Kirby Bauer Disc Diffusion Method For Antibiotic Susceptibility Testing

The aim of the Kirby Bauer disc diffusion method is to determine the antibiotics of choice to treat an infection and monitor antimicrobial resistance. Antimicrobial resistance is the ability of microorganisms to withstand the effects of antibiotics and other antimicrobial agents. It poses a serious threat to public health and can compromise the effectiveness of treatments for various infections.

The Kirby Bauer disc diffusion method is a simple and widely used technique to test the susceptibility of bacteria to different antibiotics. It involves placing antibiotic discs on an agar plate that has been inoculated with a bacterial culture. The antibiotics diffuse from the discs into the agar and create zones of inhibition around them, where the bacterial growth is inhibited. The size of the zones of inhibition indicates the level of susceptibility or resistance of the bacteria to the antibiotics.

By measuring the zones of inhibition and comparing them with standard guidelines, one can determine whether the bacteria are susceptible (S), intermediate (I), or resistant (R) to the antibiotics. This information can help clinicians to choose the most appropriate antibiotic for treating an infection and avoid using ineffective or unnecessary antibiotics that may contribute to antimicrobial resistance.

The Kirby Bauer disc diffusion method can also be used to monitor the trends and patterns of antimicrobial resistance among different bacteria and regions. This can help to identify emerging or resistant strains of bacteria and inform public health policies and interventions.

The principle of this method is based on the fact that different antibiotics have different abilities to diffuse through agar and inhibit the growth of bacteria. The diffusion rate of an antibiotic depends on its molecular size, concentration, and solubility. The larger the zone of inhibition around an antibiotic disc, the more effective the antibiotic is against the bacteria.

The organism to be tested must be incubated overnight in broth and must be compared with the 0.5 McFarland Turbidity standard. This standard is a reference to ensure that the inoculum has a uniform density and contains approximately 1.5 x 10^8^ colony forming units (CFU) per ml. A higher or lower inoculum density may affect the accuracy of the test results.

Mueller-Hinton agar must be used as it does not inhibit sulphonamides and ensures reproducibility with composition and pH of the medium. The agar when poured on Petri dishes should be 4mm thick. A thinner or thicker layer of agar may alter the diffusion rate of the antibiotics and affect the size of the zone of inhibition.

To perform the Kirby Bauer disc diffusion method, you will need the following materials:

• Mueller-Hinton agar: This is a non-selective and non-differential medium that supports the growth of most bacteria. It has a pH of 7.2-7.4 and a low concentration of thymine and thymidine, which do not interfere with the action of sulphonamides and trimethoprim. It also has a high concentration of starch, which absorbs toxic substances that may inhibit bacterial growth. The agar should be poured to a depth of 4 mm in sterile Petri dishes and stored at 4°C until use.
• Antibiotic discs: These are filter paper discs impregnated with specific concentrations of antibiotics. They are commercially available and should be stored at -20°C until use. You can choose the antibiotics based on the type of bacteria you are testing and the clinical relevance. For example, you can use penicillin, ampicillin, ceftriaxone, gentamicin, tetracycline, erythromycin, chloramphenicol, etc.
• Cotton swabs: These are sterile cotton-tipped applicators that are used to inoculate the agar plates with the bacterial suspension. They should be moistened with sterile saline or distilled water before use.
• Petri dishes: These are shallow cylindrical glass or plastic dishes with lids that are used to hold the agar plates. They should be sterilized before use and labeled with the name of the organism and the date of inoculation.
• 0.5 McFarland Turbidity standard: This is a reference solution that is used to adjust the density of the bacterial suspension to ensure a uniform inoculum. It contains 0.5% (w/v) barium chloride and 0.05 M sulfuric acid and has a turbidity equivalent to 1.5 x 10^8 CFU/ml. It can be prepared in the laboratory or purchased commercially. It should be stored at room temperature and shaken well before use.
• Inoculum: This is the bacterial suspension that is prepared from an overnight culture in broth or from a pure colony on an agar plate. It should be diluted with sterile saline or distilled water to match the turbidity of the 0.5 McFarland standard using a spectrophotometer or a nephelometer. Alternatively, you can visually compare the turbidity of the inoculum with the standard by holding them against a white background with a black line.
• Forceps: These are metal instruments with two prongs that are used to pick up and place the antibiotic discs on the agar plates. They should be sterilized with alcohol before and after each use and flamed between each disc to avoid cross-contamination.
• Metric ruler or caliper: These are measuring tools that are used to measure the diameter of the zones of inhibition around the antibiotic discs after incubation. They should have millimeter markings and be accurate to 0.5 mm.

• Before performing the test, sterilize the area with disinfectant and turn on the burner to create a sterile field.
• Take a sterile cotton swab and dip it into the inoculum, which is a broth culture of the organism to be tested. Remove excess medium by pressing the swab onto the wall of the tube.
• Swab the entire surface of a Mueller-Hinton agar plate with the inoculum, rotating the plate as you go. This technique is called lawn culture or carpet culture, as it creates a uniform layer of bacteria on the agar.
• Allow the plates to dry for 5 minutes at room temperature so that the medium absorbs the inoculum properly and prevents the antibiotic discs from sliding off.
• Sterilize a pair of forceps with alcohol and use them to pick up an antibiotic disc from its container. Do not touch the disc with your fingers or any other object.
• Place the disc on the surface of the agar plate at a distance of 24 mm from the edge and from any other disc. Lightly press the disc with the forceps to ensure that it is in good contact with the agar and avoid misplacement.
• Repeat steps 5 and 6 for each antibiotic disc that you want to test, using a different pair of forceps or sterilizing them between each disc.
• Label the plate with the name of the organism, the date, and the antibiotics tested. Incubate the plate upside down for 24 hours at 37ºC in an incubator.

After 24 hours of incubation, you will observe clear zones around the antibiotic discs where the bacteria have been inhibited. These zones are called zones of inhibition and they indicate the effectiveness of the antibiotic against the organism. To measure the zones of inhibition, you will need a metric ruler or a caliper and include the diameter of the disc in the measurement. For example, if the disc is 6 mm in diameter and the zone around it is 18 mm, then the zone of inhibition is 12 mm (18 - 6 = 12).

To interpret the results, you will need to compare the zone of inhibition with the Clinical and Laboratory Standards Institute (CLSI) guidelines. The CLSI guidelines provide standardized criteria for determining whether an organism is susceptible, intermediate, or resistant to an antibiotic based on the zone size. The criteria vary depending on the type of organism, the type of antibiotic, and the concentration of the antibiotic in the disc. You can find the CLSI guidelines online or in a reference book.

The results are reported as Susceptible (S), Intermediate (I), or Resistant (R). Susceptible means that the organism is likely to be inhibited by the antibiotic at the recommended dosage. Intermediate means that the organism may be inhibited by the antibiotic at higher dosages or in specific body sites where the antibiotic can reach higher concentrations. Resistant means that the organism is not likely to be inhibited by the antibiotic even at high dosages.

Here is an example of how to report the results:

This means that the organism is susceptible to ampicillin and ciprofloxacin, resistant to erythromycin, and intermediate to tetracycline.

The Kirby Bauer disc diffusion method is a widely used technique to determine the antibiotics of choice to treat an infection and monitor antimicrobial resistance. Antimicrobial resistance is the ability of microorganisms to withstand the effects of antibiotics and other antimicrobial agents. It poses a serious threat to public health and can compromise the effectiveness of treatments for various infections.

The Kirby Bauer disc diffusion method can help clinicians and microbiologists to:

• Identify the most suitable antibiotic for a specific infection based on the susceptibility pattern of the causative organism.
• Detect emerging or existing resistance patterns among different bacterial strains and species.
• Evaluate the impact of infection control measures and antibiotic stewardship programs on the prevalence and spread of resistant bacteria.
• Provide epidemiological data on the distribution and trends of antimicrobial resistance in different regions and settings.

The Kirby Bauer disc diffusion method is a simple, standardized, and cost-effective technique that can be performed in most clinical and research laboratories. It can provide reliable and reproducible results for most common bacterial pathogens. However, it has some limitations and cannot be used for all types of organisms or antibiotics. Therefore, it should be complemented by other methods such as broth dilution, E-test, or molecular techniques for more accurate and comprehensive antimicrobial susceptibility testing.

Advantages

The Kirby Bauer disc diffusion method has several advantages over other methods of antibiotic susceptibility testing. Some of them are:

• It is a simple and easy method that can be performed by any laboratory technician with basic skills and training.
• It is a cost-effective method that does not require expensive or specialized equipment or reagents. The materials required are readily available and inexpensive.
• It can test a wide range of antibiotics and microorganisms in a single plate, which saves time and resources.
• It can provide a visual and qualitative result that can be interpreted by any medical personnel without the need for complex calculations or software.
• It can help in determining the antibiotics of choice to treat an infection and guide the clinician in selecting the best antimicrobial agent.
• It can also help in monitoring the antimicrobial resistance patterns of microorganisms and provide information on public health importance.

Although the Kirby Bauer disc diffusion method is widely used and standardized, it has some limitations that should be considered when interpreting the results. Some of these limitations are:

• It does not provide accurate information about the minimum inhibitory concentration (MIC) of the antibiotic, which is the lowest concentration that can prevent bacterial growth. MIC is important to determine the optimal dose and duration of treatment for a patient.
• It is not suitable for testing some slow-growing or fastidious organisms, such as anaerobes, mycobacteria, fungi, or viruses, which may require special media or conditions .
• It is not considered a gold standard test, as it only reflects the susceptibility pattern of the organisms according to the CLSI guidelines, which may not correlate with the clinical outcome or the pharmacokinetics of the antibiotic .
• It may be affected by various factors, such as inoculum size, disc potency, media quality, incubation time and temperature, and measurement errors, which can lead to false results or discrepancies .
• It may not detect some mechanisms of resistance, such as efflux pumps, biofilms, or heteroresistance, which can reduce the efficacy of the antibiotic in vivo .

Therefore, the Kirby Bauer disc diffusion method should be performed and interpreted carefully and according to standard protocols. It should also be complemented by other methods, such as MIC determination or molecular tests, when necessary. The results should be reported with caution and in conjunction with other clinical and laboratory data.

The Kirby Bauer disc diffusion method is a simple and reliable technique to test the antibiotic susceptibility of bacteria. It can help clinicians to choose the most effective antimicrobial agent for treating an infection and to monitor the emergence of antimicrobial resistance. However, this method has some limitations, such as the inability to test some slow or fastidious organisms, the dependence on the quality and thickness of the agar medium, and the lack of standardization for some antibiotics. Therefore, it is important to follow the CLSI guidelines carefully and to use other methods, such as broth dilution or E-test, to confirm the results when necessary. The Kirby Bauer disc diffusion method is a valuable tool for clinical microbiology, but it should not be used alone without considering other factors, such as the patient`s condition, the site of infection, and the pharmacokinetics of the antibiotic.

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