Viability is a key metric in determining the activity or metabolism of yeast cells in solution. It is commonly measured using either brightfield microscopy or fluorescent stains like fluorescein diacetate (FDA) and propidium iodide (PI) with both manual and automated cell counters.
- Fluorescein diacetate is an esterase substrate that can freely permeate the plasma membrane of a diverse range of organisms, including bacteria and mammalian cells, to facilitate the measurement of enzymatic activity and integrity of cell membranes.
- Propidium iodide is a ubiquitous nucleic acid-binding dye used to distinguish live/dead cells and determine cell viability.
When yeast samples are mixed with an FDA/PI assay, the non-fluorescent fluorescein diacetate permeates the cell membranes. It is cleaved by the action of intracellular esterases to give the green fluorescent metabolite fluorescein. This green fluorescence is a strong indicator of viable cells. Propidium iodide is unable to permeate the cell membranes of live cells with intact cell membranes. When PI enters a cell, it generates a red fluorescence that is indicative of dead cells. Automated cell counters, like the CellDrop™ from DeNovix, record this fluorescence to rapidly calculate the viability of yeast cells in solution.
Yeast Viability Assays for Automated Cell Counters
DeNovix offers a tailored FDA-IP assay for the Yeast App on the CellDrop Automated Cell Counter. The kit is sufficient for 300 assays, containing 1.0 milliliters (mL) of fluorescein diacetate, 1.5 mL propidium iodide and 20 mL of phosphate-buffered saline (PBS) yeast dilution buffer. Each component should be stored in airtight containers and protected from light. The propidium iodide and buffer should be stored at 2 – 8°C while the fluorescein diacetate should be stored at -20°C.
- For the full Yeast Assay (FDA/PI) Protocol see Fluorescein Diacetate and Propidium Iodide Yeast Assay Protocol.
CellDrop Cell Counter: Yeast App
The CellDrop Yeast app uses the green fluorescent channel to record FDA emission from live yeast cells and a red channel to record PI emission of dead cells for yeast viability testing. Although the small size of yeast cells can be challenging for some automated cell counters, the unique CellDrop hardware combined with the CellDrop yeast viability assay provides fast, accurate viability measurements.
- For best practices when using and optimizing CellDrop see: CellDrop Best Practices