Viability/Cytotoxicity Assay kit for Bacteria Live and Dead Cells

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Remove the supernatant and resuspend the pellet of one tube in 0.3 mL of 0.85% NaCl solution and another tube in 1 mL of 0.85% NaCl. Add 0.7 mL isopropyl alcohol into the tube with 0.3 mL of 0.85% NaCl and mix well (final concentration of isopropyl alcohol: 70%) for preparing dead bacteria. Incubate both samples at room temperature for 1 hour, mixing every 15 minutes

Protocol tips
Remove the supernatant and resuspend the pellet of one tube in 0.3 mL of 0.85% NaCl solution and another tube in 1 mL of 0.85% NaCl. Add 0.7 mL isopropyl alcohol into the tube with 0.3 mL of 0.85% NaCl and mix well (final concentration of isopropyl alcohol: 70%) for preparing dead bacteria. Incubate both samples at room temperature for 1 hour, mixing every 15 minutes

Publication protocol

A combination of stains allows one to assess the relative proportions of live and dead bacteria in a culture. TS and control strains of S. enterica were grown at 30°C to mid-exponential phase. Each strain was then diluted 1:20 in duplicate in LB broth, with one duplicate incubated at 30°C and the other incubated at 39°C or 42°C, with shaking, for 12 h. After growth, each culture was diluted to an A595 of 1.0 and subsequently serially diluted to approximately 1 × 106 cells/ml. Next, 1 ml of each strain was centrifuged, washed, and resuspended in 1 ml of 0.85% NaCl solution. The fluorescent dyes DMAO and EthD-III were used alone or in combination (Biotium Viability/Cytotoxicity Assay for Bacteria Live & Dead Cells kit) according to the manufacturer's instructions.

After staining, bacteria were analyzed with a FACSCalibur flow cytometry system (BD Biosciences) equipped with an argon laser (488 nm) at 15 mW. Green fluorescence (live bacteria) was collected in the FL1 (fluorescein isothiocyanate [FITC]) channel, and red fluorescence (dead bacteria) was collected in the FL2 (phycoerythrin [PE]) channel. All parameters were collected as logarithmic signals. Population concentrations were estimated using CellQuestPro and FloJo Ver 9.6.3 software.

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Manufacturer protocol

Download the product protocol from Biotium for Viability/Cytotoxicity Assay kit for Bacteria Live and Dead Cells below.

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