SABOURAUD DEXTROSE AGAR

Code: CM0041

an acidic pH medium for the isolation of dermatophytes, other fungi and yeasts

Typical Formula*	gm/litre
Mycological peptone	10.0
Glucose (dextrose)	40.0
Agar	15.0
pH 5.6 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards

Directions
Add 65g to 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Mix well and pour in to sterile Petri dishes.

Description
This modification of Sabouraud agar (Carlier¹) is suitable for the cultivation and differentiation of fungi.

Carlier showed that the medium gives reliable results with Microsporum audouini, Microsporum canis, Trichophyton mentagrophytes, Trichophyton flavum, Trichophyton rubrum and Candida albicans. Sabouraud Dextrose Agar may be used in place of the Standard American medium of Hodges². The fungi maintain their typical cultural appearance and thus may be readily identified according to the standard macroscopic characters described by Sabouraud³.

The medium is often used with antibiotics for the isolation of pathogenic fungi from material containing large numbers of other fungi or bacteria.
Georg et al.⁴ aseptically added 0.5g cycloheximide, 20,000 units penicillin and 40,000 units streptomycin to each litre of autoclaved, cooled medium. Cryptococcus neoformans, Aspergillus fumigatus and Allescheria boydii are sensitive to cycloheximide; Actinomyces bovis and Nocardia asteroides are sensitive to penicillin and streptomycin.Alternatively, one may add 0.4g chloramphenicol and 0.05g cycloheximide to each litre of reconstituted medium before autoclaving (Ajello⁵). The same micro-organisms are sensitive to this new combination - see Dermasel Selective Supplement SR0075.

Williams Smith & Jones⁶ employed Oxoid Sabouraud Dextrose Agar, containing 20,000 units penicillin and 0.04g neomycin per litre, for the count of yeasts in the alimentary tract of the pig. Hantschke⁷ used colistin, novobiocin and cycloheximide to isolate Candida albicans. Dolan⁸ used gentamicin, chloramphenicol and cycloheximide for the selective isolation of pathogenic fungi.

Oxoid Sabouraud Dextrose Agar may also be used as the basis of a Pagano-Levin medium⁹ for the isolation of Candida albicans. 0.1g of triphenyltetrazolium chloride (as a filter sterilised solution) is added to each litre of autoclaved molten medium cooled to 55°C. The medium is usually made inhibitory to most non-pathogenic fungi and bacteria by the addition of antibiotics as above. After incubation for 3 days at 25°C, Candida albicans colonies are unpigmented or pale pink whilst other Candida species and other fungi form deeper pink or red colonies. The test is adequate for screening purposes but other diagnostic criteria should also be utilised for the identification of Candida albicans^10,11,12,13.

Technique

1. Inoculate each specimen in duplicate.
2. Incubate one set of media aerobically at 22-25°C and the other set at 35°C for 5-30 days. Loosen the caps of tubes and ensure adequate moisture for the plates to compensate for loss of water vapour. DO NOT SEAL THE PLATES.
3. Examine every 2-4 days.
4. Describe each specific type of colony morphology and sub-culture to appropriate media for further identification tests.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder.
Prepared medium: Light straw to straw coloured gel.

Quality Control

Positive controls:	Expected results
Candida albicans ATCC® 10231*	Good growth; cream colonies
Aspergillus brasiliensis ATCC® 16404 *	White mycelium; black spores
Negative control:
Uninoculated medium	No change

* This organism is available as a Culti-Loop®

Precautions
Some of the pathogenic fungi may produce infective spores which are are easily dispersed into the laboratory. Such organisms should be examined only within a protective cabinet.

The combination of cycloheximide and chloramphenicol inhibits many pathogenic fungi⁴. However, the mycelial phase of Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schoenckii and Blastomyces dermatitidis is not inhibited by these antibiotics when incubated at 25-30°C ¹⁴.

Please check relevant health and safety documentation before working with cyclohexamide