Lab

1
Lab 9

• Acid Fast Stain
• Endospore Stain
• Eosin Methylene Blue (EMB) Agar

2
Acid Fast Stain

• Differential Stain
• Ziehl Neelsen (ZN) heat method
• Acid fast cells have higher differentiation for
  primary stain and resist decolorization by acid
  alchohol
• Mycolic acid in cell walls of acid fast cells
• Used to identify bacteria in the genus
  Mycobacterium, (ie. M. leprae, M. tuberculosis)

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Bacteria for acid fast

• Mycobacterium smegmatis
• Staphylococcus aureus

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Staining procedure

• Heat fix bacteria
• Carbolfuchsin primary stain
• Steam heat enhances the entry of carbolfuchsin
  into cells
• Decolorization with acid alchohol
• Methylene Blue counterstain for acid fast
  negative stain

5
Results Acid Fast (reddish)Non Acid Fast (blue
or purple)
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Endospore stain

• Schaeffer Fulton method
• Differential stain
• Detect presence and location of spores
• Used for bacteria that form spores in the genus
  Bacillus and Clostridium
• Keratin is the protein in the outer covering of
  the spore

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Bacteria for Endospore stain

• Bacillus cereus
• Bacillus subtilis

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Staining procedure

• Heat fix bacteria
• Malachite Green primary stain
• Steam to force malachite green into the spore
• Rinse with water
• Conterstain Safranin
• Rinse with water

9
Spores of Bacillus- green Vegetative cells prior
to sporulation- red
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Eosin Methylene Blue Agar

• Selective Media
• Contain peptone, lactose, sucrose, and dyes eosin
  Y and methylene blue
• Sugars provide fermentable substances to grow
  coliform bacteria
• Green sheen serves as an indicator of lactose or
  sucrose fermentation
• Non fermenters retain the normal color or the
  color of the media

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Bacteria to inoculate EMB plate

• Staphylococcus aureus
• Escherichia coli
• Enterobactor aerogenes
• Salmonella typhimureum

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E. Coli and Enterobactor growing on EMB agar

• E. coli Enterobactor
• Green (D) coliform Pink (pi) coliform

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Salmonella on EMB

• Salmonella no growth (P) colorless colonies