ASTM D7391-20

Standard Test Method for Categorization and Quantification of Airborne Fungal Structures in an Inertial Impaction Sample by Optical Microscopy

SIGNIFICANCE AND USE
5.1 This test method is used to estimate and categorize the number and type of fungal structures present on an inertial impactor sample.  
5.2 Fungal structures are identified and quantified regardless of whether they would or would not grow in culture.  
5.3 It must be emphasized that the detector in this test 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 test 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 test 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 test method addresses only the analysis of samples. The sampling process and interpretation of results is outside the scope of this test 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 14-Mar-2020
• ICS: 07.100.99 - Other standards related to microbiology
• Technical Committee: D22 - Air Quality
• Drafting Committee: D22.08 - Assessment, Sampling, and Analysis of Microorganisms

Relations

• Replaces: ASTM D7391-17e1 - Standard Test Method for Categorization and Quantification of Airborne Fungal Structures in an Inertial Impaction Sample by Optical Microscopy
• Effective Date: 15-Mar-2020
• Refers: ASTM E691-13 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-May-2013
• Refers: ASTM E691-11 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Nov-2011
• Refers: ASTM E691-08 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Oct-2008
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM E691-05 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Nov-2005
• Refers: ASTM E691-99 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 10-May-1999
• Refers: ASTM D1193-99 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999
• Refers: ASTM D1193-99e1 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999
• Referred By: ASTM D8219-19 - Standard Guide for Cleaning and Disinfection at a Cannabis Cultivation Center
• Effective Date: 15-Mar-2020
• Referred By: ASTM D7788-14(2023) - Standard Practice for Collection of Total Airborne Fungal Structures via Inertial Impaction Methodology
• Effective Date: 15-Mar-2020
• Referred By: ASTM D1356-20a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 15-Mar-2020

Overview

ASTM D7391-20 is the internationally recognized standard test method for the categorization and quantification of airborne fungal structures in inertial impaction samples using optical microscopy. Developed by ASTM Committee D22 on Air Quality, this method provides a systematic approach to directly analyze and enumerate various types of fungal entities collected from air samples, contributing to the assessment of indoor and outdoor air quality. The standard applies to the microscopic examination of deposits on impaction samples, enabling the identification and quantification of fungal structures by their morphological characteristics.

Key Topics

• Direct Microscopy Analysis: The method employs optical microscopy to examine, categorize, and count fungal structures collected on an impaction sample substrate.
• Categorization by Morphology: Fungal structures are identified and grouped at different taxonomic levels, from genus (e.g., Cladosporium) to phylum (e.g., basidiospores), based on observable characteristics such as size, shape, color, surface texture, and septation.
• Sample Types: Procedures are provided for both slit and circular impaction samples, which differ in the shape of the deposition area-rectangular for slit and circular for circular impactors.
• Quantification Metrics: Results are reported both as fungal structures per sample (fs/sample) and as fungal structures per cubic meter of air (fs/m³), ensuring flexible application across different monitoring contexts.
• Analyst Role and Subjectivity: Human analysts perform the detection and enumeration, which introduces a degree of subjectivity dependent on training, experience, and factors such as optical or mental fatigue.
• Quality Assurance: The standard emphasizes calibration of equipment, analyst qualification, and quality control measures to mitigate subjectivity and ensure reproducibility.
• Reporting Limitations: The ability to quantify is affected by factors such as spore size, particulate matter density, air volume sampled, and percentage of the deposit examined.

Applications

• Indoor Air Quality Assessment: Used by environmental consultants and industrial hygienists to evaluate potential fungal contamination in buildings, schools, healthcare settings, or workplaces.
• Remediation Verification: Measures airborne fungal residues before and after remediation projects to determine cleaning effectiveness.
• Research and Regulatory Compliance: Supports studies related to bioaerosols and assists in compliance with guidelines for environmental health and safety.
• Method Standardization: Ensures consistent methodology across laboratories, aiding in inter-laboratory comparisons and data reliability.

Related Standards

• ASTM D1193: Specification for Reagent Water - details water purity requirements relevant to sample preparation.
• ASTM E691: Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method - supports quality control in laboratory settings.
• ISO Standards on Airborne Particulates: May provide complementary procedures for environmental and workplace air monitoring.

Practical Value

Adhering to ASTM D7391-20 provides major benefits in environmental monitoring:

• Objective Data: By standardizing how airborne fungal structures are categorized and counted, users generate comparable, defendable data.
• Versatile Use: Suitable for a range of industries and environments concerned with mold, allergens, and general bioaerosol exposure.
• Risk Identification: Helps pinpoint areas with elevated fungal presence, supporting targeted remediation and preventive strategies.
• Accountability and Transparency: Standardized documentation enhances reporting to stakeholders, clients, and regulatory agencies.

By following ASTM D7391-20, organizations can ensure that analysis of airborne fungi is thorough, reproducible, and internationally aligned, contributing to healthier indoor environments and robust scientific research.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation:D7391 −20
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 1.9 This international standard was developed in accor-
dance with internationally recognized principles on standard-
1.1 This test method is a procedure that uses direct micros-
ization established in the Decision on Principles for the
copy to analyze the deposit on an inertial impaction sample.
Development of International Standards, Guides and Recom-
1.2 This test method describes procedures for categorizing
mendations issued by the World Trade Organization Technical
and enumerating fungal structures by morphological type.
Barriers to Trade (TBT) Committee.
Typically, categories may be as small as genus (for example,
Cladosporium) or as large as phylum (for example, basidi-
2. Referenced Documents
ospores).
2.1 ASTM Standards:
1.3 This test method contains two procedures for enumer-
D1193Specification for Reagent Water
ating fungal structures: one for slit impaction samples and one
E691Practice for Conducting an Interlaboratory Study to
for circular impaction samples. This test method is applicable
Determine the Precision of a Test Method
forimpactionairsamples,forwhichaknownvolumeofair(at
a rate as recommended by the manufacturer) has been drawn,
3. Terminology
and is also applicable for blank impaction samples.
3.1 ASTM Definitions (see the ASTM Online Dictionary of
1.4 Enumeration results are presented in fungal structures/
Engineering Science and Technology ):
3 3
sample (fs/sample) and fungal structures/m (fs/m ).
3.1.1 numerical aperture.
1.5 Therangeofenumerationresultsthatcanbedetermined
3.2 Definitions of Terms Specific to This Standard:
with this test method depends on the size of the spores on the
3.2.1 circular impaction sample, n—a sample of airborne
sample trace, the amount of particulate matter on the sample
particulatemattercollectedbymeansofadevicethatdrawsair
trace, the percentage of the sample trace counted, and the
through a round aperture at a specified rate, impacting the
volume of air sampled.
particles suspended in the air onto an adhesive medium,
resulting in a circular area of deposition.Acircular impaction
1.6 Thistestmethodaddressesonlytheanalysisofsamples.
The sampling process and interpretation of results is outside sample may be collected by means of a cassette manufactured
forthatpurpose,orbymeansofasamplingdevicethatrequires
the scope of this test method.
slides to be pre-coated with impaction medium.
1.7 The values stated in SI units are to be regarded as
3.2.2 debris rating, n—a distinct value assigned to an
standard. No other units of measurement are included in this
impactor sample based on the percentage of the sample area
standard.
potentially obscured by particulate matter, and ranging from 0
1.8 This standard does not purport to address all of the
to 5.
safety concerns, if any, associated with its use. It is the
3.2.3 field blank, n—asampleslideorcassettecarriedtothe
responsibility of the user of this standard to establish appro-
sampling site, exposed to sampling conditions (for example,
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
This test method is under the jurisdiction of ASTM Committee D22 on Air contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Quality and is the direct responsibility of Subcommittee D22.08 on Assessment, Standards volume information, refer to the standard’s Document Summary page on
Sampling, and Analysis of Microorganisms. the ASTM website.
Current edition approved March 15, 2020. Published April 2020. Originally ASTM Online Dictionary of Engineering Science and Technology (Stock #:
ɛ1
approved in 2009. Last previous edition approved in 2017 as D7391 – 17 . DOI: DEFONLINE) is available on theASTM website, www.astm.org, or contactASTM
10.1520/D7391-20. Customer Service at [e-mail].
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7391−20
seals opened), returned to the laboratory, treated as a sample, forthatpurpose,orbymeansofasamplingdevicethatrequires
and carried through all steps of the analysis. slides to be pre-coated with impaction medium.
3.2.4 fungal structure (sing.), n—a collective term for 3.2.19 spore category, n—a grouping used for identification
fragmentsorgroupsoffragmentsfromfungi,includingbutnot and quantifation of fungal structures. A spore category may
limited to conidia, conidiophores, and hyphae and spores. contain a specific genus (for example, Stachybotrys), or it may
represent a combination of genera (for example, Aspergillus/
3.2.5 fungus (s), fungi, (pl.), n—eukaryotic, heterotrophic,
Penicillium-like).
absorptive organisms that usually develop a rather diffuse,
branched, tubular body (for example, network of hyphae) and 3.2.20 traverse, n—a portion of analysis of an impactor
usually reproduce by means of spores. The terms ‘mold’and sample consisting of one scan under the microscope from a
‘mildew’ are frequently used by laypersons when referring to sample-less portion of the impaction medium across the
various fungal colonization. deposittoacorrespondingsample-lessportionoftheimpaction
medium on the other side.
3.2.6 hyaline, adj—colorless.
3.3 Symbols:
3.2.7 impaction medium, n—asubstanceappliedtoamicro-
3.3.1 fs—fungal structure
scope slide used to collect (or capture) particulate matter
during sampling.
3.3.2 fs/m —fungal structures per cubic metre
3.2.8 impaction sample, n—asampletakenusingimpaction,
3.3.3 m —cubic metre
for example, slit impaction sample, circular impaction sample.
3.3.4 mm—millimetre
3.2.9 inertial impactor, n—a device for collecting particles
3.3.5 µm—micrometre
separated from an air stream by inertia to force an impact onto
an adhesive surface. Inertial impactors are available in many
4. Summary of Test Method
designs,includingthosehavingaslitjet,yieldingarectangular
4.1 Samples have been previously collected utilizing an
sample trace, and a circular jet, yielding a circular sample
impaction device operating at the device manufacturer’s rec-
trace.
ommended sample flow rate. Each sample consists of an
3.2.10 magnification/resolution combination 1, n—
optically clear substrate coated with an adhesive and optically
~150–400× total magnification and a point to point resolution
transparent medium onto which particles have been deposited
of 0.7 µm or better, as checked by a resolution check slide.
through inertial impaction.
3.2.11 magnification/resolution combination 2, n— ~400×
4.2 A sample is mounted to a microscope slide and exam-
or greater total magnification and a point to point resolution of
ined by bright field microscopy using at least two
0.5 µm or better, as checked by a resolution check slide.
magnification/resolution combinations.
3.2.12 minimum reporting limit (fs/sample); minimum re-
4.3 Spores are differentiated from each other, other fungal
porting limit (fs/m ), n—the lowest result to be reported for
structures, and from non-fungal material by color, size, shape,
total spores or any spore category. Since both fs/sample and
presence of a septum or septa, attachment scars, surface
fs/m are reported, there are two minimum reporting limits.
texture, etc., by means of a taxonomic comparison with
3.2.13 morphology, n—the form and structure of an organ-
standard reference texts or known standard samples, or both
ism or any of its parts; for fungi, the shape, form,
(see Section A1.1 for suggested references). The number of
ornamentation, or combination thereof.
spores that match each spore category are then calculated in
units of fungal structures per sample (fs/sample) and also
3.2.14 mounting medium, n—a liquid, for example, lactic
fungal structures per cubic meter of air (fs/m ).
acid or prepared stain, used to immerse the sample particulate
matter and to attach a cover slip to an impaction sample.
5. Significance and Use
3.2.15 sample trace, n—the area of particle deposition, that
5.1 This test method is used to estimate and categorize the
is, the deposit on a slit impaction sample resembling a narrow
number and type of fungal structures present on an inertial
rectangle, or the circular deposit on a circular impaction
impactor sample.
sample.
5.2 Fungalstructuresareidentifiedandquantifiedregardless
3.2.16 septum (s), septa (pl.), n—a cell wall or partition.
of whether they would or would not grow in culture.
3.2.17 slide adherent, n—an adhesive or liquid used to affix
an impaction sample substrate to a microscope slide. 5.3 It must be emphasized that the detector in this test
method is the analyst, and therefore results are subjective,
3.2.18 slit impaction sample, n—a sample of airborne par-
depending on the experience, training, qualification, and men-
ticulate matter collected by means of a device that draws air
tal and optical fatigue of the analyst.
through a linear aperture at a specified rate, impacting the
particles suspended in the air onto an adhesive medium,
6. Interferences
resulting in a rectangular area of deposition. A slit impaction
sample may be collected by means of a cassette manufactured 6.1 Differentiation of Fungal Genera/Species—Because of
the similar size and morphology of some fungal spores of
different genera and the absence of growth structures and
Kendrick, B., The Fifth Kingdom, Focus Publishing / R Pullins & Co, 2008. mycelia in airborne samples, differentiation by microscopic
D7391−20
examination alone is difficult and spores must be grouped into 6.6 Uneven Impaction Medium Uniformity—Uneven thick-
categories based strictly on morphology. In many cases, ness may be present in greased slides, pre-coated slides and
identification at the genus level is presumptive. For example, manufactured cassettes. The microscopist will compensate by
differentiation between Aspergillus and Penicillium using this adjusting the plane of focus. When grease is too thick,
test method is not typical, so a combined Aspergillus/ differentiating small spores from background artifacts (espe-
Penicillium-like category is used. When differentiation be- cially air bubbles) in the grease preparation becomes difficult.
tween such genera is desired, a different test method must be When grease is too thin, shrinkage and pooling may have
used. Unequivocal identification of every spore in each cat- occurred, causing particle loss during sampling.
egory is not possible due to optical limitations, the atypical
nature of some of the spores, overlapping morphology among 7. Apparatus
different spore types, or combination thereof, and therefore,
7.1 Marking pen, for marking sample slides.
certain spores must be categorized as Miscellaneous/
7.2 Microscope or magnification system, having a precision
Unidentifiable.
x-y mechanical stage.The microscope or magnification system
6.2 Look-Alike Non-Fungal Particles—Certaintypesofpar-
usedforanalysisshallbecapableofatleasttwomagnification/
ticles of non-fungal origin may resemble fungal spores. These
resolution combinations as follows: magnification/resolution
particles and artifacts may include air or plant resin bubbles,
combination 1 shall be ~150–400× total magnification and a
starch, talc, cosmetic particles, or combustion products. Stan-
point to point resolution of 0.7 µm or better; magnification/
dards (mounted similarly to impactor samples) should be
resolution combination 2 shall be ~400× or greater total
examined by laboratory analysts to know how to identify such
magnification and a point to point resolution of 0.5 µm or
particles. Examination of suspect particles using optical con-
better. It is recommended that at least one microscope or
ditions other than bright field microscopy (for example, polar-
magnification system in the lab be capable of magnification of
ized light microscopy, phase contrast microscopy, differential
~1000× total magnification and a point to point resolution of
interference contrast) may be helpful whenever significant
0.3 µm or better. That the resolution for combinations 1 and 2
concentrations of look-alike particles are present. In some
is suitable is to be checked using a resolution check slide (see
casesdustanddebriscanmimicthemorphologyofparticlesof
13.2.3).
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.
7.6 Resolution check slide, a microscope slide on which
6.5 Impaction Medium Stability and Clarity—Chemicals
calibrated distances, shapes, and line widths provide reliable
present in some mounting media may affect the physical
and simple image resolution and shape identification perfor-
stability or clarity of the impaction medium. For instance:
mance of the microscopic and analyst at magnification. Ex-
(1)Samples collected on silicone grease medium should
amples include: a slide onto which a variety of diatoms have
first be warmed on a hot plate at approximately 40°C to “fix”
been mounted, including examples of Stauroneis phoenicen-
thesampleinplace,whenusinglacto-phenolcottonbluestain,
teron and Pleurosigma angulatum, a brightfield resolution test
and
slide, or equivalent.
(2)Slides and cassettes using methyl cellulose ester +
7.7 Syringe or dropper, for dispensing liquid during sample
solvent adhesive medium, which is stable in lacto-phenol
preparation.
cotton blue stain, will “fog” with Calberla’s stain due to the
water and alcohol mixture; warming fogged slides may tem-
8. Reagents and Materials
porarily clear them.
The lab or analyst should develop through experimentation 8.1 Purity of Reagents—Reagent grade chemicals shall be
animpactionmedium/mountingmediumcombinationthatwill used in all tests. Unless otherwise indicated, it is intended that
result in acceptable stability, clarity, and spore visibility. all reagents conform to the specifications of the Committee on
D7391−20
Analytical Reagents of theAmerican Chemical Society where (2)Carefully pull the glass or substrate that contains the
such specifications are available. Other grades may be used, adhesive film and the sample from the cassette, and place it,
provided it is first ascertained that the reagent is of sufficiently sample side upwards, on the slide adherent. Gently lower the
high purity to permit its use without lessening the accuracy of glass slip at a slight angle to minimize air bubble formation if
the determination. using liquid adherent.
12.1.2.4 Placeonedropofmountingmediumonthesample
8.2 Purity of Water—Unless otherwise indicated, references
traceorcoverslip.Gentlylowerthecoverslipontothesample
to water shall be understood to mean reagent water as defined
trace at a slight angle to minimize air bubble formation. If the
by Type II of Specification D1193.
liquid contains stain, allow the stain to fully penetrate the
8.3 Mounting medium (with or without stain), for re-
particles before enumeration.
hydratingsporesandforholdingthecoversliptotheimpaction
12.1.2.5 (Optional)—Mark the approximate maximum ex-
sample, for example, lactic acid, lacto-cotton blue stain,
tent of scan (~3 mm larger than the visible deposit) on the
lacto-phenol-cotton blue stain, lacto-fuchsin stain (see Section
underside of the slide using a marking pen. This is especially
X2.1 for stain preparation).
useful for lightly loaded samples, in which the area to be
8.4 Microscope cover slips, large enough to cover the scanned may not be obvious when the slide is observed on the
deposit (for example, 22 mm ); for optimum performance, microscope.
choose a cover slip thickness according to the recommenda-
12.2 Preliminary Evaluation:
tions of the microscope objective lens manufacturer.
12.2.1 The purpose of this examination is to note possible
8.5 Microscope slides. sample problems and to assign a debris rating. Use
magnification/resolution combination 1.
8.6 Slide adherent, for affixing impaction cassette samples
12.2.2 Examine the entire sample trace. Note on the work-
tomicroscopeslides,forexample,clearnailpolish,immersion
sheet non-uniform deposition or other sample problems.
oil, tape.
12.2.3 Debris Rating Determination:
9. Hazards 12.2.3.1 From the amount of particulate matter present at
that part of the sample trace having approximately the greatest
9.1 Components of re-hydrating liquids and stains, for
particle load, assign the sample a debris rating on a scale from
example, lactic acid, phenol, are corrosive or hazardous.
0 to 5. (A description of the debris rating numbers is given
Consult the appropriate MSDS for any reagents used.
belowinTable1.)Sincetheamountofdebrisinafieldofview
varies with the field of view chosen and its position in the
10. Preparation of Apparatus
sample trace, choose a rating that is most representative of a
10.1 Microscope Alignment/Adjustments—Follow the
number of fields of view taken from the middle (not the more
manufacturer’s instructions.
lightly loaded edges) of the trace.
11. Calibration and Standardization
12.3 Counting Procedure for Slit Impactor:
11.1 Diameter/Width and Graduation Spacing for Ocular 12.3.1 Categorize each observed spore based on color,
morphology, size, etc.
Reticule—see 13.2.2.
12.3.2 Categorize, at a minimum, the spore categories:
12. Procedure
(1) Alternaria,
(2)ascospores (undifferentiated),
12.1 Sample Preparation:
(3) Aspergillus/Penicillium-like
12.1.1 Preparation of a Pre-Coated Slide (the impaction
(4)basidiospores (undifferentiated),
medium is already on a microscope slide).
(5) Chaetomium,
12.1.1.1 Mark each slide with a unique designation.
(6) Cladosporium,
12.1.1.2 If necessary (for example, for grease medium),
(7) Curvularia,
gently warm to no more than 40°C to “fix” impacted particles
(8) Drechslera/Bipolaris-like,
in place.
(9)smuts/Myxomycetes/Periconia,
12.1.1.3 Place one drop of mounting medium near the
deposition trace and cover with a clean cover slip. Gently (10) Stachybotrys/Memnoniella,
(11) Ulocladium, and
lower the cover slip at a slight angle to minimize air bubble
formation. If the liquid contains stain, allow the stain to fully (12)hyphal fragments
For a fungal structure having characteristics inconsistent
penetrate the particles before enumeration.
12.1.2 Preparation of a Cassette: with all reported categories, enumerate it under the general
category: Miscellaneous/Unidentified.
12.1.2.1 Cut the sealer on the cassette and dismantle into
two parts.
NOTE 1—Categories other than the above minimum may be used and
12.1.2.2 Mark each slide with a unique designation.
reported, if desired. Fungal structures in the Miscellaneous/Unidentified
12.1.2.3 If the impaction substrate is not of suitable size/
category may be grouped by characteristics and reported separately, if
desired, for example, Miscellaneous/Unidentified 1, Miscellaneous/
thickness to be examined on the microscope, it must be
Unidentified 2, etc.
mounted on a microscope slide.
(1)Place a drop of slide adherent on a clean microscope 12.3.3 Enumerate spore categories at an appropriate
slide. magnification/resolution. Enumerate the spore categories
D7391−20
TABLE 1 Debris Rating Table
Non-Microbial Particle Debris Rating (all photos taken at 600× magnification) Description Interpretation
No particulate The absence of
matter detected in particulate matter
impaction area. in the impact area
could indicate improper
sampling or a blank
sample, as most air
samples typically
contain some particles.
Such absence of
particulate matter
should be noted on
the report if the
sample was not
meant to be a blank.
Minimal (>non detect Reported values are
to approx. 5 %) minimally affected by
particulate matter particle load.
present.
Approx. 5 % toapprox. Negative bias is
25 % of the trace expected. The degree
occluded with of bias increases
particulate matter. with the percent of
the trace that is
occluded.
Aspergillus/Penicillium-like and Cladosporium at 12.3.4 Enumerate a minimum of 20% sample trace.
magnification/resolution 2 and other spore categories at either
NOTE 3—An analyst/lab could decide to enumerate the minimum of
magnification/resolution 1 or 2.
20% for all spore categories, or decide to enumerate 100% for all spore
categories, or decide to enumerate for each spore category a percentage
NOTE 2—Spores that are especially small or hyaline are best enumer-
ated at magnification/resolution 2.The categories basidiospores (undiffer- between20and100basedonexperience,onqualityobjectivesoronhow
entiated) and ascospores (undifferentiated) are so variable that some many spores in that category appeared to be present during the initial
sporesinthesecategoriescouldbeenumeratedatmagnification/resolution
screening.
1, but others should be enumerated at magnification/resolution 2. The
12.3.5 Enumerate during traverses across the sample trace.
spore categories Alternaria, Chaetomium, Curvularia, Drechslera/
Bipolaris-like, smuts/Myxomycetes/Periconia-like, Stachybotrys/
A traverse is one scan across the sample trace in a direction
Memnoniella, Ulocladium, hyphal fragments, and Miscellaneous/
perpendicular to the longest dimension of the sample trace.
Unidentified may be enumerated at either magnification/resolution 1 or 2.
D7391−20
TABLE1 Continued
Approx. 25 % to approx. Negative bias is
75 % of the trace expected. The degree
occluded with of bias increases
particulate matter. with the percent of
the trace that is
occluded.
Approx. 75 % to approx. Negative bias is
90 % of the trace expected. The degree
occluded with of bias increases
particulate matter. with the percent of
the trace that is
occluded.
Greater than approx. 90 % Quantification is not
of the trace occluded possible due to large
with particulate matter. negative bias. A new
sample should be
collected at shorter
time interval, or other
measures taken to
reduce the particle load.
12.3.5.1 Start above or below the visible apparent deposit fungalstructureappearstolieontherightboundarylineofthe
and scan across the deposit until well off the visible deposit. ocular reticule, do not count it.
This process constitutes one traverse. 12.3.5.4 An analysis consists of a number of complete
12.3.5.2 Asatraverseismade,theouteredgesoftheocular traverses. Do not use partial traverses.
reticulewillbeusedtodescribetheboundariesofarectangular 12.3.5.5 If enumerating varying percentages for each spore
counting area. category, pre-determine the % of the sample trace to be
12.3.5.3 During a traverse, identify and categorize, the enumerated, so that traverses may be chosen to cover the
fungal structures that appear to fall within the outer edges of sample trace more or less evenly.That is, do not start counting
theocularreticuleIfafungalstructureappearstolieontheleft 100% of the sample trace for all spore categories (using
boundary line of the ocular reticule, count this structure. If a adjacent traverses), and then stop counting certain spore
D7391−20
categories part of the way through the analysis, since such a
count would be biased low due to the lightly loaded first few
traverses.
12.3.5.6 If enumerating 100% of the sample trace, start the
traversesslightlyoutsidethevisibleendofthesampletrace,to
ensure that all spores are within the scanned area. If enumer-
ating less than 100% of the sample trace, start the traverses
slightly inside the visible end of the sample trace, where the
deposit appears to become uniform in order to obtain a
FIG. 2Slit Impactor Location of Traverses for Counting <100% of
representative traverse. Avoid the extreme end of the sample
the Sample Trace
trace where the deposit appears to become less dense.
12.3.5.7 If enumerating 100% of the sample trace, choose
each subsequent counting area so that it abuts the previous
counting area to provide full coverage, as shown in Fig. 1.If
12.3.8 Calculations:
enumerating less than 100% of the sample trace, separate
12.3.8.1 For each spore category:
traverses to prevent overlap. In this case, it is recommended
percentagescanned5
that the total number of traces more or less evenly cover the
widthofocularreticlein µm * 0.001mm/µm * numberoftraverses
~ ! ~ ! ~ !
trace, as shown in Fig. 2.
100*
lengthofd
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
´1
Designation: D7391 − 17 D7391 − 20
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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
ε NOTE—Research report information added editorially in May 2017.
1. 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 test 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.
3 3
1.4 Enumeration results are presented in fungal structures/sample (fs/sample) and fungal structures/m (fs/m ).
1.5 The range of enumeration results that can be determined with this test 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 test method addresses only the analysis of samples. The sampling process and interpretation of results is outside the
scope of this test 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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.9 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D1193 Specification for Reagent Water
E691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
3. Terminology
3.1 ASTM Definitions (see the ASTM Online Dictionary of Engineering Science and Technology ):
3.1.1 numerical aperture.
3.2 Definitions of Terms Specific to This Standard:
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 Assessment,
Sampling, and Analysis of MoldMicroorganisms.
Current edition approved March 15, 2017March 15, 2020. Published April 2017April 2020. Originally approved in 2009. Last previous edition approved in 20092017 as
ɛ1
D7391 – 09.17 . DOI: 10.1520/D7391-17E01.10.1520/D7391-20.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’sstandard’s Document Summary page on the ASTM website.
ASTM Online Dictionary of Engineering Science and Technology (Stock #: DEFONLINE) is available on the ASTM website, www.astm.org, or contact ASTM Customer
Service at [e-mail].
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7391 − 20
3.2.1 circular impaction sample, n—a sample of airborne particulate matter collected by means of a device that draws air
through a round aperture at a specified rate, impacting the particles suspended in the air onto an adhesive medium, resulting in a
circular area of deposition. A circular impaction sample may be collected by means of a cassette manufactured for that purpose,
or by means of a sampling device that requires slides to be pre-coated with impaction medium.
3.2.2 debris rating, n—a distinct value assigned to an impactor sample based on the percentage of the sample area potentially
obscured by particulate matter, and ranging from 0 to 5.
3.2.3 field blank, n—a sample slide or cassette carried to the sampling 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.), n—a collective term for fragments or groups of fragments from fungi, including but not limited
to conidia, conidiophores, and hyphae and spores.
3.2.5 fungus (s), fungi, (pl.), n—eukaryotic, heterotrophic, absorptive organisms that usually develop a rather diffuse, branched,
tubular body (for example, network of hyphae) and usually reproduce by means of spores. The terms ‘mold’ and ‘mildew’ are
frequently used by laypersons when referring to various fungal colonization.
3.2.6 hyaline, adj—colorless.
3.2.7 impaction medium, n—a substance applied to a microscope slide used to collect (or capture) particulate matter during
sampling.
3.2.8 impaction sample, n—a sample taken using impaction, for example, slit impaction sample, circular impaction sample.
3.2.9 inertial impactor, n—a device for collecting particles separated from an air stream by inertia to force an impact onto an
adhesive surface. Inertial impactors are available in many designs, including those having a slit jet, yielding a rectangular sample
trace, and a circular jet, yielding a circular sample trace.
3.2.10 magnification/resolution combination 1, n— ~150–400× total magnification and a point to point resolution of 0.7 μm or
better, as checked by a resolution check slide.
3.2.11 magnification/resolution combination 2, n— ~400× or greater total magnification and a point to point resolution of 0.5
μm or better, as checked by a resolution check slide.
3.2.12 minimum reporting limit (fs/sample); minimum reporting limit (fs/m ), n—the lowest result to be reported for total spores
or any spore category. Since both fs/sample and fs/m are reported, there are two minimum reporting limits.
3.2.13 morphology, n—the form and structure of an organism or any of its parts; for fungi, the shape, form, ornamentation, or
combination thereof.
3.2.14 mounting medium, n—a liquid, for example, lactic acid or prepared stain, used to immerse the sample particulate matter
and to attach a cover slip to an impaction sample.
3.2.15 sample trace, n—the area of particle deposition, that is, the deposit on a slit impaction sample resembling a narrow
rectangle, or the circular deposit on a circular impaction sample.
3.2.16 septum (pl.: septa), (s), septa (pl.), n—a cell wall or partition.
3.2.17 slide adherent, n—an adhesive or liquid used to affix an impaction sample substrate to a microscope slide.
3.2.18 slit impaction sample, n—a sample of airborne particulate matter collected by means of a device that draws air through
a linear aperture at a specified rate, impacting the particles suspended in the air onto an adhesive medium, resulting in a rectangular
area of deposition. A slit impaction sample may be collected by means of a cassette manufactured for that purpose, or by means
of a sampling device that requires slides to be pre-coated with impaction medium.
3.2.19 spore category, n—a grouping used for identification and quantifation of fungal structures. A spore category may contain
a specific genus (for example, Stachybotrys), or it may represent a combination of genera (for example, Aspergillus/Penicillium-
like).
3.2.20 traverse, n—a portion of analysis of an impactor sample consisting of one scan under the microscope from a sample-less
portion of the impaction medium across the deposit to a corresponding sample-less portion of the impaction medium on the other
side.
3.3 Symbols:
3.3.1 fs—fungal structure
3.3.2 fs/m —fungal structures per cubic metre
3.3.3 m —cubic metre
3.3.4 mm—millimetre
3.3.5 μm—micrometre
Kendrick, B., The Fifth Kingdom, Focus Publishing / R Pullins & Co, 2008.
D7391 − 20
4. Summary of Test Method
4.1 Samples have been previously collected utilizing an impaction device operating at the device manufacturer’s recommended
sample flow rate. Each sample consists of an optically clear substrate coated with an adhesive and optically transparent medium
onto which particles have been deposited through inertial impaction.
4.2 A sample is mounted to a microscope slide and examined by bright field microscopy using at least two magnification/
resolution combinations.
4.3 Spores are differentiated from each other, other fungal structures, and from non-fungal material by color, size, shape,
presence of a septum or septa, attachment scars, surface texture, etc., by means of a taxonomic comparison with standard reference
texts or known standard samples, or both (see Section A1.1 for suggested references). The number of spores that match each spore
category are then calculated in units of fungal structures per sample (fs/sample) and also fungal structures per cubic meter of air
(fs/m ).
5. Significance and Use
5.1 This test method is used to estimate and categorize the number and type of fungal structures present on an inertial impactor
sample.
5.2 Fungal structures are identified and quantified regardless of whether they would or would not grow in culture.
5.3 It must be emphasized that the detector in this test method is the analyst, and therefore results are subjective, depending on
the experience, training, qualification, and mental and optical fatigue of the analyst.
6. Interferences
6.1 Differentiation of Fungal Genera/Species—Because of the similar size and morphology of some fungal spores of different
genera and the absence of growth structures and mycelia in airborne samples, differentiation by microscopic examination alone
is difficult and spores must be grouped into categories based strictly on morphology. In many cases, identification at the genus level
is presumptive. For example, differentiation between Aspergillus and Penicillium using this test method is not typical, so a
combined Aspergillus/Penicillium-like category is used. When differentiation between such genera is desired, a different test
method must be used. Unequivocal identification of every spore in each category is not possible due to optical limitations, the
atypical nature of some of the spores, overlapping morphology among different spore types, or combination thereof, and therefore,
certain spores must be categorized as Miscellaneous/Unidentifiable.
6.2 Look-Alike Non-Fungal Particles—Certain types of particles of non-fungal origin may resemble fungal spores. These
particles and artifacts may include air or plant resin bubbles, starch, talc, cosmetic particles, or combustion products. Standards
(mounted similarly to impactor samples) should be examined by laboratory analysts to know how to identify such particles.
Examination of suspect particles using optical conditions other than bright field microscopy (for example, polarized light
microscopy, phase contrast microscopy, differential interference contrast) may be helpful whenever significant concentrations of
look-alike particles are present. In some cases dust and debris can mimic the morphology of particles of interest. When look-alike
particles are present in high concentration, accurately counting spores with similar morphology is difficult. When these conditions
exist, they should be reported in the analysis notes section of the report.
6.3 Particle Overloading—High levels of particulate matter on an impaction sample will bias the analysis in two ways:
(1) Particle capture efficiency decreases, and
(2) Debris obscures or covers spores.
Both of these factors produce a negative bias.
6.4 Staining—Staining, while optional, may help the analyst differentiate spores from debris. Without staining, clear spores
(especially small ones) may exhibit negative bias because the analyst has insufficient contrast to notice them while scanning. Also,
because spores of different fungal species absorb stains at different rates, under or over-staining makes identification difficult. The
problem can be eliminated by careful control of stain concentrations.
6.5 Impaction Medium Stability and Clarity—Chemicals present in some mounting media may affect the physical stability or
clarity of the impaction medium. For instance:
(1) Samples collected on silicone grease medium should first be warmed on a hot plate at approximately 40°C to “fix” the
sample in place, when using lacto-phenol cotton blue stain, and
(2) Slides and cassettes using methyl cellulose ester + solvent adhesive medium, which is stable in lacto-phenol cotton blue
stain, will “fog” with Calberla’s stain due to the water and alcohol mixture; warming fogged slides may temporarily clear them.
The lab or analyst should develop through experimentation an impaction medium/mounting medium combination that will result
in acceptable stability, clarity, and spore visibility.
6.6 Uneven Impaction Medium Uniformity—Uneven thickness may be present in greased slides, pre-coated slides and
manufactured cassettes. The microscopist will compensate by adjusting the plane of focus. When grease is too thick, differentiating
D7391 − 20
small spores from background artifacts (especially air bubbles) in the grease preparation becomes difficult. When grease is too thin,
shrinkage and pooling may have occurred, causing particle loss during sampling.
7. Apparatus
7.1 Marking pen, for marking sample slides.
7.2 Microscope or magnification system, having a precision x-y mechanical stage. The microscope or magnification system used
for analysis shall be capable of at least two magnification/resolution combinations as follows: magnification/resolution
combination 1 shall be ~150–400× total magnification and a point to point resolution of 0.7 μm or better; magnification/resolution
combination 2 shall be ~400× or greater total magnification and a point to point resolution of 0.5 μm or better. It is recommended
that at least one microscope or magnification system in the lab be capable of magnification of ~1000× total magnification and a
point to point resolution of 0.3 μm or better. That the resolution for combinations 1 and 2 is suitable is to be checked using a
resolution check slide (see 13.2.3).
7.3 Reference Slides—a series of mounted field samples to be used as counting references. Analysts’ results from these slides
are expected to be within laboratory acceptance limits to prove competence.
7.4 Reticule, width defining, an optical device in the light path of the microscope capable of being reproducibly set to define
a traverse width no larger than 0.75× the diameter of the ocular field of view, and having graduations of an appropriate dimension
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, procedures must be in place to ensure that the reticule is correctly positioned for each analysis.
7.5 Stage micrometer, traceable to the National Institute of Standards and Technology (NIST) or equivalent international
standard.
7.6 Resolution check slide, a microscope slide on which calibrated distances, shapes, and line widths provide reliable and simple
image resolution and shape identification performance of the microscopic and analyst at magnification. Examples include: a slide
onto which a variety of diatoms have been mounted, including examples of Stauroneis phoenicenteron and Pleurosigma
angulatum, a brightfield resolution test slide, or equivalent.
7.7 Syringe or dropper, for dispensing liquid during sample preparation.
8. Reagents and Materials
8.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society where such
specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
8.2 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean reagent water as defined by
Type II of Specification D1193.
8.3 Mounting medium (with or without stain), for re-hydrating spores and for holding the cover slip to the impaction sample,
for example, lactic acid, lacto-cotton blue stain, lacto-phenol-cotton blue stain, lacto-fuchsin stain (see Section X2.1 for stain
preparation).
8.4 Microscope cover slips, large enough to cover the deposit (for example, 22 mm ); for optimum performance, choose a cover
slip thickness according to the recommendations of the microscope objective lens manufacturer.
8.5 Microscope slides.
8.6 Slide adherent, for affixing impaction cassette samples to microscope slides, for example, clear nail polish, immersion oil,
tape.
9. Hazards
9.1 Components of re-hydrating liquids and stains, for example, lactic acid, phenol, are corrosive or hazardous. Consult the
appropriate MSDS for any reagents used.
10. Preparation of Apparatus
10.1 Microscope Alignment/Adjustments—Follow the manufacturer’s instructions.
11. Calibration and Standardization
11.1 Diameter/Width and Graduation Spacing for Ocular Reticule—see 13.2.2.
12. Procedure
12.1 Sample Preparation:
12.1.1 Preparation of a Pre-Coated Slide (the impaction medium is already on a microscope slide).
D7391 − 20
12.1.1.1 Mark each slide with a unique designation.
12.1.1.2 If necessary (for example, for grease medium), gently warm to no more than 40°C to “fix” impacted particles in place.
12.1.1.3 Place one drop of mounting medium near the deposition trace and cover with a clean cover slip. Gently lower the cover
slip at a slight angle to minimize air bubble formation. If the liquid contains stain, allow the stain to fully penetrate the particles
before enumeration.
12.1.2 Preparation of a Cassette:
12.1.2.1 Cut the sealer on the cassette and dismantle into two parts.
12.1.2.2 Mark each slide with a unique designation.
12.1.2.3 If the impaction substrate is not of suitable size/thickness to be examined on the microscope, it must be mounted on
a microscope slide.
(1) Place a drop of slide adherent on a clean microscope slide.
(2) Carefully pull the glass or substrate that contains the adhesive film and the sample from the cassette, and place it, sample
side upwards, on the slide adherent. Gently lower the glass slip at a slight angle to minimize air bubble formation if using liquid
adherent.
12.1.2.4 Place one drop of mounting medium on the sample trace or cover slip. Gently lower the cover slip onto the sample
trace at a slight angle to minimize air bubble formation. If the liquid contains stain, allow the stain to fully penetrate the particles
before enumeration.
12.1.2.5 (Optional)—Mark the approximate maximum extent of scan (~3 mm larger than the visible deposit) on the underside
of the slide using a marking pen. This is especially useful for lightly loaded samples, in which the area to be scanned may not be
obvious when the slide is observed on the microscope.
12.2 Preliminary Evaluation:
12.2.1 The purpose of this examination is to note possible sample problems and to assign a debris rating. Use magnification/
resolution combination 1.
12.2.2 Examine the entire sample trace. Note on the worksheet non-uniform deposition or other sample problems.
12.2.3 Debris Rating Determination:
12.2.3.1 From the amount of particulate matter present at that part of the sample trace having approximately the greatest particle
load, assign the sample a debris rating on a scale from 0 to 5. (A description of the debris rating numbers is given below in Table
1.) Since the amount of debris in a field of view varies with the field of view chosen and its position in the sample trace, choose
a rating that is most representative of a number of fields of view taken from the middle (not the more lightly loaded edges) of the
trace.
12.3 Counting Procedure for Slit Impactor:
12.3.1 Categorize each observed spore based on color, morphology, size, etc.
12.3.2 Categorize, at a minimum, the spore categories:
(1) Alternaria,
(2) ascospores (undifferentiated),
(3) Aspergillus/Penicillium-like
(4) basidiospores (undifferentiated),
(5) Chaetomium,
(6) Cladosporium,
(7) Curvularia,
(8) Drechslera/Bipolaris-like,
(9) smuts/Myxomycetes/Periconia,
(10) Stachybotrys/Memnoniella,
(11) Ulocladium, and
(12) hyphal fragments
For a fungal structure having characteristics inconsistent with all reported categories, enumerate it under the general category:
Miscellaneous/Unidentified.
NOTE 1—Categories other than the above minimum may be used and reported, if desired. Fungal structures in the Miscellaneous/Unidentified category
may be grouped by characteristics and reported separately, if desired, for example, Miscellaneous/Unidentified 1, Miscellaneous/Unidentified 2, etc.
12.3.3 Enumerate spore categories at an appropriate magnification/resolution. Enumerate the spore categories Aspergillus/
Penicillium-like and Cladosporium at magnification/resolution 2 and other spore categories at either magnification/resolution 1 or
2.
NOTE 2—Spores that are especially small or hyaline are best enumerated at magnification/resolution 2. The categories basidiospores (undifferentiated)
and ascospores (undifferentiated) are so variable that some spores in these categories could be enumerated at magnification/resolution 1, but others should
be enumerated at magnification/resolution 2. The spore categories Alternaria,Chaetomium,Curvularia,Drechslera/Bipolaris-like, smuts/Myxomycetes/
Periconia-like, Stachybotrys/Memnoniella,Ulocladium, hyphal fragments, and Miscellaneous/Unidentified may be enumerated at either magnification/
resolution 1 or 2.
D7391 − 20
TABLE 1 Debris Rating Table
Non-Microbial Particle Debris Rating (all photos taken at 600× magnification) Description Interpretation
No particulate The absence of
matter detected in particulate matter
impaction area. in the impact area
could indicate improper
sampling or a blank
sample, as most air
samples typically
contain some particles.
Such absence of
particulate matter
should be noted on
the report if the
sample was not
meant to be a blank.
Minimal (>non detect Reported values are
to approx. 5 %) minimally affected by
particulate matter particle load.
present.
Approx. 5 % toapprox. Negative bias is
25 % of the trace expected. The degree
occluded with of bias increases
particulate matter. with the percent of
the trace that is
occluded.
Approx. 25 % to approx. Negative bias is
75 % of the trace expected. The degree
occluded with of bias increases
particulate matter. with the percent of
the trace that is
occluded.
12.3.4 Enumerate a minimum of 20 % sample trace.
NOTE 3—An analyst/lab could decide to enumerate the minimum of 20 % for all spore categories, or decide to enumerate 100 % for all spore categories,
or decide to enumerate for each spore category a percentage between 20 and 100 based on experience, on quality objectives or on how many spores in
that category appeared to be present during the initial screening.
12.3.5 Enumerate during traverses across the sample trace. A traverse is one scan across the sample trace in a direction
perpendicular to the longest dimension of the sample trace.
D7391 − 20
TABLE 1 Continued
Approx. 75 % to approx. Negative bias is
90 % of the trace expected. The degree
occluded with of bias increases
particulate matter. with the percent of
the trace that is
occluded.
Greater than approx. 90 % Quantification is not
of the trace occluded possible due to large
with particulate matter. negative bias. A new
sample should be
collected at shorter
time interval, or other
measures taken to
reduce the particle load.
12.3.5.1 Start above or below the visible apparent deposit and scan across the deposit until well off the visible deposit. This
process constitutes one traverse.
12.3.5.2 As a traverse is made, the outer edges of the ocular reticule will be used to describe the boundaries of a rectangular
counting area.
12.3.5.3 During a traverse, identify and categorize, the fungal structures that appear to fall within the outer edges of the ocular
reticule If a fungal structure appears to lie on the left boundary line of the ocular reticule, count this structure. If a fungal structure
appears to lie on the right boundary line of the ocular reticule, do not count it.
12.3.5.4 An analysis consists of a number of complete traverses. Do not use partial traverses.
12.3.5.5 If enumerating varying percentages for each spore category, pre-determine the % of the sample trace to be enumerated,
so that traverses may be chosen to cover the sample trace more or less evenly. That is, do not start counting 100 % of the sample
trace for all spore categories (using adjacent traverses), and then stop counting certain spore categories part of the way through
the analysis, since such a count would be biased low due to the lightly loaded first few traverses.
12.3.5.6 If enumerating 100 % of the sample trace, start the traverses slightly outside the visible end of the sample trace, to
FIG. 1 Slit Impactor Location of Traverses for Counting 100 % of the Sample Trace
D7391 − 20
ensure that all spores are within the scanned area. If enumerating less than 100 % of the sample trace, start the traverses slightly
inside the visible end of the sample trace, where the deposit appears to become uniform in order to obtain a representative traverse.
Avoid the extreme end of the sample trace where the deposit appears to become less dense.
12.3.5.7 If enumerating 100 % of the sample trace, choose each subsequent counting area so that it abuts the previous counting
area to provide full coverage, as shown in Fig. 1. If enumerating less than 100 % of the sample trace, separate traverses to prevent
overlap. In this case, it is recommended that the total number of traces more or less evenly cover the trace, as shown in Fig. 2.
12.3.6 Stopping Rule:
12.3.6.1 Start an analysis for that spore category as if less than 100 % of the sample trace is to be enumerated, that is, not at
the extreme end and separating the traverses.
12.3.6.2 Discontinue counting after the completion of the traverse during which either: Option (1) 100 fungal structures are
reached for that category (and continue counting for the remaining categories), or Option (2) 300 total fungal structures are
reached.
NOTE 4—Option 1 may be useful when one or two fungal
...