Preparation of fungal culture media

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) Aim, Introduction, Principle, Materials required, composition, Procedure Results interpretation ( i.e : colony morphology) Clinical significance 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 SDA agar Petri dishes / test tubes Autoclave Weighing machine Incubator Buffers HCL, NaOH to adjust pH to 5.6 Other PPEs

9. Composition of SDA

10. Procedure for Preparation of Sabouraud Agar Suspend 65 gm of the medium in one liter of purified water. Heat with frequent agitation and boil for one minute to completely dissolve the medium. Autoclave at 121° C for 15 minutes. Cool to 45 to 50°C and pour into Petri dishes or tubes for slants. For the processing of specimens, streak the specimen onto the medium with a sterile inoculating loop in order to obtain isolated colonies. Incubate the plates at 25 – 30°C in an inverted position (agar side up) with increased humidity. 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 It does not promote the conidiation of filamentous fungi. Antimicrobial agents added into a medium to inhibit bacteria may also inhibit certain pathogenic fungi. Avoid overheating a medium with an acidic pH; this may result in a soft medium.