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A modified AATCC 30—1993 method to test fungicide treated fabrics against

dermatophytes

Article  in  Mycological Research · January 1999

DOI: 10.1017/S0953756298006832

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Mycol. Res. 103 (1) : 88–90 (1999) Printed in the United Kingdom 88

A modified AATCC 30–1993 method to test fungicide treated

fabrics against dermatophytes

K. W. G R E G O R Y, A. B. H A R R I S O N A N D W. B. B E T TS*
Microbiology Research Unit, Department of Biology, University of York, PO Box 373, York, YO1 5YW, U.K.

An agar plate test is described to determine the growth inhibition of dermatophytes by fungicides applied to fabrics. Trichophyton
rubrum and Epidermophyton floccosum, two major causative agents of tinea pedis (athlete’s foot), were used as test organisms. The
assay was capable of distinguishing between direct contact effects and diffusion of fungicide into the agar medium, and could be
used semi-quantitatively. The method included an essential sterilization stage to prevent interference by micro-organisms
contaminating the test materials. Loss of fungicide efficacy due to the sterilization procedure required careful control.

Over recent years considerable interest has developed in
antimicrobial finishes (biocidal and biostatic) applied onto or
incorporated into materials, including textiles. Such finishes
can reduce microbially-induced odour and staining, increase
the half-life of materials by reducing biodeterioration and rot,
assist good hygiene practices for the preparation and storage
of food, and be useful adjuncts or treatments in clinical
practice (e.g. on wound dressings, prosthetics and clothing).
There is consequently a need for relatively rapid and easily
performed procedures to measure the level of antimicrobial
activity incorporated into or applied onto textiles at source,
and possibly during simulated usage (e.g. wash and wear)
during product development. Methods for the measurement
of both diffusible and contact antimicrobial activity, using
representatives of both Gram positive and Gram negative
bacteria, are well established (Anon., 1990 ; 1993 b). There are
also methods available to measure the susceptibility of fabrics
to mildew and rot, and to measure the efficiency of fungicides
in textiles exposed to conditions under which these occur
(Anon., 1993 a). There are, however, no standard methods
available for testing the efficiency of fungicides in or on
textiles against parasitic fungi such as the dermatophytes.
Tinea pedis, or athlete’s foot, is the most common human
fungal infection world-wide (Masifridling, 1996). There have
been many studies to determine the causative agents of tinea
pedis, and most have demonstrated that Trichophyton rubrum
is the most frequent fungus involved in the infection (Terragni
et al., 1993 ; Aly, 1994 ; Manzanogayosso et al., 1994 ; Kemna
& Elewski, 1996). Other Trichophyton species have also been
implicated, but to a lesser extent. Several other dermatophyte
genera have also been isolated from clinical samples. In
particular, a long term study in Finland found that Epider-
* Corresponding author.
mophyton floccosum was important in tinea pedis (Lehenkari &
Silvennoinenkassinen, 1995).
The purpose of the present study was to develop an
analytical method to indicate and quantify the potency of
non-specific antimicrobial agents or fungicides, applied to a
range of textile materials, against both T. rubrum and E.
floccosum. The test procedure was based upon the American
Association of Textile Chemists and Colorists test method
30–1993 (Anon., 1993 a) originally developed to measure
mildew and rot resistance of treated textiles.
MATERIALS AND METHODS
Clinical isolates of T. rubrum (Castell.) Sabour. and E. floccosum
(Harz) Langeron & Miloch. obtained from York District
Hospital, York, U.K., were maintained on Sabouraud Dextrose
Agar (SDA) (Oxoid CM41).
In order to obtain sufficient mycelial biomass for experi-
ments the fungi were grown in Sabouraud Dextrose Broth,
containing 40 gl−" glucose and 10 gl−" mycological peptone
(pH 5n6), in a shaking water bath at 26 mC for 10–14 d. Four
fungal pellets (approx. 8–10 mm diam.) were transferred
aseptically into 100 ml sterile distilled water in a 500 ml glass
bottle containing " 50 g 9 mm diam. glass beads. The pellets
were disrupted by vigorous shaking for 5 min and the
suspension left to stand for 15 min to allow larger fragments
to settle. The resulting aqueous layer above the settled
material was aseptically transferred to a sterile 100 ml glass
bottle. Sufficient material was obtained for 80 to 100 plates.
SDA plates, which had been allowed to dry for 3 d at
ambient temperature (21p2m) in a sterile environment, were
inoculated with 0n5 ml of the settled fungal suspension which
was spread evenly over the plate using a sterile glass spreader.
Plates were then left to dry for 1 h at ambient temperature.
K. W. Gregory, A. B. Harrison and W. B. Betts 89

The test was conducted with two different fabric types

which had been treated with an antimicrobial finish (Amolden
a b
MG590, Daiwa Chemical Industries Ltd, Japan). Replicate
sample pieces (6) from textiles were cut into 20 mm squares
which were then laid onto the centre of the spread plates. The
sample piece was then placed at the centre of the spread plate
and a further 0n1 ml clear inoculum was pipetted onto the
sample piece. The process was repeated with fabric samples
which had not been treated with the antimicrobial finish, as
controls.
If the inoculum did not adequately soak into thicker sample
pieces they were first moistened by submerging in 10 ml c d
sterile 0n05 % Triton X-100 (Boehringer Mannheim, Germany)
in a universal bottle. Separate bottles were prepared for each
set of samples to avoid cross contamination. As much surplus
liquid as possible was removed from the sample by dragging
it against the side of the universal bottle.
Plates were then incubated at 26m with the sample piece
uppermost for 6 d (T. rubrum) or 8 d (E. floccosum). At the end
of the incubation period the plates were inspected for growth
on and under the textile sample, as well as for the presence or
absence of growth in a halo around the sample. If present, the
width of the halo was measured across the centre line of the Fig. 1. Growth of T. rubrum (left) and E. floccosum (right) on treated
sample, both horizontally and vertically. An average of these (above) and untreated (below) non-sterile sock fabric at 26m.
two values was then taken to give an estimate of the diffusible
activity of the fungicidal treatment. Table 2. Halo sizes for socks and tights treated with Amolden MG590
which had been sterilized before being tested against E. floccosum and T.
rubrum
RESULTS
Halo size (mm)
The method described has been used to demonstrate the
fungicidal activity of the antimicrobial agent Amolden MG590 Sterilization method E. floccosum T. rubrum
when applied to two different textiles.
Socks 70m, 2h 4n67p0n32 (12)* 2n79p0n50 (12)
The results for the experiment using treated fabric samples
120m, 15 min 5n08p0n22 (12) 3n79p0n22 (12)
and the two test organisms are presented in Table 1. There Tights 70m, 2h No halo No halo
was a halo of inhibition around the sock fabric and no visible 120m, 15 min No halo No halo
growth on top of the material. The treated tights, however, * Mean valuesp.. (sample size).
showed no halo of inhibition but no growth on the top of the
sample.
Untreated sock samples had a contaminating bacterial the large error. This was compounded by contaminating
growth around the perimeter of the test pieces which organisms (bacterial and\or fungal) from the test piece
prevented the growth of both test organisms. Untreated inhibiting the growth of the test organisms.
tights fabric samples showed growth surrounding, and on top The problems of growth of contaminating organisms
of, the sample for both T. rubrum and E. floccosum. present on the non-sterile samples hindered observations and
Representative photographs showing sock samples exposed measurements of the effect of the antimicrobial agent. To
to T. rubrum and E. floccosum are presented in Fig. 1. overcome this, samples of control and test materials were
When E. floccosum was used as the test organism the mean sterilized either by autoclaving at 120m for 15 min or by dry
halo width was greater than that for T. rubrum, suggesting heat at 70m for 2 h. The results for fungicide treated samples
that the former is more sensitive to this particular compound. are presented in Table 2.
The results must, however, be regarded with caution due to In the case of the treated sock fabric there was both a zone
of inhibition around, and no visible growth on, the samples.
Table 1. Halo sizes for non-sterile socks and tights treated with Amolden
For both sterilization treatments the mean halo width was less
MG590, tested against E. floccosum and T. rubrum than that obtained when non-sterile sample pieces were used.
This suggested that the sterilization procedure reduced the
Halo size (mm) fungicide’s efficacy. In the case of the treated tights fabric
E. floccosum T. rubrum
there was no zone of inhibition around, but no visible growth
on, the sample (Figs 2–5). Both untreated fabrics showed
Socks 5n80p0n33 (10)* 3n79p0n81 (12) growth of the test organisms adjacent to and on top of the
Tights No halo No halo samples following either sterilization procedure. Contaminant
* Meanp.. (sample size). fungal growth was still observed on some of the samples
 Fungicide efficacy
2 3
4 5
Fig. 2–5. Growth of T. rubrum on tights fabric at 26m. Fig. 2.
Treated, non-sterilized. Fig. 3. Treated, dry heat sterilized. Fig. 4.
Treated, autoclaved. Fig. 5. Untreated, autoclaved for reference.
sterilized at 70m, whilst no contaminants were observed with
autoclaved samples.
DISCUSSION
The original aim of the experiments conducted was to
develop a routine testing procedure to measure the efficacy of
fungicides when applied to fabrics. The modification of the
AATCC 30–1993 method proved to be unsuccessful due to
problems of the growth of contaminants on the non-sterile
test pieces, which was related to the long incubation times
required for the test organisms. Using similarly treated fabric
samples to measure antibacterial activity against Staphylococcus
aureus and Klebsiella pneumoniae using AATCC method
147–1993 (Anon., 1993 b) problems of contaminant growth
were not encountered (unpublished data). The provision of
uncontaminated treated samples may not be possible due to
either the method by which the antifungal agent is applied or
in the case of wash\wear trials contamination by the person
wearing the socks. This led to a modification to the test
method.
The further experiments showed that it was necessary to
sterilize samples in order to remove contaminating organisms.
Of the two sterilization methods investigated autoclaving at
120m for 15 min proved to be the most successful in terms of
(Accepted 4 March 1998)
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90
eliminating the contaminant growth. The choice of autoclaving
as a sterilization method for routine use in a standard testing
procedure may, however, be compromised by the sensitivity
of the antifungal compound to high temperatures. The choice
of sterilizing method is dependent upon both the textile
material and the applied antimicrobial agent. This may be
established as part of the test procedure or from data supplied
by the manufacturer. The method chosen should cause the
minimum loss of efficacy of the fungicide while ensuring that
contaminant interference is minimized.
The production of a halo of inhibition around a test piece
can only be regarded as a semi quantitative measurement. This
is because the diffusibility of the antifungal compound will
principally govern the size of the halo. It may be more
appropriate to report the results of the test procedure as
positive or negative based upon the contact inhibition of
fungal growth on top or beneath the fabric sample.
The method described here using sterilized samples provides
both a simple and effective means of testing materials treated
with antifungal agents for their activity against two of the
principal causative agents of tinea pedis. It is able to
distinguish between the direct contact activity and diffusible
activity of the compound leaching from the fabric although
care needs to be taken in the interpretation of these results.
The authors gratefully acknowledge Dr A. Anderson, York
District Hospital for the identification and supply of T. rubrum
and E. floccosum isolates ; and LJ Specialities, Wigan, U.K. for
providing the samples of biocide treated fabrics.
REFERENCES
Aly, R. (1994). Ecology and epidemiology of dermatophyte infections. Journal
of the American Academy of Dermatology 31, 521–525.
Anon. (1990). Testing Method for Antibacterial of Textiles JIS L 1902–1990.
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Anon. (1993 a). Antifungal Activity, Assessment on Textile Materials : Mildew
and Rot Resistance of Textile Materials. AATCC Test Method 30–1993.
American Association of Textile and Chemists and Colorists : North
Carolina, U.S.A.
Anon. (1993 b). Antibacterial Activity Assessment of Textile Materials : Parallel
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