Potato Dextrose Agar (PDA)

steps involved in potato dextrose agar preparation

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    Introduction

    Potato dextrose agar often abbreviated as PDA. Most mycologists widely use it as a general-purpose medium for yeasts and molds that can be identified to some extent based on their morphological features and their pigmentation in culture often being important for identification of cultures. PDA thus aids in cultivating and differentiating pathogenic and non-pathogenic fungi.

    Principle

    Potato Dextrose Agar is a frequently used microbial growth media for cultivation of molds, yeasts and other fungi. It is made up of potato infusion and dextrose (a.k.a glucose). The potato infusion and dextrose as a carbohydrate source support the luxuriant growth of fungi and bacteria and is observed to encourage mold sporulation and pigment production in certain dermatophytes. However, the media may be supplemented with selective agents such as acids (e.g. tartaric acid) or antibiotics (e.g. chloramphenicol, or chlortetracycline) to inhibit the growth of bacteria that can impede the yeasts and mold. Agar acts as a solidifying agent.

    In 2007, an article published in FEMS Microbiology Letters, stated importance of copper as suppliment to to achieve good coloration of fungal cultures on PDA media. The authors reasoned that the sufficient amount of copper is essential for the activity of enzymes involved in pigment production. They also showed that there is a batch to batch variation in copper amount in both commertially available and lab made PDA media. For more information follow the reference.

    Applications

    1. Potato Dextrose Agar (PDA) is commonly used in the dairy industry for detecting the presence of yeasts and molds in product samples. For this purpose, the addition of tartaric acid is recommended.
    2. PDA supplemented with chlortetracycline is recommended for the microbial enumeration of yeast and mold from cosmetics.
    3. PDA supplemented with Chloramphenicol is recommended for the selective cultivation of fungi from mixed samples.
    4. PDA is used for isolation and enumeration of yeasts and molds in clinical samples. 
    5. Stock cultures of certain dermatophytes can be maintained on PDA media.
    6. PDA is also used for stimulating sporulation and differentiating typical varieties of dermatophytes based on their pigment production.

    pH

    The pH of Potato Dextrose Agar medium is about 5.6+/- 0.2. Use HCl and KOH for the pH adjustment.

    Composition

    Composition for PDA media with potato infusion

    IngredientsFor 100mLFor 500mLFor 1000ml
    Potato (infusion form)20 gm100 gm200 gm
    Dextrose2 gm10 gm20 gm
    Agar-Agar1.5 gm7.5 gm15 gm
    Distilled waterUpto 100mLUpto 500mLUpto 1000mL

    Composition for PDA media with commercially available potato infusion powder

    IngredientsFor 100mLFor 500mLFor 1000ml
    Potato extract0.4 gm2 gm4 gm
    Dextrose2 gm10 gm20 gm
    Agar-Agar1.5 gm7.5 gm15 gm
    Distilled waterUpto 100mLUpto 500mLUpto 1000mL

    Preparation of potato infusion

    Steps involved in potato infusion preparation:

    1. Take 200 gm of potato for 1L of PDA media preparation.
    2. Wash the potato to remove dirt.
    3. Peel off the skin and dice them.
    4. Add the pieces to 1L of distilled water.
    5. Boil for 20-25 min on a hot plate.
    6. Collect the extract through the muslin cloth.

    The preparation of the media by using the above raw materials is rather tedious. Hence in recent times, the infused form of potato is being replaced with commercially available potato starch/extract powder.

    4 gm of the potato extract powder is equivalent to 200 gm of potato infusion.

    Reagents

    Along with the reagents mentioned in the table

    • 1N KOH
    • 1N HCl

    Instruments and other requirements

    • Glass beaker
    • Conical Flask / Erlenmeyer Flask
    • Spatula
    • Measuring Cylinder
    • pH meter
    • Weighing balance
    • Distilled Water
    • Butter Paper
    • Magnetic stirrer and pellet
    • Pipettes and tips
    • Petri plates and/or test tubes
    • Hot plate

    Procedure

    1. Weigh the ingredients separately with respect to the volume of the media. (Here, we are considering 1L of the media).
    2. Suspend the ingredients such as potato infusion (200 gm) or potato extract (4 gm) and glucose (a.k.a dextrose) 20 gm in a glass beaker containing about 900mL of ddH2O.
    3. Dissolve the components in the beaker using a magnetic stirrer. (Heat may be applied to dissolve the medium completely).
    4. Adjust the pH of the medium to 5.6 using 0.1N HCl and 0.1N KOH.
    5. Adjust the broth to a final volume of 1L using ddH2O.
    6. Transfer the broth to conical flask or aliquot into smaller volumes.
    7. Now add agar accordingly with respect to the volume of the media (i.e., 15 gms agar for 1L of the media., 3.75 gm for 250 ml).
    8. Close the mouth of the flask with a cotton plug. Seal it further with paper and rubber band.
    9. Autoclave for 20 min at 15 psi (1.05kg/cm2) on liquid cycle.
    10. However, if antibiotics are to be included, their stock solutions should be filter sterilized prior to addition to the media. These antibiotics must be added after the media is cooled to about 45-50ºC.
    11. Some formulations prefer the addition of sterile tartaric acid (10%) instead (or in combination) of antibiotics. Tartaric acid decreases the pH to about 3.5. The lowered pH inhibits bacteria growth.
    12. Mix well and pour into sterile Petri plates or tubes for slants.

    Alternatively, the commercially available Potato Dextrose Agar media powders can be used. Weigh the mixture of content as prescribed by the manufacturer.

    Storage

    Store in dark at 2-8ºC. Plates may be used for one week when stored in a clean sterile area.

    Caution

    Heating Potato Dextrose Agar after acidification should be avoided as it may hydrolyze the agar and may destroy the solidifying properties.

    Limitations

    Since PDA is a general-purpose and not a differential medium only presumptive identification is possible by observing colony morphology. Microscopic examination and biochemical tests should be followed to identify isolates to genus and species.

    Reference

    1. DIFCO
    2. Himedia
    3. Microbiologyinfo

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