A simple colorimetric method for viable bacteria detection based on cell counting Kit-8

Abstract

In this study, Cell Counting Kit-8 (CCK-8) was introduced to detect the concentration of live bacteria for the first time depending on the redox reaction between CCK-8 solution and dehydrogenase. CCK-8 solution can be reduced to form water soluble orange-yellow formazan by the dehydrogenase present in bacterial cells, and the concentration of live bacteria is proportional to the absorbance value of formazan at 450 nm. Based on this principle, <i>Staphylococcus aureus</i> and <i>Escherichia coli</i> were chosen as the model bacteria. The optimal detection conditions were investigated and a good linear relationship was obtained in the concentration range from 2.600 × 10² to 1.160 × 10⁹ CFU mL^-1 with a linear equation of <i>Y</i> = 0.06305 log₁₀ <i>X</i>-0.1153 (<i>X</i> in CFU mL^-1, <i>R</i>² = 0.9747) for <i>S. aureus</i> and 9.750 × 10² to 6.000 × 10⁸ CFU mL^-1 with a linear equation of <i>Y</i> = 0.06122 log₁₀ <i>X</i>-0.1358 (<i>X</i> in CFU mL^-1, <i>R</i>² = 0.9958) for <i>E. coli</i>. The CCK-8 based viable bacteria detection method can be completed within 2 h with a wide bacterial detection concentration range. Satisfactory results were obtained when applied to an actual sample analysis and there is a good consistency between the proposed CCK-8 based method and the traditional plate counting method. More importantly, this method can realize the one-time detection of a large number of samples with high sensitivity, which suggests its great potential in high-throughput bacterial detection.