Plate bioassay for antimicrobial activity

You can help OMP wiki by editing the content of this page. For information about becoming a registered user and obtaining editing privileges, see Help:Accounts.

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

Notes

This protocol is used for Dr. James Smith's lab at TAMU to test for activity in strains of Burkholderia contaminans. Make the necessary changes to the protocol to suit your lab's needs.

References

See Help:References for how to manage references in OMPwiki.