungal structures: one for slit impaction samples and onefor circular impaction samples. This test method is applicablefor impaction air samples, for which a known volume of air (ata rate
5、 as recommended by the manufacturer) has been drawn,and is also applicable for blank impaction samples.1.4 Enumeration results are presented in fungal structures/sample (fs/sample) and fungal structures/m3(fs/m3).1.5 The range of enumeration results that can be determinedwith this test method depend
6、s on the size of the spores on thesample trace, the amount of particulate matter on the sampletrace, the percentage of the sample trace counted, and thevolume of air sampled.1.6 This test method addresses only the analysis of samples.The sampling process and interpretation of results is outsidethe s
7、cope of this test method.1.7 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.8 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
8、tandard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.1.9 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles
9、for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterE691 Practice for Conducting an Interlaboratory Study toDetermi
10、ne the Precision of a Test Method3. Terminology3.1 ASTM Definitions (see the ASTM Online Dictionary ofEngineering Science and Technology3):3.1.1 numerical aperture.3.2 Definitions of Terms Specific to This Standard:3.2.1 circular impaction sample, na sample of airborneparticulate matter collected by
11、 means of a device that draws airthrough a round aperture at a specified rate, impacting theparticles suspended in the air onto an adhesive medium,resulting in a circular area of deposition. A circular impactionsample may be collected by means of a cassette manufacturedfor that purpose, or by means
12、of a sampling device that requiresslides to be pre-coated with impaction medium.3.2.2 debris rating, na distinct value assigned to animpactor sample based on the percentage of the sample areapotentially obscured by particulate matter, and ranging from 0to 5.1This test method is under the jurisdictio
13、n of ASTM Committee D22 on AirQuality and is the direct responsibility of Subcommittee D22.08 on Sampling andAnalysis of Mold.Current edition approved March 15, 2017. Published April 2017. Originallyapproved in 2009. Last previous edition approved in 2009 as D7391 09. DOI:10.1520/D7391-17E01.2For re
14、ferenced 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.3ASTM Online Dictionary of Engineering Science and Technology (Stock
15、 #:DEFONLINE) is available on the ASTM website, www.astm.org, or contact ASTMCustomer Service at serviceastm.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with international
16、ly recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.13.2.3 field blank, na sample slide or cassette carried t
17、o thesampling site, exposed to sampling conditions (for example,seals opened), returned to the laboratory, treated as a sample,and carried through all steps of the analysis.3.2.4 fungal structure (sing.), na collective term forfragments or groups of fragments from fungi, including but notlimited to
18、conidia, conidiophores, and hyphae and spores.3.2.5 fungus (s), fungi, (pl.), neukaryotic, heterotrophic,absorptive organisms that usually develop a rather diffuse,branched, tubular body (for example, network of hyphae) andusually reproduce by means of spores. The terms mold andmildew are frequently
19、 used by laypersons when referring tovarious fungal colonization.3.2.6 hyaline, adjcolorless.3.2.7 impaction medium, na substance applied to a micro-scope slide used to collect (or capture) particulate matterduring sampling.3.2.8 impaction sample, na sample taken using impaction,for example, slit im
20、paction sample, circular impaction sample.3.2.9 inertial impactor, na device for collecting particlesseparated from an air stream by inertia to force an impact ontoan adhesive surface. Inertial impactors are available in manydesigns, including those having a slit jet, yielding a rectangularsample tr
21、ace, and a circular jet, yielding a circular sampletrace.3.2.10 magnification/resolution combination 1, n150400 total magnification and a point to point resolutionof 0.7 m or better, as checked by a resolution check slide.3.2.11 magnification/resolution combination 2, n 400or greater total magnifica
22、tion and a point to point resolution of0.5 m or better, as checked by a resolution check slide.3.2.12 minimum reporting limit (fs/sample); minimum re-porting limit (fs/m3), nthe lowest result to be reported fortotal spores or any spore category. Since both fs/sample andfs/m3are reported, there are t
23、wo minimum reporting limits.3.2.13 morphology, nthe form and structure of an organ-ism or any of its parts; for fungi, the shape, form,ornamentation, or combination thereof.3.2.14 mounting medium, na liquid, for example, lacticacid or prepared stain, used to immerse the sample particulatematter and
24、to attach a cover slip to an impaction sample.3.2.15 sample trace, nthe area of particle deposition, thatis, the deposit on a slit impaction sample resembling a narrowrectangle, or the circular deposit on a circular impactionsample.3.2.16 septum (pl.: septa), na cell wall or partition.3.2.17 slide a
25、dherent, nan adhesive or liquid used to affixan impaction sample substrate to a microscope slide.3.2.18 slit impaction sample, na sample of airborne par-ticulate matter collected by means of a device that draws airthrough a linear aperture at a specified rate, impacting theparticles suspended in the
26、 air onto an adhesive medium,resulting in a rectangular area of deposition. A slit impactionsample may be collected by means of a cassette manufacturedfor that purpose, or by means of a sampling device that requiresslides to be pre-coated with impaction medium.3.2.19 spore category, na grouping used
27、 for identificationand quantifation of fungal structures. A spore category maycontain a specific genus (for example, Stachybotrys), or it mayrepresent a combination of genera (for example, Aspergillus/Penicillium-like).3.2.20 traverse, na portion of analysis of an impactorsample consisting of one sc
28、an under the microscope from asample-less portion of the impaction medium across thedeposit to a corresponding sample-less portion of the impactionmedium on the other side.3.3 Symbols:3.3.1 fsfungal structure3.3.2 fs/m3fungal structures per cubic metre3.3.3 m3cubic metre3.3.4 mmmillimetre3.3.5 mmicr
29、ometre4. Summary of Test Method4.1 Samples have been previously collected utilizing animpaction device operating at the device manufacturers rec-ommended sample flow rate. Each sample consists of anoptically clear substrate coated with an adhesive and opticallytransparent medium onto which particles
30、 have been depositedthrough inertial impaction.4.2 A sample is mounted to a microscope slide and exam-ined by bright field microscopy using at least twomagnification/resolution combinations.4.3 Spores are differentiated from each other, other fungalstructures, and from non-fungal material by color,
31、size, shape,presence of a septum or septa, attachment scars, surfacetexture, etc., by means of a taxonomic comparison withstandard reference texts or known standard samples, or both(see Section A1.1 for suggested references). The number ofspores that match each spore category are then calculated inu
32、nits of fungal structures per sample (fs/sample) and alsofungal structures per cubic meter of air (fs/m3).5. Significance and Use5.1 This test method is used to estimate and categorize thenumber and type of fungal structures present on an inertialimpactor sample.5.2 Fungal structures are identified
33、and quantified regardlessof whether they would or would not grow in culture.5.3 It must be emphasized that the detector in this testmethod is the analyst, and therefore results are subjective,depending on the experience, training, qualification, and men-tal and optical fatigue of the analyst.6. Inte
34、rferences6.1 Differentiation of Fungal Genera/SpeciesBecause ofthe similar size and morphology of some fungal spores ofdifferent genera and the absence of growth structures andmycelia in airborne samples, differentiation by microscopicD7391 1712examination alone is difficult and spores must be group
35、ed intocategories based strictly on morphology. In many cases,identification at the genus level is presumptive. For example,differentiation between Aspergillus and Penicillium using thistest method is not typical, so a combined Aspergillus/Penicillium-like category is used. When differentiation be-t
36、ween such genera is desired, a different test method must beused. Unequivocal identification of every spore in each cat-egory is not possible due to optical limitations, the atypicalnature of some of the spores, overlapping morphology amongdifferent spore types, or combination thereof, and therefore
37、,certain spores must be categorized as Miscellaneous/Unidentifiable.6.2 Look-Alike Non-Fungal ParticlesCertain types of par-ticles of non-fungal origin may resemble fungal spores. Theseparticles and artifacts may include air or plant resin bubbles,starch, talc, cosmetic particles, or combustion prod
38、ucts. Stan-dards (mounted similarly to impactor samples) should beexamined by laboratory analysts to know how to identify suchparticles. Examination of suspect particles using optical con-ditions other than bright field microscopy (for example, polar-ized light microscopy, phase contrast microscopy,
39、 differentialinterference contrast) may be helpful whenever significantconcentrations of look-alike particles are present. In somecases dust and debris can mimic the morphology of particles ofinterest. When look-alike particles are present in highconcentration, accurately counting spores with simila
40、r mor-phology is difficult. When these conditions exist, they shouldbe reported in the analysis notes section of the report.6.3 Particle OverloadingHigh levels of particulate matteron an impaction sample will bias the analysis in two ways:(1) Particle capture efficiency decreases, and(2) Debris obsc
41、ures or covers spores.Both of these factors produce a negative bias.6.4 StainingStaining, while optional, may help the analystdifferentiate spores from debris. Without staining, clear spores(especially small ones) may exhibit negative bias because theanalyst has insufficient contrast to notice them
42、while scanning.Also, because spores of different fungal species absorb stains atdifferent rates, under or over-staining makes identificationdifficult. The problem can be eliminated by careful control ofstain concentrations.6.5 Impaction Medium Stability and ClarityChemicalspresent in some mounting m
43、edia may affect the physicalstability or clarity of the impaction medium. For instance:(1) Samples collected on silicone grease medium shouldfirst be warmed on a hot plate at approximately 40C to “fix”the sample in place, when using lacto-phenol cotton blue stain,and(2) Slides and cassettes using me
44、thyl cellulose ester +solvent adhesive medium, which is stable in lacto-phenolcotton blue stain, will “fog” with Calberlas stain due to thewater and alcohol mixture; warming fogged slides may tem-porarily clear them.The lab or analyst should develop through experimentationan impaction medium/mountin
45、g medium combination that willresult in acceptable stability, clarity, and spore visibility.6.6 Uneven Impaction Medium UniformityUneven thick-ness may be present in greased slides, pre-coated slides andmanufactured cassettes. The microscopist will compensate byadjusting the plane of focus. When gre
46、ase is too thick,differentiating small spores from background artifacts (espe-cially air bubbles) in the grease preparation becomes difficult.When grease is too thin, shrinkage and pooling may haveoccurred, causing particle loss during sampling.7. Apparatus7.1 Marking pen, for marking sample slides.
47、7.2 Microscope or magnification system, having a precisionx-y mechanical stage. The microscope or magnification systemused for analysis shall be capable of at least two magnification/resolution combinations as follows: magnification/resolutioncombination 1 shall be 150400 total magnification and apo
48、int to point resolution of 0.7 m or better; magnification/resolution combination 2 shall be 400 or greater totalmagnification and a point to point resolution of 0.5 m orbetter. It is recommended that at least one microscope ormagnification system in the lab be capable of magnification of1000 total m
49、agnification and a point to point resolution of0.3 m or better. That the resolution for combinations 1 and 2is suitable is to be checked using a resolution check slide (see13.2.3).7.3 Reference Slidesa series of mounted field samples tobe used as counting references. Analysts results from theseslides are expected to be within laboratory acceptance limits toprove competence.7.4 Reticule, width defining, an optical device in the lightpath of the microscope capable of being reproducibly set todefine a traverse wid
