ASTM D7391-09
(Test Method)Standard Test Method for Categorization and Quantification of Airborne Fungal Structures in an Inertial Impaction Sample by Optical Microscopy
Standard Test Method for Categorization and Quantification of Airborne Fungal Structures in an Inertial Impaction Sample by Optical Microscopy
SIGNIFICANCE AND USE
This test method is used to estimate and categorize the number and type of fungal structures present on an inertial impactor sample.
Fungal structures are identified and quantified regardless of whether they would or would not grow in culture.
It must be emphasized that the detector in this method is the analyst, and therefore results are subjective, depending on the experience, training, qualification, and mental and optical fatigue of the analyst.
SCOPE
1.1 This test method is a procedure that uses direct microscopy to analyze the deposit on an inertial impaction sample.
1.2 This test method describes procedures for categorizing and enumerating fungal structures by morphological type. Typically, categories may be as small as genus (for example, Cladosporium) or as large as phylum (for example, basidiospores).
1.3 This method contains two procedures for enumerating fungal structures: one for slit impaction samples and one for circular impaction samples. This test method is applicable for impaction air samples, for which a known volume of air (at a rate 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 determined with this method depends on the size of the spores on the sample trace, the amount of particulate matter on the sample trace, the percentage of the sample trace counted, and the volume of air sampled.
1.6 This method addresses only the analysis of samples. The sampling process and interpretation of results is outside the scope of this method.
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation:D7391 −09
Standard Test Method for
Categorization and Quantification of Airborne Fungal
Structures in an Inertial Impaction Sample by Optical
Microscopy
This standard is issued under the fixed designation D7391; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
1.1 This test method is a procedure that uses direct micros- 2.1 ASTM Standards:
copy to analyze the deposit on an inertial impaction sample. D1193Specification for Reagent Water
1.2 This test method describes procedures for categorizing
3. Terminology
and enumerating fungal structures by morphological type.
Typically, categories may be as small as genus (for example, 3.1 ASTM Definitions (see ASTM Terminology Dictionary):
Cladosporium) or as large as phylum (for example, basidi- 3.1.1 numerical aperture
ospores).
3.2 Definitions of Terms Specific to This Standard:
1.3 This method contains two procedures for enumerating 3.2.1 circular impaction sample, n—a sample of airborne
particulate matter collected via a device that draws air through
fungal structures: one for slit impaction samples and one for
circular impaction samples. This test method is applicable for a round aperture at a specified rate, impacting the particles
impaction air samples, for which a known volume of air (at a suspended in the air onto an adhesive medium, resulting in a
rate as recommended by the manufacturer) has been drawn, circularareaofdeposition.Acircularimpactionsamplemaybe
and is also applicable for blank impaction samples. collectedbymeansofacassettemanufacturedforthatpurpose,
or by means of a sampling device that requires slides to be
1.4 Enumeration results are presented in fungal structures/
pre-coated with impaction medium.
3 3
sample (fs/sample) and fungal structures/m (fs/m ).
3.2.2 debris rating, n—a distinct value assigned to an
1.5 Therangeofenumerationresultsthatcanbedetermined
impactor sample based on the percentage of the sample area
with this method depends on the size of the spores on the
potentially obscured by particulate matter, and ranging from 0
sample trace, the amount of particulate matter on the sample
to 5.
trace, the percentage of the sample trace counted, and the
3.2.3 field blank, n—asampleslideorcassettecarriedtothe
volume of air sampled.
sampling site, exposed to sampling conditions (for example,
1.6 Thismethodaddressesonlytheanalysisofsamples.The
seals opened), returned to the laboratory, treated as a sample,
sampling process and interpretation of results is outside the
and carried through all steps of the analysis.
scope of this method.
3.2.4 hyaline, adj—colorless.
1.7 The values stated in SI units are to be regarded as
3.2.5 impaction medium, n—asubstanceappliedtoamicro-
standard. No other units of measurement are included in this
scope slide used to collect (or capture) particulate matter
standard.
during sampling.
1.8 This standard does not purport to address all of the
3.2.6 impaction sample, n—asampletakenusingimpaction,
safety concerns, if any, associated with its use. It is the
for example, slit impaction sample, circular impaction sample.
responsibility of the user of this standard to establish appro-
3.2.7 inertial impactor, n—a device for collecting particles
priate safety and health practices and determine the applica-
separated from an air stream by inertia to force an impact onto
bility of regulatory limitations prior to use.
an adhesive surface. Inertial impactors are available in many
This test method is under the jurisdiction of ASTM Committee D22 on Air
Quality and is the direct responsibility of Subcommittee D22.08 on Sampling and For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Analysis of Mold. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Current edition approved May 1, 2009. Published June 2009. DOI: 10.1520/ Standards volume information, refer to the standard’s Document Summary page on
D7391-09. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7391−09
designs,includingthosehavingaslitjet,yieldingarectangular ommended sample flow rate. Each sample consists of an
sample trace, and a circular jet, yielding a circular sample optically clear substrate coated with an adhesive and optically
trace. transparent medium onto which particles have been deposited
through inertial impaction.
3.2.8 magnification/resolution combination 1, n—~150-
400× total magnification and a point to point resolution of 0.7
4.2 A sample is mounted to a microscope slide and exam-
µm or better, as checked by a resolution check slide.
ined by bright field microscopy using at least two
magnification/resolution combinations.
3.2.9 magnification/resolution combination 2, n—~400× or
greatertotalmagnificationandapointtopointresolutionof0.5
4.3 Spores are differentiated from each other, other fungal
µm or better, as checked by a resolution check slide.
structures, and from non-fungal material by color, size, shape,
presence of a septum or septa, attachment scars, surface
3.2.10 minimum reporting limit (fs/sample); minimum re-
texture, etc., by means of a taxonomic comparison with
porting limit (fs/m ), n—the lowest result to be reported for
standard reference texts and/or known standard samples (see
total spores or any spore category. Since both fs/sample and
A1.1 for suggested references). The number of spores that
fs/m are reported, there are two minimum reporting limits.
matcheachsporecategoryarethencalculatedinunitsoffungal
3.2.11 morphology, n—the form and structure of an organ-
structurespersample(fs/sample)andalsofungalstructuresper
ism or any of its parts; for fungi, the shape, form, and/or
cubic meter of air (fs/m ).
ornamentation.
3.2.12 mounting medium, n—a liquid, for example, lactic
5. Significance and Use
acid or prepared stain, used to immerse the sample particulate
5.1 This test method is used to estimate and categorize the
matter and to attach a cover slip to an impaction sample.
number and type of fungal structures present on an inertial
3.2.13 sample trace, n—the area of particle deposition, that
impactor sample.
is, the deposit on a slit impaction sample resembling a narrow
5.2 Fungalstructuresareidentifiedandquantifiedregardless
rectangle, or the circular deposit on a circular impaction
of whether they would or would not grow in culture.
sample.
5.3 Itmustbeemphasizedthatthedetectorinthismethodis
3.2.14 septum (pl.: septa), n—a cell wall or partition.
the analyst, and therefore results are subjective, depending on
3.2.15 slide adherent, n—an adhesive or liquid used to affix
the experience, training, qualification, and mental and optical
an impaction sample substrate to a microscope slide.
fatigue of the analyst.
3.2.16 slit impaction sample, n—a sample of airborne par-
6. Interferences
ticulate matter collected via a device that draws air through a
linear aperture at a specified rate, impacting the particles
6.1 Differentiation of Fungal Genera/Species—Because of
suspended in the air onto an adhesive medium, resulting in a
the similar size and morphology of some fungal spores of
rectangular area of deposition.Aslit impaction sample may be
different genera and the absence of growth structures and
collected via a cassette manufactured for that purpose , or via
mycelia in airborne samples, differentiation by microscopic
a sampling device that requires slides to be pre-coated with
examination alone is difficult and spores must be grouped into
impaction medium.
categories based strictly on morphology. In many cases,
identification at the genus level is presumptive. For example,
3.2.17 spore category, n—a grouping used for identification
differentiation between Aspergillus and Penicillium using this
and quantifation of fungal structures. A spore category may
method is not typical, so a combined Aspergillus/Penicillium-
contain a specific genus (for example, Stachybotrys), or it may
like category is used. When differentiation between such
represent a combination of genera (for example, Aspergillus/
genera is desired, a different method must be used. Unequivo-
Penicillium -like).
calidentificationofeverysporeineachcategoryisnotpossible
3.2.18 traverse, n—a portion of analysis of an impactor
due to optical limitations, the atypical nature of some of the
sample consisting of one scan under the microscope from a
spores, and/or overlapping morphology among different spore
sample-less portion of the impaction medium across the
types, and therefore, certain spores must be categorized as
deposittoacorrespondingsample-lessportionoftheimpaction
Miscellaneous/Unidentifiable.
medium on the other side.
6.2 Look-alike Non-fungal Particles—Certain types of par-
3.3 Symbols:
ticles of non-fungal origin may resemble fungal spores. These
3.3.1 fs—fungal structure
particles and artifacts may include air or plant resin bubbles,
3.3.2 fs/m —fungal structures per cubic metre
starch, talc, cosmetic particles, or combustion products. Stan-
3.3.3 m —cubic metre dards (mounted similarly to impactor samples) should be
examined by laboratory analysts to know how to identify such
3.3.4 mm—millimetre
particles. Examination of suspect particles using optical con-
3.3.5 µm—micrometre
ditions other than bright field microscopy (for example, polar-
ized light microscopy, phase contrast microscopy, differential
4. Summary of Test Method
interference contrast) may be helpful whenever significant
4.1 Samples have been previously collected utilizing an concentrations of look-alike particles are present. In some
impaction device operating at the device manufacturer’s rec- casesdustanddebriscanmimicthemorphologyofparticlesof
D7391−09
interest. When look-alike particles are present in high 7.3 Reference Slides—a series of mounted field samples to
concentration, accurately counting spores with similar mor- be used as counting references. Analysts’ results from these
phology is difficult. When these conditions exist, they should
slidesareexpectedtobewithinlaboratoryacceptancelimitsto
be reported in the analysis notes section of the report.
prove competence.
6.3 Particle Overloading—High levels of particulate matter
7.4 Reticule, width defining, an optical device in the light
on an impaction sample will bias the analysis in two ways:
path of the microscope capable of being reproducibly set to
(1)Particle capture efficiency decreases, and
defineatraversewidthnolargerthan0.75×thediameterofthe
(2)Debris obscures or covers spores.
ocular field of view, and having graduations of an appropriate
Both of these factors produce a negative bias.
dimension to allow measurement of spore size, for example,
6.4 Staining—Staining,whileoptional,mayhelptheanalyst
Walton-Beckett reticule (round) or 100 divisions in 10 mm
differentiate spores from debris. Without staining, clear spores
(linear or square). If a non-round reticule is used, procedures
(especially small ones) may exhibit negative bias because the
must be in place to ensure that the reticule is correctly
analyst has insufficient contrast to notice them while scanning.
positioned for each analysis.
Also,becausesporesofdifferentfungalspeciesabsorbstainsat
7.5 Stage micrometer, traceable to the National Institute of
different rates, under or over-staining makes identification
Standards and Technology (NIST) or equivalent international
difficult. The problem can be eliminated by careful control of
standard.
stain concentrations.
6.5 Impaction Medium Stability and Clarity—Chemicals
7.6 Resolution check slide, a microscope slide on which
present in some mounting media may affect the physical
calibrated distances, shapes, and line widths provide reliable
stability or clarity of the impaction medium. For instance:
and simple image resolution and shape identification perfor-
(1)Samples collected on silicone grease medium should
mance of the microscopic and analyst at magnification. Ex-
first be warmed on a hot plate at approximately 40°C to “fix”
amples include: a slide onto which a variety of diatoms have
thesampleinplace,whenusinglacto-phenolcottonbluestain,
been mounted, including examples of Stauroneis phoenicen-
and
teron and Pleurosigma angulatum, a brightfield resolution test
(2)Slides and cassettes using methyl cellulose ester +
slide, or equivalent.
solvent adhesive medium, which is stable in lacto-phenol
7.7 Syringe or dropper, for dispensing liquid during sample
cotton blue stain, will “fog” with Calberla’s stain due to the
preparation.
water and alcohol mixture; warming fogged slides may tem-
porarily clear them.
The lab or analyst should develop through experimentation 8. Reagents and Materials
animpactionmedium/mountingmediumcombinationthatwill
8.1 Purity of Reagents—Reagent grade chemicals shall be
result in acceptable stability, clarity, and spore visibility.
used in all tests. Unless otherwise indicated, it is intended that
6.6 Uneven Impaction Medium Uniformity—Uneven thick-
all reagents conform to the specifications of the Committee on
ness may be present in greased slides, pre-coated slides and
Analytical Reagents of theAmerican Chemical Society where
manufactured cassettes. The microscopist will compensate by
such specifications are available. Other grades may be used,
adjusting the plane of focus. When grease is too thick,
provided it is first ascertained that the reagent is of sufficiently
differentiating small spores from background artifacts (espe-
high purity to permit its use without lessening the accuracy of
cially air bubbles) in the grease preparation becomes difficult.
the determination.
When grease is too thin, shrinkage and pooling may have
occurred, causing particle loss during sampling. 8.2 Purity of Water—Unless otherwise indicated, references
to water shall be understood to mean reagent water as defined
7. Apparatus
by Type II of Specification D1193.
7.1 Marking pen, for marking sample slides.
8.3 Mounting medium (with or without stain), for re-
7.2 Microscope or magnification system, having a precision
hydratingsporesandforholdingthecoversliptotheimpaction
x-y mechanical stage.The microscope or magnification system
sample, for example, lactic acid, lacto-cotton blue stain,
usedforanalysisshallbecapableofatleasttwomagnification/
lacto-phenol-cottonbluestain,lacto-fuchsinstain(seeX2.1for
resolution combinations as follows: magnification/resolution
stain preparation).
combination 1 shall be ~150-400× total magnification and a
8.4 Microscope cover sli
...
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