AgarDiffusion Assay Well Diffusion Assay MIC Determination Developing Resistance Soil Screening Isolating Active Compounds

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Agar Diffusion Assay

Materials Cultures of indicator organism(s). (These are bacteria or fungi that will be inhibited by the test chemical.) Leaves or other plant parts to test Nutrient agar plate (One for each indicator organism) Sterile cotton swab Scissors or hole-punch Forceps Alcohol in a small beaker Procedure Aseptically swab the indicator organism onto a nutrient agar plate. Swab in three directions to ensure complete plate coverage. Let the plate stand for 5 minutes. Cut 5-mm squares or disks from the leaves to be tested. Using a wax pencil, mark the underside of the Petri plate into 5 or 6 sections; one for each leaf to be tested. Label the sections. Sterilize forceps by dipping in alcohol and burning off the alcohol. Place a leaf disk on the surface of the agar. Gently tap the disk to ensure better contact with the agar. Repeat, placing five to six different disks the same distance apart on the Petri plate. Record the leaf species used and its location on the plate. Incubate the plate inverted at 35°C for 24 to 48 hrs. Measure the zones of inhibition in millimeters, using a ruler on the underside of the plate. Record the zone size. Discard all cultures in the "To be autoclaved" area. Repeat this procedure with different test organisms (bacteria or fungi) to find the range of antimicrobial activity.

Well Diffusion Assay

Materials Cultures of indicator organism(s). (These are bacteria or fungi that will be inhibited by the test chemical.) Leaves or other plant parts to test Mortar and pestle Solvent Pasteur pipettes (2) Nutrient agar plate (One for each indicator organism) Cork borer (4-mm diameter) Sterile cotton swab Procedures Prepare plant tissue extracts by grinding the appropriate tissue in a few milliliters of solvent in a mortar and pestle. Possible solvents are sterile water; methyl alcohol, ethyl alcohol, or acetone. Sterilize a cork borer by autoclaving or disinfect it by rising in alcohol followed by sterile water. Obtain a nutrient agar plate and aseptically punch (4-mm) holes in the agar using a cork borer. Using a wax pencil, mark the underside of the Petri to label the wells. Aseptically swab the indicator organism onto a nutrient agar plate. Swab in three directions to ensure complete plate coverage. Let the plate stand for 5 minutes.. Place 1, 2, or 3 drops of the filtered plant extract in the appropriate wells. Fill one well with the solvent used to prepare the tissue extracts. This is a control. Incubate the plate at 35°C for 24 to 48 hrs. Measure the zones of inhibition in millimeters, using a ruler on the underside of the plate. Record the zone size. Discard all cultures in the "To be autoclaved" area.