Raghuramulu G et. al. / JPBMS, 2011, 5 (17)
Available online at www.jpbms.info
ISSN NO- 2230 - 7885
Original Research Article
JPBMS
JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES
Identification of two species Trichophyton mentagropytes and Trichophyton rubrum on the
basis of biochemical tests and cultural characteristics.
Mrs. G. Indira1,Prof G.Raghuramulu1, Prof.S.Ramreddy2, Prof.Kondal Rao3.
Asst Prof .in microbiology, Dept. of Microbiology. Pingle govt Degree and PG college,Warangal ,Andhra Pradesh, India.
1 Dept. of Zoology, Kakatiya university, Warangal, Andhra Pradesh,India.
2 Dept. of Microbiology, Kakatiya university, Warangal, Andhra Pradesh, India.
3 Dept. of Microbiology, Kakatiya Medical College, Warangal, Andhra Pradesh, India.
*
1*
Abstract: Trichophyton is the commonest dermatophyte which is geophilic, zoophilic and anthrophilic in nature. The
genus Trichophyton includes 24 species; some of these are saprophytes. The most common human pathogenic species of
Tricophyton genus are T.mentagrophytes 1 and T.rubrum2. These causes tinea corporis, tinea cruris, tinea pedis, tinea
capitis, tinea unguium by infecting skin, hair and nail respectively. The two species can be differentiated on the basis of
biochemical tests like urease test 3 invitro hair perforation test4 . These are also identified on the culture characters like
macro and micro conidia. These rarely produce macro conidia which may be clavate or cigar shaped or elongated, pencil
shaped. Microconidia are abundant and may be globose or pyri form and are born singly along the sides of hyphae or in
grape like clusters.
Key Words: Trichophyton mentagrophytes, T.rubrum, Sabouraud agar medium, urease test, hair perforation test,
microconidia .
Introduction:
Dermatophytes are not a particular fungus but rather
common shorthand label for group of three genera of
fungus that causing skin diseases of people and animal.
These causes’ ringworm infections called tinea and are
named as tinea corporis, tinea cruris, tinea capitis, tinea
pedis, tinea manum and tinea anguium according to their
site of infection.1 There are three types of dermatophytes
viz Epidermophyton, Trichophyton and Microsporum. In
these Trichophyton is considered as the most common
dermotophyte and includes about 22 species. As the
species are closely related it is often difficult to identify
but can be speciated basing on biochemical tests and
studying the micromorphology.
Objective:
To study the identification of two common species of
Trichophyton i.e., T.rubrum and T.mentagrophytes on
the basis of biochemical and cultural characteristics. These
are the most common species in Trichophyton according
to surveillance by Gupta et al. (1993) , Ranganathan etal
(1995) and Mishra et al. (1998)[5-7]
Specimen collection[13]:-Depending upon presumptive
diagnosis, diseased specimens were collected from skin,
nail and hair as skin scrapings, nail clippings and hair
samples.
Culturing:
Sabouraud’s dextrose agar medium was used for culturing
dermatophytes by adding chloramphenicol to prevent
bacterial contamination and cycloheximide for prevention
of saprophytic molds (odds). These specimens were
inoculated into a pair of medial plates are slants. One set
was incubated in BOD incubated at 22 degrees centigrade
and other set at 37 degrees centigrade. The cultures were
examined daily and after 6 days growth was observed.
Biochemical tests: The cultures of dermatophyte species
were subjected to the following tests for identity
confirmation.
Urease test: (Philpot 1967, sinki 1981)[ 8,3].
Figure 1&2: This test is conducted to differentiate T.rubrum and
T.mentagrophytes by employing urease test medium.
Materials and Methods:
Study group:- A study w as conducted on 100 clinically
diagnosed patients of tinea infections attending as out
patients in Dept. of Dermatology, Mahatma Gandhi
Memorial Hospital, Warangal. (AP), for a period of 6
months from January 2009 to July 2009.
*Corresponding Author
G. Indira
*Asst
Prof .in microbiology, Dept. of Microbiology. Pingle
govt Degree and PG college,Warangal ,Andhra Pradesh.
1
UREASE TEST SHOWING NEGATIVE RESULTS
Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 05, Issue 05
Raghuramulu G et. al. / JPBMS, 2011, 5 (17)
perforations , But T.rubrum grows on hair and
disintegrates it without making perforation.
Pigment production test (Kane 1980, Chander 2002)[9, 10]:Pigment production test was conducted to differentiate T
rubrum and T mentagrophytes. The two species were
inoculated in corn meal agar with 1% dextrose medium.
Corn Meal Agar Medium:
Corn meal
8g
Agar
4g
Dextrose
2g
Distilled water
200ml
After inoculation with test fungi,incubate the slants at
room temperature. The isolates of T rubru produce dark
red vinaceous pigment which appears as red colour on the
reverse side. The isolates of T mentagrophytes fail to
produce this pigment.
UREASE TEST SHOWING POSITIVE RESULTS
Urease test medium (Christensen’s urea agar medium)
Urea agar base 29 grams
Agar
15 grams
Distilled water 1000 ml
During the preparation of medium phenol red indicator is
used and medium was inoculated with test fungi. T.
mentagrophytes a urease positive organism shows urease
activity within 7 days and the colour of medium changed
to pink. T.rubrum isolates are urease negative and did not
produce urease enzyme.
Invitro hair perforation test: Figure 3: (Padhye 1980,
Bahuguna 1989)[8,4]
Hair perforation test was conducted for differentiating
T.rubram and T.mentagrophytes. A filter paper disk was
Micromorphology:Tease slide mounts from colonies were made under
lactophenol cotton blue (LPCB) to study the
morphological details by microscopic examinations. The
morphology of hyphae and structures of asexual spores
like microconidia and macroconidia were observed.
Trichophyton colonies were grown at room temperature
and growth rate was slow. From the front the colour is
white to bright yellowish beige or reddish violet. Reverse
side was pale yellowish brown to reddish brown.
Trichophyton was differentiated from microsporum and
Epidermophyton based on the shape of macroconidia,
which were clavate to cigar shape thin wall to thick wall
and smooth.
T.rubrum:- It is identified by the presence of flat colonies
wooly or granular. Colour ranges from cottony white to
cream. Reverse side was blood red colour. Hyphae were
septate and branched. Microconidia were numerous,
single celled, pyriform (tear drop shaped) and present
singly along the hyphae.
Macroconidia were rare, long, pencil shaped with 3 to 5
septa.
T.mentagrophytes
Colonies are flat spreading powdery white to cream
colour.. Presence of characteristic spiral hyphae was the
identification character. Microconidia were single cell and
arranged in grape like clusters. Macroconidia were long
and appear like rat tail filaments or club shaped to cigar
shape. Multicelled mostly 2 to 5 celled with thin smooth
walls.
HAIR PERFORATION WITH WEDGE SHAPED PERFORATION
IN T. MENTAGROPHYTES
placed into the bottom of standard 100mm sterile
petridish and 15ml of distilled water added.
Then a lock of child’s hair were placed into the water and
sterilized in an autoclave. 2 or 3 drops of 2% yeast extract
was added and hairs were inoculated with test fungi.
Incubate the plates at room temperature and hair strands
are examined periodically over a period of 4 weeks under
microscope 45X) for wedge shaped perforations. Isolates
of T.mentagrophtytes produce hair perforating organs
that penetrate hair radially and cause wedge shaped
2
Results:Trichophyton can infect skin, nail and hair but
Epidermophyton infects skin and nail whereas
Micorsporum infects skin and hair. In 100 clinical samples
49 samples were collected from inea corporis and 32 from
tinea capitis and 19 from tinea anguium. Out of 49 tinea
corposis cases 33 samples shown positive growth in
which 09 were reported as T.rubrum and 6 as
T.,mentagrophytes. In tenia anguium out of 19 samples
16 samples showed positive growth and in these 5 were
T.rubrum and 2 were T.mentagrophytes. In tinea capitis
infection out of 32 samples 27 shown positive growth in
which 6 reported as T.rubrum and 4 as T.mentagrophytes.
This report reveals the incidence of T.rubrum and
Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 05, Issue 05
Raghuramulu G et. al. / JPBMS, 2011, 5 (17)
T.mentagrophytes in all the 3 infected areas hence these 2
species were concluded as the most ommonest species
isolated from the deceased specimens.
Discussion:The dermatophyte species isolated and their incidence in
dermatophytoses was presented in Table 1. This data
shows out of 100 cases, isolation rate of dermatophytes is
76 and among them T.rubrum is 26.32%. So it was the
predominant isolate followed by T.mentagrophytes this is
15.75%. These two were followed by other species like
M.audouinii with 13.16%, E.floccosum with 14.47%,
M.gypseum with 11.84%, T.tonsurans with 7.89%,
T.violaceum with 6.58% and M.canis with 3.95%. Thus in
present study T.rubrum was the predominant species
isolated from all types of clinical specimens i.e., skin, hair
and nail from a variety of clinical conditions of ringworm.
Table 1:Isolation dermatophytic species and their incidence in dermatiphytoses
SPECIES
CASES
Total. No. of
cases
T. rubrum
No. of cases
percentage
TINEA CORPORIS
(49)
TINEA CAPITIS
TINEA ANGUIUM
(32)
(19)
09
18.37
06
18.75
05
26.32
TOTAL
100
20
26.32
T. mentagroph
ytes
No. of cases
percentage
06
12.24
04
12.5
02
10.53
12
15.7
M. audouinii
No. of cases
percentage
06
12.24
04
12.5
-------
10
13.16
E. floccosum
No. of cases
percentage
04
8.16
-----
07
34.9
11
14.47
M. gypseum
No. of cases
percentage
02
4.08
07
21.88
-------
09
11.84
T. violaceum
No. of cases
percentage
02
4.08
03
9.38
-------
05
6.58
M. canis
No. of cases
percentage
03
6.12
-------
03
3.95
T. tonsurans
No. of cases
percentage
01
2.04
02
10.53
06
7.89
No. of cases
percentage
33
67.35
TOTAL
----03
9.38
27
84.38
16
84.21
76
10
Aknowledgements:
My sincere thanks to Prof.S. Ramreddy,Department of
Microbiology,Kakatiya University,Warangal.A.P ,Prof.G.
Raghuramulu, Department of Zoology, Kakatiya
University, Warangal.A.P and Prof. Kondal Rao, Kakatiya
Medical Colloge, Warangal,A.P for their continuous
encouragement guidance throughout this work.
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Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 05, Issue 05