1、Designation: D7658 17Standard Test Method forDirect Microscopy of Fungal Structures from Tape1This standard is issued under the fixed designation D7658; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A n
2、umber 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 uses optical microscopy for thedetection, semi-quantification, and identification of fungalstructures in tape lift prep
3、arations.1.2 This test method describes the preparation techniquesfor tape-lift matrices, the procedure for confirming the pres-ence of fungal structures, and the reporting of observed fungalstructures1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are
4、included in thisstandard.1.4 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 applica-bility of regulatory limitations pri
5、or to use.1.5 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalB
6、arriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterD1356 Terminology Relating to Sampling and Analysis ofAtmospheres3. Terminology3.1 DefinitionsFor definitions of other terms used in thistest method, refer to Terminology D1356.3.2 Defini
7、tions of Terms Specific to This Standard:3.2.1 fungal structure (sing.), na collective term for afragment- or groups of fragments from fungi, including but notlimited to conidia, conidiophores, hyphae and spores.3.2.2 magnification/resolution combination 1, n100400 total magnification and a point to
8、 point resolutionof 0.7 m or better.3.2.3 magnification/resolution combination 2, n 400 orgreater total magnification and a point to point resolution of 0.5m or better.3.2.4 mounting medium, na liquid, for example, lacticacid or prepared stain, used to immerse the sample particulatematter and to att
9、ach a cover slip to the sample.3.2.5 tape lift sample, nmaterial lifted from a surfaceusing clear, transparent, single sided, adhesive collectionmedium, typically tape or commercially available preparedslides.4. Summary of Test Method4.1 A tape lift sample is prepared.4.2 The prepared sample is exam
10、ined on an optical micro-scope for the presence, type and semi-quantification of fungalstructures and reported.5. Significance and Use5.1 The significance of this test method is to standardize theanalysis of the detection of removable fungal structures liftedfrom a surface with tape to improve consi
11、stency betweenlaboratories and analysts.5.2 This test method is intended to ensure consistent data tothe end user.5.3 Fungal structures are identified and semi-quantifiedregardless of whether they would or would not grow in culture.5.4 It must be emphasized that the detector in this testmethod is th
12、e analyst, and therefore results are subjective,depending on the experience, training, qualification, opticalacuity, and mental fatigue of the analyst.5.5 This test method can be used to assess the presence andcharacteristics of fungal material on a surface.6. Interferences6.1 Look-Alike Non-Fungal
13、ParticlesCertain types of par-ticles of non-fungal origin may resemble fungal structures.1This test method 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 March 1, 2017. Pub
14、lished April 2017. DOI: 10.1520/D7658-17.2For 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.Copyright ASTM Inter
15、national, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards,
16、Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1These particles and artifacts may include air or plant resin,bubbles, starch, talc or cosmetic particles, or combustionproducts. Non-fungal reference slides (mounted similarly totape-lift s
17、amples) should be examined by laboratory analysts toknow how to differentiate such particles. Examination ofsuspect particles using optical conditions other than bright fieldmicroscopy (for example, polarized light microscopy, phasecontrast microscopy, differential interference contrast) may behelpf
18、ul whenever significant concentrations of look-alike par-ticles are present. In some cases dust and debris can mimic themorphology of particles of interest.6.2 Particle OverloadingHigh levels of non-fungal back-ground particulate may obscure or cover fungal structures.6.3 StainingFungal structures o
19、f different fungal speciesabsorb stains at different rates, under or over-staining makesidentification difficult. The problem can be minimized withcareful control of stain concentrations.NOTE 1Staining, while optional, may help the analyst differentiatefungal structures from debris. Without staining
20、, clear spores (especiallysmall ones) may exhibit negative bias because the analyst has insufficientcontrast to detect them while scanning.7. Apparatus7.1 Microscope or magnification system, having a precisionx-y mechanical stage. The microscope or magnification systemused for analysis shall be capa
21、ble of at least two magnification/resolution combinations as follows: magnification/resolutioncombination 1 shall be 100400 total magnification and apoint to point resolution of 0.7 um or better; magnification/resolution combination 2 shall be 400 or greater totalmagnification and a point to point r
22、esolution of 0.5 um orbetter.Acceptable resolutions for combinations 1 and 2 shall bechecked using a resolution check slide.NOTE 2It is recommended that at least one microscope or magnifi-cation system be available that is capable of magnification of 1000 totalmagnification and a point to point reso
23、lution of 0.3 um or better.7.2 Syringe or dropper, for dispensing liquid during samplepreparation.7.3 Stage micrometer, traceable to the National Institute ofStandards and Technology (NIST) or equivalent internationalstandard.7.4 Forceps, for manipulating adhesive tape, cleaned toprevent cross conta
24、mination.7.5 Scalpel, or other cutting tool, if needed for cutting tape,cleaned to prevent cross contamination.8. Reagents and Materials8.1 Mounting medium (with or without stain), for re-hydrating spores, optimal resolution, and securing the coverslip to the sample. For example, lactic acid, lacto-
25、cotton bluestain, lacto-phenol-cotton blue stain, lacto-fuchsine stain, glyc-erin jelly (see Appendix X2 for examples of stain prepara-tions).8.2 Microscope cover slips, large enough to cover the tapepreparation. For optimum performance, choose a cover slipthickness according to the recommendations
26、of the microscopeobjective lens manufacturer.8.3 Microscope slides, glass slides to be used when samplesare not taken on commercially available lift-samples.8.4 Disinfectant, for cleaning of forceps or scalpel.9. Hazards9.1 Components of re-hydrating liquids and stains may becorrosive or hazardous.
27、Consult the appropriate Safety DataSheet for any reagents used.9.2 Sharp instruments used in sample preparation may causeinjury if not handled with care. These sample instruments may,at times, be contaminated with biological material capable ofintroducing organisms to the user.9.3 Samples shall be h
28、andled using good laboratory tech-nique to minimize exposure.10. Preparation of Apparatus10.1 Microscope Alignment/Adjustments/Lens CleaningFollow the manufacturers instructions.11. Calibration and Standardization11.1 Graduation Spacing for Ocular Reticule:11.1.1 Measuring Gradations on the Ocular R
29、eticuleForeach magnification/resolution combination, verify the m pergraduation, using a stage micrometer, at the magnification(s)used for counting at least once per year, and after any serviceor repair to the microscope. The graduations are used tomeasure the size of spores as an aid to identificat
30、ion.11.1.2 Resolution CheckcCheck the resolution ofmagnification/resolution combinations 1 and 2 at least annuallyand after servicing, as in accordance with the manufacturersinstructions for the resolution check slide used.12. Procedure12.1 Preparation of Tape Lift Samples:12.1.1 If the tape lift wa
31、s not submitted on clear tape orprepared adhesive slide, then the sample may not be analyzedusing this test method.12.1.2 Remove the tape lift from its container.12.1.3 Mark each slide with a unique designation.12.1.4 Mount sample in one of these three ways:12.1.4.1 For samples submitted affixed to
32、a microscopeslide, gently lift one end of the tape from the slide with forcepsand place a drop of mounting medium under the sample area.Additional manipulation of the sample may be necessary toattain uniform contact with the glass slide. Return the liftedportion of the tape to the slide taking care
33、to minimize theamount of bubbles.12.1.4.2 For samples submitted on a prepared adhesiveslide, place a drop of mounting medium to the center of thesample.Acover slip is applied at such an angle that bubbles areminimized.12.1.4.3 For all other submitted samples, cut a representa-tive portion of a tape
34、lift with scalpel and mount sample-sideup on a microscope slide with forceps. Anchor on each side ifneeded.Adrop of mounting medium is applied to the center ofD7658 172the sample, and a cover slip is placed at such an angle thatbubbles are minimized. This technique can be used regardlessof whether t
35、he tape lift sample was submitted sample side upor down, and regardless of what the tape was affixed to whensubmitted.12.2 Sample Evaluation:12.2.1 Place prepared slide on the stage of the microscope.Center the sample deposit over the light source.12.2.2 Align/adjust microscope following the manufac
36、tur-ers instructions.12.2.3 Using magnification/resolution 1, examine entiresample preparation to detect fungal matter.12.2.4 If no fungal matter is detected at magnification/resolution 1, switch to magnification/resolution 2. Examine aminimum of 20 fields of view if fungal material is not detected.
37、12.2.5 If no fungal structures were detected, record lack ofdetection.12.2.6 If fungal matter is identified at low magnification,switch to magnification/resolution 2 and relocate the area foridentification and determination of fungal loading.12.2.7 Determine and record each fungal type as encoun-ter
38、ed.12.2.7.1 The minimum categories to be reported are:(1) Alternaria,(2) ascospores (undifferentiated),(3) Aspergillus/Penicillium-like,(4) basidiospores (undifferentiated),(5) Chaetomium,(6) Cladosporium,(7) Curvularia,(8) Drechslera/Biopolaris-like,(9) smuts/Myxomycetes/Periconia,(10) Stachybotrys
39、/Memnoniella,(11) Ulocladium, and(12) hyphal fragments.NOTE 3Depending on the fungal type and the scan magnification, itmay be necessary to employ greater magnification or oil immersion, orboth, for identification.12.2.8 Record the presence of hyphae, fruiting bodies, orclumps and chains of spores f
40、or each fungal type detected, orcombination thereof.12.2.9 Determine the fungal loading category. (Fungal load-ing categoriesdefined in 12.2.10.1.)12.2.10 Determine and record the non-fungal loading cat-egory of the sample. (Non-fungal loading categories defined in12.2.10.2.)12.2.10.1 Fungal Loading
41、 Categories:(1) The loading of fungal material is reported using a scaleof Categories 05. Category 0 is assigned when no fungalmaterial is observed. Category 1 is assigned when the fungalmaterial loading covers less than5%ofarepresentative fieldof view. Category 2 is assigned when the fungal materia
42、lloading covers between approximately5%and25%ofarepresentative field of view. Category 3 is assigned when thefungal material loading covers between approximately 25 %and 75 % of a representative field of view. Category 4 isassigned when the fungal material loading covers betweenapproximately 75 % an
43、d 90 % of a representative field of view.Category 5 is assigned when the fungal material loading coversgreater than approximately 90 % of a representative field ofview. Refer to the visual representations of particle loadingcategories (Fig. 1).12.2.10.2 Non-Fungal Particle Loading Categories:(1) The
44、 loading of non-fungal background debris is re-ported by using a scale of Categories 05. Particle Category 0is assigned when no background debris is observed. ParticleCategory 1 is assigned when debris loading covers less than5 % of a representative field of view. Particle Category 2 isassigned when
45、 debris loading covers between approximately5 % and 25 % of a representative field of view. ParticleCategory 3 is assigned when debris loading covers betweenapproximately 25 % and 75 % of a representative field ofview. Particle Category 4 is assigned when a debris loadingcovers between approximately
46、 75 % and 90 % of a represen-tative field of view. Particle Category 5 is assigned when debrisloading covers greater than approximately 90 % of a repre-sentative field of view. Refer to the visual representations ofparticle loading categories (Fig. 1).13. Quality Assurance/Quality Control13.1 Establ
47、ish and maintain a quality assurance/qualitycontrol system for this analysis.NOTE 4Accreditation bodies may have specific QA/QC requirements.13.2 Contamination Control:13.2.1 HousekeepingKeep preparation and analysis areasclean, for example, routinely wet-wipe to minimize transfer oflab dust to samp
48、les.13.2.2 Process/Medium BlankAt a defined frequency,place in the sample preparation area during sample preparationa piece of clear adhesive tape mounted tacky side up onto aglass slide. When the sample batch has been prepared, place adrop of mounting medium on the adhesive tape followed by acover
49、slip, to create a process blank that includes all glass andliquid components of a typical sample. Analyze in the samemanner as a sample. Establish acceptance criteria for suchblanks.13.2.3 Cross ContaminationNo more than one samplemay be prepared at a time.13.3 Precision and Accuracy:13.3.1 Analyst Training and QualificationQualify an ana-lyst to be competent to perform this test method by acombination of background and education, aerobiological andmycological training, experience, and performance on bulksamples of known/reference content (for e
