Plate bioassay for antimicrobial activity

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Purpose

This assay can be used to test for production of or sensitivity to antimicrobial agents by measuring a zone of inhibition around an antimicrobial compound.

Materials Needed

(For testing four plates)

8 NBY Agar plates

25-30 mL NBY Top Agar

750 mL water

Sterile toothpicks

Millimeter measuring stick

Indicator Fungus-Geotrichum candidum

30 C incubator

65 C incubator

Protocol

-Inoculate culture to be tested on NBY agar plates and incubate overnight at 30 C

-Use a sterile toothpick to stab inoculate another NBY agar plate with a single colony from the culture

-Incubate at 30 C overnight

-Incubate the plates at 65 C for one hour to kill the bacteria, leaving just the antimicrobial compound it produced behind

-Mix a colony of Geotrichum candidum into 750 microliters of water

-Mix the water-bacteria mixture with 25-30 mL of molten NBY Top Agar. Make sure the molten agar is not too hot for the fungus, or too cold that it solidifies early

-Pour the top agar solution onto the NBY plates that were stab inoculated, insuring the entire plate is covered with agar

-Let the top agar harden and incubate at 30 C overnight

-Measure the diameter of the clearing of the bacterial lawn around each stab, this is called the zone of inhibition

References

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