Sabouraud Dextrose Agar (SDA) Composition, Preparation, Principle, Uses, Results

What is Sabouraud Dextrose Agar (SDA)?

• Sabouraud Dextrose Agar (SDA) is a selective medium formulated by Raymond J. A. Sabouraud in 1892 for the cultivation of fungi, particularly those associated with skin infections. It is primarily used for the isolation and identification of dermatophytes, yeasts, and various pathogenic and non-pathogenic fungi.
• The medium consists of peptones derived from meat and casein, which provide nitrogenous compounds necessary for fungal growth. Dextrose monohydrate serves as the energy source, and the high concentration of dextrose combined with the low pH (approximately 5.6) creates an environment favorable for fungal growth while inhibiting the growth of bacteria.
• Sabouraud Dextrose Agar is commonly referred to as “Sabouraud’s dextrose agar” due to the preference of using glucose (dextrose) instead of maltose in the original formulation. This adjustment allows for a higher osmotic stability and better growth of fungi while most bacteria cannot tolerate the high sugar concentration.
• SDA is widely used in clinical laboratories to isolate and culture fungi responsible for skin, hair, and nail infections, such as dermatophytes and yeasts. It is also utilized in microbial limit tests for food, pharmaceuticals, cosmetics, and other clinical specimens.
• In some variations of SDA, selective agents like chloramphenicol, gentamicin, or tetracycline may be added to inhibit the overgrowth of bacteria and promote the successful isolation of fungi and yeasts.
• When working with heavily contaminated samples, additional inhibitory agents may be incorporated into the agar to suppress bacterial growth and facilitate the isolation of fungi.
• It is important to note that certain pathogenic fungi may produce infective spores that can be easily dispersed in the air during laboratory procedures. Therefore, examinations should be conducted in a safety cabinet or under appropriate containment measures to prevent the spread of infectious spores.

Purpose of Sabouraud Dextrose Agar (SDA)

The purpose of Sabouraud Dextrose Agar (SDA) can be understood based on its historical development and selective properties. The key purposes of SDA are as follows:

1. Culturing Dermatophytes and Fungi: Sabouraud agar was originally developed to support the cultivation of dermatophytes, which are fungi that cause infections of the skin, hair, and nails. SDA allows for the growth of these fungi, which often require longer incubation periods of several weeks.
2. Inhibition of Bacterial Growth: Sabouraud agar serves as a selective medium by inhibiting the growth of bacteria while promoting the growth of fungi. The acidic nature of the medium, with a pH of approximately 5.6, was initially used to prevent bacterial contamination. Over time, the addition of antibiotics to the acidic medium became common practice to further inhibit bacterial growth, as well as the growth of certain saprophytic fungi, depending on the specific antibiotics used.
3. Reliable Fungal Identification: Sabouraud agar was designed to yield consistent and reliable results for fungal identification across different laboratories. By providing an environment conducive to fungal growth and inhibitory conditions for bacteria, Sabouraud agar standardizes the observations and reduces differences in appearance that could potentially lead to identification errors.
4. Promotion of Fermentation and Acid Production: The presence of a high concentration of glucose (4%) in Sabouraud’s formulation serves to support vigorous fermentation by any bacteria present. This fermentation leads to the production of acid, which further inhibits bacterial growth over time.

Overall, the purpose of Sabouraud Dextrose Agar (SDA) is to provide a selective medium that allows for the growth of fungi, particularly dermatophytes, while inhibiting the growth of bacteria. It was developed to prevent bacterial contamination, enable reliable fungal identification, and promote the fermentation and acid production that eventually inhibits bacterial growth.

Different types of Sabouraud agar

There are different types of Sabouraud agar formulations designed for specific purposes. Here are a few examples:

1. Sabouraud Agar with Antibiotics: This variation of Sabouraud agar incorporates antibiotics to inhibit bacterial growth and target specific fungal groups. Commonly used antibiotics include gentamicin, chloramphenicol, and cycloheximide. Gentamicin targets gram-negative bacteria, chloramphenicol inhibits a wide range of fungi including pathogenic fungi, and cycloheximide primarily targets saprophytic fungi. The addition of antibiotics is done after autoclaving and cooling the medium to approximately 45-50°C.
2. Sabouraud Agar with Methyl Blue: Sabouraud Methyl Blue Agar contains 0.01% methyl blue along with the standard components of Sabouraud Agar. This modification is used to distinguish between Candida albicans and Candida dubliniensis based on the yellow fluorescence exhibited by C. albicans under a Woods lamp.
3. Emmon’s Modification of Sabouraud Agar: In 1977, Emmons developed an alternative formulation of Sabouraud’s agar. This modification involves adjusting the pH and reducing the dextrose concentration, resulting in a neutral pH medium. This modification aims to promote the recovery of certain pathogenic fungi that may not grow optimally under the standard acidic conditions of Sabouraud agar.
4. Sabouraud Broth: Sabouraud Broth has the same composition as Sabouraud Agar but does not contain agar. It is a liquid medium used for the cultivation of fungi, particularly for yeast and mold species. It provides a favorable environment for fungal growth without the solidifying properties of agar.

These different types of Sabouraud agar formulations allow for specific applications and provide variations in selectivity, pH, and growth promotion of different fungal species. The choice of formulation depends on the specific needs of the laboratory and the target fungi being cultured.

Principle of Sabouraud Dextrose Agar (SDA)

The principle of Sabouraud Dextrose Agar (SDA) can be understood based on its composition and the specific conditions it provides for fungal growth. The key principles of SDA are as follows:

1. Nutrient Source: SDA contains peptones derived from animal tissues, which serve as a source of amino acids and nitrogenous compounds, providing nutrition for the growth of fungi and yeasts.
2. Energy and Carbon Source: Dextrose (glucose) is added to SDA as the energy and carbon source for fungal metabolism and growth.
3. Solidifying Agent: Agar is included as the solidifying agent in SDA, allowing the medium to solidify and provide a suitable surface for fungal growth.
4. Antimicrobial Agents: SDA may incorporate broad-spectrum antimicrobials such as chloramphenicol, tetracycline, and gentamicin. These antimicrobials inhibit the growth of a wide range of gram-positive and gram-negative bacteria, preventing their overgrowth and allowing fungal colonies to develop.
5. pH Adjustment: The pH of SDA is adjusted to approximately 5.6. This slightly acidic pH enhances the growth of fungi, especially dermatophytes, while inhibiting the growth of bacteria present in clinical specimens.
6. Fungal Growth Promotion: The combination of high dextrose concentration, low pH, and inhibitory antimicrobials creates an environment that favors fungal growth and suppresses the growth of contaminating bacteria from test samples.
7. Ingredient Variations: SDA can vary slightly in terms of peptone sources, which can influence the growth and morphology of fungal colonies. The peptones used in SDA, such as pancreatic digests of casein, contribute to the variability in pigmentation and sporulation observed in different formulations of SDA.

In summary, Sabouraud Dextrose Agar (SDA) provides a nutrient-rich environment with dextrose as the energy source, solidified by agar. The addition of antimicrobial agents and adjustment of pH promote the growth of fungi, particularly dermatophytes, while inhibiting bacterial growth. The variability in peptone sources can lead to slight differences in fungal morphology and pigmentation. SDA serves as a selective medium for the isolation and cultivation of various fungi and is widely used in clinical laboratories for diagnostic purposes.

Composition of Sabouraud Dextrose Agar (SDA)

Ingredients	Gms/Liter
Dextrose	40.0
Peptone	10.0
Agar	15.0

Final pH ( at 25°C) 5.6±0.2

In addition,

• Sabouraud Dextrose Broth is the same formula as above but without agar. Final pH 5.6 +/– 0.2 at 25oC
• Sabouraud Dextrose agar with Chloramphenicol has 50.0 mg chloramphenicol. Final pH 5.6 +/– 0.3 at 25oC
• Sabouraud Dextrose agar with Chloramphenicol & Gentamicin contains 50.0mg of chloramphenicol, and 5.0mg gentamicin. Final pH at 25°C: 5.6 +/– 0.3
• Sabouraud Dextrose agar with Chloramphenicol & Tetracycline has 50.0 mg chloramphenicol, and 10.0 mg tetracycline. Final pH at 25oC: 5.6 +/– 0.3
• Sabouraud Dextrose Agar Emmons contains only 20.0 grams of dextrose. Final pH 6.9 +/– 0.2 at 25oC

Preparation and Method of Use of Sabouraud Dextrose Agar (SDA)

To prepare Sabouraud Dextrose Agar (SDA), you can follow these steps:

1. Measure and Suspend: Measure 65 grams of the SDA medium and add it to one liter of purified water.
2. Heat and Dissolve: Heat the mixture with frequent agitation and boil for approximately one minute to ensure complete dissolution of the medium components.
3. Autoclave: Transfer the mixture to autoclave-safe containers and autoclave at 121°C for 15 minutes. This process ensures the sterilization of the SDA medium, eliminating any potential contaminants.
4. Cooling: Allow the sterilized SDA medium to cool down to a temperature of approximately 45 to 50°C.
5. Pouring into Petri Dishes or Tubes: Once the SDA medium has cooled to the desired temperature, pour it into sterile Petri dishes or tubes for slants. Ensure aseptic technique during this step to prevent contamination.
6. Specimen Processing: To process specimens, streak the specimen onto the SDA medium using a sterile inoculating loop. This technique helps to obtain isolated colonies for further analysis and identification.
7. Incubation: Place the inoculated plates in an inverted position (agar side up) in an incubator set at a temperature of 25 to 30°C. Maintain increased humidity in the incubator, which can be achieved by using humidifying devices or placing a water-filled tray inside the incubator.
8. Examination and Reporting: Check the SDA plates weekly for the presence of fungal growth. Fungal colonies may take time to develop, so it is recommended to hold the cultures for 4 to 6 weeks before reporting them as negative for fungal

Result Interpretation of Sabouraud Dextrose Agar (SDA)

The interpretation of results on Sabouraud Dextrose Agar (SDA) involves examining the colonies that have grown on the medium. Here are the key points to consider:

1. Isolated Colonies: On SDA plates, isolated colonies should be observed in the streaked areas. These colonies indicate the growth of individual fungal organisms. They may vary in size, shape, and color, depending on the specific fungal species.
2. Confluent Growth: In areas of heavy inoculation, you may observe confluent growth, where the fungal colonies merge together to form a continuous layer. This can occur when the initial inoculum is dense or when the growth conditions are optimal for fungal proliferation.
3. Color and Morphology: Fungal colonies on SDA exhibit distinct color and morphology characteristics. Yeasts typically appear as creamy to white colonies, while molds form filamentous colonies that can vary in color. Molds may display a wide range of colors, including green, blue, brown, or black, depending on the species.
4. Confirmatory Procedures: While the color and morphology of colonies on SDA can provide initial clues about the fungal species present, additional procedures are usually required for accurate identification. These may include microscopic examination of fungal structures, biochemical tests, or molecular methods.

It is important to note that the interpretation of results on SDA should be performed by trained personnel familiar with fungal morphology and identification. Specialized techniques and additional media may be necessary to further characterize the isolated colonies and confirm the presence of specific fungal species.

Colony morphology of some fungi on SDA

Fungi	Colony morphology
Candida albicans	Pasty opaque slightly domed, smooth, and cream or white colonies
Aspergillus flavus	Yellow-green powdery on front and pale yellowish on reverse
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
Aspergillus fumigatus	Bluish green powdery colonies on front  and pale yellow on reverse .
Trichosporon mucoides	White to cream, yellowish, wrinkled
Geotrichum candidum	White to cream colored, flat with aerial mycelium

Quality Control of SDA

Quality control of Sabouraud Dextrose Agar (SDA) involves assessing various aspects of the medium to ensure its effectiveness and reliability. Here is the information on the quality control parameters:

1. Appearance: The prepared SDA medium should have a cream to yellow color and a granular texture.
2. Gelling: The medium should form a firm gel comparable to a 1.5% agar gel.
3. Colour and Clarity: The gel in the Petri plates should be light amber in color and clear to slightly opalescent.
4. pH: The pH of a 6.14% w/v aqueous solution of SDA, after sterilization, should be approximately 5.6 ± 0.2.
5. Growth Promotion Test: The growth promotion test is performed according to the method specified in the relevant pharmacopoeia (IP). It involves incubating the medium at 30-35°C for 24-48 hours to assess the recovery rate of bacteria and fungi.
6. Growth Promoting Properties: The growth of microorganisms on the SDA medium should be comparable to that obtained with a previously tested and approved lot of the medium. The inoculated colonies should exhibit growth and indicative reactions within the specified incubation temperature and period.
7. Cultural Response: Various microorganisms are used for additional microbiological testing to evaluate the cultural response of SDA. The growth and recovery of specific organisms, such as Candida albicans, Aspergillus brasiliensis, Saccharomyces cerevisiae, Escherichia coli, Trichophyton rubrum, and Lactobacillus casei, are assessed based on the incubation temperature, incubation period, and the percentage of growth observed.

These quality control measures ensure that the SDA medium performs as expected and provides optimal conditions for the growth of specific microorganisms. By assessing its appearance, gelling, pH, and the growth response of indicator organisms, the quality and reliability of SDA can be confirmed.

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

Modifications of Sabouraud Agar

One modification of Sabouraud Agar is SabHI Agar. SabHI Agar is formulated by combining Sabouraud Dextrose Agar (SDA) and Brain Heart Infusion Agar (BHIA). This modification is designed to enhance the recovery of pathogenic fungi compared to using either medium individually.

The combination of SDA and BHIA provides a nutrient-rich environment that supports the growth of a wide range of fungi, including pathogenic species. SDA provides the selective properties and low pH that favor fungal growth, while BHIA contributes additional nutrients and growth factors from the infusion of brain and heart tissues.

The use of SabHI Agar can improve the recovery and isolation of pathogenic fungi from clinical specimens or other sources. It offers a more comprehensive and supportive medium for the growth and identification of a broader spectrum of fungal pathogens compared to using SDA or BHIA alone.

By combining the strengths of both SDA and BHIA, SabHI Agar provides a versatile and effective option for the cultivation and identification of pathogenic fungi in various laboratory settings.

Precautions

When working with Sabouraud Agar or any fungal cultures, it is important to take certain precautions. Here are the key precautions to consider:

1. Growth Factors for Trichophyton Species: Some Trichophyton species may require additional growth factors, such as thiamine, inositol, or nicotinic acid, to grow optimally. Therefore, their growth on Sabouraud Agar may be limited or absent.
2. Terminology: Sabouraud Agar may be referred to as Sabouraud’s Dextrose Agar or Sabouraud’s Agar. These terms are used interchangeably.
3. Mold Morphology: When observing mold growth on Sabouraud Agar, it is important to examine both the top (obverse) and bottom (reverse) surfaces. Different molds can exhibit variations in appearance on each surface.
4. Safe Fungi for Undergraduate Students: Certain fungi, such as Penicillium camemberti, P. roqueforti, Rhizopus stolonifer, Aspergillus species (excluding A. fumigatus and A. flavus), Saccharomyces cerevisiae, Rhodotorula rubrum, and Neurospora crassa, are considered safe for undergraduate students to handle. However, caution should still be exercised to prevent dispersion of spores.
5. Handling Plates and Opening: Wrap the plates in parafilm to securely close them and minimize the risk of spore dispersal. Plates should be incubated with the lid on the top to prevent spreading of spores upon opening. Opening culture plates should only be done within a biosafety cabinet to minimize the risk of contamination or allergic responses.
6. Biosafety Precautions: For laboratories involving environmental enrichment and handling of enriched microorganisms or cultures, appropriate biosafety precautions should be followed. This may include working at biosafety level 2, handling known infectious agents according to their biosafety level, using a biosafety cabinet for manipulation of cultures with a high probability of containing infectious agents, and incorporating antifungal agents in the enrichment media when necessary.
7. Endemic Fungi: Instructors should be aware of endemic fungi capable of causing systemic infections in the region they are teaching in and should avoid environmental isolations in such cases.

By following these precautions, the risk of contamination, infection, and allergic responses can be minimized when working with Sabouraud Agar and fungal cultures. It is important to prioritize biosafety and adhere to appropriate guidelines to ensure a safe laboratory environment.

Uses of Sabouraud Dextrose Agar (SDA)

Sabouraud Dextrose Agar (SDA) has various uses in microbiology. Here are the key applications of SDA:

1. Selective Cultivation: SDA is primarily used for the selective cultivation of yeasts, molds, and aciduric bacteria. Its formulation promotes the growth of these organisms while inhibiting the growth of bacteria and other contaminants.
2. Isolation of Pathogenic Fungi: SDA is commonly used in combination with antibiotics for the isolation of pathogenic fungi from samples that contain a large number of other fungi or bacteria. The addition of antibiotics helps suppress the growth of unwanted microorganisms, allowing the targeted pathogenic fungi to thrive and be isolated.
3. Microbial Contamination Testing: SDA is employed to determine the presence of microbial contamination in various substances, including food, cosmetics, and clinical specimens. The medium provides a favorable environment for the growth of fungi, allowing for the detection and enumeration of fungal contaminants.

It’s important to note that SDA is not only used in clinical and research settings but also plays a role in quality control and monitoring processes in industries such as food production and cosmetic manufacturing. By providing a suitable medium for the growth of yeasts, molds, and aciduric bacteria, SDA enables the detection and assessment of microbial contamination in these products.

Overall, the primary uses of Sabouraud Dextrose Agar (SDA) include the selective cultivation of yeasts, molds, and aciduric bacteria, the isolation of pathogenic fungi, and the assessment of microbial contamination in various samples. Its versatility and effectiveness make it a valuable tool in microbiology laboratories and quality control settings.

Limitations of Sabouraud Dextrose Agar (SDA)

Sabouraud Dextrose Agar (SDA) has certain limitations that should be taken into consideration. Here are the key limitations:

• Poor Growth of Certain Strains: Some fungal strains may exhibit poor growth or fail to grow on SDA. This can be due to variations in nutritional requirements or other factors specific to those strains.
• Inhibitory Effect on Pathogenic Fungi: The addition of antimicrobial agents, such as antibiotics, to inhibit bacterial growth in SDA can also have an inhibitory effect on certain pathogenic fungi. This can lead to incomplete or restricted growth of specific fungal species.
• Avoid Overheating: Overheating a medium with an acidic pH, such as SDA, can result in a soft medium consistency. This can affect the physical properties of the agar and impact its ability to support fungal growth.
• Pure Culture Requirement: For accurate identification, organisms must be isolated in pure culture. Mixed or contaminated cultures can hinder the interpretation of results and the accurate identification of fungal species.
• Additional Tests for Identification: While SDA provides a favorable environment for fungal growth, further morphological, biochemical, and/or serological tests are often required for the final identification of fungal isolates. These additional tests help to differentiate between closely related species or confirm the identification.
• Limited Conidiation Promotion: SDA may not promote the conidiation (formation of spores) of filamentous fungi to the same extent as other specialized media. This can limit the observation and study of certain fungal life stages and characteristics.

Understanding these limitations is important to ensure appropriate use and interpretation of SDA results. Depending on the specific requirements of the study or identification process, additional techniques or media may need to be employed to overcome these limitations and obtain accurate and comprehensive results.

FAQ

References

• https://www.himedialabs.com/eu/coasdstds/index/download/id/GMM063/source/tds/lang/EN
• https://www.thermofisher.com/order/catalog/product/CM0041T
• https://microbiologie-clinique.com/sabouraud-dextrose-agar.html
• https://sharebiology.com/sabouraud-dextrose-agar-sda/
• https://asm.org/ASM/media/Protocol-Images/Sabouraud-Agar-for-Fungal-Growth-Protocols.pdf?ext=.pdf
• https://microbeonline.com/sabouraud-dextrose-agar-sda-principle-composition-uses-colony-morphology/