1. Add 300 μl (300 μl) culture to 1.2 ml (0.9 ml) 100% ethanol in Eppendorf (volume not critical, therefore depends how much culture can bespared).
2. 1h-2+ weeks at 4ºC or -20ºC.
3. Spind down cells.
4. Prepare n x 1 ml SSC (n = number of samples).
5. From that, prepare n x 0.25 ml SSC + 0.25 μl RNaseA 10 mg/ml.
6. Resuspend cells in 250 μl SSC + RNaseA by vortexing.
7. Incubate at 37ºC overnight (or at least 1h if in a rush – but overnightgives better results).
8. Prepare n x 50 μl SSC + 1.5 μl proteinase K 40 mg/ml.
9. Add 50 μl SSC + protK to samples.
10. Incubate for exactly 1h at 50ºC.
11. Prepare n x 0.5 ml SSC + propidium iodide (final concentration is 3μg/ml).
12. Add 0.5 ml SSC propidium iodide 100X to samples (workingconcentration: 3 μg/ml).
13. Incubate at room temperature for 1+ h then store at 4ºC until use.(Overnight or over weekend gives best results but can store for atleast a month and still get results).