HNF analysis

GUIDELINE FOR ENUMERATION OF HETEROTROPHIC NANOFLAGELLATES (HNFs)

Materials:

- Black PC membrane filters 0.8 μm

- Formaldehyde 36% 

- Pipets and tips

- DAPI 0.1mg/ml working solution

- Waste container for DAPI

- Pump

- Microscope slides + cover glass

- Immersion oil

Sample preparation:

1. Add 1 ml formaldehyde to 20 ml sample (2% final solution)

2. Incubate in the dark in the fridge (4°C) for 1-2h, maximum 24h

3. Filter the sample on the black 0.8 μm membrane filter (hint: use an old GFF filter under it, it holds better)

4. Add 100-200 μl DAPI working solution

5. Incubate for 10 minutes in the dark!!!!

6. Filter out the remaining liquid and leave it to dry in the dark

7. Freeze the filters in -20°C (or in -80°C for longer storage)

Sample analysis:

- Place the filter on the slide and “glue" it with one drop of the immersion oil

- Cover the filer with the cover glass, again with a drop of the immersion oil

- Use epifluorescence microscope for counting using UV excitation for heterotrophs (blue DAPI stained cells) and blue excitation for autotrophs (red chlorophyll a autofluorescence)

- Count at least 20 fields of view