Introduction
Sabouraud agar or Sabouraud dextrose agar (SDA) is a selective medium for fungal culture and primarily used for the isolation of Dermatophytes, Yeasts and various other Pathogenic and Non- pathogenic fungi as well as it also supports the growth of Nocardia spp. and other filamentous bacteria. Moreover, the slightly acidic pH of this SDA medium inhibits the growth of most of the bacteria and permits the growth of almost all the fungi with various modifications applicable.
It was created by, and is named after, Raymond Sabouraud in 1892. The formulation was later adjusted by Chester W. Emmons when the pH level was brought closer to the neutral range and the dextrose concentration lowered to support the growth of other microorganisms. The acidic pH (5.6) of traditional Sabouraud agar inhibits bacterial growth.
Principle
Fungi, in contrast with bacteria, are routinely cultured on a solid medium i.e. Sabouraud Dextrose Agar Medium (SDA) to obtain the discrete colonies of the Fungi present in the specimen or to get the information about cultural characteristics of Fungi on a solid medium, colony morphology and patterns of growth etc.
The three essential components of the SDA medium, which confer the growth of fungi are peptone, dextrose (glucose) and pH. The mycological peptone (a mixture of animal and plant peptones) provides a nourishing source of amino acids and nitrogenous compounds while dextrose acts as a source of carbon and energy. Both of these components help the rapid growth of the fungi. Whereas, agar (solidifying agent) aids to get morphological details of the colony. The components mentioned above can be modified by adding the various substances as per the requirements of the fungi.
The final pH of the medium is adjusted to 5.4 – 5.8, preferably the 5.6 at 25 °C. pH plays an essential role in the selective growth of fungi. pH 5.6 is preferred for composing culture media in the pre-antibiotic era to minimise bacterial contamination. At pH ~5.6, most types of bacterial growth is inhibited. However, bacteria that can thrive in an acidic environment manage to grow on this media. Hence, besides the pH, broad-spectrum antibiotics (e.g. chloramphenicol, tetracycline and Gentamycin) are used to augment the antibacterial effect. Percentage of glucose used in the media also helps in minimising bacterial contamination. A high percentage of glucose leads to the vigorous growth of bacteria, which leads to higher fermentation and acid production, later inhibiting bacterial growth.
Preparation of Sabouraud Dextrose Agar (SDA)
Composition Of Sabouraud Agar
| COMPONENTS | QUANTITY (in grams) |
|---|---|
| Dextrose (Glucose) | 40 |
| Mycological Peptone | 10 |
| Agar-Agar | 15 |
Material Required
- Sterile Conical Flask / Erlenmeyer Flask
- Spatula
- Dextrose powder
- Mycological Peptone
- Agar-Agar powder
- Measuring Cylinder
- 1N HCl
- 1N NaOH
- pH Strip
- Weighing Scale
- Distilled Water
Preparation Procedure
- Weigh the quantity of Mycological Peptone, Dextrose, and Agar using the weighing scale for 1000 ml of Sabouraud Dextrose Agar (SDA).
- Take a clean and dry Conical Flask/ Erlenmeyer flask.
- Pour 500 ml of distilled water to the flask and add the weighed quantity of Mycological Peptone, and Dextrose. Mix well the content by swirling the flask.
- Now add the weighed quantity of Agar-Agar.
- Mix well the content and Heat it with continuous agitation to dissolve the constituents.
- Now add more distilled water to the medium and make the volume 1000 ml.
- Check the pH of the solution using pH strip, it should be 5.6 ±2. If required, adjust the pH by adding either 1N HCl (acid) or 1N NaOH (base) as per the case.
- Mix well the content and apply the Non-absorbent cotton plug to the flask.
- Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.
- Allow the content to cool down to 40-45 °C and pour in the empty media plates under a strict aseptic atmosphere (preferably in Laminar Air Flow) and allowed it to cool at room temperature.
- Use the prepared media plates to inoculate the specimen to be cultured and then place in the incubator at optimum temperature.
Sabouraud Dextrose Agar Result Interpretation
After sufficient incubation, SDA plates should show isolated colonies in streaked areas and confluent growth in areas of heavy inoculation. Examine plates for fungal colonies exhibiting typical color and morphology. Additional procedures should be performed to confirm findings.Yeasts will grow as creamy to white colonies. Molds will grow as filamentous colonies of various colors.
Colony Characteristics
| 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 |
Limitations Of Sabouraud Dextrose Agar (SDA)
- Some strains may be encountered that grow poorly or fail to grow on this medium.
- Apart from bacteria, certain pathogenic fungi may also be inhibited by the antimicrobial agents added into the medium.
- For identification purpose, organisms must be in pure culture.
- Morphological, biochemical, and serological tests should be performed for final identification.
- Use of protective cabinets is recommended while handling the samples with pathogenic fungi to avoid the spread of spores.
- For identification, organisms must be in pure culture.
Uses of Sabouraud Dextrose Agar (SDA)
- SDA is primarily used for the selective cultivation of yeasts, molds and aciduric bacteria.
- The medium is often used with antibiotics for the isolation of pathogenic fungi from material containing large numbers of other fungi or bacteria.
- It is also used for the recovery and total counting of yeasts and moulds in environmental monitoring.
- This medium is also employed to determine microbial contamination in food, cosmetics, and clinical specimens.