What Is a Kirby Bauer Test?

The Kirby-Bauer test has been around for decades, and it is still relatively misunderstood. While many know about measuring colony forming units of bacteria, still others are in the dark about how to quantify the bacterial responses of antibiotics. Thus, you may find yourself asking, “what is a Kirby Bauer test anyway?”

Bacteria and antibiotics

For millennia, we have known that pathogens can grow, make us sick, and spread from person to person. We can get sick from food, from the environment, and even from our own systems. What we did not know was that those pathogens were microscopic living organisms called bacteria. While less than one percent of bacteria are harmful to humans, our study of them will help us determine how they grow and how they respond to chemicals aimed at stopping their growth.

Enter antibiotics. Antibiotics are actually inherent to nature’s process and can be found in abundance from various sources. Though Alexander Fleming discovered the power of penicillin in moldy bread in 1928, ancient cultures like the Egyptians had been using natural sources of antibiotics thousands of years earlier.

The connection between naturally occurring bacteria that harms humans and naturally occurring antibiotics that stop the growth of that bacteria and actually heals humans is an ancient one, and one that is finally being studied in labs.

Enter the Kirby-Bauer test.

What Is a Kirby-Bauer Test?

Alexander Fleming discovered penicillin in 1928, and by the 1950s, microbiologists had invented the disk diffusion method to test how effective a particular antibiotic is against a particular strain of bacteria.

Then, in the 1960s, researchers W.M.M Kirby and A.W. Bauer perfected the disk diffusion test that has now become standard practice in all labs not using automation, and even some that do.

To perform what is now known commonly as the Kirby-Bauer test, a petri dish with an agar plate is cultured with bacteria. Then, disks soaked in an antibiotic are arranged on top of the bacteria culture on the plate. As time goes by, it is possible to witness clear space forming around the disk, where no bacteria can grow because of the presence of the antibiotic, this space is known as the zone of inhibition.

As the distance from the disk becomes greater, the strength of the antibiotic grows weaker, and you will see bacteria forming in those interstices.

Obviously, if no space is created around the disk, you know the antibiotic is ineffective against the bacteria, and the measurement of the zone of inhibition gives you an indication as to how effective an antibiotic is against a strain of bacteria.

To perform the Kirby-Bauer test, you would need:

• Agar plates
• 24 hour-old bacterial cultures
• Antibiotic disks
• Ethanol
• Forceps

Swap the agar plates with your culture and be sure to cover the entire area so you don’t confuse blank space with antibiotic space.

Allow the plate to dry for 5 minutes.

Place the antibiotic disks on your agar plate over the bacteria you swabbed. Be sure your forceps are sterilized in ethanol, so you don’t introduce foreign bacteria.

Lightly press down on your disks with a sterile inoculating loop to be sure the antibiotics are in contact with the agar plate.

Incubate your agar plates upside down at 37 degrees Celsius.

When you are ready to measure the zone of inhibition, the primary calculation you are making, you will take a metric ruler across the zone of inhibition at its widest point and measure from one edge to the other, using millimeters.

If there is no zone around the disk, report the zone of inhibition as zero.

Looking at the guidelines chart, you will be able to tell, based on the measurement, if the bacteria is sensitive, resistant, or intermediate when in contact with the antibiotic.

Human Error

The biggest problem with the disk diffusion test is human error, of course. Measurements can be taken inaccurately, making an antibiotic seem more or less effective than it actually is.

The Automated Alternative

Oculyze has developed an automated system for quickly and accurately measuring the size of the zones of inhibition on Agar plates for the disc diffusion (aka Kirby-Bauer) test. As already discussed, the susceptibility of each antimicrobial can only be quantified by measuring and comparing the size of the zones of inhibition. However, performing this step manually is tedious and error-prone. And this is where Oculyze comes in.

The Oculyze Disk Diffusion Test Results Analyzer makes accurate calculations for you. You just inoculate and incubate your agar plates and then upload the image of the plate to the Oculyze app. The results show you the diameter of each zone of inhibition, with the largest white number indicating the best reacting one.

In this way, you can not only trust in the measurement of the zone of inhibition, but you can also compare zone sizes across your plate or across multiple plates.

Test our Disk Diffusion Test Results Analyzer! Completely free of charge and with no commitment to purchase. Just please note that the recognition provided here is solely for demonstration purposes and may not accurately represent the performance of our product. Our customers receive customized recognitions tailored to their specific needs, which ensures high levels of accuracy.

Want to know more? Contact us and we’ll be happy to help!

Sources:

1. https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Microbiology_Labs_I/09%3A_Kirby-Bauer_(Antibiotic_Sensitivity)
2. https://www.acs.org/education/whatischemistry/landmarks/flemingpenicillin.html
3. https://asm.org/getattachment/2594ce26-bd44-47f6-8287-0657aa9185ad/Kirby-Bauer-Disk-Diffusion-Susceptibility-Test-Protocol-pdf.pdf