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

Hello,

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.

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?

Hello,

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.