The well diffusion method is a common procedure for evaluating the antibacterial activity of substances, such as antibiotics or plant extracts. Here’s a step-by-step procedure for conducting the well diffusion assay:
Materials Needed:
- Nutrient agar plates (previously inoculated with a standardized bacterial culture)
- Sterile paper discs (6-8 mm in diameter)
- Antibiotic solutions or test substances
- Sterile forceps
- Sterile pipettes
- Incubator set at the appropriate temperature for bacterial growth
- Aseptic workspace (laminar flow hood or biosafety cabinet)
- Calipers (for measuring inhibition zones)
- Bacterial culture broth
- Mueller-Hinton agar (if testing antibiotics)
Procedure:
- Inoculate Agar Plates:
- Using a sterile loop, streak the surface of nutrient agar plates with a standardized bacterial culture. Ensure even distribution of the culture.
- Prepare Discs:
- Sterilize paper discs by autoclaving or using a dry heat sterilizer.
- Impregnate the discs with the antibiotic solution or the test substance of interest. Ensure that the concentration is consistent on each disc.
- Create Wells:
- Use sterile forceps to place the impregnated paper discs on the surface of the inoculated agar plates.
- Gently press the discs onto the agar to ensure good contact.
- Incubation:
- Incubate the agar plates inverted (lid down) at the appropriate temperature for bacterial growth (usually 37°C) for 16-24 hours.
- Examine Inhibition Zones:
- After incubation, observe the plates for zones of inhibition around the discs. Inhibition zones are areas where bacterial growth is suppressed due to the action of the tested substance.
- Measure Zones:
- Use calipers to measure the diameter of the clear zones of inhibition in millimeters. This provides a quantitative assessment of antibacterial activity.
- Control Plates:
- Always include control plates, such as a positive control (known antibiotic) and a negative control (sterile disc impregnated with the solvent). This helps validate the assay.
- Interpretation:
- Larger inhibition zones indicate greater antibacterial activity.
- Compare the test substance’s inhibition zones to those of the positive control to determine its effectiveness.
- Record and Report:
- Record the results, including the diameter of inhibition zones and other relevant observations.
- Prepare a report or document the findings as needed.
The well diffusion method is a valuable tool for preliminary screening of antibacterial activity. It provides a visual representation of the effectiveness of substances against specific bacterial strains.