Preparing a Smear Exercise 10

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Preparing a Smear (Exercise 10)

• Smear thin layer of cells adhered to slide for
  staining
• 1) Make a 1/2 diameter circle on bottom of
  slide
• 2) Flame inoculating loop
• 3) Resuspend culture
• 4) Transfer culture to slide
• 5) Spread culture over the area of the circle
• 6) Flame loop
• 7) Allow slide to air dry
• 8) Pass slide through flame to heat fix

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Negative Staining (Exercise 12)

• Negative stains (India ink or nigrosin) used to
  study morphology
• Cells repel dye appear transparent against a
  dark background
• Add sample to drop of stain at end (Method 1) or
  center (Method 2) of slide
• Method 1 Use 2nd slide to spread suspension to
  other end
• Method 2 Use loop or toothpick to evenly spread
  sample
• Allow the slide to air dry (1-5 minutes). Do NOT
  heat slide.

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Gram StainChristian Gram (1884)

• Gram stain differential stain that divides
  bacteria into 2 groups
• Based on chemical differences in cell wall
  peptidoglycan
• Peptidoglycan glycan chains cross-linked by
  peptide fragments
• Provides rigid structure to cell wall
• Penicillin disrupts peptide bonds causes cell
  to rupture

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Gram Staining(Exercise 14)

• Primary stain crystal violet (1 min) rinse
  with water
• Mordant Grams Iodine (1 min) to lock in
  the dye
• Rinse with Decolorizer (NOT with alcohol or
  water)
• Decolorize/Counterstain safranin (20-60 sec)
  then rinse with water

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• S. aureus B. megaterium gram
• P. aeroginosa M.B. catarrhalis gram -

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Table 4.6a
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Table 4.6b