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Testing an Antibiotic Using a Disk Diffusion Assay - Kirby Bauer Method - YouTube
Channel: Gold Biotechnology, Inc.
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Ah, Gold!
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Hi everybody, its Patrick from GoldBio here
One of our goals is to save your lab money.聽
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So we're here today to show you聽
an assay that will allow you to聽聽
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easily test the efficacy of your antibiotics.
Like most chemical reagents antibiotics will聽聽
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slowly degrade over time and lose potency,聽
whether in a dry powder or in solution.聽
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So how do you know if that ampicillin you found聽
in the back of the freezer was any good? Easy!
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To test the efficacy we'll be using聽
a modified disc diffusion assay also聽聽
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known as a Kirby Bauer assay.
In this assay a filter paper聽聽
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disc is loaded with a specific amount of聽
antibiotic and placed on a lot of bacteria.聽
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The antibiotic will diffuse out of聽
the disc and into the media where聽聽
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it's taken up by the growing cells.
If the bacteria are susceptible to聽聽
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the antibiotic they will die off.
The area with no bacterial growth聽聽
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surrounding the disc is called the zone of聽
inhibition and by looking at the size of this聽聽
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region the researcher can say if the strain is聽
susceptible or resistant to a certain antibiotic.聽
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For this test, we'll use a known strain of聽
bacteria to make sure a specific amount of聽聽
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antibiotic is giving us the correct聽
zone of inhibition. Let's get started
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You will need: 6 millimeter filter paper disks,
sterile petri dishes,聽聽
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agar plates with no added antibiotic,
sensitive and resistant bacterial cultures,聽
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an antibiotic to test and
an antibiotic control
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You'll need two bacterial cultures: one聽
that's susceptible to the antibiotic聽聽
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you're testing, and one that's resistant.
The day before you want to test grab a聽聽
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isolated colony from a plate and grow overnight.
The day of the test dilute your cultures back and聽聽
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grow to about mid log phase around an OD of 0.5
While your cultures are growing out, you can make聽聽
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up your antibiotic test disks.
For testing out an antibiotic聽聽
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powder start with a fresh solution of聽
each antibiotic being tested and then聽聽
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diluting them down to the desired concentration.
The disks are able to hold about 20 microliters of聽聽
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liquid and for the ampicillin test we'll need聽
a final amount of 10 micrograms on the disk.聽
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So we'll need a solution with a final聽
concentration of 0.5 mix per mil.
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Making sure to keep everything as sterile聽
as possible add a few 6 millimeter聽聽
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filter paper discs to a petri dish.
I always add a few in case I drop one.聽
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Next add 20 microliters of聽
ampicillin solution to each disk.
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After the solution has been聽
added let the disc dry.聽
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This can be done covered at room temp on聽
the bench and it can take up to three hours.聽
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For faster drying I leave my dishes open聽
in a laminar flow hood which will dry the聽聽
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disks in as little as 30 minutes.
Label your auger plates in a way聽聽
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that sections off each test disk so you聽
can differentiate them after incubation.聽
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I use six sections because I use a blank聽
disc, two control discs and three discs聽聽
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for the antibiotic I'm testing, in order聽
to make sure that I get a better estimate聽聽
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of the antibiotics functionality.
Once your cultures have reached the聽聽
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correct density, we're going to plate聽
around 200 microliters onto each plate.
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Make sure to spread the bacteria聽
evenly so that all areas are covered.聽
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Once there's no visible liquid on the聽
plate you're ready to place your disks.聽
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Working quickly you'll use tweezers to place the聽聽
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disks on to the center of the section聽
area pressing them gently into place.聽
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You want to make sure to sterilize your聽
tweezers in 70% ethanol between disks.聽
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For each plate I always include a blank disc with聽
no antibiotic and a purchased disc as a control.
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After all the discs are placed incubate聽
the plates inverted overnight and then聽聽
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after around 16 to 20 hours measure the聽
zones of inhibition around each disc.聽
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If the test discs match or exceed聽
the standard then your product is聽聽
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working correctly and you'll be able聽
to use them for other experiments.
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And that's it, now you can easily check聽
the antibiotic sitting in the back of聽聽
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your freezer for efficacy, before you聽
worry about spending money on more.聽
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And by manipulating the different variables聽
in the test, you can use the Kirby Bauer聽聽
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assay for a variety of other lab tests.
If you have any questions please email me聽聽
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at [email protected]
Thanks for watching!
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