Fig. 1 Bacterial cell viability detection mechanism.

Assay DataFig. 2 Correlation between initial number of E. coli and time-dependent O.D. increase. The initial number of viable E.coli were determined by a colony counting method.

Fig. 3 Correlation between the initial number of SA and time-dependent O.D. increase. The initial number of viable SA were determined by the colony counting method.

Comparison Chart

Fig. 4 Influence of culture media or substances used for bacterial cell culture.The data indicated that WST is less sensitive to various culture media or substances which are used for bacterial cell culture. WST is a better tetrazolium salt than XTT for bacterial cell viability assays.

General Procedure 2Determination of the susceptibility of Staphylococcus aureus to oxacillinOxacillin: antimicrobial agent: 0-64 μg/mlMicroorganism: Staphylococcus aureus (SA)Methicillin-resistant Staphylococcus aureus (MRSA)1. Culture SA or MRSA with Mueller-Hintonmedium containing various concentrations of Oxacillin for 6 hours at 35ºC.2. Add Microbial Viability Assay solution equal to 1/20 the volume of the culture medium.3. Incubate for 2 hours at 35ºC.4. Measure the O.D. at 450 nm to determine the MIC (Minimum inhibitory concentration).

Fig. 4 Susceptibility test of SA and MRSA against Oxacillin*.The data indicated that MRSA has lower susceptibility than SA. The MICs of MRSA (32 μg/ml) and SA (0.5 μg/ml) are close to the MICs determined by the CLSI (Clinical and Laboratory Standards Institute) method.

1. T. Tsukatani, et al., Colorimetric cell proliferation assay for microorganisms in microtiter plate using water-soluble tetrazolium salts. J Microbiol Methods. 2008;75:109-116.2. T. Tsukatani, et al., Colorimetric microbial viability assay based on reduction of water-soluble tetrazolium salts for antimicrobial susceptibility testing and screening of antimicrobial substances. Anal Biochem. 2009;393:117-125.3. T. Tsukatani, et al., Determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts. Food Chem.2011;127:711-7154. T. Tsukatani, et al., Comparison of the WST-8 colorimetric method and the CLSI broth microdilution method for susceptibility testing against drug-resistant bacteria. J Microbiol Methods. 2012;90:160-1665. Jeffrey C. Pommerville. 2014. Fundamentals of Microbiology: Body Systems Edition (3rd Third Edition), p.339. Burlington, MA: Jones & Bartlett Learning.

Appropriate Cell DensityTable 6 Initial cell number can reach O.D. 0.5 with 1-hour and 4-hour incubation.The initial cell number of each microorganism was determined by colony counting. Each microorganism cell culture was diluted with medium and 190 μl of the cell culture was added to each well. Then 10 μl of assay solution was added. The cells were incubated at 30^oC or 37^oC for 1 hour and 4 hours to determine how many cells are required to reach O.D.=0.5 at 460 nm.