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Two-color assay distinguishes live and dead yeast

This kit combines a novel two-color fluorescent probe for yeast viability, FUN® 1, with a fluorescent fungal surface labeling reagent, Calcofluor® White M2R. Plasma membrane integrity and metabolic function of fungi are required to convert the yellow-green fluorescent intracellular staining of FUN® 1 into red-orange intravacuolar structures; Calcofluor® White M2R labels cell-wall chitin with blue-fluorescence, regardless of metabolic state.

This protocol can be used for:

  • Identifying live and dead yeast using a fluorescence microscope

This protocol should not be used for:

  • Flow cytometry

You will need the following for this protocol:

Protocol

1. Preparation of yeast suspensions

1. Grow yeast to late log phase (107–108 cells/mL) in medium such as yeast extract peptone dextrose (YPD)
2. Add 50 µL of the yeast culture to 1 mL of wash buffer
3. Centrifuge at 10,000 × g for 5 minutes
4. Remove supernatant and resuspend pellet in 1 mL of wash buffer

2. Staining bacteria

1. Add 1 µL of Component A (FUN® 1 cell stain) and 5 µL of Component B (Calcofluor® White M2R) to 1 mL of yeast suspension (10 µM and 25 µM final concentrations of each of the dyes, respectively)
2. Incubate at 30°C in the dark for 30 minutes
3. Put 5 µL of the stained yeast suspension onto a glass slide and cover with a coverslip
4. Image cells with the appropriate filters listed below
Spectral information and storage
  FUN® 1 cell stain Calcofluor® White M2R
Excitation/Emission (in nm) 488/530 365/435
Standard filter set FITC DAPI
Storage conditions ≤20°C, protect from light ≤20°C, protect from light

 

Protocol tips

  • Warm vials to room temperature and centrifuge briefly before opening
  • Wash to remove all growth medium from yeast before staining
  • Phosphate wash buffers may decrease staining efficiency and are not recommended
  • If precipitate is observed in Calcofluor® dye, briefly centrifuge at 10,000 x g to clear solution

image of yeast cells stained using the LIVE/DEAD® Yeast Viability Kit
Saccharomyces cerevisiae stained with FUN® 1 cell stain, which generates red-fluorescent intravacuolar structures, and with Calcofluor® White M2R, a blue-fluorescent cell wall stain.