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is a good starting point. If needed, centrifuge the sample, remove the supernatant and resuspend the
pellet with the yeast dilution buffer. After the dilution or washing step, proceed to the next step.
IMPORTANT
If your yeast cells were grown in the YPD medium, be sure to dilute the sample at least
1:100 with the Cell Dilution Buffer before counting. The component of the YPD medium
has a strong esterase activity and inhibits the uptake of FDA into the yeast cells,
leading to weak fluorescence signal.
6.1.2.3 If the answers are all no, just proceed to the next step.
6.1.3 Transfer 1 µl of FDA and 1 µl of PI to a new 1.5 mL microfuge tube.
Note : Whereas AO and PI measure the amount of nucleic acid of cells, FDA measures the activity of
metabolic enzymes (Esterase) of cells. Whereas the amount nucleic acid does not vary greatly between
different yeast strains, the activity of metabolic enzymes can vary in much higher degree between
different yeast strains. Even the same strain of yeast can have different levels of enzymatic activity
depending on their growth status. The variability of the signal strength of FDA is somewhat inevitable
between different strains and growth status. In some strains, the difference becomes significant (for
example, Brettanomyces) and in some strains, the difference is minimal (for example, Cerevisiae).
6.1.4. Add 18 µl of the sample to the tube and mix by pipetting up and down or flicking the bottom of
the tube while holding with the other hand.
Note : Any solutions containing FDA and PI should be protected from strong light.
6.1.5 Incubate the mixed sample for 10 min.
Note : Due to the working principle of FDA, please make sure to incubate the mixed sample properly at
room temperature.