DRBC agar: Principle and preparation

Enumeration of fungi is possible on DRBC media. The image shows growth of Rhizopus spp. as distinct countable units on DRBC media. On the other-hand on PDA, it is spread all over the plate.
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    Introduction

    DRBC (Dichloran-rose bengal chloramphenicol) agar or DRBC media was first formulated by Douglas King, JR et al. in 1979. DRBC media is an improvement of RBC media to facilitate the enumeration of fungi that is not possible/difficult in RBC media.

    Douglas King, JR, and his colleagues screened 31 compounds known for their fungi static/fungicidal effects. The main aim of the screening is to reduce the spreading of fungi on culture media (better enumeration). Among those, they have selected two compounds (dichloran and pentachloronitrobenzene) with the desired effect, such as slowing the spreading of fungi but not altering the germination of spores. However, they proceeded to test only dichloran as the other drug is a suspected carcinogen.

    The researchers tested various dichloran and rose-Bengal concentrations to find a suitable one where enumeration is possible without disturbing CFU count. In general, dichloran concentration inversely affects the fungal colony diameter. So it is possible to alter the concentrations based on the spreading nature of the fungal species.

    Principle

    Dichloran & Rose-Bengal: Reduces the spreading of the fungus. In general, the relationship is inversely proportional. The following table gives a glimpse of the relation of colony size to the dichloran concentration.

    Table 1. Average colony diameter of molds at different dichloran concentrations on MA (maltose agar) media.

    Dichloran concentration Reduction of colony diameter (in %)
    1 13
    5 41
    10 66
    25 74
    50 78
    100 81

    Although dichloran effectively reduces mold colony diameter, it is less effective on yeast as the colony diameter change is less than 10%.

    Needless to say, the size and growth rate of the fungal colony depend on the species (Table 2).

    Table 2. growth rate of fungi varies in DRBC media.

    Table 2. growth rate of fungi varies in DRBC media.
    Fungus used Growth rate (mm per day at 25°C)
    Rhizopus stolonifer 8.1
    Aspergillus flavus 4.5
    Penicillium cyclopium 3.5

    Chloramphenicol: is a broad-spectrum antibiotic. It inhibits bacterial protein synthesis by binding to ribosomes.

    Dipotassium Phosphate: buffering agent.

    Magnesium sulfate: trace elements.

    Peptone: source of carbon and nitrogen.

    Dextrose: energy source.

    Applications

    • For enumeration of fungi from food samples.
    • Selective isolation of yeasts and molds from food and soil samples.

    Advantages

    • It effectively prevents bacterial growth to some extent due to low pH and chloramphenicol.
    • Some fungal and bacterial colonies take up rose bengal (bacterial colonies can be observed in media lacking chloramphenicol). This makes colony counting easy.
    • It is superior media than RBC as most fungal species give more colony count c

    Disadvantages

    • At higher concentrations of dichloran, conidiogenesis is affected ( niger). At a concentration of 5 ug/ml, conidiogenesis was inhibited completely. However, at 2.5 ug/ml, conidiogenesis is almost normal.
    • Colony morphology is affected for molds at higher concentrations (>5ug/ml). It makes identification difficult. In these cases, subculturing is recommended.

    Composition

    Table 3. Composition of DRBC agar
    Component For 1L media (in grams)
    Peptone 5g
    Dextrose (Glucose) 10g
    Potassium dihydrogen phosphate 1g
    Magnesium sulphate 0.5g or 500mg
    Rose Bengal 0.025g or 25mg
    Chloramphenicol 0.100g or 100mg
    Dichloran 0.002g or 2mg
    Agar 15g

    * Chloramphenicol is optional. Use it to avoid bacterial colony presence.

    pH

    pH 5.40 - 5.80 (5.6) is recommended to reduce bacterial growth. The media can also be used at pH 7.2. However, higher pH can increase bacterial contamination, increase the diameter of fungal colonies, and faster growth.

    Procedure

    1. Add the components except for agar and chloramphenicol in the required quantities in 800 ml of distilled water.
    2. Adjust the pH and make up the volume to 1L.
    3. Aliquot the solution if necessary.
    4. Add agar.
    5. Autoclave.
    6. Add chloramphenicol while the media is cooling down.
    7. Pour in plates and seal with parafilm.

    Storage

    • Wrap the media bottles with aluminum foil to avoid light exposure.
    • Store the media flasks at 4°C for a maximum of 4 weeks.
    • Use freshly poured plates within 2 weeks.
    • If plates are made from stored media, use them within a week.

    Caution

    • Add agar after adjusting the pH of the media to avoid hydrolysis.
    • Keep the media/plates away from light. Rose-bengal is light sensitive, and its photodegradation product is toxic to fungi.
    • Do not use media after recommended time as colony count can change.

    Reference