1、Designation: D 7391 09Standard Test Method forCategorization and Quantification of Airborne FungalStructures in an Inertial Impaction Sample by OpticalMicroscopy1This standard is issued under the fixed designation D 7391; 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.1. Scope1.1 This test method is a procedure that uses direct micros-copy to ana
3、lyze 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) or as large as phylum (for example, basid-iospores).1.3 This me
4、thod contains two procedures for enumeratingfungal structures: one for slit impaction samples and one forcircular impaction samples. This test method is applicable forimpaction air samples, for which a known volume of air (at arate as recommended by the manufacturer) has been drawn,and is also appli
5、cable 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 method depends on the size of the spores on thesample trace, the amount of particulate
6、matter on the sampletrace, the percentage of the sample trace counted, and thevolume of air sampled.1.6 This method addresses only the analysis of samples. Thesampling process and interpretation of results is outside thescope of this method.1.7 The values stated in SI units are to be regarded asstan
7、dard. 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 standard to establish appro-priate safety and health practices and determine the appl
8、ica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent Water3. Terminology3.1 ASTM Definitions (see ASTM Terminology Dictionary):3.1.1 numerical aperture3.2 Definitions of Terms Specific to This Standard:3.2.1 circular impaction
9、sample, na sample of airborneparticulate matter collected via a device that draws air througha round aperture at a specified rate, impacting the particlessuspended in the air onto an adhesive medium, resulting in acircular area of deposition.Acircular impaction sample may becollected by means of a c
10、assette manufactured for that purpose,or by means of a sampling device that requires slides to bepre-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 fro
11、m 0to 5.3.2.3 field blank, na sample slide or cassette carried to 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 hyaline, adjcolorless.3.2.5 impaction medium, na substanc
12、e applied to a mi-croscope slide used to collect (or capture) particulate matterduring sampling.3.2.6 impaction sample, na sample taken using impac-tion, for example, slit impaction sample, circular impactionsample.1This test method is under the jurisdiction of ASTM Committee D22 on AirQuality and i
13、s the direct responsibility of Subcommittee D22.08 on Sampling andAnalysis of Mold.Current edition approved May 1, 2009. Published June 2009.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volu
14、me information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.2.7 inertial impactor, na device for collecting particlesseparated from an air stream by inertia to for
15、ce an impact ontoan adhesive surface. Inertial impactors are available in manydesigns, including those having a slit jet, yielding a rectangularsample trace, and a circular jet, yielding a circular sampletrace.3.2.8 magnification/resolution combination 1, n150-4003 total magnification and a point to
16、 point resolution of 0.7m or better, as checked by a resolution check slide.3.2.9 magnification/resolution combination 2, n4003 orgreater total magnification and a point to point resolution of 0.5m or better, as checked by a resolution check slide.3.2.10 minimum reporting limit (fs/sample); minimum
17、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 two minimum reporting limits.3.2.11 morphology, nthe form and structure of an organ-ism or any of its parts; for fungi, the shape, form, and/orornam
18、entation.3.2.12 mounting medium, na liquid, for example, lacticacid or prepared stain, used to immerse the sample particulatematter and to attach a cover slip to an impaction sample.3.2.13 sample trace, nthe area of particle deposition, thatis, the deposit on a slit impaction sample resembling a nar
19、rowrectangle, or the circular deposit on a circular impactionsample.3.2.14 septum (pl.: septa), na cell wall or partition.3.2.15 slide adherent, nan adhesive or liquid used to affixan impaction sample substrate to a microscope slide.3.2.16 slit impaction sample, na sample of airborne par-ticulate ma
20、tter collected via a device that draws air through alinear aperture at a specified rate, impacting the particlessuspended in the air onto an adhesive medium, resulting in arectangular area of deposition. A slit impaction sample may becollected via a cassette manufactured for that purpose , or viaa s
21、ampling device that requires slides to be pre-coated withimpaction medium.3.2.17 spore category, na grouping used 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
22、, Aspergillus/Penicillium-like).3.2.18 traverse, na portion of analysis of an impactorsample consisting of one scan 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
23、.1 fsfungal structure3.3.2 fs/m3fungal structures per cubic metre3.3.3 m3cubic metre3.3.4 mmmillimetre3.3.5 mmicrometre4. 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 con
24、sists of anoptically clear substrate coated with an adhesive and opticallytransparent medium onto which particles 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 combinatio
25、ns.4.3 Spores are differentiated from each other, other fungalstructures, and from non-fungal material by color, size, shape,presence of a septum or septa, attachment scars, surfacetexture, etc., by means of a taxonomic comparison withstandard reference texts and/or known standard samples (seeA1.1 f
26、or suggested references). The number of spores thatmatch each spore category are then calculated in units of fungalstructures per sample (fs/sample) and also fungal structures percubic meter of air (fs/m3).5. Significance and Use5.1 This test method is used to estimate and categorize thenumber and t
27、ype of fungal structures present on an inertialimpactor sample.5.2 Fungal structures are identified and quantified regardlessof whether they would or would not grow in culture.5.3 It must be emphasized that the detector in this method isthe analyst, and therefore results are subjective, depending on
28、the experience, training, qualification, and mental and opticalfatigue of the analyst.6. Interferences6.1 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, di
29、fferentiation by microscopicexamination alone is difficult and spores must be grouped intocategories based strictly on morphology. In many cases,identification at the genus level is presumptive. For example,differentiation between Aspergillus and Penicillium using thismethod is not typical, so a com
30、bined Aspergillus/Penicillium-like category is used. When differentiation between suchgenera is desired, a different method must be used. Unequivo-cal identification of every spore in each category is not possibledue to optical limitations, the atypical nature of some of thespores, and/or overlappin
31、g morphology among different sporetypes, and therefore, certain spores must be categorized asMiscellaneous/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 bubble
32、s,starch, talc, cosmetic particles, or combustion products. 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, p
33、olar-ized light microscopy, phase contrast microscopy, 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 hig
34、h concen-tration, accurately counting spores with similar morphology isdifficult. When these conditions exist, they should be reportedin the analysis notes section of the report.D73910926.3 Particle OverloadingHigh levels of particulate matteron an impaction sample will bias the analysis in two ways
35、:(1) Particle capture efficiency decreases, and(2) Debris obscures 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 bia
36、s because theanalyst has insufficient contrast to notice them 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 M
37、edium Stability and ClarityChemicalspresent in some mounting media 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
38、-phenol cotton blue stain,and(2) Slides and cassettes using methyl 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 sho
39、uld develop through experimentationan impaction medium/mounting 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 microsc
40、opist will compensate byadjusting the plane of focus. When grease 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 sa
41、mpling.7. Apparatus7.1 Marking pen, for marking sample slides.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/resol
42、utioncombination 1 shall be 150-4003 total magnification and apoint to point resolution of 0.7 m or better; magnification/resolution combination 2 shall be 4003 or greater totalmagnification and a point to point resolution of 0.5 m orbetter. It is recommended that at least one microscope ormagnifica
43、tion system in the lab be capable of magnification of10003 total magnification 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
44、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 width no larger than
45、0.753 the diameter of theocular field of view, and having graduations of an appropriatedimension to allow measurement of spore size, for example,Walton-Beckett reticule (round) or 100 divisions in 10 mm(linear or square). If a non-round reticule is used, proceduresmust be in place to ensure that the
46、 reticule is correctlypositioned for each analysis.7.5 Stage micrometer, traceable to the National Institute ofStandards and Technology (NIST) or equivalent internationalstandard.7.6 Resolution check slide, a microscope slide on whichcalibrated distances, shapes, and line widths provide reliableand
47、simple image resolution and shape identification perfor-mance of the microscopic and analyst at magnification. Ex-amples include: a slide onto which a variety of diatoms havebeen mounted, including examples of Stauroneis phoen-icenteron and Pleurosigma angulatum, a brightfield resolutiontest slide,
48、or equivalent.7.7 Syringe or dropper, for dispensing liquid during samplepreparation.8. Reagents and Materials8.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytic
49、al Reagents of the American Chemical Society wheresuch specifications are available. Other 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.8.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water as definedby Type II of Specification D 1193.8.3 Mounting medium (with or without stain), for re-hydrating spores and for holding the cover slip to the impactionsample, for examp
