Previously MIC was 6.25 mg/mL — now even 100 mg/mL doesn’t kill. What could be going wrong?

Hi everyone,

I’m running antibacterial assays with an insoluble powder and I’m facing a major reproducibility issue.

In my earlier experiments, my MIC was 6.25 mg/mL, and I observed clear killing at higher concentrations like 50 mg/mL.

Now I’ve repeated the experiment — and even at 100 mg/mL I’m not seeing killing. Growth looks very similar to the untreated control.

Experimental setup:

•	Bacteria grown overnight for \~16–18 hours before use

•	Based on my OD–CFU calibration, final inoculum per well should be \~5 × 10⁵ CFU/mL

•	Each well contains 150 µL of drug suspension

•	I inoculate by pipetting 5 µL of bacterial culture into each well

•	Incubation at 37°C with shaking

•	Plates read after 24 hours (standard MIC/MBC style setup)

What’s confusing is that based on my calculations, 100 mg/mL should expose each bacterial cell to far more active compound than 6.25 or 50 mg/mL — so I would expect at least similar or stronger killing, not complete loss of effect.

I’m trying to troubleshoot what might be happening:

•	Could it be that my overnight culture (\~16–18 h) is too old and the bacteria are in stationary phase, making them more tolerant?

•	Could this be a mixing issue since the powder is insoluble and may settle during incubation?

•	Could burst-release kinetics mean that a classic 24-hour MIC/MBC setup isn’t appropriate, and that exposure needs to be synchronized or measured at shorter time points?

•	Could small differences in inoculum prep drastically change results?

Has anyone experienced something similar — where initial MIC looked strong, but later repeats at even higher concentrations show little to no activity?

Any insights would be really appreciated. I’m trying to figure out whether this is a biological tolerance issue, a physical distribution problem, or an assay design flaw.

Thanks in advance.