Quantification of metabolically active biomass using Methylene Blue dye Reduction Test (MBRT): measurement of CFU in about 200 s

J Microbiol Methods. 2006 Apr;65(1):107-16. doi: 10.1016/j.mimet.2005.06.010. Epub 2005 Jul 22.


Quantification of viable cells is a critical step in almost all biological experiments. Despite its importance, the methods developed so far to differentiate between viable and non-viable cells suffer from major limitations such as being time intensive, inaccurate and expensive. Here, we present a method to quantify viable cells based on reduction of methylene blue dye in cell cultures. Although the methylene blue reduction method is well known to check the bacterial load in milk, its application in the quantification of viable cells has not been reported. We have developed and standardized this method by monitoring the dye reduction rate at each time point for growth of Escherichia coli. The standard growth curve was monitored using this technique. The Methylene Blue dye Reduction Test (MBRT) correlates very well with Colony Forming Units (CFU) up to a 800 live cells as established by plating. The test developed is simple, accurate and fast (200 s) as compared to available techniques. We demonstrate the utility of the developed assay to monitor CFU rapidly and accurately for E. coli, Bacillus subtilis and a mixed culture of E. coli and B. subtilis. This assay, thus, has a wide applicability to all types of aerobic organisms.

MeSH terms

  • Bacillus subtilis / growth & development*
  • Bacillus subtilis / metabolism
  • Biomass*
  • Cell Survival / physiology
  • Colony Count, Microbial
  • Coloring Agents / metabolism*
  • Escherichia coli K12 / growth & development*
  • Escherichia coli K12 / metabolism
  • Hydrogen-Ion Concentration
  • Methylene Blue / metabolism*
  • Oxidation-Reduction
  • Reproducibility of Results
  • Spectrophotometry, Ultraviolet


  • Coloring Agents
  • Methylene Blue