Sabouraud Dextrose Agar + Chloramphenicol EP/USP/ISO

The formula is based on the European Pharmacopoeia. Dextrose is the fermentable carbohydrate providing carbon and energy. Peptone mixture provides nitrogen, vitamins, minerals and amino acids essential for growth. Bacteriological agar is the solidifying agent. The high dextrose concentration and acidic pH make this medium selective for fungi.

This medium is a modification of the Dextrose Agar described by Sabouraud, with the addition of chloramphenicol that inhibits a great majority of bacterial contaminants. Chloramphenicol is an antibiotic which is used for isolating pathogenic fungi from heavily contaminated material, as it inhibits most contaminating bacteria. It is a recommended antibiotic for use with media due to its heat stability and wide bacterial spectrum.

This medium has a lower concentration of chloramphenicol than the medium of the same name (LC-1090), making it less inhibitory to contaminant bacteria.

Sabouraud Dextrose Agar + Chloramphenicol is recommended by the European Pharmacopoeia when the total yeast and molds count (TYMC) is expected to exceed the acceptance criterion due to the bacterial growth.

Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.

Preparation

Suspend 65 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Distribute and sterilize in autoclave at 118-121 °C for 15 minutes. AVOID OVERHEATING.

According to the European Pharmacopoeia

According to the European Pharmacopoeia for the examination of TYMC in products.

Membrane filtration:

1. Prepare the sample.
2. Transfer the appropriate amount of the sample to a membrane filter.
3. Place the membrane on the surface of Sabouraud Dextrose Agar.
4. Incubate the plate of Sabouraud Dextrose Agar at 20-25 °C for 5-7 days.

Plate-count methods:

1. Prepare the sample.
2. Inoculate the plates of Sabouraud Dextrose Agar according to the pour-plate method or the surface-spread method.
3. Incubate the plates of Sabouraud Dextrose Agar at 20-25 °C for 5-7 days.
4. Select the plates corresponding to a given dilution and showing the highest number of colonies less than 50.

According to the European Pharmacopoeia for the test of Candida albicans in products:

1. Prepare the product to be examined and use 10 mL or the quantity corresponding to not less than 1 g or 1 mL to inoculate 100 mL of Sabouraud Dextrose Broth.
2. Incubate at 30-35 °C for 3-5 days.
3. Subculture on a plate of Sabouraud Dextrose Agar.
4. Incubate at 30-35 °C for 24-48 hours.
5. Growth of white colonies may indicate the presence of C. albicans. Confirm by identification tests.
6. The product complies with the test if such colonies are not present or if the confirmatory tests are negative.

According to ISO 16212

For the enumeration of yeast and molds according to ISO 16212:

1. Prepare the initial suspension from a sample of at least 1 g or 1 mL of the product. If needed, additional serial dilutions (e.g. 1:10 dilution) may be performed from the initial suspension using the same diluent (according to the expected level of contamination of the product).
2. The enumeration of the yeast and molds may be made by plate count methods and membrane filtration method.
3. Inoculate the initial suspension and/or sample dilution in Sabouraud Dextrose Agar + Chloramphenicol plates.
4. Incubate at 25 ± 2.5 °C for 3-5 days.
5. After incubation, count the colonies.

• Sabouraud R. 1892. Ann. Dermatol. Syphilol. 3:1061.
• Jarett, L., and A.C. Sonnenwirth (ed) 1980. Gradwohl's clinical laboratory methods and diagnosis, 8th ed. CV Mosby.
• Curry, A. S., J. G. Graf, and G. N. McEwen, Jr. (ed) 1993. CTFA Microbiology Guidelines. The Cosmetic, Toiletry, and Fragrance Association, Washington, D.C.
• European Pharmacopoeia 9.3
• ISO 16212:2017 Cosmetics. Microbiology. Enumeration of yeast and molds.