Acid Fast stain

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Acid Fast stain
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Mycobacteria

• There are gt70 species of mycobacteria
• Two are major pathogens
• Mycobacterium tuberculosis (Koch, 1882)
• Mycobacterium leprae (Hansen, 1874)

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Mycobacteria structure

• Contain large amount of fatty waxes (mycolic
  acid) within their cell wall ? resist staining by
  ordinary methods
• Require a special stain for diagnostic ? Acid
  Fast stain.

http//www.med.yale.edu/labmed/casestudies/images/
cs4_mycolic_acid.jpg
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Acid Fast Stain

• Carbolfushin at high concentration ? penetrate
  the lipid cell wall ? reach the cytoplasm
• Cytoplasm once stained resist decolorization ?
  acid-alcohol can not dissolve the cell wall and
  penetrate beneath it
• ? hence the name acid fast
• Bacteria that do not have mycolic acid are
  readily decolorized by the acid-alcohol

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Acid Fast stain

• Place the slide on the slide rack over the metal
  staining tray ? concentrated carbolfuchsin 5 min
  ? rinse very well
• Cover with the acid-alcohol solution ? 2 minutes
  ? rinse
• Flood with methylen blue ? let stand 1 minute ?
  rinse
• Blot dry ? observe
• Note Carbolfuchsinacid-alcoholblue color

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Acid Fast Stain

• Mycobacterium smegmatis (pink) ? acid fast retain
  the carbolfuchsin dye
• Micrococcus luteus (blue) ? not acid fast ?
  decolorized with acid-alcohol ? conterstained
  with methylen blue

http//www.bact.wisc.edu/Microtextbook/images/book
_3/chapter_3/3-17.jpg
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Objectives

• You have 2 bacteria Mycobacterium and E. coli
• On each you perform 1 Gram stain and 1 Acid fast
  stain
• Each student should perform each stain at least
  once