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Comment on fungal tape lift reporting frameworks

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consensus unit used for reporting. The method of using sticky tape
to transfer fungal colonies from one surface onto microscope slides
was rst reported for dermatophyte fungi in the 1970’s and later
for other fungi.8,9 This was shown to be an excellent technique that
preserved for example the conidium and conidiophore morphology.
Many researchers still use sticky tape sampling to evaluate biological
contaminants like indoor fungi.10‒12 while the method has proven
valuable in criminal and civil forensics where mould growth or
absence has been used as evidence linking people and objects with
places;13 or for sampling of other contaminants like chemical threat
agents.14 The commercial development of readily available and
inexpensive tape lifts,15,16 offer a consistent sample area on a exible
plastic slide for the determination of mould, other microbial, settled
bioaerosols, and inorganic dust contamination. However, the problem
of ‘describing what is seen’ remains. This has a lot to do with the
diversity of locations that samples may come from and from the type
of information that is sought. Eight methods have been identied. Each
has more or less merit depending on the amount and quality of fungal
material versus background debris. The rst aim of this comment
is therefore to review the literature and offer an opinion about a
standardized protocol for fungal surface testing using tape lifts. The
second aim is to offer the analyst a exible framework when choosing
an appropriate qualitative or semi-quantitative reporting index that is
matched both to the quality and objectives of the sampling. Usually
the goal is to identify a healthy or unhealthy building microbiome and
to guide the scope or validate any remediation effort that has or should
or must occur (IICRC S500/S520/R520).17,18
Method 1
Two current Standards focus on sampling surfaces for fungi using
tape lifts. The rst19 (D7910-14) is a Standard Practice that states that
tape lifts may be used for both “qualitative or quantitative analysis by
direct microscopy” of “material present at one specic location on a
surface for fungal content”. The signicance of use is for “qualitative
analysis or to quantify fungal material per sample or per unit area”.
These statements are entirely sensible since the recovery efciency
is a source of uncertainty and the Standard does not comment on
sampling objectives or how the method is used to address building
occupant exposure or occupant health risk. The second20 (D7658-17),
is a Standard Test Method and the signicance of use is to ensure
consistency between laboratories and analysts, where “Fungal
structures are identied and semi-quantied regardless of whether
they would or would not grow in culture”. As well, the intention of the
Standard “is to standardize the analysis of the detection of removable
fungal structures lifted from a surface with tape”. The analyst will
then “determine and record each fungal type as encountered” into 12
minimum categories including into Genus and spore type morphologies
as seen. The problem is that depending on how the sample was taken,
where it was taken from, and how much vegetative material is present,
will all impact on the type and accuracy of information that is possible
to meaningfully obtain from each slide. The Standard (D7658-17) is
unclear since the only quantitative scale offered is a semi-quantitative
fungal loading category scale from 0-5 where: Category 0=no fungal
material present; Category 1 = fungal material covers <5% of a
representative eld of view; Category 2=5-25%; Category 3=25–
75%; Category 4=75–90% and Category 5=>90%. The reference in
the Standard D7658-17 is for particle loading of debris but seems to
be used interchangeably. Notably, this same approach was adopted for
spore counting in the superseded21 and current Standard22 (D7391–09
and D7391-17e1) where the percentage debris scale dened above was
used for recording background debris, not to be mistaken for the spore
numbers. To some extent, the objectivity goal of the two Standards for
tape lifts falls short since in practice there are several other approaches
for microscope-image classication.
Method 2
The second method for describing contamination according to
the Australian Mould Guideline6 proposes a Hygiene Rating using
5 categories: Low=<50 spores/cm2; Normal = 50 – 500 spores/cm2;
Elevated=500–1000 spores/cm2 + prevailing species; Contaminated
= >1000 spores/cm2 + dominant species + propagules; Extreme
Contamination =>5000 spores/cm2 + dominant species + propagules
+ conuent spores. Implementations of this scale have sometimes
described the last two categories as ‘High’ and ‘Extremely High’
J Bacteriol Mycol Open Access. 2019;7(6):155157. 155
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Comment on fungal tape lift reporting frameworks
Volume 7 Issue 6 - 2019
Cameron L Jones1,2
1Biological Health Services, Australia
2National Institute of Integrative Medicine, Australia
Correspondence: Dr. Cameron L. Jones, Biological Health
Services, Level 1, 459 Toorak Rd, Toorak, Victoria, 3142, Australia,
Tel +61414998900, Email
Received: November 26, 2019 | Published: December 10,
Journal of Bacteriology & Mycology: Open Access
Short Communication Open Access
The microbiology of the built environment and its relationship to
occupant health indoors is an increasingly active area of scholarship.1
For example, adverse human-microbe interactions are often claimed
as the cause of sick-building syndrome-type complaints; while it is
argued that different indoor habitats including inanimate surfaces
confer selection pressure on common environmental fungi leading
towards increasing virulence.2 In turn, measuring actual or suspect
indoor fungal contamination is increasingly common especially on
indoor damp surfaces3 or following water damage,4 and is an ongoing
area of occupational health and safety and the focus of building
disputes and litigation.5 While there are established metrics for
assessing the microbiological component of air using viable colony
counts or viable and non-viable fungal spore counts in units per cubic
meter of air;6,7 there is some difculty with how best to measure and
report surface fungal contamination using tape lifts where there is no
Comment on fungal tape lift reporting frameworks 156
©2019 Jones
Citation: Jones CL. Comment on fungal tape lift reporting frameworks. J Bacteriol Mycol Open Access. 2019;7(6):155157. DOI: 10.15406/jbmoa.2019.07.00262
Method 3
A third method for evaluating contamination was proposed by
Krause & Hammad23 who used 4 categories: Category I = described
as a “clean surface” =<10 fungal structures/cm2; Category II=
described as “light deposition of fungal structures including hyphal
fragments and spores” =100–1000 fungal structures/cm2; Category III
= described as “accumulation of fungal structures” =100–1000 fungal
structures/cm2; Category IV = described as “heavy accumulation
of fungal structures and possible amplication” =>1000 fungal
structures/cm2. The presence of conidiophores and hyphae are used as
indicators of past or present fungal growth.
Method 4
Another approach used by the American Industrial Hygiene
Association (see: Clarke, GA. 2001)24 used 3 categories: Normal
background = no signicant fungal material or no signicant fungal
biomass showing no more than 1-5%; Possible contamination source
=5-25%; and Probable contamination source = 25-100%. This has
also been adopted by the NYCOSH.25
Method 5
Another percentage-based method26 to determine if contents
are contaminated by mould and that has been suggested for use as
part of post-remediation evaluation sampling or third-party post-
remediation verication is the following. Surface samples by tape lift
can be “analyzed so that the quantity of fungal spores is presented as
a percentage of the sample area”, “rather than a raw count” where:
Normal fungal ecology = ≤1%; Indoor environment contaminated
with settled spores that were dispersed directly or indirectly (Condition
2) = between 1 and 3%; Indoor environment contaminated with the
presence of actual mold growth and associated spores (Condition 3)
= ≥ 3% The presence of target spore types (Chaetomium, Fusarium,
Memnoniella, Stachybotrys, and Trichoderma) is an automatic
indication of fungal contamination, regardless of the percentage of
Method 6
Similar interpretations for evaluating toxigenic fungi and
mycotoxins in outdoor, recreational environments have been used,27
where “the amount of fungal spores was rated based on the coverage
of spores on the tape samples observed”: Trace =<5%; Light=5-25%;
Moderate =26-75%; Heavy =76-90% and Very Heavy =>90%.
Another variation uses: Low = 1-25%, Medium = 26-50%, High =
51-75%, Very High = 76-100% that broadly follows the D7391-09
Standard for estimation of non-microbial particle debris rating.21
Method 7
Other methods28 describe the benets of tape lifts where they are
used to test “discolorations resulting from moisture damage [that] may
imply mould growth” and are “used to conrm that fungal growth
has been removed following remediation”. The overall objective is
to determine whether mould is present or not? The authors stress that
care must be taken not to inappropriately extrapolate from the tape lift
area to larger areas that may have different moisture or fungal growth
conditions. The method is understood to be “qualitatively specic”
and “semi-quantitative at best”. Notably, results from interpretation
should be “approached with caution” and “laboratory reports
should not state results in terms of number of spores per unit area,
because the measure is meaningless.” The value of the method lies
in being able to differentiate between normal accumulation of mould,
unusual accumulation of spores linked to adjacent fungal growth or
conrmation of fungal growth on a surface. Two sets of categories are
proposed: No spores detected; No abundance of unusual types (those
types often associated with growth on building materials); minimal
abundance of unusual types; moderate abundance of unusual types,
and; high abundance of unusual types. The second category scale is:
0=no growth; 1+= minimal fungal growth; 2+= low to moderate fungal
growth; 3+= moderate fungal growth; 4+= heavy fungal growth.
Method 8
One nal descriptive scale for tape lifts was advanced by Horner
et al.29 where the presence or absence of spores, hyphae and fruiting
structures were recorded against common Taxa from: Alternaria,
Chaetomium, Eurotium, Cladosporium, Penicillium/Aspergillus,
Stachybotrys. Spore amounts were noted as: S = scattered single, F
= few, A = Abundant or M = massive. Amounts of hyphae/fruiting
structures were noted as: S = scattered single, F = few, or A = abundant.
From the above 8 methods, we see that describing tape lift
observations is sometimes objective and sometimes subjective. There
is uncertainty in tape lift data because there is variance in: (i) the area
or region of interest being sampled, (ii) the number of samples, (iii)
the hypothesis being tested (i.e. is the expected outcome clean or
unclean)30 (iv) the experience of the sampler, (v) how much pressure
was used to take the sample, (vi) how much background debris was
present, (vii) the relationship between the tape lift data and other
metrics of microbial exposure (surface and airborne) and potentially
(viii) cost factors. Sampling too few surfaces or selectively choosing
or avoiding locations could skew or bias the data set. Similarly, only
relying on tape lifts for surface contamination measurement without
performing companion testing like RODAC contact plates or swab
testing to viable culture or surrogate surface cleanliness metrics
like ATP bioluminescence could allow for a lack of convergence
when assessing all the surface data and lead to interpretation and
recommendation errors. With this in mind, I propose that tape lift
fungal assessments should use a combination of several (at least 3)
of the above methods that are well-matched to the observed structures
seen under the microscope. Including representative micrographs
in reports could also be helpful. This will improve reliability and
validity of surface sampling for fungi. There is also considerable
risk in over-objectifying subjective microscope-data, especially
when image-analysis is not used. This can occur when lab reports
are produced showing extensive fungal Speciation categorized into
ne-grained spore or fungal structures. Apart from going against the
advice from the AIHA,28 the relationship between the human observer
and how biased judgment and misrepresentation can occur should not
be underestimated. Excepting for tape transfers made from surfaces
showing abundant visual mould growth (like biolms or surfaces
showing visual bulk), there is little chance that strict numerate
taxonomic rankings can be reported. It is undisputed that tape lifts
are useful in industrial hygiene water damage investigations or for
environmental screening of contaminated surfaces and objects31,32 or to
assess for potential fungal particle transmission.33 However, the value
of this method of measurement is a continuum between the objective
and subjective and depends on the overall image quality of the fungi
that can be removed by the adhesive from any chosen surface.
Comment on fungal tape lift reporting frameworks 157
©2019 Jones
Citation: Jones CL. Comment on fungal tape lift reporting frameworks. J Bacteriol Mycol Open Access. 2019;7(6):155157. DOI: 10.15406/jbmoa.2019.07.00262
Funding details
Conicts of interest
The author declares no conict of interest.
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... 2,10,[14][15][16] The presence of fungal fragments which have also been indicated as potential contaminants 17,18 and inactive fungal material can still release toxins. 19 Surface sampling for non-viable mould is typically conducted using tape lift surface samples [20][21][22] (e.g. Zefon Bio-Tape) in a technique first described by Flegel. ...
... Non-viable mould samples are analysed by microscopy, often aided by a staining procedure to improve contrast. 25 Several methods for analysis of nonviable surface samples exist 22 but reliable quantitative methods are typically considered more useful. 26 Meider and Messal 26 have recently published data outlining a quantitative method of analysis of tape lift samples. ...
... While it cannot provide information on the species of mould present, it can provide insight into if a potential water damage issue exists, if a hidden mould issue is contributing to poor air quality or if remediation works have significantly reduced mould levels. 3,5,22 Microscopic analysis of nonviable samples can also provide insight about the activity of mould in samples through assessing for fungal structures such as conidiophores, whereas a molecular based technique does not. 37 The prohibitive costs or time limitations of molecular techniques will likely slow uptake of these techniques -but present a significant improvement in care for sufferers of chronic mould exposure investigations such as for CIRS patients. ...
Surface sampling techniques for non-viable fungi in building environments are useful tools for investigators in determining hazards to occupants. However, data regarding capture efficiency in this context is limited. Our data demonstrates that collection efficiency of Bio-Tape surface capture medium on paper-faced gypsum board only captures between half and three-quarters of mould present on the surface. Surface sampling using a dry-swab technique showed similar efficiency of capture to tape lift samples. ‘Surface air’ samples had poor collection efficiency and should be avoided where possible in preference to other sampling options. Finally, we propose a sampling strategy based on non-viable microscopy techniques followed by molecular analysis for validation and speciation of samples of interest. Improvements in sampling and data analysis techniques for mould sampling of buildings will aid in providing meaningful results to help building inspectors evaluate health hazards.
... These use surrogate indicators to measure cleaning success and if required, implement educational intervention. They include: (i) ATP bioluminescence [28], (ii) swab or replicate organism detection and counting (RODAC) press-plate [29] or similar culture-based methods, (iii) tape lifts [30] or (iv) fluorescent or photoreactive inks, powders or gels [31]- [32]. ATP works by testing for the presence of a cell enzyme called adenosine triphosphate (ATP) that is present in both viable and non-viable organic debris, meaning that if it is present, then so must DNA-containing cell waste (the biological load). ...
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Mycology can contribute to a variety of forensic investigations, including the determination of postmortem intervals from mold growth on corpses, psychoactive substances and toxins, hazards from mold growth in buildings, and providing trace evidence linking people and objects with places. Studies are also starting to be undertaken to explore the use of molecular data on fungi in the characterization of soils. In addition, where there are health concerns, possibly associated with mold growth in buildings, guidance is presented with respect to the collection of samples, and some caveats are given which must be considered in interpretation of data. Attention is drawn to pertinent publications which either appeared, or came to the attention of the authors, since the review they prepared in 2010. This is supplemented by examples from their own recent casework. In order to avoid valuable information being overlooked, there is a need for investigating officers, and those involved in forensic medicine, especially pathologists and toxicologists, to be aware of the evidential value of fungi. In particular, they should not overlook opportunities to recover spores from human remains, to examine any mold colonies growing on corpses, and to analyze gut contents for fungal material and spores.
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Specific indoor environments select for certain stress-tolerant fungi and can drive their evolution towards acquiring medically important traits. Here we review the current knowledge in this area of research, focussing on the so-called black yeasts. Many of these melanised stress-tolerant organisms originate in unusual ecological niches in nature, and they have a number of preadaptations that make them particularly suited for growth on human-made surfaces and substrates. Several pathogenic species have been isolated recently from various domestic habitats. We argue that in addition to enriching for - potentially - pathogenic species, the selection pressure and stress acting on microorganisms in indoor environments are driving their evolution towards acquiring the missing virulence factors and further enhancing their stress tolerance and pathogenic potential. Some of the polyextremotolerant fungi are particularly problematic: they can grow at elevated temperatures, and so they have a higher potential to colonise warm-blooded organisms. As several species of black fungi are already implicated in health problems of various kinds, their selection and possible evolution in human environments are of concern.
Published guidelines on mold remediation do not specify sampling protocols to measure the efficacy of remediation efforts. The purpose of this study was to evaluate fungal remediation of contaminated ducts by comparing the amount of residual surface contamination to the amount in new ducts. Fungal contamination of galvanized metal and rigid fibrous glass ducts were evaluated using fluorometric and microscopic methods. Fungal contamination was measured in newly installed ducts in addition to pre- and post-remediation. Newly installed ducts had low levels of fungal debris. Findings demonstrated that both fluorometry and microscopy methods detected fungal surface contamination. After cleaning, metal ducts were statistically less contaminated than when new. Fungal contamination of rigid fibrous glass ducts was reduced by ~90%, but was statistically more contaminated than when new. The fluorometric method performed as well as the microscopic method in detecting fungal contamination and provided faster results for monitoring efficacy of fungal remediation.
This report describes the sequence of fungal colonization and the influence of biocide incorporation on paint films, determined using quantitative methods. Two buildings were painted with an acrylic paint, with and without an experimental biocide formulation containing a carbamate (carbendazin), N-octyl-2H-isothiazolin-3-one and N-(3,4-dichlorophenyl)N,N-dimethyl urea (total biocide concentration 0.25% w/w). One week after painting, the major groups of organisms detected were yeasts and Cladosporium. The yeast population fell to undetectable levels after the third week and this microbial group was not detected again until the 31st week, after which they increased to high levels on the 42nd week. Aureobasidium showed a pattern similar to the yeasts. The main fungal genera detected over the 42-week period were Alternaria, Curvularia, Epicoccum, Helminthosporium, Coelomycetes (mainly Pestalotia/Pestalotiopsis), Monascus, Nigrospora, Aureobasidium and Cladosporium. The latter was the main fungal genus detected at all times. The physiological factors controlling colonization are discussed. Cladosporium, Aureobasidium, Tripospermum and yeasts on the painted surfaces were all able to grow on mineral salts agar containing 10% sodium chloride. This is the first time that the genus Tripospermum has been reported on painted buildings. The fungal population on biocide-containing surfaces was significantly lower than on non-biocide-containing paint after 13 weeks and continued so to 42 weeks after painting, but there was no statistically significant difference in the level of fungal biodiversity.
A technique is described for making semipermanent microscope slides of fungi using sticky tape. After being touched to a fungal colony, a modified segment of sticky tape is touched to ethyl alcohol and then immersed in a 50% glycerine solution containing cotton blue stain. Finally, it is transferred (sticky side up) to a microscope slide, covered with a cover with a cover glass, and sealed.