I'm currently working on a project with antibiotic resistance in bacteria, and using a disc-diffusion method; however we're trying to switch to Minimum Inhibitory Concentration, but I'm looking for an alternative that uses less material. Does anyone use a 96-well microplate to do it?
Because I work with Streptococcus agalactiae (GBS), I need a way to add blood to the mix
Dear Cicilia
you can find several protocol reporting MIC determination in 96 well plates.
(e.g http://www.boddylab.ca/file/MIC_Determination_by_Microtitre_Broth_Dilution_Method.pdf) and with GBS is also easier because it growth anaerobically therefore you do not need areation therefore you can avoid shaking. However MIC determination in microtiter plate require a OD measurment and therefore you need to growth the bacteria in a media as THB, TSB probably also with 5% of blood but you need to make a blank and be sure that the media itself do not show strong absorbance at 600nm.
good luck
Manuele
Dear Cicilia
you can find several protocol reporting MIC determination in 96 well plates.
(e.g http://www.boddylab.ca/file/MIC_Determination_by_Microtitre_Broth_Dilution_Method.pdf) and with GBS is also easier because it growth anaerobically therefore you do not need areation therefore you can avoid shaking. However MIC determination in microtiter plate require a OD measurment and therefore you need to growth the bacteria in a media as THB, TSB probably also with 5% of blood but you need to make a blank and be sure that the media itself do not show strong absorbance at 600nm.
good luck
Manuele
Hi Cilicia,
In fact, MIC determination is the most common way of assessing antibiotic inhibitory concentrations. I would suggest you to follow the international standards like EUCAST (http://www.eucast.org/ast_of_bacteria/mic_determination/?no_cache=1) or CLSI guidelines. As an example I have attached you some documents, that might help.
Good luck!
Determination of MIC values using the 96-well plate or broth microdilution is the most accurate method of determining the MIC. You need to prepare 0.5 McFarland from your bacteria and prepare doubling dilutions of the antibiotic you are using. add 100 microliters of a sterile broth preferably MH to each well and then add 100 microliters f the antibiotics dilutions. 5 microliters of the bacterial suspension used to inoculate each well. incubate overnight and the MIC is the lowest concentration of the antibiotic that inhibited bacterial growth. hope that helps.
You can as well use MIC test strips, that's also easier. That allows you to select the antimicrobials you want to tested.
Yes,
I also use a 96 well plates with help to less amount of use material and showing a good result and different different cont. To know O.D.
Can I get information about model of spectrometer best for MIC OD measurement ? Thank you
@Mohammad Hossen you can use an indicator dye, this will save you the stress of having to measure the OD. tetrazolium salts are appropriate and preferably INT
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