What Does the Kirby-Bauer Test Assess

If you’ve been studying or working on biology, microbiology, and/or antibiotics, you may have begun practicing disc diffusion without realizing its origins. You’re hearing about a Kirby-Bauer test, and maybe you feel like it’s too late to ask anyone in the lab about it for fear of looking inept. Fear not. This article will help you answer the question, “what does the Kirby-Bauer test asses?” It will also give you an in-depth look at the history and the process.

Also read:
What is a Kirby Bauer Test?
How to Measure the Zone of Inhibition: Kirby-Bauer Test

The Kirby-Bauer Disc Diffusion Method Procedure

Way back in the early 1800s when Louis Pasteur first got his hands on a microscope and started examining germs at close range, science began to wonder about antibiosis. The thinking went that if there was a bacterium that could cause harm, there had to be a way to combat that bacterium with its counterpoint.

In essence, something that occurred in nature, bacteria, had to have something that could wrestle with it, an antibiotic. This rule is almost always true in nature in order to keep the delicate balance we have.

But how to investigate this phenomenon?

Scientists began to expose bacteria they were studying to other naturally occurring organisms to see what the effect might be.

Then, 100 years later, a Scottish bacteriologist named Alexander Fleming came back to the hospital where he worked after a vacation to find a mess of Petri dishes.

In those dishes, he discovered a penicillium mold effectively fighting off a bacterium he had been examining – Staphylococcus aureus.

It is worth noting that Fleming has said of his discovery that he did not invent the first antibiotic but that nature had done that. All he did was discover it.

And a wonderful discovery it was.

But the truth is that Fleming did not have the equipment necessary to properly examine and study the interaction between penicillium and bacteria. He had to call upon a colleague at Oxford, Dr. Howard Florey, who was a professor of pathology and much more experienced in chemistry, for help.

From there, the study of how an antibiotic can combat a bacterium began.

But it was not easy.

Scientists would place bacteria on a petri dish and then place antibiotics over the bacteria to see how the bacteria would behave. They figured out how to dilute bacteria samples, how to place the bacteria on agar so they could identify and count individual colony forming units (CFUs), and how to place small doses of a particular antibiotic onto the bacteria to engage and examine the reactions.

But there was no standard, so a lot of lab mishaps and human error came into play.

Things went on in this way until the 1960s, when William Kirby and Alfred Bauer cowrote a paper on disc diffusion and the zone of inhibition. It was a standardized, streamlined approach to the mess that had been taking place in laboratories across the globe.

The World Health Organization adopted the method outlined in this paper, and the Kirby-Bauer test has since been adopted in labs around the world as the standard.

Purpose of the Kirby-Bauer Test

The purpose of the Kirby-Bauer test is to measure how effective an antibiotic is against a bacterium.

To perform the test, you must have:

A Petri dish with agar (use the growth nutrient best suited for the bacteria you are studying)
A bacteria sample
Sterile water
Sterile vials for dilutes
Pasteur pipettes
Cotton swabs
Rubber gloves
An incubator
A black marker
Antibiotic discs (preferably in an automatic release mechanism, so the discs are spaced out evenly and placed exactly)

To begin, you will need to dilute the bacteria sample enough to count the CFUs on the Petri dish.

You can do this dilution by taking 1 ml of the bacteria and placing it in a sterile vial then adding 9 ml of sterile water. This dilution will give you a 1:10 ratio and can be performed one or two more times if necessary. Be sure to keep track of how many times you dilute.

Gently shake the vial with the stopper on it to be sure the bacteria mix well.

Next, take a cotton swab, dip it into the vial, and spread the bacteria on the agar plate on the Petri dish. Swipe the swab back and forth in a tight jagged motion, rotate the dish 90 degrees, and repeat the swiping until the entire dish has been covered with the medium to each edge and up along the sides.

Next, use your automated disc dispenser to place antibiotic discs strategically on top of your bacteria medium.

Place the lid on your Petri dish and place the dish in the incubator at the temperature required for you particular bacteria, for 24 hours.

After 24 hours, you will see “zones of inhibition,” which are the areas around the discs where the bacteria have receded, letting you know how effective the antibiotic is against the bacteria. This procedure is the Kirby-Bauer test.

How to Measure the Zone of Inhibition

To measure the zone of inhibition, or the space around the disks, you will take a small ruler that measures in millimeters. From the bottom of the Petri dish, place the “0” part of the ruler in the center of the disk and measure from that 0 to the outer edge of the inhibition zone.

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All you’ll need to do is upload an image of the underside of the Petri dish, and you will get an accurate measurement of the zone of inhibition back almost immediately.

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