Disk diffusion assay: Kirby-Bauer Test

Summary

The disc diffusion method, or Kirby-Bauer test, is a widely used technique to assess bacterial resistance to antibiotics. This procedure involves inoculating a Muller Hinton agar plate with bacteria, applying antibiotic-impregnated discs, and incubating the plate. After incubation, the zones of inhibition around the discs are measured to determine the bacterium's susceptibility to various antibiotics. This method helps physicians select the most effective treatment for infections by identifying resistant strains, making it a crucial tool in clinical microbiology.

Takeaways

• 😀 A physician must understand the sensitivities of the pathogen causing a patient's infection.
• 😀 The disc diffusion method, also known as the Kirby-Bauer test, is the most widely used method for testing bacterial sensitivities.
• 😀 The National Committee for Clinical Laboratory Standards recommends using Muller-Hinton agar as the nutrient medium.
• 😀 Each bacterium should be tested separately for accurate results.
• 😀 The agar plate is inoculated with bacteria to create a lawn, which is used for testing antibiotic efficacy.
• 😀 Label the bottom of the plates with your name, date, and bacterium for proper identification.
• 😀 Proper swabbing technique involves using a sterile cotton swab and rotating the plate to ensure an even lawn of bacteria.
• 😀 After inoculating the plate, let it stand for five minutes before proceeding with antibiotic testing.
• 😀 Standardized antibiotic-impregnated paper discs are used, with five different antibiotics being tested.
• 😀 The plate is incubated at 35°C for 24-48 hours, and the zones of inhibition are measured to determine antibiotic effectiveness.
• 😀 The size of the inhibition zones is compared to clinical standards to evaluate bacterial susceptibility to each antibiotic.

Q & A

What is the purpose of the disc diffusion method?

The disc diffusion method, also known as the Kirby-Bauer test, is used to test the sensitivity of bacteria to different antibiotics, helping determine which antibiotics are effective against a specific bacterial infection.

Why is Muller Hinton agar used in the disc diffusion method?

Muller Hinton agar is recommended because it provides an ideal medium for bacterial growth and allows for the accurate diffusion of antibiotics, which is necessary to evaluate the effectiveness of the drugs.

How is the bacterial inoculum prepared for the disc diffusion method?

The inoculum is prepared by dipping a sterile cotton swab into a broth culture of the bacteria, then streaking it across the surface of Muller Hinton agar in a systematic manner to ensure even distribution.

Why is it important to label the bottom of the plates?

Labeling the bottom of the plates with your name, date, and the bacterium helps ensure proper identification and tracking of the samples, preventing mix-ups during the experiment.

What is the purpose of rotating the plate while swabbing?

Rotating the plate while swabbing ensures that the bacteria are evenly distributed across the agar surface in both directions, creating a uniform bacterial lawn for accurate results.

What should be done after inoculating the plate with bacteria?

After inoculating the plate, it should be left to stand for five minutes to allow the bacteria to settle and adhere to the agar surface before applying the antibiotic discs.

How are the antibiotic discs prepared for placement on the agar?

Sterilized forceps are used to pick up the antibiotic discs, ensuring that they are placed on the agar without contamination. The forceps are sterilized by dipping them in alcohol and burning off the excess alcohol.

How is the effectiveness of the antibiotics measured?

The effectiveness is measured by observing the zones of inhibition, which are clear areas around the antibiotic discs where bacteria have been inhibited or killed. The size of these zones is measured with a ruler.

What is the significance of comparing the zone sizes to clinical standards?

Comparing the zone sizes to clinical and laboratory performance standards helps determine whether the bacterium is susceptible or resistant to the antibiotics being tested, guiding treatment decisions.

What should be done after completing the experiment?

After recording the data, the plates should be discarded for autoclaving, ensuring proper disposal and sterilization of potentially harmful materials.