Experiment 5 acid-fast stain,Culture and stain of fungus

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Experiment 5 acid-fast stain,Culture and stain
of fungus

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Content

• 1.acid-fast stain of M.tuberculosis
• 2. Culture of fungus
• 3.Simple stain of fungus

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acid-fast stain of M.tuberculosis
Cell wall of M.tuberculosis is impermeability to
stains and dyes. But M.tuberculosis can be
stained by acid-fast stain with long time
heating. Bacteria except M.tuberculosis can be
decolorized by 3 acid alcohol . So the color of
M.tuberculosis is red and that of other
Mycolic acid negtive bacteria is blue after
Counterstain with methylene blue
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• 1. Make a smear of the sputum, dry and fix it.
• 2. Ziehl-Neelsen acid-fast stain
• (1) Flood the slide with carbolfuchsin. Heat the
  slide to steaming for 5mins. Do not boil or allow
  the smear to dry. As stain evaporated from the
  slide, replenish with additional carbolfuchsin.
  Allow the slide to cool and rinse it thoroughly
  with water.
• (2) Decolorize the slide with acid alcohol until
  the red color no longer comes off in the
  decolorizer. It takes about 30secs. Rinse the
  slide with water.
• (3) Counterstain with methylene blue, allow the
  stain to react for 1min, rinse as above.
• (4) Bolt the slide carefully. Examine under
  microscope.

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• Culture of Fungi
• Sabourauds dextrose agar is commonly used medium
  for cultivation of fungi.
• Colony types
• (1) Yeast-form colony e.g. Yeast Cryptococcus
  neoformans.
• (2) Yeast-like colony e.g. Candida albicans.
• (3) Hyphomycete-form colony e.g. Dermatophytes.

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Microslide cultivation technique (1)Place a block
of Sabourauds dextrose agar (about 1cm2) on a
sterile slide. (2)Using a straight inoculating
needle, inoculate the specimen to be assayed on
each side of the block. (3)Gently heat a
coverslip by passing it quickly through the flame
of an alcohol burner and immediately place it
directly on the surface of the inoculated agar
block. (4)Place the slide in a petri dish on a
slide supporter glass rods above a moistened
filter paper disk, incubate at 28? for 35
days. (5)The slide can be microscoped at
intervals during cultivation to observe the
dynamics of hyphae and spore growth.
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• Simple stain of fungus
• (Candida albicans )
• precedure
• 1.Smear preparation (same to gram stain)
• 2.stainadd crystal violet (or methylene blue),
  stain for 1 min,rinse,blot, Microscopic
  examination ?

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• Experimental report
• 1.acid-fast staining method
• 2.Simple stain of fungus
• experimental principle , procedures, and analysis
  of experimental results, drawing