Improved Formazan Dissolution for Bacterial MTT Assay

Area of Science:

• Microbiology
• Biotechnology
• Cell Biology

Background:

• The MTT assay is a common method for assessing cell viability and proliferation.
• Accurate MTT assay results depend on complete cell and formazan solubilization and solution stability.
• Solubilization solvents can introduce artifacts, leading to misinterpretation of MTT assay data.

Purpose of the Study:

• To compare different solubilization solutions for the MTT assay.
• To identify solvents that minimize artifacts in MTT assays for microbiological cultures.
• To ensure accurate spectrophotometric detection of formazan.

Main Methods:

• Comparison of various solubilization solutions for MTT assay.
• Evaluation of solvent performance based on solubilization efficiency, background absorbance, reproducibility, sensitivity, and color stability.
• Testing MTT assay linearity with cell density for Gram-negative and Gram-positive microorganisms.
• Utilizing dual-wavelength measurements (570 nm and 700 nm) to correct for artifacts.

Main Results:

• Dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), buffered with ammonia buffer (pH 10) and containing 5% SDS, provided the best results.
• These solvents ensured rapid and complete solubilization of formazan and cells.
• Optimal conditions yielded minimal background absorbance, good reproducibility, high sensitivity, and color stability for at least 24 hours.
• A linear relationship between viable cell number and formazan absorbance was maintained up to 1 × 10^9 cells/mL for both Gram-negative and Gram-positive bacteria.

Conclusions:

• DMF and DMSO are effective solubilization solutions for the MTT assay in microbiological cultures.
• These solvents minimize artifacts, improving the accuracy of cell viability and proliferation assessments.
• Proper methodology, including simultaneous blank controls and dual-wavelength measurements, is crucial for reliable MTT assay results.