Attuluri Vamsi Kumar, Assistant Professor, Division: MTL, SMAS, Galgotias University, UP.

1. Fungal culture media

2. Experiment No 2.
Prepare routine culture used in mycology laboratory

3. Sabouraud Dextrose Agar (SDA)
1. Aim,
2. Introduction,
3. Principle,
4. Materials required,
5. composition,
6. Procedure
7. Results interpretation (i.e: colony morphology)
8. Clinical significance
9. Limitations for SDA

4. Aim
• To prepare Sabouraud Dextrose agar media for isolation of
clinical fungus.

5. Introduction
• Sabouraud Dextrose Agar (SDA) is used for
the isolation, cultivation, and maintenance of
non-pathogenic and pathogenic species
of fungi and yeasts.
• SDA was formulated by Sabouraud in 1892
for culturing dermatophytes.
• The pH is adjusted to approximately 5.6 in
order to enhance the growth of fungi,
especially dermatophytes, and to slightly
inhibit bacterial growth in clinical specimens.

6. Principle
The SDA media is comprised of enzymatic digest of casein (BHI) and animal tissues which
provide a nutritious source of amino acids and nitrogenous compounds for the growth of fungi
and yeasts.
Dextrose is the fermentable carbohydrate incorporated in high concentration as a carbon and
energy source.
Agar is the solidifying agent.
The addition of antibiotics like Chloramphenicol and/or tetracycline acts as broad-spectrum
antimicrobials to inhibit the growth of a wide range of gram-positive and gram-negative bacteria.
Cyclohexamide is added to further inhibit the growth of gram-negative bacteria.

7. According to textbook

8. Materials Required
1. SDA agar
2. Petri dishes / test tubes
3. Autoclave
4. Weighing machine
5. Incubator
6. Buffers HCL, NaOH to adjust pH to 5.6
7. Other PPEs

9. Composition of SDA

10. Procedure for Preparation of Sabouraud
Agar
1.Suspend 65 gm of the medium in one liter of purified water.
2.Heat with frequent agitation and boil for one minute to
completely dissolve the medium.
3. Autoclave at 121° C for 15 minutes.
4.Cool to 45 to 50°C and pour into Petri dishes or tubes for
slants.
5.For the processing of specimens, streak the specimen onto the
medium with a sterile inoculating loop in order to obtain
isolated colonies.
6.Incubate the plates at 25 – 30°C in an inverted position (agar
side up) with increased humidity.
7.Cultures should be examined at least weekly for fungal growth
and should be held for 4 – 6 weeks before being reported as
negative.

11. Result and interpretation
• After sufficient incubation, SDA plates should show isolated colonies
in streaked areas and confluent growth in areas of heavy inoculation.
Examine plates for fungal colonies exhibiting typical color and
morphology. Additional procedures should be performed to confirm
findings.
• Yeasts will grow as creamy to white colonies. Molds will grow as
filamentous colonies of various colors.

12. Results & interpretation
Fungi Colony morphology
1 Aspergillus flavus Yellow-green, powdery and pale yellowish on reverse
2 Aspergillus niger
The initial growth is white, becoming black later on giving “salt and
pepper appearance” which results from darkly pigmented conidia
borne in large numbers on conidiophores and reverse turning pale
yellow
3 aspergillus fumigatus Blue – green, powdery and pale yellow on reverse .
4 Trichosporon mucoides White to cream, yellowish, wrinkled

13. Limitations of Sabouraud Agar
1.It does not promote the conidiation of filamentous fungi.
2.Antimicrobial agents added into a medium to inhibit bacteria
may also inhibit certain pathogenic fungi.
3.Avoid overheating a medium with an acidic pH; this may result
in a soft medium.