Making the great leap out of the 19th Century…”Acid-Fast” staining

The “standard” acid-fast differential staining process for the “High G+C Gram-Positive” bacteria[1] as we learned it is pretty archaic.

It goes something like this:

  • Smear and heat-fix the slide
  • flood the slide with Carbolfuschin
  • Heat the slide for 5 minutes in steam over a boiling water bath
  • Rinse with “Acid Alcohol
  • Stain for a minute with Methylene blue.

At the end, anything “Acid-Fast” (having a “waxy” outer layer) will show up red, anything else will be blue.

My objection is the messy and time-consuming steam-bath.

Today’s lab included one “unknown” which we suspected to be a Mycobacterium, so while one of us was going through the tedious 19th-century-style procedure, I decided to try something.

The steambath heat is just intended to (I believe) slightly “melt” the waxy layer of the cell and otherwise help “drive” the dye into it. So, instead of dealing with the time to make a water bath, heat until it steams, and then wait for the slide to sit there and hope the bubbling bath doesn’t splatter the slide with crud, I just stuck the flooded slide in the lab microwave and cooked it for 20 seconds.

It worked. Quite well, actually (other than letting the slide dry out, leaving some crystals on the slide) – the bright red mycobacterial cells showed up nicely. I’m annoyed that my ‘stick the camera up to the eyepiece’ technique came out slightly out of focus (I may see if I can enhance it later – if so, I’ll post it.). Somebody commented that it looked as good as a “textbook” example, which was nice for my ego…

Unfortunately, I guess I’m far from the first person to think of this. I don’t know if anyone’s done this exactly the same way, but This procedure describes directly heating the Carbolfuschin in the microwave and soaking the slides directly in it. There is also apparently an old Lancet[2] article which I don’t currently have access to – I’ll have to check it out later.

Next thing to do is try the endospore stain this way. Behold, the miracles of applying last century’s technologies to the problems of century-before-last!

[1] Ehrlich P. Zur Fa¨rbung der Tuberkelbakterien. Aus dem Verein fu¨r innere Medizin zu Berlin. Deutsche Med Wochenschr 1882; 8:269?270
[2] Hafiz, S., R. C. Spencer, M. Lee, H. Gooch, and B. I. Duerden. 1984 . Rapid Ziehl-Neelsen staining by use of microwave oven. Lancet ii:1046.

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The Author is (currently) an autodidactic student of Industrial and Environmental microbiology, who is sick of people assuming all microbiology should be medical in nature, and who would really like to be allowed to go to graduate school one of these days now that he's finished his BS in Microbiology (with a bonus AS in Chemistry). He also enjoys exploring the Big Room (the one with the really high blue ceiling and big light that tracks from one side to the other every day) and looking at its contents from unusual mental angles.

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