I agree with Nour Elhouda Bekkar . Elaborating that, you adjust the optical density of bacteria (against which you want to calculate the MIC) at 600 nm approximately 0.4-0.5. This OD equals to almost 4-5x10^8 cfu/ml for most of the bacteria like E.coli, S. aureus, etc. Then dilute this culture 1000 times in Mueller Hinton Broth (MHB) and it would be your working inoculum for MIC. Now use microbroth dilution method as per CLSI guidelines. Add 100 micro liter of MHB containing two-fold diluted concentration of your extratcs in the wells of 96-well plate. Add 100 ul of above mentioned inoculum (Total volume 200 ul) and incubate the plates at 37 degree Celsius. Check the growth after 18-20 h. The well with no visible growth will be the MIC for your extract.
Dear R. Saad,
If the extract is powder, you can make different dilutions (known concentrations) of the extract and use culture tubes (broth) or disc diffusion (agar plates) for MIC. Estimate the growth inhibition by O.D. - CFU/mL (for broth) or zone of inhibition (agar plates).
The media for bacterial growth can be selected based on the strain you wish to check.
I hope it will help you.
Best
Prasun
Using microdilution method. Making the Microbial suspensions adjusted to 0,5 McFarland with a volume of increased concentrations. The well in the microplate with no visible growth culture is considered as the MIC value.
I agree with Nour Elhouda Bekkar . Elaborating that, you adjust the optical density of bacteria (against which you want to calculate the MIC) at 600 nm approximately 0.4-0.5. This OD equals to almost 4-5x10^8 cfu/ml for most of the bacteria like E.coli, S. aureus, etc. Then dilute this culture 1000 times in Mueller Hinton Broth (MHB) and it would be your working inoculum for MIC. Now use microbroth dilution method as per CLSI guidelines. Add 100 micro liter of MHB containing two-fold diluted concentration of your extratcs in the wells of 96-well plate. Add 100 ul of above mentioned inoculum (Total volume 200 ul) and incubate the plates at 37 degree Celsius. Check the growth after 18-20 h. The well with no visible growth will be the MIC for your extract.
Be very careful to evaluate the solubility (in water) of the extract before testing. Sometimes the solubility is very low. you can use some solubilizing agents if not soluble but the problem is those will interfere with the assay. One option for insoluble compounds is to dissolve in soybean oil, then make emulsions and perform the test
I agree the solubility of extracts is very important. To have a good antimicrobial activity results, we have to ensure the solubility of extracts.
Thanks for your answers which are really valuable. as mentioned above there is a need to use a solvent like DMSO, but there is a confusion regarding the technique in application of the solvent as its not clear in which step its applied and at which conc. that is should applied. and can we use another solvents as ethanol??
waiting for your answers and grateful for your interest
First of all, use solvent extraction method to extract the active components of the herbal powder,leaves or roots. Use different solvents such as water, ethanol, acetone etc.once you have extracted this, you can prepare a micro dilution of the extract to obtain different concs. Then use agar well diffusion method to test.
Use different solvents as dmso, ethanol and water and test each of them. Do sensitivity test or agar well diffusion. Later procced to micro dillution method to determine MIC thn MBC.
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