Hi all,
I am now working on Actinobacillus pleuropneumoniae and do the MIC test with my crude extract. However, after a few hours, the result of OD600 of the higher concentration crude extract is higher than that of lower one.
This is my method: the MIC of crude extract against A. pleuropneumoniae was determined by the modified microbroth dilution method. A. pleuropneumoniae was growed overnight in Brain heart infusion with β-Nicotinamide adenine dinucleotide 10 µg/mL, and then were the diluted in fresh broth medium to get the concentration of A. pleuropneumoniae was 5 × 10^3 CFU/mL. Equal volumes (50 μL) of bacterial culture and crude extract were mixed in the wells of a 96-well microplate. Negative control wells contain bacteria culture and medium only. Positive control wells contain bacteria culture and Chloramphenicol 2 µg/mL. A 96-well plate was placed in a 37 °C incubator for 16−18 h after dilution, and each dilution was replicated 6 times
The result in the attachment.
Has anyone encountered this situation?
Thanks for your consideration
OD600 is quite sensitive to settling, cell size, and shape. Do you stir the wells before measurements? Have you tried doing a cell count with a hemocytometer?
Ron Reiserer Thanks for your answer. I do stir the plate before measurements but I have not counted the bacterial number. A hypothesis I suppose is that the higher OD600 comes from the bacterial precipitation but I do not know how the deal with it
You could try running some samples on a standard cuvette and photometer. Since the standard photometer operates horizontally, you can get a better understanding of the settling rate of your culture by taking several readings over a couple minutes. I typically use a small volume cuvette for this since settling is seen much faster in the smaller volume.
You may want to correlate your OD with a viable plate count since you are interested in the live/dead condition of your culture. This will also inform you about the effectiveness of OD600 in your specific case.
You may also want to try using the agar dilution method.
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