I have to quantify forest soil bacteria. As a standard method I chose the
DAPI direct count method (DAPI is a DNA specific fluorescent dye) my phase
contrast counts gave numbers about two magnitudes higher than the accepted
values for bacteria per gram soil.
The problem is, that the bacteria so far resisted all of my tries to get
them stained with DAPI. It seems likely that remnants of humic acids cause
the disturbance (e.g. I get perfect signals for cultured cells).
To reduce humic acids content, I already tried (di-)phosphate buffer at pH
9, Crombach buffer, even NaOH at pH > 11 (which killed my bacteria off but
otherwise was of little use).
Has anyone got similar problems with DAPI or could propose alternative
Would it be worth a try with acridine orange??