Journal of Occupational and Environmental Hygiene, 1: 442–447
ISSN: 1545-9624 print / 1545-9632 online
2004 JOEH, LLC
An Investigation into Techniques for Cleaning
Mold-Contaminated Home Contents
S.C. Wilson, T.L. Brasel, C.G. Carriker, G.D. Fortenberry, M.R. Fogle,
J.M. Martin, C. Wu, L.A. Andriychuk, E. Karunasena, and D.C. Straus
Center for Indoor Air Research, Department of Microbiology and Immunology, Texas Tech University
Health Sciences Center, Lubbock, Texas
This study examined the efﬁcacy of the following treatments
to reduce selected fungal spore and mycotoxin levels on materi-
als commonly found in home contents: (1) gamma irradiation
at a 10–13 kiloGray exposure, (2) a detergent/bleach wash,
and (3) a steam cleaning technique. A minimum of six repli-
cates were performed per treatment. Paper, cloth, wood, and
carpet were inoculated with either fungal spores (Stachybotrys
chartarum, Aspergillus niger, Penicillium chrysogenum,or
Chaetomium globosum) at 240,000 spores/2.54 cm
rial or with the mycotoxins roridin A, T-2, and verrucarin A at
10 µg per 2.54 cm
of material. Treatments were evaluated with
an agar plating technique for fungal spores and a yeast toxicity
culture assay for mycotoxins. Results showed that gamma ir-
radiation inactivated fungal spores, but the treatment was not
successful in inactivating mycotoxins. The washing technique
completely inactivated or removed spores on all materials
except for C. globosum, which was reduced on all items except
paper (p < 0.05). Washing inactivated all mycotoxins on paper
and cloth but not on carpet or untreated wood (p < 0.001). The
steam cleaning treatment did not completely eliminate any fun-
gal spores; however, it reduced P. chrysogenum numbers on all
materials, C. globosum was reduced on wood and carpet, and
S. chartarum was reduced on wood (p < 0.05). Steam cleaning
was unsuccessful in inactivating any of the tested mycotoxins.
These results show that the bleach/detergent washing technique
was more effective overall in reducing spore and mycotoxin
levels than gamma irradiation or steam cleaning. However, the
other examined techniques were successful in varying degrees.
Keywords contents, fungi, mycotoxin, sick building syndrome,
Address correspondence to: S.C. Wilson, Department of Micro-
biology and Immunology, TTUHSC 3601 4th Street, Lubbock, TX
79430; e-mail: Stephen.Wilson@tthusc.edu.
here is a great deal of confusion and misinformation
both on the Internet and in other media regarding the
status of household contents in mold-contaminated
structures. This is because there are no studies that
show a relationship between items from a mold-contaminated
structure and human health. Additionally, there are no accurate
and comprehensive testing methodologies for determining
what levels of spores and/or mycotoxins are present on contents
inside structures. For example, the identiﬁcation of fungal
colonies on contents can be performed using tape lift and swab
however, some items may have spores hidden
inside them or in hard to reach areas, and these sampling
techniques are often inadequate to extract the spores. Sam-
pling techniques for spores and hyphae on contents are there-
fore best viewed as being approximate and not comprehensive
Despite the limitations in determining the extent of fungal
contamination on contents, and the lack of knowledge regard-
ing the relationship between levels of fungal spores and/or
mycotoxins and human ill-health, there is a requirement for a
solution regarding the appropriate treatment of contents in a
mold-contaminated dwelling. The purpose of this study was
to investigate some sterilizing procedures that may be effec-
tive in eliminating fungal spores and mycotoxins on home
MATERIALS AND METHODS
epresentative materials of household contents used in the
study were wood, cloth, paper, and carpet. Fungal spores
or mycotoxins were inoculated separately onto the materials.
Three techniques were used for cleaning/sterilizing the ma-
terials: (1) gamma irradiation, (2) a washing technique using
a commercially available detergent/bleach combination, and
(3) a commercial steam cleaner.
The paper was general purpose printing paper (Hp multipur-
pose paper; Hewlett Packard Co., Palo Alto, Calif.). The cloth
was a white 65% cotton/35% polyester material (No. 556092;
Landau Uniforms, Memphis, Tenn.). The wood was untreated
5-ply plywood, 63 mm thick. The carpet was a white 100%
nylon pile with a woven polypropylene backing (Williamsville
06600 Color 127 ﬂeece; Lowe’s Home Improvement Center,
442 Journal of Occupational and Environmental Hygiene July 2004
Lubbock, Texas). The materials for sampling were cut into
1-inch squares (2.54 cm
) and sterilized by autoclaving before
Fungal Spore Inoculation onto Materials
The fungi Aspergillus niger, Stachybotrys chartarum, Peni-
cillium chrysogenum, and Chaetomium globosum were recov-
ered from contaminated building materials and subcultured
onto malt extract agar (MEA) plates.
These plates were in-
cubated at 25
C until conﬂuent growth was achieved, typically
between 6–7 days. Spores were collected from these plates and
inoculated into phosphate buffered saline (PBS), pH 7.0 at a
rate of 240,000 spores/50 µL of PBS. The 50 µL PBS spore
suspension was inoculated onto the materials and allowed to
dry at room temperature before the experimental treatments
of washing and steam cleaning were applied. This level of
spores, if untreated, would result in conﬂuent fungal growth
on the material after 7 days of incubation.
Mycotoxin Inoculation onto Materials
Because trichothecene mycotoxins are produced by
sick building syndrome-associated organisms such as
in this trial, two macrocyclic trichothecenes
(roridin A and verrucarin A) and one simple trichothecene
(T-2) were used. The mycotoxins were inoculated at a level
of 10 µg per square inch of material. This was achieved by
suspending 200 µg of the mycotoxins into a 1.0 mL solution
of 99.9% high-performance liquid chromatography (HPLC)
grade methanol giving a ﬁnal concentration of 10 µg/50 µL.
The 50 µL was inoculated onto the materials and allowed to
dry at room temperature before the experimental treatments
Experimental Treatments: Washing
The spore- and mycotoxin-inoculated materials were placed
separately into sterile plastic tubes containing 15 mL of a 2%
solution of a commercial detergent (Xtra; USA Detergents Inc.,
North Brunswick, N.J.) and a 2% solution of chlorine bleach
(Ultra Clorox Regular; The Clorox Co., Oakland, Calif.) in
deionized water. The temperature of the solution was 39
The tubes were agitated vigorously, three times per min for a
total of 10 min. The tubes were then emptied of the solution
and rinsed three times with deionized water for 5 min each in
the same manner washing was performed. Positive and nega-
tive controls were included, that is, materials were inoculated
but did not receive the experimental treatment, and materials
received the experimental treatment but were not inoculated.
This procedure was replicated six times.
Experimental Treatments: Steam Cleaning
A commercial “dry steam vapor” cleaner with a 3 L ca-
pacity was used (Eurosteam, Crowley, Texas). The cleaner
was operated at a high-heat, low-moisture setting. The steam
was directed onto the spore and mycotoxin inoculated materials
from a distance of 6 inches for 10 sec. This procedure was
replicated six times.
Experimental Treatments: Gamma Irradiation
Because the fungicidal activity of gamma irradiation is well
in this trial the main focus was its effects on
mycotoxins; however, its effect on the spores of the selected
organisms was also determined. Sterile cotton swabs were used
to retrieve 2.54 cm
of growth of the four different fungi from
conﬂuent growth on MEA plates. In terms of mycotoxins, the
inoculation technique was the same as used for the washing
and steam cleaning techniques.
After the materials and swabs had been inoculated, they
were transported to a commercial nuclear facility and irra-
diated at a dosage between 10–13 kiloGrays. Ten kiloGrays
are equivalent to one megaRad. Positive and negative controls
were employed, that is, materials and swabs were inoculated
but did not receive an experimental treatment, and materials
and swabs received the experimental treatment but were not
inoculated. This process was replicated six times.
Determination of Treatment Efﬁcacy: Fungal Spores
After the washing and steam cleaning treatments, the mate-
rials were retrieved from the tubes, dried, and placed in 10 mL
of PBS. After 5 hours the samples were serially diluted using
PBSata1in10dilution rate three times. One hundred µLof
each dilution was then added to potato dextrose agar (PDA)
plates. After the gamma irradiation treatment, the inoculated
swabs were directly plated onto PDA media.
All agar plates
were incubated for 7 days at 25
C. After 7 days, colony forming
units (CFU) of fungi were identiﬁed using macroscopic and
Determination of Treatment Efﬁcacy: Mycotoxins,
After the washing, steam cleaning, and gamma irradiation
treatments, all materials were suspended in 15 mL of 99.9%
HPLC grade methanol and allowed to soak for 18 hours. The
methanol was then poured into 20-mL glass scintillation vials.
The crude toxin extract was allowed to dry to completion under
a fume hood. The dried remnants were resuspended in 1 mL
of 99.9% HPLC methanol and ﬁltered through polyvinylidene
ﬂuoride membrane ﬁlters with a pore size of 0.22 microns.
The resulting ﬁltrates were then used for toxicity testing using
a yeast toxicity assay.
Yeast Toxicity Assay
This assay is based on the procedure of Engler et al.
principle of this assay is as follows: cultures of Kluyveromyces
marxianus (No. 8554; American Type Culture Collection,
Manassas, Va.) are incubated with the ﬁltrates for 8 hours.
The yeast culture is sensitive to the presence of trichothecene
mycotoxins and will not grow in their presence even at very
small quantities (100–250 nanograms/mL). Optical density
(OD) readings are made every 2 hours. The assay is terminated
at 8 hours. A high OD is a result of increased turbidity, which
is a consequence of the growth of the organism. A low OD is
due to little or no growth of the organism.
Journal of Occupational and Environmental Hygiene July 2004 443
K. marxianus was grown at 37
C and stored at 4
C on yeast-
peptone-glucose (YPG) agar. Cultures for inoculation of the
assay were prepared by adding a single colony from an agar
plate to 5 mL YPG-50 media in a culture tube. The tube was
incubated in a rotary incubator for approximately 16 hours at
C to give the culture a ﬁnal density of 1 × 10
For the assay procedure, YPG-50 was supplemented with a
stock solution of polymixin B sulfate (PMBS) (ICN Biomed-
icals, Aurora, Ohio) to give a ﬁnal bioassay PMBS concentra-
tion of 15 mg/mL. Tests were run in triplicate. One hundred
and thirty-six µL of PMBS-supplemented YPG-50 medium
was added to the wells of a 96-well polystyrene microtiter
plate. Eight µL of test sample or control was added to each
well, followed by 16 µL of yeast inoculum to yield an initial
cell density of approximately 1 × 10
cells/mL. Blank wells
contained 152 µL of medium and 8 µL of water. Control wells
consisted of 144 µL of medium and 16 µL of yeast inoculum.
The plates were sealed and incubated on a plate shaker at 37
for 8 hours (when cells reached stationary phase). Cell density
was measured every 2 hours by measuring the absorbance
in a microtiter plate reader at a wavelength of 550 nm. The
absorbance was correlated with a K. marxianus 8-hour growth
curve to determine cell density.
With regard to fungal spores, mean CFU were determined
from the plates inoculated with the treated materials and were
compared using a Mann Whitney Rank Sum Test (SigmaStat,
software, version 2.0; SPSS Inc., Chicago, Ill.) with the mean
CFU from the plates inoculated with the untreated materials. In
this analysis, because of the difference in efﬁcacy between the
two techniques, the washing and steam cleaning techniques
were compared separately with the controls. With regard to
mycotoxins, mean OD readings from the positive controls
were compared with the mean OD readings from the gamma
irradiated, washing, and steam treated materials using a one-
way analysis of variance test. Conditions of normality and
equal variance were met for this analysis.
Washing Technique: Fungal Spores
Table I shows the results for the washing technique for
fungal spores. All spores except those of C. globosum were
either inactivated or removed from the materials. Mean CFU
of C. globosum were reduced on all items except paper (p <
Washing Technique: Mycotoxins
Table II shows the results with regard to mycotoxin levels.
The mean ODs of the cloth and paper ﬁltrates were higher
than the mean ODs of the ﬁltrates of the positive controls (p <
0.001) and similar to the mean ODs of the negative controls,
indicating that the ﬁltrates were not toxic to K. marxianus
cultures. The mean ODs of the carpet and wood ﬁltrates were
no different from the positive controls (p > 0.05) and lower
than the mean ODs of the negative controls, indicating that the
ﬁltrates were toxic to the K. marxianus cultures.
TABLE I. The Effects of Washing with a Detergent/Bleach Combination on Fungal Spore Activity
Washing Technique N = 9 Control N = 5
Organism Material Mean (CFU/2.54 cm
) SE Mean (CFU/2.54 cm
) SE p Value
C. globosum Wood 72
S. chartarum Wood 0 0 1300 748.3 NA
Cloth 0 0 56 33.6 NA
Paper 0 0 1328 2089 NA
Carpet 0 0 1100 1043 NA
P. chrysogenum Wood 0 0 8020 4346 NA
Cloth 0 0 15800 4693 NA
Paper 0 0 32600 5455 NA
Carpet 0 0 5560 1689 NA
A. niger Wood 0 0 8740 6328 NA
Cloth 0 0 1420 542.6 NA
Paper 0 0 8600 6853 NA
Carpet 0 0 12236 991.8 NA
Notes: Controls received spores but no treatment. Different superscripts indicate differences at the level of signiﬁcance given in the far column.
SE = standard error.
NA = not applicable.
444 Journal of Occupational and Environmental Hygiene July 2004
TABLE II. Optical Density (Yeast Toxicity Assay for Mycotoxin Activity) Readings After Washing, Steam
Cleaning, or Gamma Irradiation Treatment Techniques
Positive Control Washing Steam Gamma Iradiation
Mycotoxin Material Mean SE Mean SE Mean SE Mean SE
Roridin A Wood 0.4
0.009 0.4A 0.05 0.14
0.012 0.3A 0.019 0.12
0.009 0.3A 0.054 0.13
0.014 0.3A 0.007 0.23
T-2 Wood 0.46
0.03 0.51A 0.009 0.34
0.034 0.32A 0.007 0.13
0.028 0.25A 0.02 0.16
0.052 0.56A 0.023 0.29
Verrucarin A Wood 0.34
0.037 0.35A 0.011 0.16
0.023 0.29A 0.011 0.12
0.027 0.27A 0.015 0.15
0.012 0.32A 0.011 0.27
Notes: N = Six per treatment. Positive controls were materials that had toxins added, but did not receive any treatment. Different superscripts indicate differences
at the p < 0.001 level of signiﬁcance.
SE = standard error.
Steam Cleaning Technique: Spores
Table III shows the results for the steam cleaning technique
for fungal spores. Results were mixed, with no organisms being
completely eliminated as with some of the washing results.
Mean CFU of P. chrysogenum were reduced on all materials
(p < 0.05). Mean CFU of S. chartarum were reduced on wood
only (p < 0.01). Mean CFU of C. globosum were reduced on
wood and carpet (p < 0.05). Mean CFU of A. niger were no
different from the positive controls (p > 0.05).
Steam Cleaning Technique: Mycotoxins
Table II shows the results with regard to mycotoxin levels.
The mean ODs of the ﬁltrates from all treated materials were no
different from the positive controls (p > 0.05) and lower than
TABLE III. Effects of a Steam Cleaning Technique on Spore Activity
Steam Cleaning Control
Organism Material Mean (CFU/2.54 cm) SE Mean (CFU/2.54 cm) SE
C. globosum Wood 48
S. chartarum Wood 36
P. chrysogenum Wood 321
A. niger Wood 1201
Notes: Different superscripts indicate a signiﬁcant difference at the p < .05 level.
SE = standard error.
Journal of Occupational and Environmental Hygiene July 2004 445
the negative controls, indicating that the ﬁltrates were toxic to
K. marxianus cultures.
Gamma Irradiation: Spores
The MEA plates from the inoculated swabs showed no
growth after 7 days incubation at 25
Gamma Irradiation: Mycotoxins
Table II shows the results with regard to mycotoxin levels.
The mean ODs of the ﬁltrates from all treated materials were
no different from those of the positive controls (p > 0.05) and
lower than the negative controls, indicating that the ﬁltrates
were toxic to K. marxianus cultures.
he results show that all three techniques had varying de-
grees of success on the tested materials, spores, and my-
cotoxins. With regard to the gamma irradiation results, many
studies have been performed on gamma irradiation and myco-
toxins, particularly mycotoxins on grains.
In these stud-
ies, results have been mixed with a range of levels of irradi-
ation being required to inactivate different mycotoxins. Ad-
justing relative humidity during irradiation appears to play a
although some studies have not shown any signiﬁcant
Possibly, the increase needs to be large for
the treatment to become effective. In this study the technique
was not successful with dry items, although other studies
have shown gamma irradiation to be effective in inactivating
fungal spores and colonies. If mycotoxin inactivation is not
a concern, then this technique appears to be quite effective,
although attention must be given to the degrading effect of
gamma irradiation on items.
With regard to the washing technique, the failure of the
technique to inactivate C. globosum could be due to a number
of factors, for example, the ascospores of C. globosum are
retained inside a structure known as a perithecium
release, and it is possible that the perithecium played a protec-
tive role in this situation. The bleach used in this trial contained
sodium hypochlorite as the active agent. Sodium hypochlorite
has been shown to have bactericidal properties.
It is also
fungicidal to certain organisms.
One study showed that it
had mixed results at 20
C against Penicillium roqueforti.
In terms of toxins, 20 mL of a 1.3% concentration of sodium
hypochlorite is sufﬁcient to inactivate 20 µg of pure
However, this technique has a proviso that 5%
acetone is used as a rinse afterward to either remove or in-
activate a carcinogenic byproduct, 2,3-dichloro aﬂatoxin B
which is formed out of the inactivation process. Sodium
hypochlorite has also been used to inactivate trichothecene
mycotoxins at a concentration between 3 and 5%.
trial it was successfully used in conjunction with a commercial
detergent on paper and cloth. The failure of this treatment to
inactivate mycotoxins on carpet and wood could be because
the carpet ﬁbers presented an effective physical barrier to the
detergent/bleach, and the wood absorbed the alcohol-based
mycotoxin preparations. Both features could prevent the wash-
ing treatment from accessing the mycotoxins.
With regard to the steam cleaning technique, it was not
as effective as the washing technique but was successful in
signiﬁcantly reducing C. globosum and S. chartarum spore
burdens on wood. The technique involved a 10-sec spraying
time. It is conceivable that longer time periods could result in
effective removal of spore burdens. Also, using a biocide in
the steam cleaner may provide an effective treatment.
In this study the focus was on the effects of the treatments
on fungal spores and mycotoxins. However, a recent study
showed that fungal colonies can aerosolize large numbers of
antigenic fungal fragments into the environment simultane-
ously with fungal spores. These fragments were shown to
be much smaller than fungal spores. It is possible that these
fragments also carry mycotoxins and contribute to fungal con-
tamination of contents. The cleaning techniques tested in this
trial may be effective against fungal fragments but this remains
to be examined.
While the techniques described have shown varying degrees
of success in inactivating mycotoxins and fungal culturability,
it must be noted that fungal spores can still have allergenic
properties even when they are nonviable. High efﬁciency par-
ticulate air [ﬁlter] vacuuming is often used in mold remediation
situations and may have application with regard to the removal
of spores from contents.
n this trial, gamma irradiation at a 10–13 Kgy exposure
was successful in inactivating spores of Aspergillus niger,
Stachybotrys chartarum, Penicillium chrysogenum, and
Chaetomium globosum, but not the mycotoxins roridin A, ver-
rucarin A, and T-2 that had been inoculated onto carpet, cloth,
paper, and wood. Washing with bleach and a commercial de-
tergent was effective against spores of A. niger, S. chartarum,
and P. chrysogenum, but not C. globosum. The treatment was
successful against the tested mycotoxins for cloth and paper
materials, but not carpet and wood. The steam cleaning tech-
nique was successful only with spores of S. chartarum and
C. globosum on wood material.
inancial support was provided by Assured Indoor Air
Quality Ltd., Dallas, Texas, and Texas Tech University
Health Sciences Center (TTUHSC). Drs. Stephen Wilson and
David Straus were supported by a Center of Excellence grant
from TTUHSC; Dr. David Straus, Mr. Gary Fortenberry,
Mr. Trevor Brasel, and Ms. Enusha Karunasena were supported
by a grant from the Texas Higher Education Coordinating
Board. The authors would also like to gratefully acknowledge
the technical and material support from Mr. Mark LeGarda of
Steris Isomedix Services, El Paso, Texas.
446 Journal of Occupational and Environmental Hygiene July 2004
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