Sabouraud Dextrose

Intended Use

Sabouraud Dextrose Agar is used in qualitative procedures for cultivation of pathogenic and nonpathogenic fungi, particularly dermatophytes. The medium is rendered more selective for fungi by the addition of antimicrobics. Sabouraud Dextrose Broth and Sabouraud Maltose Agar and Broth are also used for culturing yeasts, molds and aciduric microorganisms.

Sterile Pack RODAC™ environmental sampling plates, containing Sabouraud Dextrose Agar with Lecithin and Polysorbate 80, are used for the detection and enumeration of microorganisms present on surfaces of sanitary importance.

Sterile Pack plates are particularly useful for monitoring surfaces in clean rooms and other environmentally-controlled areas and are also recommended for use in air sampling equipment, such as the Surface Air System.

Sterile Pack Finger Dab™ Isolator plates are intended for sampling gloved hands.

Sabouraud Dextrose Agar and Sabouraud Dextrose Broth meet United States Pharmacopeia (USP), European Pharmacopoeia (EP) and Japanese Pharmacopoeia (JP)^1-3 performance specifications, where applicable.

Summary and Explanation

Sabouraud Dextrose Agar is a general-purpose medium devised by Sabouraud for the cultivation of dermatophytes.⁴ The low pH of approximately 5.6 is favorable for the growth of fungi, especially dermatophytes, and slightly inhibitory to contaminating bacteria in clinical specimens.^5-8 Sabouraud Dextrose Agar is also recommended for the testing of cosmetics⁹ and food.^10,11 General Chapters and of the USP describe test methods for using Sabouraud Dextrose Agar when performing the microbial enumeration tests and tests for isolating Candida albicans from nonsterile pharmaceutical products.¹

The addition of antimicrobics is a modification designed to increase bacterial inhibition.

Sabouraud Dextrose Agar is also available in RODAC™ (Replicate Organism Detection and Counting) environmental sampling plates. These plates are specially constructed so that an agar medium can be over-filled, producing a meniscus or dome-shaped surface that can be pressed onto a surface for sampling its microbial burden. RODAC plates are used in a variety of programs to establish and monitor cleaning techniques and schedules.12-16 After touching the surface to be sampled with the medium, the environmental sampling dish is covered and incubated at an appropriate temperature. The presence and number of microorganisms is determined by the appearance of colonies on the surface of the agar medium. Collection of samples from the same area before and after cleaning and treatment with a disinfectant permits the evaluation of the efficacy of sanitary procedures. The RODAC SL (secure lid) has three lugs on the base, providing a tight fit between lid and base to reduce accidental contamination.

Sabouraud Maltose Agar is a modification of Sabouraud Dextrose Agar with maltose substituted for the dextrose. It is a selective medium due to the acid pH. Davidson et al. reported that Sabouraud Maltose Agar was a satisfactory medium in their studies of infections caused by Microsporum audouini, M. lanosum and Trichophyton gypseum.¹⁷ Davidson and Dowding also used this medium in isolating T. gypseum from a case of tinea barbae.¹⁸

Sabouraud Dextrose Broth is used for culturing yeasts and molds in cosmetics.⁹ General Chapter of the USP recommends the use of Sabouraud Dextrose Broth when isolating Candida albicans from nonsterile pharmaceutical products.¹

Sabouraud Maltose Broth is a modification of Sabouraud Dextrose Broth in which maltose is substituted for dextrose. It is selective due to its acid pH and is used for the detection of fungi.

Principles of the Procedure

Sabouraud dextrose media are peptone media supplemented with dextrose to support the growth of fungi. Sabouraud agar is also available with maltose substituted for the dextrose. Peptones are sources of nitrogenous growth factors. The carbohydrate provides an energy source for the growth of microorganisms. Gentamicin is an aminoglycoside antibiotic that inhibits the growth of gram-negative bacteria. Chloramphenicol is inhibitory to a wide range of gram-negative and gram-positive bacteria, and cycloheximide is an antifungal agent that is primarily active against saprophytic fungi and does not inhibit yeasts or dermatophytes.¹⁹

Lecithin neutralizes quaternary ammonium compounds, and polysorbate 80 neutralizes substituted phenolic disinfectants. ^10,20-22

For the Sterile Pack products, the entire double-bagged product is subjected to a sterilizing dose of gamma radiation, thus the contents inside the outer bag are sterile.²³ This allows the inner bag to be aseptically removed and brought into an environmentallycontrolled area without introducing contaminants. A third sterile bag is included as a transport device. Since the agar medium has been sterilized after packaging, the presence of microbial growth after sampling and incubation can be relied upon to represent the presence of environmental contaminants and not pre-existing microorganisms in the medium that may have been introduced during manufacture. The RODAC plates have a marked grid to facilitate counting organisms. The Sterile Pack Finger Dab Isolator plates are triple-bagged and are intended for sampling gloved hands.

User Quality Control

Identity Specifications
Difco™ Sabouraud Dextrose Agar
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution:                       6.5% solution, soluble in purified water upon
                                   boiling. Solution is light to medium amber, very
                                   slightly to slightly opalescent.
Prepared Appearance:    Light to medium amber, slightly opalescent.
Reaction of 6.5%
Solution at 25°C:           pH 5.6 ± 0.2
Difco™ Sabouraud Dextrose Broth
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution:                       3.0% solution, soluble in purified water. Solution
                                    is light amber, clear.
Prepared Appearance:     Light amber, clear.
Reaction of 3.0%
Solution at 25°C:            pH 5.6 ± 0.2
BBL™ Sabouraud Dextrose Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous material,
                                    may contain a large number of minute to small
                                    tan specks.
Solution:                        6.5% solution, soluble in purified water upon
                                    boiling. Solution is pale to medium, yellow to
                                    tan, clear to slightly hazy.
Prepared Appearance:     Pale to medium, yellow to tan, clear to slightly hazy.
Reaction of 6.5%
Solution at 25°C:            pH 5.6 ± 0.2
Cultural Response
Difco™ Sabouraud Dextrose Agar
Prepare the medium per label directions. Inoculate and incubate at 30± 2°C for 18-48 hours, or up to 7 days for Trichophyton. Incubate (*) cultures at 20-25°C for up to 5 days. Incubate (**) culture at 30-35°C for 48 hours.

Difco™ Sabouraud Dextrose Broth
Prepare the medium per label directions. Inoculate tubes and incubate at 30± 2°C for 18-48 hours or up to 7 days if necessary. For (*) culture inoculate a 125 mL bottle and incubate at 30-35°C for 48 hours. For (**) culture inoculate a 125 mL bottle and incubate at 20-25°C for 3 days.

BBL™ Sabouraud Dextrose Agar
Prepare the medium per label directions. Inoculate and incubate at 25 ± 2°C for 7 days. Incubate (*) cultures at 20-25°C for up to 5 days. Incubate (**) culture at 30-35°C for 48 hours.

Formula

Difco™ Sabouraud Dextrose Agar
Approximate Formula* Per Liter
Peptic Digest of Animal Tissue........................................ 5.0 g
Pancreatic Digest of Casein............................................ 5.0 g
Dextrose.................................................................... 40.0 g
Agar.......................................................................... 15.0 g
BBL™ Sabouraud Dextrose Agar
Approximate Formula* Per Liter
Pancreatic Digest of Casein............................................ 5.0 g
Peptic Digest of Animal Tissue........................................ 5.0 g
Dextrose.................................................................... 40.0 g
Agar.......................................................................... 15.0 g
Difco™ Sabouraud Dextrose Broth
Approximate Formula* Per Liter
Peptic Digest of Animal Tissue........................................ 5.0 g
Pancreatic Digest of Casein............................................ 5.0 g
Dextrose.................................................................... 20.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend/dissolve the powder in 1 L of purified water:
     Difco™ Sabouraud Dextrose Agar – 65 g;
     BBL™ Sabouraud Dextrose Agar – 65 g;
     Difco™ Sabouraud Dextrose Broth – 30 g;
     Mix thoroughly.
2. Heat the agar media with frequent agitation and boil for 1 minute to completely dissolve the powder. Avoid overheating which could cause a softer medium.
3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using stable, typical control cultures.

Sample Collection and Handling

For clinical specimens, refer to laboratory procedures for details on specimen collection and handling.^6-8
For cosmetic, food or environmental monitoring samples, follow appropriate standard methods for details on sample collection and preparation according to sample type and geographic location. ^9-12
For pharmaceutical samples, refer to the USP for details on sample collection and preparation for testing of nonsterile products.¹

Procedure

For clinical specimens, refer to appropriate standard references for details on testing protocol to obtain isolated colonies from specimens using Sabouraud Dextrose Agar and Sabouraud Dextrose Broth.^6-8
For cosmetic, food or environmental monitoring samples, refer to appropriate standard references for details on test methods using Sabouraud Dextrose Agar or Sabouraud Dextrose Broth.^9-12
For pharmaceutical samples, refer to USP General Chapters and for details on the examination of nonsterile products and performing microbial enumeration tests and the isolation of Candida albicans using Sabouraud Dextrose Agar and Sabouraud Dextrose Broth.¹

For isolation of fungi from potentially contaminated specimens, a selective medium should be inoculated along with the nonselective medium. Incubate the containers at 25-30°C with increased humidity. All cultures should be examined at least weekly for fungal growth and should be held for 4-6 weeks before being reported as negative.

Liquefy the medium in pour tubes by heating in boiling water. Cool to 45-50°C and pour into sterile Petri dishes. Allow to solidify for a minimum of 30 minutes.

Prepared tubed slants primarily are intended for use with pure cultures for maintenance or other purposes. With prepared plates and Mycoflask™ bottles, streak the specimen as soon as possible after it is received in the laboratory, using a sterile inoculating loop to obtain isolated colonies. Consult appropriate references for information about the processing and inoculation of specimens.^6-8

For the Sterile Pack media, sample selected surfaces by firmly pressing the agar medium against the test area. Hold the plate with thumb and second finger and use index finger to press plate bottom firmly against surface. Pressure should be the same for every sample. Do not move plate laterally as this spreads contaminants over the agar surface making resolution of colonies difficult. Slightly curved surfaces may be sampled with a rolling motion.

Areas (walls, floors, etc.) to be assayed may be divided into sections or grids and samples taken from specific points within the grid.

Incubate exposed plates at 35-37°C for 48 hours, and 25°C for 7 days or as required.

Expected Results

After sufficient incubation, the containers should show isolated colonies in streaked areas and confluent growth in areas of heavy inoculation. Transfer of growth from slants to plated media may be required in order to obtain pure cultures of fungi.  Examine containers for fungal colonies exhibiting typical color and morphology.24 Biochemical tests and serological procedures should be performed to confirm findings.

In the RODAC procedure, colonies are counted (fewer than 200 colonies for accurate counts) and expressed as either the number of colonies per RODAC plate or the number of colonies per cm.5,10,11 Criteria for cleanliness of equipment and environment (surfaces) can be developed by using a database derived from repeated routine sampling of specific sites.²⁵

Subculture colonies of interest so that positive identification can be made by means of biochemical testing and/or microscopic examination of organism smears.

Limitations of the Procedure

Some fungi may be inhibited by the acidic pH of the medium and by the antimicrobics in the selective media.^5-7

*Store at 2-8° C.

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