Microbial Viability Assay Kit-WST

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	10 μl of a 1 : 10 (for E. coli) or 1 : 80 (for L. innocua) mixture of the electron mediator and add WST‐8 immeaditely. Incubate the plate in the incubator at 30C for 1 hr

Protocol tips
10 μl of a 1 : 10 (for E. coli) or 1 : 80 (for L. innocua) mixture of the electron mediator and add WST‐8 immeaditely. Incubate the plate in the incubator at 30C for 1 hr

Publication protocol

The metabolic activity of L. innocua and E. coli after the pulsed light treatment was determined with the Microbial Viability Assay kit (Dojindo Molecular Technologies, Kumamoto, Japan). This kit contains the tetrazolium salt WST‐8 and the electron mediator 2‐methyl‐1·4‐naphthoquinone. WST‐8 is cleaved to a water‐soluble formazan dye by active dehydrogenases, indicating metabolic activity of microbial cells. This reaction leads to the formation of a yellow colour at which the intensity is proportional to the number of viable cells (Tsukatani et al. 2008). 100 μl of the sample suspensions were transferred to a 96‐well microplate and mixed with 100 μl twofold‐concentrated Mueller–Hinton broth. 10 μl of a 1 : 10 (for E. coli) or 1 : 80 (for L. innocua) mixture of the electron mediator and WST‐8 was added immediately. Each sample was measured in triplicates, and blanks (PBS) as well as untreated controls were included in every assay. The absorption was measured at 450 nm and recorded every minute in a microplate reader (Spectramax 190; Molecular Devices, Sunnyvale, CA) for 1 h at 30°C. Within this time frame, a linear increase in absorption could be observed. The relative metabolic activity after the pulsed light treatment was determined by calculating the ratio of absorbance between the treated samples and the untreated controls after 60 min. All samples were analysed immediately after the pulsed light treatment and again after a storage period of 24 h at 5°C.

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Manufacturer protocol

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