Sabouraud Dextrose Agar Preparation

Sabouraud dextrose agar is a mycological culture media which is used for the selective cultivation and isolation of fungi from both clinical and non-clinical samples. Sabouraud dextrose agar (SDA) is unique and different from most bacteriological culture media (used for cultivation and isolation of bacteria) because SDA is usually supplemented with antimicrobial agents particularly antibiotics such as chloramphenicol – which inhibits the growth of bacteria. Cyclohexamide is also included as a supplement in SDA during its preparation; and cycloheximide helps to inhibit the growth of saprophytic fungi, while allowing only the pathogenic fungi being sought for in the sample to grow. Sabouraud dextrose agar is generally recommended for cultivation of fungi in the microbiology laboratory. Another mycological media that performs similar function as SDA is potato dextrose agar PDA), which can also be used in place of SDA. In principle or in practice; you will see: SDA+Chloramphenicol; SDA+ Gentamicin; SDA+Chloramphenicol+Cyclohexamide; SDA+Chloramphenicol+Tetracycline et cetera. These different names of SDA shows the different formulation of SDA, and they indicate the types of antimicrobial agents that was incorporated as a supplement during its preparation by the researcher. So depending on your choice of antibiotics to use, the name of your SDA after preparation is up to you to define. In this section, the preparation of Sabouraud dextrose agar will be elaborated.

This is how Sabouraud dextrose agar looks like after preparation

Components of Sabouraud dextrose agar

The components of Sabouraud dextrose agar base required for Sabouraud dextrose agar preparation include:

  1. Agar – which is the solidifying agent
  2. Digest of soyabean meal
  3. Dextrose / glucose – which is the source of energy and carbon
  4. Peptone – which is the source of nitrogen and vitamin
  5. pH – which is usually adjusted to 7.0 at 25 °C (or 77 °F).

Additional requirements: These include antibiotics such as:

  1. Vial of chloramphenicol (5 mg 0r 50 mg)
  2. Vial of cyclohexamide (5 mg 0r 50 mg)

MATERIALS

You require these materials to prepare your Sabouraud dextrose agar: Sabouraud dextrose agar powder (usually comes in 500 g), vial of chloramphenicol (5 mg), vial of cyclohexamide (5 mg), autoclave, conical flask, measuring cylinder, beaker, stirring rod, Bunsen burner, incubator, refrigerator, wire gauze, spatula, weighing balance, timer, cotton wool, aluminium foil, distilled water, Petri dish

STEP BY STEP PROTOCOL TO PREPARE SABOURAUD DEXTROSE AGAR

  1. Weigh out 35 g of Sabouraud dextrose agar powder using the weighing balance.
  2. Suspend the 35 g of Sabouraud dextrose agar powder in 1 litre (1000 ml) of distilled water in a conical flask.
  3. Mix the solution by stirring to dissolve the agar.
  4. Bring the mixture to boil, by mild boiling of the mixture over a Bunsen burner flame. This helps to dissolve the agar completely. Monitor the boiling process closely in order to avoid charring the agar.
  5. Transfer the conical flask containing the boiled/mixed Sabouraud dextrose agar suspension to the autoclave.
  6. Sterilize the medium at 121 degrees Celsius at 15 psi (or 15 lbs of pressure) for 15 min in the autoclave.
  7. At the end of sterilization, allow the autoclave to return to normal (zero point) before opening. Otherwise the pressure built up in the autoclave will affect the quality and quantity of the prepared molten medium. More so, you may be affected by the steam from the autoclave due to the high pressure built up in the autoclave.
  8. Allow molten medium to cool to about 45-50 degrees Celsius.
  9. Add the required concentration of antibiotic / antimicrobial agent supplement of your choice; and stir the solution properly. NOTE: antibiotics should be added after sterilization; and not before or during sterilization of the media.
  10. Pour prepared molten Sabouraud dextrose medium into sterile Petri dish plates.
  11. Allow the poured plates on the bench to solidify.
  12. Do sterility check by incubating the poured plates at room temperature. Fungi grow at room temperature (25-28 degrees Celsius) for 18-24 h.
  13. At the end of incubation, check the plates for any sign of microbial growth (which is usually indicated by the presence of colony).
  14. Absence of colony on the plate means that your sterilization is good.
  15. You can now use your prepared Sabouraud dextrose agar plates for your experiment OR store in the refrigerator at 4 degrees Celsius until use.

REFERENCES

  1. Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. Part 2 . Cambridge University Press, UK.

 

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