DEPARTMENT OF MICROBIOLOGY

MGM MEDICAL COLLEGE, INDORE

STANDARD OPERATING PROCEDURE

SOP NO. : 1 Date of issue:
Procedure for isolation Effective date :
Department of and identification of Page 1 of 1
Microbiology dermatophytes

1. Purpose- This procedures describe the process for isolation and identification of
species of dermatophytes from the samples taken from infected skin , hair and nails.
2.Scope- This SOP provides guidlines for sample collection, storage and transportation along
with the procedures for identification of fungal species.

3.Responsibilty- Department of Microbiology

STANDARD OPERATING PROCEDURE

1.Sample Collection

A) Skin Scrapings:

Clean the site with 70% alcohol

Take samples by scraping across inflamed margin of the lesion in to the

apperently healthy tissue using a curved disposable scalpel blade

Then collect it in sterile petri dish or sterile paper packages.

B) Nail:
Friable material should be removed from under the nail
 Clippings will be taken from the border with scissors or nail clippers

C) Hair:
Infected hair will be removed by plucking with epilating forceps
or
scraping the scalp with a blunt scalpel

2. Storage and transport of samples

 If processing is to be delayed for more than several hours, it is recommended that

specimens should be stored at 15-30°C (dermatophytes survival is best at this
temperature).
 Specimens should be transported in sterile, dry and leak-proof container.

IDENTIFICATION OF DERMATOPHYTES

Identification of Dermatophytes

Microscopy Culture

Hyphae Conidia Growth rate Colony

characterstics

Colour Texture
 Surface Reverse

STANDARD OPERATING PROCEDURE

SOP NO. : 2 Date of issue:
Procedure for isolation Effective date :
Department of and identification of Page 3 of 10
Microbiology dermatophytes

1. DIRECT MICROSCOPY:

Take a clean grease-free glass slide

Place a large drop of 20% KOH on slide

Transfer small quantity of the specimen with a loop or the tip of a scalpel
into the KOH drop

Apply a coverslip, after a few minutes,tap gently to achieve a thin

preparation
Place the slide in sterile petri dish lined with damp filter paper and left it
for 20-30 minutes

Then examine under the microscope 10x lens, then 40x objective lens
(condensor, iris and diaphragm should be sufficiently closed)

Observation
1. Examine the clear specimen under low power (10X or 20X objective).
Scan the entire cover slip from end to end in a zigzag fashion.
2. If any fungal elements are suspected, examine under high power (40X
objective).
3. Reduce the light coming into the condenser while examining at high
power.
4. Look for branching hyphae, type of branching, the colour, septation
and thickness of hyphae, and spores.

Name Signature
Prepared by Dr. Ananya Verma
Checked by Dr. Suneel Kumar Ahirwar
Approved by Dr. Anita Mutha

STANDARD OPERATING PROCEDURE

SOP NO. : 4 Date of issue:
Procedure for isolation Effective date :
Department of and identification of Page 5 of 10
Microbiology dermatophytes
2.FUNGAL CULTURE:

The Specimen is inoculated onto Sabouraud dextrose agar (SDA) and

Dermatophyte Test Media (DTM) .

Specimen is reduced in size to pieces ~ 1mm

At least 4 inocula should be gently implanted into the agar on each

plate at well spaced intervals.

The plates are incubated aerobically at 25-30ºc for upto 21 days and
are checked daily for appearance of fungal colonies.

Name Signature
Prepared by Dr. Ananya Verma
Checked by Dr. Suneel Kumar Ahirwar
Approved by Dr. Anita Mutha

STANDARD OPERATING PROCEDURE

SOP NO. : 4 Date of issue:
Procedure for isolation Effective date :
Department of and identification of Page 6 of 10
Microbiology dermatophytes
3.TEASE MOUNT:

PRINCIPLES: A tease prepration is the quickest and most common technique for
mounting fungi for microscopic examination.Since the mould’s growth is teased apart
with dissecting needles, conidia or spores may be disloged from the conidiogenous or
sporogenous cells.
Precise identification is done by examine the colony under microscope.

MATERIALS:
1.Long handled inoculating needle
2.two dissecting needles
3.Lactophenol
4.Clean microscopic slide and cover slip
5.Clear fingernail polish

PROCEDURE:

Place a small drop of lactophenol cotton blue (mounting medium) on a clean

microscopic slide.

Remove aseptically a small portion of growth midway between the colony center and
edge. Place the removed colony on a drop of lactophenol cotton blue on a slide

Tease the fungus using a pair of dissecting needles so as to have a thin spread out

Gently place a cover slip at the edge of the drop of mounting fluid.
Avoid trapping air bubbles. Excess of lactophenol can by wiped out using a blotting
paper

For preserving the mount, seal the edges of coverslip with nail polish/varnish

Examine the slide under the microscope using 10x objective lens, then 40x objective
lens.

Name Signature
Prepared by Dr. Ananya Verma
Checked by Dr. Suneel Kumar Ahirwar
Approved by Dr. Anita Mutha
 STANDARD OPERATING PROCEDURE
SOP NO. : 5 Date of issue:
Procedure for isolation Effective date :
Department of and identification of Page 8 of 10
Microbiology dermatophytes

4. SLIDE CULTURE:

Whenever it is difficult to identify moulds with tease mounts, slide cultures can be put
up. Nutritionally deficient media like corn meal agar is good for enhancing sporulation.

PROCEDURE:
Aseptically cut 1 cm square agar blocks from CMA

Transfer the agar block on to the slide

Transfer very small amount of colony to the four sides of the agar block

With sterile forceps, place the coverslip on the inoculated agar block

Add 1 – 1.5 mL, of sterile water to the petri dish; humid atmosphere does not allow the
agar block to dry out
(5-20% glycerine can be added to the sterile water to prevent condensation of moisture
on the slide)

Place the slide culture in a petridish canister and incubate in the dark.
Slide culture is ready to be taken down when mature conidia or spores are observed.

“Taking – down” the slide culture:

 Take a small drop of mounting medium (LCB) on a microscopic slide.

 With the forceps, carefully remove the coverslip from the agar block. Do not push
or pull the coverslip.
  Pass the coverslip through the blue portion of flame very quickly. This will heat
fix the fungus and its spores (overheating can result in collapse of the hyphae).
 Carefully place the coverslip on the mounting medium so as to avoid trapping any
air bubbles.
 Wipe off the excess of mounting medium. Seal the edges of coverslip with nail
polish.
 Second mount can also be prepared from the microscope slide of the slide culture
setup, by removing the agar block. Put a drop of lactophenol and a coverslip. The
coverslip can be sealed with nail polish.
 A drop of 95% alcohol or ethyl acetate may be used to soak the colony before
using lactophenol blue to avoid air bubble.
 The block can be dropped aseptically on a suitable media for the growth. This
would help to maintain the fungus in the laboratory.

Result:

After performing the above procedures ,most common fungal species from different
infected sites like skin , hair and nails are identified.

Name Signature
Prepared by Dr. Ananya Verma
Checked by Dr. Suneel Kumar Ahirwar
Approved by Dr. Anita Mutha