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Acid fast staining: need help - (Aug/20/2013 )


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!



Please describe your fixation procedure.

-Phil Geis-

Dear Phil,


after spreading the material on the slide I air dried it at either room temperature or 65 °C. Then I moved it three times through a flame of a Bunsen burner.


Can you see the microbial cells under the scope?  Are they reasonably spread out?

-Phil Geis-


yes,in brightfield I could see the bacteria under the microscope.They were well separated.

The cells don't take up the dye. Even if I apply heat until steaming (5min) during the staining process (25 min in total) the majority of my M. phlei cells do not fluoresce. I could not find a difference to the negatove control (Staph. aureus). I expected that all Mycobateria cells would light up.


Does the culture age for acid-fast staining matter and do you know a suitable incubation period? I used approximately 4 days at 37 °C on Columbia sheep blood agar.


Many thanks from a beginner in both staining methods and fluorescence microscopy!


Based on this protocol you are overexposing the sample to the permanganate, and exposures over 2 min quench the fluorescence. Try this one or look for other sources for the same staining method, people use to include useful comments on the protocols.

-El Crazy Xabi-