Hi everyone,
I am working on finding minimal inhibitory concentration of a target compound to E. coli harboring resistance plasmid. MICs were determined using turbidity of bacterial culture. The method is described as follow:
In each 150 μL well of 96-well plates, I introduced a 10 μL of selected clone cultures (106 CFU/mL.), 10 μL of serially 2-fold diluted compounds, and 130 μL of fresh LB medium
Studied concentration of target compound is from 10 ppm to 1000 ppm.
PBS was used as blank.
LB broth containing only bacteria was used as control sample.
Followed growth by measuring absorbance over 20 h at 30 degC. Absorbance was measured every 30 min after 5-second shaking.
My problem is that the absorbance of control sample is only 0.2 after 20 hour, while in the batch study, after 20 hours, the absorbance can reach more than 1. For other sample on 96-well plates, the absorbance still can increase to 1 after 20 hours.
I am really wonder whether there is any limitation existed when I doing MIC test. Could you please help me?
The standard procedure for broth microdilution MICs does not include shaking of the plate. It is simply incubated for 20 hours at the required temperature (usually 35oC), and the absorbance is read after 20 hours.
Thank you so much for your information, Adam. I will redo my experiment without shaking. Since I will study E.coli species at 30 DegC, do you think I should do MIC test at the same temperature?
As I know, MIC test is conducted at the same condition that we use for process of a study.
If the 30oC temperature is important, then I suppose it makes sense to do the incubation at that temperature.
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