currently conducting a study on antibiotic resistance and using MIC assay to evaluate the effectiveness of colistin antibiotics and using standard broth microdilution assay but encountering inconsistent results. I used the following methods-
1. Concentration of antibiotics were 256µg/ml . The Ab is diluted in broth to create a series of 2 fold concentrations. Here is the result of MIC assay and we found inconsistent results after measuring ELISA machine.
2. I standardized amount of microbial broth culture was added to each well containing different concentrations of Antibiotics.
3. The microtiter plates were inoculated at 37° for 18-24 hrs.
4. Microtiter plates were examined for microbial growth by visual inspection of turbidity by ELISA machine.
When interpreting MIC values, be aware of: 1. Test variability: Intrinsic (biological) and extrinsic (methodological) factors. 2. Antibiotic degradation or instability. 3. Inoculum effect: Incorrect bacterial concentration. 4. pH and temperature deviations. 5. Medium composition and cation concentration (e.g., Ca2+ and Mg2+). 6. Incubation time and conditions. 7. Reader variability (ELISA machine). Troubleshooting steps: 1. Verify antibiotic stock solution concentration and stability. 2. Check inoculum density (CFU/mL) and purity. 3. Ensure proper broth preparation and pH control. 4. Validate ELISA machine calibration and performance. 5. Repeat tests with fresh reagents and controls. 6. Consider parallel testing with alternative methods (e.g., agar dilution). 7. Analyze results using appropriate statistical methods. For broth microdilution: 1. Use standardized broth (e.g., Mueller-Hinton). 2. Maintain consistent incubation conditions (37°C, 16-20 hours). 3. Ensure accurate antibiotic dilutions. 4. Include controls (positive, negative, and sterility). 5. Follow CLSI guidelines
Please follow CLSI guidelines for procedure and interpretation including quality control.
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