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71abdullah 71abdullah
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13 years ago
I am doing a project in which I have to test some plant extract for antibiotic resistance. I have been told to perform disk diffusion antibiotic sensitivity test, which involves soaking small paper disk in the extract and placing the extract-laden disk on a solid culture of certain bacteria. I am supposed to try different concentration and look for clear zone - which is indicative of antibiotic activity of the extract.

But I wonder why i have to use disk. Why don't use pipette to drop (and pinpoint) small amount of extract on the culture media and look for the clear zone. Why soaking a disk with extract? specially when it can be done more simply (presumably)?
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wrote...
Educator
13 years ago
This is called the Kirby-Bauer test and it's just as simple as pipetting small amounts of extract directly unto the agar. It's an inexpensive test widely used to reveal which drug is most effective against a particular pathogen. The procedure involves, like you described, inoculating a Petri plate uniformly with a standardized amount of the pathogen in question and arranging on the plate disks soaked in the drugs to be tested. In general, the larger the zone of inhibition around a disk, the more effective the drug.

Link: https://biology-forums.com/definitions/index.php/Diffusion_Susceptibility_Test
Link: https://biology-forums.com/definitions/index.php/Kirby-Bauer_test

According to the links I provided, it states that the disk (the inoculum) is sufficiently heavy, resulting in a lawn of bacteria after incubation. This, I'm assuming, does not happen with pipetting liquid onto the medium because eventually it will spread out and be absorbed by the agar. Also, pathogens at the centre of the liquid drop may not get enough oxygen and so your reducing oxygen content at that one area.
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Valued Member
13 years ago
In this test, also known as the Kirby-Bauer test, a bacterial culture is inoculated on an agar medium,
and filter paper disks impregnated with chemotherapeutic agents are overlaid on the culture. After incubation, the absence of microbial growth around a disk is called a zone of inhibition. The diameter of the zone of inhibition, when compared with a standardized reference table, is used to determine whether the organism is sensitive, intermediate, or resistant to the drug.

The diameter is produces is different for different bacteria and so based on the diameter you get, you can see just how effect it is based on other results, for example:

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71abdullah Author
wrote...
13 years ago
Bio_man, disk does not contain inoculum. The link you provided simply says the agar was heavily inoculated before adding the disk (which contains antibiotic).
wrote...
Educator
13 years ago
I was thinking about this today I found came up with a conclusion; I think the reason why they use this test is because it provides the researcher a quantitative understanding depicting the affects of the extract, and this is done by calculating the diameter of the zone. Otherwise, with what you suggested, all you would get are colonies growing randomly and areas where growth is inihibited. But by knowing the diameter of the zone of inhibition, you know how effective the extract is as you can compare its diameter with other known bactericidal extracts.
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