DAPI staining (cell counts) - meyermicrobiolab/Meyer_Lab_Resources GitHub Wiki

Filtration for DAPI Staining (Cell Counts)

  1. Fix desired volume of sample with 37% Formaldehyde to a final concentration of 2-4% (i.e. 1 mL of culture and 100uL 37% Formaldehyde = 3.7%, 18 ml of sea water with 1.8 ml 37% Formaldehyde) for at least one hour at 4°C.

  2. Place prefilter (i.e. backing filter – GF 25 mm Type AE) on grid. Wet with filtered MilliQ water through filter syringe.

  3. Place back membrane filter (Isopore Membrane polycarbonate, 0.22 μm, 25 mm) on top of backing filter with clean forceps – shiny side up.

  4. Place lid on top tightly with nut. Plug any used wholes with stoppers. Connect pump to vacuum with hose.

  5. With valves closed, add ~5 ml of filter sterilized 1X SSB to each unit.

  6. Add fixed volume of sample (i.e. 500 μl of culture) and mix up and down with a 1 ml pipette a couple of times.

  7. Add 7 drops of DAPI (1 μg/ml concentration) through filter syringe to unit and mix up and down with a 1 ml pipette a couple of times.

  8. Let sit for 10 minutes covered in aluminum foil.

  9. Open valve, turn on vacuum, and allow sample to filter through.

  10. Wash filter and vacuum, and allow sample to filter through three times with ~ 5 ml of filtered MilliQ water to wash off excess DAPI.

  11. Turn off vacuum, close valves, and remove filters with clean forceps.

  12. Allow filters to dry for a few minutes on slide in dark.

  13. Place a small drop of vectashield on top of the filter and cover with a cover slip. Gently press down on cover slip to spread vectashield across filter. Place a drop of immersion oil on top of cover slip and view under oil immersion on scope.

  14. View under UV filter on scope.

0.1 mg/mL = 100 μg/mL