I have Mycobacterium phlei cultured on blood agar for several days. I took some colony material and spread it with water on an object slide and heat fixed it.
I tried the Auramin-Rhodamin method. I used our Texas Red and GFP filters to evaluate the slide for any fluorescence at 4x, 10x, 20x and 40 magnification. Unfortunately, the bacteria do not exhibit fluorescence. I stained 15min, then cleaned the slide for 20sec with running tap water. Then, I flooded the slide with acid-alcohol for 1min. Afterwards, I cleaned for 20sec with tap water, counterstained for 5min with KMNO4, then I washed again using water.
I have no idea what the problem is. Fluorescence is working as some debris fluoresces.
Can anyboda help?
Thanks in anticipation!