Technique Tuesday: MIC Broth Dilution

Sep 29, 2020
By Jenna Beam

Technique Name: Minimum Inhibitory Concentration (MIC) tests.

This article will focus on broth dilution tests, however other tests (like the E-test) are also used to determine the MIC.

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MIC tests are super quick and simple to set up and pretty easy to interpret, as well. We give this a solid 1 bacterium out of 5 on a difficulty scale.  What is the general purpose? This technique is used to figure out what “concentration” (meaning a measure of how much of a given substance is present in a solution or mixture) of an antibiotic stops the growth of various types of bacteria.

Why do we use it? We use MIC tests to figure out how effective certain antibiotics are at fighting bacteria. For example, if someone has an infection that won’t go away, a doctor can order an MIC test to see if the bacteria causing the infection are resistant to the antibiotic. If bacteria are resistant, then the doctor may need to prescribe a different antibiotic. It can also be used to test how effective new antibiotics will be. 

How does it work? 

An MIC test works by exposing bacteria to increasing concentrations of a particular antibiotic and identifying which concentration prevents the bacteria from growing. There are three main components of an MIC: the antibiotic being tested, growth media (more on this below), and the bacteria. To set up an MIC, the first thing we need to do is make the antibiotic solution. In the diagram below, you can see that we start with the highest concentration of antibiotic we want to test, and then reduce the concentration as we move across the plate, otherwise known as “diluting.” We then add the bacteria to the different antibiotic concentrations and put the plate in a nice, warm incubator to let the bacteria grow overnight. 

After about 16-20 hours, we look first at how well the bacteria  grew without any antibiotic, also known as the “growth control”. We include this so we know what growth in the plate looks like without any restrictions. This may seem silly, but controls are super important for making sure you correctly interpret your results. Without the growth control, we wouldn’t have anything to compare the antibiotic-treated bacteria samples to and therefore have no way of knowing if the antibiotics are actually working. Next, we look at our “media control”. The media we use in MIC tests is a type of broth (think chicken broth) called Mueller-Hinton broth (fun fact: Dr. Jane Hinton was one of the first two Black women to earn a Doctor of Veterinary Medicine degree in the United States). We include the media control so we know what the media alone looks like and to make sure that we didn’t accidentally contaminate our plate. We then look at the wells containing the bacteria with antibiotics. As you can see in the image below, we look for the first well that looks like the media control well – that’s your MIC!

An example of an MIC test. The media control is in Column #1. We don’t put any bacteria in this column to make sure that our media is free from contamination. All the way on the right in column #12 is the growth control. We don’t put any antibiotic in this column. In column #2, we put the highest concentration of antibiotic we want to test and then dilute the antibiotic as we move to the right. Our lowest concentration will be in column #11. We determine the MIC by looking at the first well where we don’t see the bacteria growing. Based on the information above, what do you think the MIC is for each row A-H? See the table below for the answers! Diagram adapted from Emery Pharma.

*MICs based on the diagram:

Lane MIC Lane MIC
A Column 8 E Column 4
B Column 7 F Column 8
C Column 7 G Column 9
D Column 6 H Column 5
Edited by Allison Woods and Alec Chaves