DOI: 10.5454/mi.8.3.

7
Vol.8, No.3, September 2014, p 135-139

SHORT COMMUNICATION

Modified Slide Culture Method for Faster and Easier Identification of

Dermatophytes
YEVA ROSANA1*, TETSUHIRO MATSUZAWA2, TOHRU GONOI2,
1
AND ANIS KARUNIAWATI
1
Department of Microbiology, Medical Faculty, University of Indonesia
Jl. Pegangsaan Timur no.16, Jakarta 10320, Indonesia
2
Medical Mycology Research Centre, Chiba University, Japan

Basic slide culture as a morphological identification was known as the most common method for the
identification of pathogenic mold fungi. This method preserved the morphological features relatively
undisturbed compared with adhesive tape preparations. However, it was necessary to modify method of basic
slide culture to improve its usability and shorten the time it needed to identify mold fungi. There were four kinds
of method carried out in this study; two kinds of modified slide culture, one kind of direct culture on slant agar
plate, and a basic slide culture for identifying mold fungi, which result would be compared with each other. These
four methods were tested to 4 species of dermatophytes which were known as mold fungi that could infect skin,
hair, and nails in human; those were Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum,
and Epidermophyton floccosum. Result of this study showed that both modified slide culture and direct culture on
slant agar plate could visualize the structure of dermatophytes faster than basic slide culture method. These
methods were also easier to prepare compared to basic culture method. Conclusion of this study showed that
basic slide culture method needed to be modified for better identification of mold fungi.

Key words: Identification, Dermatophytes , Modified Slide Culture

Slide culture konvensional sebagai suatu identifikasi morfologi dikenal sebagai metode yang paling umum
untuk identifikasi jamur kapang patogen. Metode ini relatif lebih tahan lama untuk menyimpan gambaran
morfologi dibandingkan dengan menggunakan metode selotip. Walaupun demikian, perlu dilakukan modifikasi
metode slide culture konvensional untuk meningkatkan kegunaannya dan mempersingkat waktu yang
diperlukan untuk mengidentifikasi jamur kapang. Pada penelitian ini dilakukan empat metode; dua metode slide
culture yang dimodifikasi, satu metode slide culture pada plat agar miring, dan satu metode slide culture
konvensional untuk jamur kapang, yang hasilnya akan dibandingkan satu sama lain. Keempat metode ini
diujikan pada 4 spesies dermatofita yang dikenal sebagai jamur kapang yang bisa menginfeksi kulit, rambut, dan
kuku pada manusia; yaitu Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum, dan
Epidermophyton floccosum. Hasil penelitian menunjukkan bahwa kedua metode slide culture yang dimodifikasi
dan slide culture langsung pada plat agar miring dapat memvisualisasikan struktur dermatofita lebih cepat
dibandingkan metode slide culture konvensional. Metode ini juga lebih mudah pada tahap persiapannya
dibandingkan slide culture konvensional. Kesimpulan dari penelitian ini menunjukkan bahwa metode slide
culture konvensional perlu dimodifikasi untuk identifikasi jamur kapang yang lebih baik.

Kata kunci : Jamur Dermatofita, Identifikasi, Modifikasi Slide Culture

Superficial fungi was known to live on the dead occurred in both healthy and immunocompromised
horny layer of the skin and elaborate an enzyme that patients, and their etiologic agents consisted of
enabled them to digest keratin, causing the superficial dermatophytes, yeasts and nondermatophyte molds
skin to scale and disintegrate, nails to crumble, and (Mims et al. 2008; Richardson et al. 2000).
hairs to break off. Superficial fungi were also capable Dermatophytes were responsible for most
of eliciting an allergic or id reaction. Superficial fungal superficial fungal infections. Dermatophytes could be
infections were common skin diseases, affecting classified according to their natural habitats into three
millions of people worldwide. These infections categories: (1) geophilic, which normally live in the
soil, (2) zoophilic, which primarily parasitize the body
*Corresponding author; +6221 3160492. Fax: +6221 surfaces of animal, (3) anthropophilic, which generally
3100810, E-mail : [email protected]
infect human (Hiruma et al. 2003). There were three
136 ROSANA Microbiol Indones

genera of dermatophytes could infect the human, placed on the sterile medium while the other was put
including Trichophyton , Microsporum and after the growth of the dermatophytes. Method of direct
E p i d er m o p h yt o n . O n ly M i cro s p o r u m an d culture on slant agar only need half volume of standard
Tr i c h o p h y t o n i n v ad e d t h e h ai r, w h e r e as media for culture, and can visualize structure the
Epidermophyton and Trichophyton caused nail dermatophytes directly under the microscope. These
infections. These species of dermatophytes were four kinds method were tested to 4 species of
correlated with the clinical diseases, which are d er m atop hy tes , w hi ch w er e Tri cho ph yton
classified according to the location of the infection; the mentagrophytes, Microsporum canis, Microsporum
clinical lesion, the species of fungi, and therapy varied gypseum, and Epidermophyton floccosum.
for different sites (Pakshir et al. 2009). Identification of
this species was very important because of their Basic Slide Culture. A bent glass rod was placed in
chronic nature had a risk for significant morbidity. sterile petri plate side, and a sterile glass slide was put
Microscopic examination using KOH preparation on the glass rod. A 1-by-1-cm block potato dextrose
was a simple laboratory method for detection of fungal agar (PDA) cut with a sterile scalpel was then
organisms present in skin and nails or hair. It was transferred to the glass slide (Fig. 1A). Using sterile
accomplished by scraping the skin, nail lesion or hair wire needle, the fungus would then be inoculated from
and examining the material with the microscope. Using culture plate to the four sides of the agar block. Sterile
this method we could show the two structural elements coverslip was put over the block with slight pressure to
of fungi such as the spore and hyphae, but it could not ensure adherence. Approximately 2 mL of sterile water
identify the species of dermatophytes (Larone 2011). was put on the bottom of petri plate, and then the plate
To identify or confirm the diagnosis of superficial cover was replaced. The whole procedure was repeated
fungal infections, fungal culture must be done using for each of culture used in this study. When everything
appropriate medium (Difco 2003). It was best to set, the plates were put on clean plastic basket and
examine dermatophytes microscopically when the incubated at 300 Celcius.
culture had form conidia or spores. The best method for
preserving and observing the actual structure of Modified Slide Culture. A 1-by-1-cm block PDA cut
dermatophytes were the slide culture (Fujita 2013). with a sterile scalpel was transferred to a plate of PDA
This method preserved the morphological features (Fig. 1B). Fungus was inoculated using sterile wire
relatively undisturbed compared with tease mounts and needle onto the four sides of the agar block. Sterile
cello-tape mounts (Hughes et al. 2004). Slide culture coverslip was put over the block with slight pressure to
was not a rapid method, but it was unsurpassed as a ensure adherence, and the plate cover was replaced
routine means of studying the fine points of the afterwards. The whole procedure was repeated for each
microscopic morphology of dermatophytes (Patrick et of culture used in this study. After it finished, the plates
al. 2010, Wijedasa et al. 2012). Because basic slide were put on clean plastic basket and incubated at 300
culture method was quite difficult to carry out and Celcius.
requires a long time to get a result, it was needed to Another modified slide culture method used
modify slide culture method to gain a better result. fungus that was grown on potato dextrose agar
In this study, there were carried out four methods (Fig. 1C). A 1-by-1-cm block PDA was put on fungus
plate to identify dermatophytes fungi that could infect culture. Sterile coverslip was put over the block with
skin, hair, and nails in human: a basic slide culture slight pressure to ensure adherence, then the plate
method, two kinds of modified slide culture and one cover was replaced. The whole procedure was repeated
kind of direct culture on slant agar. Although basic slide for each of culture used in this study. Then, the plates
culture was a standard method for mold identification, were put on clean plastic basket and incubated at 300
but it needs space to incubate and not easy to prepare. Celcius.
The method of modified slide culture, agar block was
put directly to a plate agar to produce of reproductive Direct Culture on Slant Agar Plate. Approximately
structure more rapid. It did not require bent glass rod 10 mL PDA was poured on sterile petri plate and was
and glass slide, and without adding sterile water on the spread on the plate. Then the petri plate was put on its
petri plate. Moreover it could be put more than two cover to gained about half of the plate covered with thin
slides. The difference between two kinds of modified layer of PDA. After potato dextrose agar was solid, the
slide culture were time to put agar block, the one was petri plate was covered and kept in room temperature.
Volume 8, 2014 Microbiol Indones 137

Fig 1 Basic slide culture, agar block PDA on the glass slide put on the bent glass rod (A); Modified slide culture 1, agar block
PDA was put on the sterile medium (B); Modified slide culture 2, agar block PDA was put after the growth of the
dermatophytes (C); Direct culture on slant agar plate, visualize structure the dermatophytes directly under the
microscope (D).

Fig 2 Macroconidia of Microsporum canis (A); Spiral hyphae and microconidia of Trichophyton mentagrophytes (B);
Macroconidia of Epidermophyton floccosum (C), Macroconidia of Microsporum gypseum (D).
138 ROSANA Microbiol Indones

Fungus was directly inoculated on slant agar plate. The For direct culture plates, the examination was done
whole procedure was repeated for each of culture used on the slant agar plate under a microscope (Fig. 1D).
in this study. When done, the whole plates were put on The dermatophytes structures which are key features
clean plastic basket and incubated at 300 Celcius. for species identification were observed through thin
layer of PDA directly.
Reading and Interpretation of the Results. The Earlier visualization of structures related to
growth of fungus was examined periodically. The identification was possible for all 4 species as shown in
fungus could grow on the surface of the slide and also Figures 2. All of the methods allowed observation of
under the surface of the coverslip. The closed petri very delicate fungal structures of diagnostic
plate could be placed on the microscope stage, and the significance. This method could overcome three major
slide culture could be examined with the low-power drawbacks that were associated with adhesive tape
(10x) objective. When reproduced structures had preparations. First, fungal structures were be prevented
developed well, forceps was used carefully to remove from crushed and damaged during the preparation
the coverslip and to put it on a second slide filled with a procedures, affecting the accurate identification of the
drop of LPCB. Agar block was removed using flamed fungus. Second, as a result of the smear was not dry,
wire needle and put into container of antifungal slide culture can be used for microscopic examination
disinfectant. A drop of LPCB was put on the remaining for long period from the initial preparation. Third,
slide and the slide was then covered with a new fungal structures embedded in the agar can be
coverslip. Each of the microscopic slide culture was observed. Slide cultures was sealed around the edges
sealed around the edges with the nail polish and kept on with the nail polish were also suitable for long-term
room temperature. storage on room temperature.

Table 1 Comparison of basic slide cultures, modified slide sulture and direct culture on slant agar plate

Species * Day 5 Day 7 Day 10

Microsporum canis BSC Macroconidia (-) Macroconidia (-) Macroconidia (+)
MSC1 Macroconidia (-) Macroconidia (+)
MSC2 Macroconidia (-) Macroconidia (+)
DSC Macroconidia (-) Macroconidia (+)
Trichophyton BSC Spiral hyphae (-) Spiral hyphae (-), Spiral hyphae (+),
mentagrophytes macroconidia (-) macroconidia (-), macroconidia (+),
Microconidia (-) Microconidia (+) microconidia (+)
MSC1 Spiral hyphae (-) Spiral hyphae (+)
macroconidia (-) macroconidia (+)
Microconidia (+) microconidia (+)
MSC2 Spiral hyphae (-) Spiral hyphae (+)
macroconidia (-) macroconidia (+)
Microconidia (+) microconidia (+)
DSC Spiral hyphae (+)
macroconidia (+)
microconidia (+)
Epidermophyton BSC Macroconidia (-) Macroconidia (-) Macroconidia (+)
floccosum MSC1 Macroconidia (-) Macroconidia (+)
MSC2 Macroconidia (-) Macroconidia (+)
DSC Macroconidia (+)
Microsporum BSC Macroconidia (-) Macroconidia (-) Macroconidia (+)
gypseum MSC1 Macroconidia (-) Macroconidia (+)
MSC2 Macroconidia (-) Macroconidia (+)
DSC Macroconidia (-) Macroconidia (+)
* BSC (basic slide cultures); (MSC1) modified slide sulture 1; (MSC2) modified slide culture 2; DSC (direct culture on slant agar plate)
Volume 8, 2014 Microbiol Indones 139

The comparison of each method could be seen in Department Faculty of Medicine University of
Table 1. Basic slide culture required more days to Indonesia and Medical Mycology Research Centre
visualize morphology of dermatophytes, compared to Chiba University Japan for technical assistance and
modified slide culture 1, modified slide culture 2, and supporty.
direct culture on slant agar plate. Microsporum canis
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ACKNOWLEDGEMENT an alternative slide culture method for the
morphological identification of fungal species. Sri
This study is funded by Takeda Science Lanka J Infec Dis . 2(2): 47-52.
Foundation. We thanks to team of Microbiology