How to Calculate the Zone of Inhibition
Calculating the zone of inhibition is an important step in the Kirby-Bauer test, which is used to analyze the sensitivity of bacteria to antibiotics. First developed in the 1960s, the test is still used today in clinical labs to compare the effectiveness of various antibiotics. Science students practicing their research skills can also perform this simple test. Although setting up the test takes time, calculating the zone of inhibition itself is fairly easy.
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Instructions
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1
Gather the prepared samples. In the test, a strain of bacteria is spread onto a dish of agar with small circular pieces of antibiotic-soaked filter paper pressed on top. The set-up is incubated overnight, allowing the antibiotic to infuse into the agar.
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2
Place the agar dish on a black sheet of paper. This will make the zone of inhibition easier to see.
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3
Identify the zone of inhibition around one piece of filter paper. The zone will be a circular area around the filter paper where there is no bacteria growth.
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4
Place a ruler under the dish and measure the diameter of the zone of inhibition. Measure to the nearest millimeter. If bacteria growth was not inhibited around this piece of filter paper, the zone of inhibition is the diameter of the filter paper.
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5
Repeat steps 3 and 4 for each piece of filter paper.
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6
Label the filter papers and their respective antibiotics with the smallest zones of inhibition as "Resistant," the ones with the largest zones as "Sensitive" and those in between "Intermediate."
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Tips & Warnings
When determining the borders for the zone of inhibition, look for a clear line of growth. A faint haze or tiny colonies near the edge can be ignored. However, if large colonies in an otherwise clear zone appear, contamination should be suspected.
Working with bacteria can be dangerous. Do not open an agar filled dish with bacteria in it. Make sure to dispose of materials in a biohazard bag and always wash your hands after handling the prepared dishes.
