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Acid fast staining: need help


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5 replies to this topic

#1 czernobill

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Posted 20 August 2013 - 02:10 AM

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!

 



#2 Phil Geis

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Posted 20 August 2013 - 08:14 AM

Please describe your fixation procedure.



#3 czernobill

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Posted 20 August 2013 - 10:12 AM

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.



#4 Phil Geis

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Posted 21 August 2013 - 04:03 AM

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



#5 czernobill

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Posted 21 August 2013 - 08:01 AM

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!



#6 El Crazy Xabi

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Posted 22 August 2013 - 03:44 AM

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.






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