Antimicrobial Susceptibility Testing (AST) Protocol | Kirby-Bauer Disk Diffusion
Introduction to Antimicrobial Susceptibility Testing (AST)
This laboratory protocol details the Kirby-Bauer disk diffusion method for Antimicrobial Susceptibility Testing (AST), designed to help our students and researchers accurately assess antimicrobial resistance (AMR). Antimicrobial susceptibility testing is an important laboratory procedure used to determine the effectiveness of antimicrobial agents against isolated microorganisms. It plays an indispensable role in guiding both empirical and definitive therapy for infectious diseases. By identifying specific antimicrobial agents to which a microorganism is susceptible, AST aids clinicians in selecting appropriate treatments, optimizing patient outcomes, and preventing the emergence and spread of antimicrobial resistance (AMR).
AMR is a global health crisis that occurs when microorganisms evolve mechanisms to resist the effects of antimicrobial drugs. This phenomenon poses a significant challenge to public health, limiting therapeutic options and increasing morbidity and mortality rates. Consequently, understanding the mechanisms of antimicrobial resistance and implementing effective AST protocols are essential steps in combating this growing threat. The protocol detailed below is used in our lab and provided here for the convenience of our students to ensure consistency and accuracy in their results.
Learning Outcomes:
Upon completing this training protocol, our lab students will be able to:
Standardize a bacterial suspension to a 0.5 McFarland standard
Inoculate a Mueller-Hinton agar plate to obtain confluent growth
Perform the Kirby-Bauer disk diffusion test
Measure zones of inhibition accurately
Interpret AST results using standardized breakpoints
Principle of antimicrobial susceptibility testing
The principle underlying AST is to expose microorganisms to standardized concentrations of antimicrobial agents and assess their growth or inhibition under controlled conditions. Various methods are employed, but the core concept remains the same: if a microorganism is susceptible to an antimicrobial agent, its growth will be inhibited or killed, while resistant microorganisms will continue to grow despite the presence of the drug.
Core mechanisms of antimicrobial action
Antimicrobial agents exert their effects through diverse mechanisms, targeting different cellular components or processes of microorganisms. These mechanisms include:
Inhibition of cell wall synthesis: This mode of action is characteristic of many antibiotics, such as penicillins and cephalosporins. They interfere with the synthesis of peptidoglycan, an essential component of the bacterial cell wall.
Disruption of cell membrane integrity: Agents like polymyxins target the bacterial cell membrane, leading to leakage of cellular contents and eventual cell death.
Inhibition of protein synthesis: Antibiotics such as aminoglycosides and tetracyclines bind to ribosomes and disrupt protein synthesis, hindering bacterial growth.
Inhibition of nucleic acid synthesis: Quinolones and fluoroquinolones inhibit DNA replication by targeting DNA gyrase, an essential enzyme for bacterial DNA replication.
Metabolic inhibition: Sulfonamides and trimethoprim interfere with folic acid metabolism, which is vital for bacterial growth.
Understanding these mechanisms is essential for accurately interpreting AST results and selecting appropriate therapeutic options.
Procedure for antimicrobial susceptibility testing (AST)
Disk diffusion method (Kirby-Bauer test)
This protocol details the disk diffusion method for antimicrobial susceptibility testing (AST) used in our lab, and is intended primarily for our students to ensure accuracy and consistency. The disk diffusion method, commonly known as the Kirby-Bauer test, is a standardized technique used to assess the susceptibility of bacteria to antimicrobial agents. This method involves inoculating a standardized bacterial suspension onto an agar plate and placing antibiotic-impregnated disks on the surface. After incubation, the diameter of the zone of inhibition surrounding each disk is measured and compared to established interpretive standards to determine susceptibility.
Materials:
Mueller-Hinton agar plates
Antimicrobial susceptibility disks (containing known concentrations of antibiotics)
Sterile cotton swabs
Inoculation loop
Sterile saline solution (0.85%)
Graduated cylinder
Spectrophotometer or densitometer
Non-selective culture medium
Incubator
Calipers or ruler
Step-by-Step Kirby-Bauer Disk Diffusion Procedure:
Preparation of bacterial suspension
Select a pure, well-isolated colony of the test organism from a non-selective culture medium.
Suspend the colony in sterile saline solution to match the turbidity of a 0.5 McFarland standard. This can be visually compared or measured using a spectrophotometer or densitometer.
Inoculation of agar plate
Dip a sterile cotton swab into the bacterial suspension and remove excess fluid by pressing the swab against the inside of the tube.
Inoculate the entire surface of the Mueller-Hinton agar plate by streaking the swab back and forth in three directions, rotating the plate 60 degrees after each pass.
Application of antibiotic disks
Using sterile forceps, place the desired antimicrobial disks onto the inoculated agar plate at predetermined distances.
Ensure that the disks are evenly spaced and do not overlap.
Incubation
Invert the agar plate and incubate at 35 ± 2°C for 16-18 hours.
Measurement of zone of inhibition
After incubation, measure the diameter of the zone of inhibition around each antibiotic disk in millimeters.
Use calipers or a ruler for accurate measurement.
Interpretation
Compare the measured zone diameters to established interpretive standards provided by clinical and laboratory standards institutes (e.g., CLSI, EUCAST).
Interpret the results as susceptible, intermediate, or resistant based on the zone diameter and the breakpoint for each antimicrobial agent:
Susceptible: The organism is inhibited by the standard therapeutic dose of the antimicrobial agent.
Intermediate: Susceptibility is uncertain; clinical efficacy may be variable.
Resistant: The organism is not inhibited by the standard therapeutic dose of the antimicrobial agent.
