Agar Diffusion Assay
Well Diffusion Assay
MIC Determination
Developing Resistance
Isolating Active Compounds

Discover a New Antibiotic
Soil Screening

Christine L.Case

Isolating An Antibiotic-Producing Bacterium



The rough, circular Bacillus sp. colonies are inhibiting growth of the filamentous Bacillus cereus var. mycoides. PhotoİCLCase


Prepare a a soil slurry by mixing a pinch of soil in ~5 ml water. Mix on a vortex mixer. Place 0.1 - 0.5 ml of soil slurry on a sterile nutrient agar plate. Spread the slurry with a spreading rod.

Incubate the plate inverted at 25°C for 5 to 7 days. Look for evidence of antibiosis (see figure to the left). Or look for actinomycete colonies. Actinomycetes produce powdery colonies with hyphae embedded in the agar (see below).


Streptomyces. Actinomycetes produce powdery colonies with hyphae embedded in the agar. PhotoİCLCase


Testing Isolates





Isolate one colony and inoculate it in a single line across a nutrient agar plate. Incubate the plate inverted under good growth appears.

Inoculate different bacteria perpendicular to the line of growth. Be careful not to touch the existing bacterial growth.

Incubate the plate inverted at 35°C for 24-48 hr. Look for zones of inhibition near the original growth.

The Bacillus shown in the figure is producing an antibiotic that inhibited the Streptococcus.