Sabouraud Dextrose Agar (SDA): Introduction, Principle, Composition, Preparation, Testing Procedure, Result-Interpretation, Colony Characteristics, Uses, Keynotes, and SDA Footages

Introduction of Sabouraud Dextrose Agar (SDA)

Sabouraud Dextrose Agar (SDA) is a common fungal culture medium and  Sabouraud is the surname of creator Raymond Jacques Adrien Sabouraud a French physician born in Nantes. He formulated SDA in 1892 for culturing dermatophytes. SDA  is the most common fungal medium to recover the growth of fungi in Mycology. It is different from bacterial medium due to having main two properties, comparatively low pH (5.6) and high concentration of sugar. It is useful for the isolation, cultivation, and maintenance of non-pathogenic and pathogenic species of fungi ( yeasts and molds). 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.

Principle of SDA

Carlier’s modification of SDA contains ingredients like dextrose, mycological, peptone, agar, and pH 5.6. Mycological peptone provides nitrogen and vitamin source required for organisms in this medium. Dextrose provides an energy source while agar acts as a solidifying agent. High dextrose concentration and low pH favor fungal growth and inhibit contaminating bacteria from test specimens. This modification is useful for the cultivation of fungi (yeasts, molds), particularly useful for the fungi associated with skin infections. This medium is also employed to determine microbial contamination in food, cosmetics, and clinical samples.

Composition of Sabouraud Dextrose Agar (SDA)

Sabouraud  original formulation

Ingredients	Gm/Litre
Peptone	10
Glucose	40
Agar	15
Distilled Water (D/W)	1000 ml

Note:  The acidic pH of traditional Sabouraud agar inhibits bacterial growth.

Emmon’s modification

Ingredients	Gm/Litre
Neo-peptone	10
Glucose	20
Agar	20
Distilled Water (D/W	1000 ml

Note: Shifting pH towards neutral and lowered concentration of sugar of the Emmon’s modification enhances to support the growth of other microorganisms and some pathogenic fungi also, such as dermatophytes.

Carlier’s modification

Ingredients	Gms / Litre
Dextrose (Glucose)	40
Mycological, peptone	10
Agar	15
Final pH ( at 25°C)	5.6±0.2

  Note: High sugar (dextrose) concentration and low pH(5.6) favor fungal growth and inhibit contaminating bacteria from test specimens.

Modified SDA with the incorporation of antimicrobial agents

(Gentamicin, chloramphenicol, tetracycline, cycloheximide)

1. SDA with chloramphenicol contains 50.0 mg of chloramphenicol and the final pH of the medium should be 5.6 +/- 0.3 at 25ºC. Chloramphenicol inhibits a wide range of gram-positive negative bacteria.
2. SDA with chloramphenicol and gentamicin contains 50.0 mg of chloramphenicol and 5.0 mg gentamicin. The final pH of the medium should be  5.6 +/- 0.3 at 25ºC. Chloramphenicol inhibits a wide range of gram-positive negative bacteria whereas gentamicin inhibits gram-negative bacteria.
3. SDA with chloramphenicol and tetracycline contains 50.0 mg of chloramphenicol and 10.0 mg of tetracycline. The final pH of the medium should be 5.6 +/- 0.3 at 25ºC. Chloramphenicol inhibits a wide range of both gram-positive negative bacteria while tetracycline inhibits a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites.
4. SDA with cycloheximide: Cycloheximide inhibits primarily saprophytic fungi but not dermatophytes or yeasts. It is incorporated as an amount of 0.5 g/liter.

Note: Antimicrobial agents should only be added after media has been autoclaved and then cooled to 45 to 50°C. Keep all plates at 4°C until they are used, regardless of whether they contain antibiotics.

Preparation of SDA various modifications

SDA original (Sabouraud formulation)

1. Add all ingredients in nearly 900 ml of distilled water or deionized water.
2. Adjust pH to 5.6 with hydrochloric acid.
3. Then adjust the final volume to 1 liter.
4. Autoclave 20 minutes at 121°C, 15 lbs.
5. Cool to 45 to 50°C and pour into Petri plates or tubes for slants.

Note: The addition of antimicrobial agent, chloramphenicol is optional and its use depends on the user’s preference. If desired add 5 mg to the above recipe.

Emmons modification SDA

• Add all ingredients in nearly 900 ml of distilled water.
• Adjust pH to 6.8 to 7.0 with hydrochloric acid (HCl).
• Then adjust the final volume to 1 liter.
• Sterilize by autoclaving 20 minutes at 121°C, 15 lbs.
• Cool to 45 to 50°C and pour into Petri plates or tubes for slants.

Carlier’s modification of SDA

1. Suspend 65.0 grams in 1000 ml distilled water.
2. Heat to boiling to dissolve the medium completely.
3. Autoclave at 15 lbs pressure (121°C) for 15 minutes.
4. Cool to 45-50°C.
5. Mix well and pour into sterile Petri plates or test tubes.
6. Store prepared SDA plates or tubes at 2-8°C until any defects appear on them.

Requirements for SDA

• SDA
• Suspected fungal specimen
• Quality control strains ( as positive controls: Positive controls:
  Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404 while as negative control uninoculated medium)
• Incubator/s
• Biological safety cabinet (BSC)
• Inoculating loop  or wire
• Bunsen burner
• Personal protective equipment (PPE) as required

Test procedure of SDA

1. Inoculate the specimens on SDA.
2. Incubate the medium ( according to the required temperature and time depending on the nature of fungi to be recovered e.g. as you know, dimorphic fungi occur in two forms-yeast forms ( parasitic phase) and mold ( spores and filamentous form). Typically, molds are incubated at room temperature (22 to 25°C) and yeasts are incubated at 37°C if suspected of being dimorphic fungi. Incubation times will vary, from approximately 2 days for the growth of yeast colonies such as Malassezia species to 2 to 4 weeks for growth of dermatophytes ( Trichophyton, Microsporum, and Epidermophyton) or dimorphic fungi such as Histoplasma capsulatum. Indeed, the incubation time required to acquire fungal growth is one of the  diagnostic tools used to identify or confirm fungal species.)
3. Observe for fungal growth.

Result Interpretation of fungal growth on Sabouraud Dextrose Agar

• Yeasts: Creamy to white colonies
• Molds: Filamentous colonies of various color
• Positive controls: Candida albicans: Luxuriant
  (white colonies)
• Aspergillus brasiliensis : White mycelium; black spores
• Negative control:  No change in Uninoculated medium( SDA)

Uses of Sabouraud Dextrose Agar

1. SDA is recommended for the cultivation of yeasts, molds, and aciduric bacteria from clinical samples.
2. The medium with the addition of antimicrobial agents (antibiotics) makes it more selective for the isolation of pathogenic fungi from material containing large numbers of microbial load i.e. other fungi or bacteria.
3. This medium is also applicable to determine microbial contamination in food and cosmetics specimens.

Limitations of Sabouraud Dextrose Agar

1. For heavily contaminated specimens, the plate must be supplemented with inhibitory agents for inhibiting bacterial growth with lower pH.
2. Avoid overheating SDA medium during preparation with an acidic pH, this may result in a soft medium
3. SDA is not recommended as a primary isolation medium because it is insufficiently rich to recover certain fastidious pathogenic species, particularly most of the dimorphic fungi like e.g. Blastomyces dermatitidis, coccidioides immitis, Histoplasma capsulatum.
4. Some pathogenic fungi may produce infective spores which are easily dispersed in the air, so examination should be carried out in the safety cabinet.
5. It does not promote the conidiation of filamentous fungi.
6. Further biochemical or serological tests should be performed for confirmation
7. For identification, organisms must be in pure culture.
8. Antibiotics added into a medium to inhibit bacteria may also inhibit certain pathogenic fungi.

Keynotes of Sabouraud Dextrose Agar

• Colony characteristics,  microscopic structures, rate of growth, media which support the growth of the organism, and source of the specimen are very helpful for the isolation of fungi.
• A variety of biochemical tests are available for the identification of yeasts.
• Sabouraud Dextrose Broth (SDB) is the same formulation as SDA, without agar.

SDA Footages

Candida Colony Characteristics on Sabouraud dextrose agar (SDA) Demonstration

Cryptococcus Colony Morphology on Sabouraud dextrose agar (SDA) Demonstration

Candida and Cryptococcus Colony Morphology on Sabouraud dextrose agar (SDA) Demonstration

Geotricum growth on SDA

Penicillium Colony Morphology on SDA

Acremonium on SDA

Acremonium Reverse Pigment on SDA

Trichophyton mentagrophyte colony characteristics on SDA

Aspergillus niger colony characteristics on Sabouraud dextrose agar (SDA) Demonstration

Aspergillus fumigatus colony morphology on SDA

Black mould Growth on SDA

Sporothrix schenckii colony characteristics on SDA

Trichoderma growth on SDA

Further Readings on SDA

1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
2. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
3. Rippon’s JW: Medical Microbiology. The pathogenic fungi and the Pathogenic Actinomycetes. 3^rd ed 1988 Publisher WB Saunder co, Philadelphia.
4. A Text-Book of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
5. Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
6. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
7. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
8. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmons, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Francisco 1996.
9. https://www.thomassci.com/Laboratory-Supplies/Microbiological-Media/_/Sabouraud-Dextrose-Agar
10. https://en.wikipedia.org/wiki/Sabouraud_agarhttps://drfungus.org/knowledge-base/sda/
11. http://himedialabs.com/TD/M063.pdf