I want to set different ODs as blank for different antibiotic dilutions on 384 well plate and measure it after 24 hours and report MIC 50% (Minimum Inhibition Concentration) automatically by OD metery not visually .
Anyone has the same experience?
Which formulation you used for final OD in comparison to primary OD to find MIC ?(the light is scattered in different concentration of bacteria and doesn't follow logarithmic curves)
Please, write about your experience.
Thank you
Hi Hesam Mahmodi,
If you want to calculate the MIC or IC50%, IC90% etc, a precise and accurate way is to use a fluorometric method (Resazurin assay). If you follow this technique, you don't need to set different ODs.
If you are using the 96 well plates:
1. Add 100 ul of media
2. Perform a serial dilution of different concentrations of antibiotics.
3. Add 10 ul of bacterial culture maintained at 0.5 McFarland standard (i.e. 1.5 x 10^8 CFU/mL or OD0.1)
4. Incubate for 18 h. Add 30 uL of 0.5% Resazurin solution and incubate for 2 h to check bacterial viability.
5. Use a plate reader to check the fluorescence (530/590). Wells with viable cells will turn pink from the blue.
6. Keep media and bacteria+media as controls.
7. Take Media+bacteria control as 100% bacterial growth and normalize your data.
8. You can use GraphPad prism or SPSS to find the IC50%.
If you want, you can follow:
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Hi Hesam Mahmodi,
The minimum inhibitory concentration (MIC) of the compounds are determined by means of the two-fold serial broth microdilution assay following Clinical Laboratory Standards Institute guidelines (CLSI). In aseptic conditions 100 μL of MHB or SDB medium is distributed in each well of a 96-well V-bottom plate. To the first well of each row is added 100 μL of the disinfectant/antibiotic formulation, positive control (commercial antimicrobial) or negative control (sterile culture media) solutions at 1 mg/mL concentration. Using a multichannel micropipette, a serial dilution was made to 1:2 proportion between each row of wells (50 000- 0.49 mg/L range). Lastly 10 μL of bacterial suspension [0.5 McFarland standard (≈108 CFU/mL)] is added to every well and plates are covered and incubated at 37 °C for 24 h. The bacterial growth is measured with an absorbance microplate reader set to 595 nm. Assays are carried out at least in triplicate for each tested microorganism.
You can use GraphPad prism or SPSS to find The MIC values.
Kind regards
Hi @Satwik Majumder
Thank you for your respond.
I have three questions.
1. Can I use MTT or XTT instead of Resauzurin? Which one is the best?
2.Some antibiotics such as Nitrofurantoin makes color. Does it affect the OD?What is the solution?
3.What is the formula or method in SPSS or other softwares to calculate MIC? because in some experiences I saw that OD decreases gradually and it doesn't drop in one or two consecutive wells. How I set one of that wells to MIC by OD?