Antimicrobial Testing

Antimicrobial Testing: Disk Diffusion Method (Kirby-Bauer Assay)

Introduction to Antimicrobials and Antibiotics

• A true antibiotic is an antimicrobial chemical produced by microorganisms that affects other microorganisms.
• These antimicrobials are crucial in combating infectious diseases.
• Bacteria exhibit varying responses to antibiotics and synthetic drugs, even within the same species.
• Example: Staphylococcus aureus isolates from different individuals are likely to be different strains with varying antibiotic sensitivities.

The Kirby-Bauer Test

• Also known as the disc diffusion test, it is a standard method for assessing antibiotic susceptibility.
• Developed in the 1950s, refined by W. Kirby and A. Bauer, and standardized by the World Health Organization in 1961.
• While largely superseded by automated tests in clinical labs, it remains in use for certain bacteria or in some labs.
• The test determines bacterial resistance or sensitivity to specific chemicals, guiding treatment decisions.
• The presence or absence of an inhibitory area (zone of inhibition) around the disc indicates the bacterial sensitivity to the drug.

Key Terminology (from OpenStax Microbiology)

• Biostatic/Bacteriostatic agents: Prevent microbial growth without killing.
• Biocidal/Bactericidal agents: Kill microorganisms.
• Sterilization: Kills all organisms, including spores (e.g., autoclaving).
• Disinfectants: Reduce pathogenic microorganisms to safe levels, typically used on inanimate objects.
• Antiseptics: Disinfectants safe for use on living tissue, inhibiting microbial growth (e.g., iodine solution, rubbing alcohol).
• Antibiotics: Antimicrobial substances used in humans and animals to combat bacterial infections.
• Antimicrobials: A broad category including disinfectants, antiseptics, and antibiotics.

Filter Paper Method Overview

• Tests the susceptibility or resistance of bacteria to antimicrobial substances using paper discs on agar plates.
• Discs are either impregnated with antibiotics or dipped into test solutions.
• Zone of Inhibition: A clear area around the disc indicates bacterial susceptibility.
• Resistance: Bacterial growth up to the disc edge indicates resistance.
• This method is referred to as the Kirby-Bauer method (for antibiotics) or disk diffusion method.
• The purpose is to test different chemicals and antibiotics to see if the bacteria is resistant or susceptible to each.

Laboratory Activity Protocol

Overview

• The experiment involves testing the effectiveness of antibiotics and household chemicals on bacterial growth.
• Day 1: Inoculate agar plates with bacteria and place antimicrobial disks on the plates, followed by incubation.
• Day 2: Assess the impact of antimicrobials and measure zones of inhibition.

Materials Needed

• 2 nutrient agar plates
• Sterile cotton swabs
• Broth culture of bacteria
• Tweezers/forceps
• Antibiotic paper discs
• Sterile paper discs
• Antimicrobial solutions

Day 1 Protocol

1. Divide each agar plate into four sections using a wax pencil or marker.
   • Label each plate with initials and the bacteria being tested.
2. Inoculate plates with bacteria:
   • Moisten a sterile cotton swab with bacterial broth.
   • Remove excess fluid.
   • Streak the swab across the entire plate while rotating it to create an even bacterial lawn (confluent growth).
   • Dispose of used swabs in biohazard trash.
3. Choose four chemicals to test per plate and label each quadrant.
   • One plate is for antibiotics (pre-embedded on disks).
   • The other plate is for household chemicals (dip clean paper disks).
4. After the bacteria swabbed onto the plate is dry, place the paper discs impregnated with chemicals on the center of each correspondingly labeled quadrant using sterile forceps (wipe with alcohol and briefly flame before use and between uses).
   • Gently press the disc to ensure it sticks to the agar.
5. Incubate the plates overnight at the appropriate temperature for the bacterial species.
   • Do not invert the plates to avoid dislodging the paper discs. A figure showing an agar plate divided into quadrants is available for reference.