An improved MTT colorimetric method for rapid viable bacteria counting

Area of Science:

• Microbiology
• Biotechnology

Background:

• The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay is used for bacterial growth analysis.
• Bacterial MTT assays face more interfering factors than mammalian cell assays, impacting accuracy.

Purpose of the Study:

• To develop an improved MTT assay protocol for bacterial growth analysis.
• To establish a correlation equation (CFU=kF) between colony-forming units (CFU) and formazan conversion (F).
• To determine the scale factor 'k' for specific bacterial species.

Main Methods:

• Developed an improved MTT assay protocol for bacteria.
• Established the equation CFU=kF correlating bacterial count with formazan production.
• Determined the scale factor 'k' using Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli).
• Utilized High-Performance Liquid Chromatography (HPLC) for formazan analysis.

Main Results:

• Optimal MTT reduction time for S. aureus and E. coli at room temperature is approximately 30 minutes.
• E. coli exhibits approximately four times greater MTT reduction capacity per cell than S. aureus.
• HPLC analysis effectively mitigates interferences in formazan dissolution and quantification.
• Identified a narrow linear range (0-125 μg/mL) for formazan concentration-absorbance and proposed strategies to overcome this limitation.

Conclusions:

• The improved MTT assay protocol and CFU=kF equation enhance the accuracy of bacterial growth quantification.
• Understanding bacterial MTT reduction kinetics and optimizing analysis methods are crucial for reliable results.
• HPLC and addressing the linear range limitation are key to successful bacterial MTT assays.