Jpn. J. Med. Mycol.

Vol. 49, 125 − 128, 2008

ISSN 0916 − 4804

Original Article

Dimethyl Sulfoxide（DMSO）Inhibits the Germination of Candida

albicans and the Arthrospores of Trichophyton mentagrophytes
Muhammad Akram Randhawa

Department of Pharmacology, College of Medicine, King Faisal University

P.O Box 2114 Dammam 31451, Saudi Arabia
〔Received: 23, July 2007．Accepted: 17, January 2008〕

Abstract
Dimethyl sulfoxide（DMSO）is commonly used as a solvent for antifungal drugs. Earlier the author has
reported the inhibitory effect of DMSO on the growth of many strains of dermatophytes’colonies in dermasel
agar and proposed that this could cause the variations between results of different studies for the evaluation
of the activities of antifungal drugs. In studies regarding the determination of the effect of antifungal drugs on
the germination of arthrospores of dermatophytes it was observed that relatively higher concentrations of
DMSO were being used as a solvent for the antifungal drugs, the final concentration in the media being 5%.
Therefore, the present study was aimed at determining the effect of different concentrations of DMSO（1.25
to 10%）on the growth of germ tubes of arthrospores of Trichophyton mentagrophytes and Candida albicans, in
glucose peptone broth. With DMSO 10% there was a negligible growth of germ tubes of both the
arthrospores and yeast; between 2.5 and 7.5% there was a rather linear dose-related inhibitory effect; whereas
1.25% had insignificant effect from controls. The present study shows that besides other factors, variations in
the results of the susceptibility tests of antifungal drugs might occur due to the effect of DMSO on the
growth of fungi and differences in the final concentration of DMSO in the medium.
Key words ： DMSO, Trichophyton mentagrophytes arthrospores, Candida albicans germ tubes, glucose pep-
tone broth

（Trichophyton, Epidermophyton and Microsporum）４）.

Introduction
DMSO is a highly polar and stable substance with
exceptional solvent property. It also acts as a pene-
trant of drugs through the skin １）. Five percent DMSO
has also been added in fungal suspensions, as a cry-
oprotectant, for storage at very low temperature, − 80
℃２）.
DMSO 2% has been reported to significantly inhibit
the growth of Candida species by broth dilution
method, while 1% and below had insignificant effect ３）.
Recently, the author has reported the inhibitory effect
of DMSO, even below 1%, on the growth of different
strains of three important genera of dermatophytes
Address for correspondence ： Dr. Muhammad Akram Randhawa
Department of Pharmacology, College of Medicine, King
Faisal University, P.O Box 2114 Dammam 31451, Saudi Ara-
bia
In published studies regarding the germination of
arthrospores of dermatophytes it was observed that
relatively higher concentrations of DMSO were being
used as a solvent for the antifungal drugs, the final
concentration in the growth media being up to 5%５−７）.
Therefore, the present study was aimed at determin-
ing the effect of different concentrations of DMSO
（1.25 to 10%）on the growth of germ tubes of
arthrospores of different strains of a dermatophyte,
Trichophyton mentagrophytes and a yeast, Candida albi-
cans, in glucose peptone broth.
Materials and Methods
a. DMSO, glucose peptone agar & glucose peptone
broth:
DMSO was obtained from SIGMA, USA. From 100%
DMSO, 10, 7.5, 5, 2.5 and 1.25% dilutions were prepared
in sterile distilled water.
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Glucose peptone agar was obtained from OXOID,
England and contained: glucose 2%, mycological pep-
tone 1% and agar 1.45%. Forty-four and one half grams
was suspended in 1 liter of distilled water and gently
heated to dissolve completely. Then it was sterilized
by autoclaving at 121 ℃ for 15 minutes.
Glucose peptone broth was also obtained from
OXOID, England containing: 4% glucose, 1% peptone
broth. After mixing with an appropriate amount of dis-
tilled water, it was sterilized by autoclaving at 121 ℃
for 15 minutes.
b. Arthrospores of Trichopyton mentagrophytes
Trichopyton mentagrophytes（ var interdigitale）
arthrospores were produced in glucose peptone agar,
maintained at 37 ℃ in the presence of 20% CO2 in air
for two weeks. Surface growth was removed, suspend-
ed in phosphate buffered saline（PBS）and shaken on
a vortex mixer. The suspension was filtered through
glass wool to remove unbroken chains of arthrospores.
The filtered arthrospore suspension was washed three
times in PBS at 3000 g for 5 minutes and adjusted to a
concentration of 1 × 10 7 /ml. The viability of
arthrospores was checked by using 0.3 ml of
arthrospore suspension in 0.9 ml glucose peptone
broth incubated at 37 ℃ for 12 hours on a rotary shak-
er at 100 rpm. An arthrospore was considered to have
germinated when a visible germ tube had developed,
as seen in the microscope under high power（× 40）.
The method was essentially the same as that of
Hashimoto and Blumenthal, with some modifications ８）.
c. Yeasts
Candida albicans, obtained from the clinical samples,
was grown on glucose peptone agar. Surface growth
was removed, suspended in PBS and shaken on a vor-
tex mixer. The suspension was filtered through glass
wool. The filtered yeast suspension was washed three
times in PBS at 3000 g for 5 minutes and adjusted to a
final concentration of 1 × 10 7/ml. The viability of
yeasts was checked by using 0.3 ml of yeast suspen-
sion in 0.9 ml glucose peptone broth incubated at 37 ℃
for 3 hours on a rotary shaker at 100 rpm. A yeast
was considered to have germinated when a visible
germ tube had developed, as seen in the microscope
under high power（× 40） . proposed that besides other factors which could cause
d. Anti-germination assay:
To 0.3 ml of the arthrospore/yeast suspension was
added 0.3 ml glucose peptone broth plus 0.6 ml of each
dilution of DMSO mentioned above. For control pur-
poses 0.3 ml of the arthrospore suspension was added
真菌誌 第 49 巻 第２号 平成 20 年
to 0.3 ml glucose peptone broth plus 0.6 ml of sterile
distilled water to make a total of 1.2 ml, corresponding
to the volume of test preparations. Incubation was car-
ried out at 37 ℃ on a rotary shaker（100 rpm）for 12
hours for arthrospores and 3 hours for yeast. Three
germination bottles were used for each concentration
of DMSO.
e. Observations & statistical analysis:
At the end of the incubation period 100 microliters
of glutaraldehyde（25%）was added to each germina-
tion bottle to stop the process of germination. From
each germ bottle three mounts were made on glass
slides and examined microscopically for germination
（thus making nine observations for each dilution of
DMSO）. The % of germinating arthrospores/yeasts
was counted in each slide from a total of 100 cells
observed in different fields of microscope. Thus a
mean（with standard deviation）of nine observations
was calculated for each concentration of DMSO tested.
The results of different concentrations of DMSO were
compared with the controls by Student’ s t-test. The
study was conducted on three strains of T. mentagro-
phytes as well as three strains of C. albicans at different
occasions.
Results
The effect of different concentrations of DMSO on
the germination of arthrospores of T. mentagrophytes
and the growth of C. albicans germ tubes is given in
Table 1 and 2, respectively. In both the species the
growth of germ tubes was inversely related to the
concentration of DMSO. With 10% DMSO there was
almost complete inhibition of the growth. The growth
gradually increased lowering concentration and the
results of DMSO 7.5, 5 and 2.5% were significantly dif-
ferent from controls. However, with 1.25% DMSO the
effect was insignificant.
Discussion
Poor agreement between different methods for the
evaluation of anti-fungal drugs is commonly reported,
especially for their activity against dermatophytes9 − 11）.
Some of the possible factors causing this variability
had also been investigated ３）. Earlier, the author has
poor agreement among these methods, one important
factor might be the effect of dimethyl sulfoxide
（DMSO）on the growth of fungi, which was shown to
inhibit the growth of many strains of dermatophytes
in very low concentrations（less than 1%）４）.
Arthrospores of dermatophytes remain dormant in
 Jpn. J. Med. Mycol. Vol. 49（No. 2）
, 2008 127

Table 1．% of germination of three strains of arthrospores of T. mentagrophytes in the presence of differ-
ent concentrations of DMSO in glucose peptone broth（mean+SD, n=9）

Arthrospores of % DMSO in glucose peptone broth

T. mentagrophytes

10 7.5 5 2.5 1.25 Controls

4.11 17.78 54.56 81.44 93 97.33
Strain 1 ± 1.45 ± 2.59 ± 3.43 ± 4.36 ± 2.35 ±1
<0.001* <0.001* <0.001* <0.001* <0.1*
4.56 33.44 50.22 83.66 95.17 95.33
Strain 2 ± 1.81 ± 4.03 ± 3.77 ± 2.66 ± 2.79 ± 2.88
<0.001* <0.001* <0.001* <0.001* >0.1*
2.67 23.83 42.17 79.67 89.83 92.83
Strain 3 ± 1.03 ± 2.79 ± 3.31 ± 5.05 ± 3.13 ± 3.37
<0.001* <0.001* <0.001* <0.01* >0.1*
* P values when the results were compared with the controls by Student’
s‘t’test.

Table 2．% of germ tubes of yeasts of C. albicans in the presence of different concentrations of DMSO in
glucose peptone broth（mean+SD, n=9）

% DMSO in glucose peptone broth

Candida albicans

10 7.5 5 2.5 1.25 Controls

0.89 32.67 59.89 64.56 67.89 74.67
Strain 1 ± 0.93 ±8 ± 3.62 ± 3.91 ± 5.44 ± 3.6
<0.001* <0.001* <0.001* <0.001* <0.1*
0.67 39 62.83 81.5 90.5 95.83
Strain 2 ± 1.21 ± 2.9 ± 5.12 ± 3.73 ± 3.94 ± 2.14
<0.001* <0.001* <0.001* <0.001* <0.1*
1 37.83 59.5 77.33 87.83 95.5
Strain 3 ± 0.89 ± 4.71 ± 3.78 ± 3.88 ± 3.6 ± 2.07
<0.001* <0.001* <0.001* <0.001* <0.02*
*P values when the results were compared with the controls by Student’
s ‘t’test.

exfoliated skin for a long time12, 13）. Their germination is
an initial step in the pathogenesis of dermatophyto-
sis13 − 15）. Drugs that could inhibit the germination of
arthrospores would be useful for prophylaxis from der-
matophytosis. Only a few studies have investigated
antifungal drugs for anti-germination effects on
arthrospores of dermatophytes5 − 7）.
Many factors have been determined which could
affect the germination of dermatophyte arthrospores,
including: temperature, pH of the medium, oxygen,
etc.8, 16）. From the previous observations of the effect of
DMSO on the filamentous growth of dermatophytes ４）
it was thought that changes in the concentration of
DMSO as a solvent for antifungal drugs might also
affect the germination of their arthrospores and cause
variations in the results. In the present study we
found that 10% DMSO almost completely inhibited
growth of germ tubes of both the arthrospores and
yeast; between 2.5 and 7.5% there was a rather linear
dose-related inhibitory effect, whereas the effect of
1.25% differed insignificantly from the controls.
The production of arthrospores was tried on many
species of dermatophytes at different temperatures
（25, 30, 37 ℃）. However, T. mentagrophytes gave the
maximum yield of arthrospores at 37 ℃ and 20% CO2.
In a few studies reported in the literature regarding
the investigation of antifungal drugs for anti-germina-
tion effects arthrospores of only T. mentagrophytes have
been used5 − 7, 16）.
In the antigermination assay, incubation was carried
 128
out at 37 ℃ on a rotary shaker（100 rpm）for 12
hours for arthrospores and 3 hours for yeast. More
than 90% germination of arthrospores has been report-
ed in 7-8 hours ８）. Similarly, in our initial experiments
near 90% growth of germ tubes of the yeast was
observed in 3-4 hours. Slowing the speed to below 50
rpm favoured clumping of yeast and arthrospores.
The lower concentrations of DMSO which had negli-
gible effect on the germination of arthrospores and
yeasts might have an additive/synergistic effect with
the antifungal drugs, and this needs further investiga-
tion.
Acknowledgements
The author gratefully acknowledges the financial help of
the King Faisal University Research Grants Commission.
He is also very grateful to Dr. Salih Hamad Mohamad
Aljabre, Professor of Dermatology, College of Medicine,
King Faisal University for guidance and to Mr. Lari
Bartholomew for technical help.
References
1）Champion RH, Burton JL, Burns DA, Breathnach SM:
Topical Therapy. In Textbook of Dermatology, 6th Ed.
Blackwell Science, Ltd. UK, pp.3529, 1998.
2）Arthington-Skaggs BA, Motley M, Warnock DW, Mor-
rison CJ: Comparative evaluation of PASCO and
National Committee for Clinical Laboratory Standards
M27-A broth dilution methods for antifungal drug sus-
ceptibility testing of yeast. J Clin Microbiol 38（6）: 2254-
2260, 2000.
3）Rodriguez-Tudela JL, Cuenca-Estrella M, Diaz-Guerra
TM: Standardization of antifungal susceptibility vari-
ables for a semi-automated methodology. J Clin Micro-
biol 39（7）:2513-2517, 2001.
4）Randhawa MA: The effect of dimethyl sulfoxide
（DMSO）on the growth of dermatophytes. Jpn J Med
Mycol 47: 313-318, 2006.
5）Wright LR, Scott EM, Gorman SP: The sensitivity of
mycelium, arthrospores and microconidia of Trichophy-
真菌誌 第 49 巻 第２号 平成 20 年
ton mentagrophytes to imidazole determined by in vitro
tests. J Antimicrob Chemother 12: 317-327, 1983.
6）Scott EM, Gorman SP, Wright LR: The effects of imida-
zoles on germination of arthrospores and microconidia
of Trichophyton mentagrophytes. J Antimicrob Chemother
13: 101-110, 1984.
7）Aljabre SHM, Scott EM, Shankland GS, et al.: In vitro
susceptibility of Trichophyton mentagrophytes arthroconi-
dia to clotrimazole and griseofulvin in human corneo-
cyte suspension. Mycoses 34: 479-482, 1991.
8）Hashimto T, Blumenthal HJ: Factors affecting germina-
tion of Trichophyton mentagrophytes arthrospores. Infect
Immun 18（2）:479-486, 1977.
9）Ramani R, Charturvedi V: Proficiency testing program
for clinical laboratories performing antifungal suscepti-
bility testing of pathogenic yeast species. J Clin Micro-
biol 41（3）:1143-1146, 2003.
10）Santos DA, Hamdan JS: Evaluation of broth microdilu-
tion antifungal susceptibility testing conditions for Tri-
phyton rubrum. J Clin Microbiol 43 （4）:1917-1920, 2005.
11）Ghannoum MA, Arthington-Skaggs B, Chaturvedi V,
Espinel-Ingroff A, et al.: Interlaboratory study for quali-
ty control isolates for a broth microdilution method
（modified CLSI M38-A）for testing susceptibilities of
dermatophytes to antifungals. J Clin Microbiol 44（12）:
4353-4356, 2006.
12）Dovrak J, Hubalek Z, Otcenasek M: Survival of der-
matophytes in human skin scale. Archiv Dermatol 98:
540-542, 1968.
13）Aljabre SHM, Richardson MD, Scott EM, et al.: Dor-
mancy of Trichophyton mentagrophytes arthroconidia. J
Med Vet Mycol 30: 409-412, 1992.
14）Aljabre SHM, Richardson MD, Scott EM, et al.: Adher-
ence of arthroconidia and germlings of anthropophilic
and zoophilic varieties of Trichophyton mentagrophytes to
human corneocytes as an early events in the pathogen-
sis of dermatophytosis. Clin Exp Dermatol 18: 231-235,
1993.
15）Richardson MD, Aljabre SH: Pathogenesis of dermato-
phytosis. Curr Top Med Mycol 5: 49-77, 1993.
16）Hashimoto T, Blumenthal HJ: Survival and resistance
of Trichophyton mentagrophytes arthrospores. Appl Envi-
ronment Microbiol 35（2）:274-277, 1978.