Hello,

I’m currently conducting MIC assays using tetracycline and have encountered some issues that I’d appreciate your input on.

We began testing at 1000 µg/mL, but observed bacterial growth in all wells, indicating that this concentration was insufficient to inhibit growth. To align with the concentrations used for other antibiotics in our study, we increased the starting concentration to 5000 µg/mL. However, this led to a different issue.

In our serial dilution plates at 5000 µg/mL, the first 3 wells (with the highest tetracycline concentrations) showed unexpectedly high OD600 readings, despite no visible microbial growth. The OD values gradually dropped around the 4th or 5th well, eventually matching our negative control. Wells 6–12 displayed low-level microbial growth as expected.

We suspect that either:

Given this, I have a few questions for those with experience in antibiotic MIC assays:

• What methods have you found effective for improving tetracycline solubility (e.g., filtration, heating, pH buffering)?
• How do you control for colorimetric interference during OD-based readings, especially with visibly pigmented antibiotics?
• And importantly, what would be a recommended or standard maximum concentration of tetracycline to use for MIC testing in environmental or resistant isolates?

Any guidance, shared experiences, or relevant publications would be greatly appreciated. I'm happy to connect and collaborate with others investigating antimicrobial resistance or assay optimization.

Best regards,

Eshaan Patel

Marian University Wood College of Osteopathic Medicine