Gram Stain

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Gram Stain

• Experiment 11

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Gram Stain

• DevelopedDanish bacteriologist Christian Gram in
  1884
• Most important, commonly used differential stain
• Differentialdistinguished between different cell
  wall compositions 2 groups
• Based on ability of some cells to resist
  destaining (decolor) with alcohol

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4 reagents

•  
• 1. Crystal VioletPrimary stain (purple) colors
  all cells
• 2. Grams IodineMordant a killing agent,
  substance that increased cell affinity for
  stains, will intensify color stain. The mordant
  and violet can bind and form a large complex.
  The retention of Crystal Violet-Iodine (CV-I)
  complex is based on cell wall composition. This
  complex will not decolorize.
• 3. 95 ethyl alcoholdecolorizing agentlipid
  solvent--most critical stepmay or may not remove
  primarybased on cell wall composition
• 4. SafraninCounterstain (red) stains if primary
  washed out by ETOH NO CV-I

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Gram Stain

• Thick smears do not work well
• Young cultures only (18-24 hrs) due to older
  cells begin to break down cellular structure and
  will give mixed results due to cell wall damage
• Overheating or heating before the smear is dry
  will shrink the cells
• Mixed or erroneous results are mainly due to
• old cultures, overheating at fixation step,
  decolor to long or rinsed to long-water

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Gram Stain

• What info you can obtain
• Gram reaction
• Cell shape, arrangement, size
• Cell wall compositionindication of what
  antibiotics will or wont effect organism
• Endospores will not stainhollow balls appear in
  center of cells, spore walls

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Gram

• Thick, multi-layered peptidoglycan outer wall,
  low lipid concentrations
• These retain the CV-I complex because the
  molecule is too large to pass thru the layer or
  small openings created by the alcohol
• Examples diphtheria, strep throat, scarlet
  fever, tetanus

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Gram -

• Thin peptidoglycan outer wall, outer membrane
  composed of lipoproteins, lipopolysaccharides and
  phospholipids.
• Lose CV-I complex--alcohol disrupts the outer
  membrane due to the high lipid content--they can
  pass thru the thin layer due to disrupted
  membrane--washed away
• Examples, dysentery, typhoid fever, E. coli,
  salmonella

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Gram Stain Procedure

•  
• 1. Fix a smear SMALL air dry, heat fix
• 2. Crystal Violet 20-30 sec
• 3. Water wash
• 4. Grams Iodine 20-30 sec
• 5. Water wash
• 6. 95ETOH 5 sec or until the stream stops
• 7. Water wash
• 8. Safranin 20-30 sec
• 9. Water wash
• 10. Gently blot dry and observe

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Gram Stain Results

• Gram Purple/bluish
• CV-I Retained
• Gram- Pink/reddish
• CV-I Washed out
• THE TIMES IN YOUR PROCEDURE HAVE BEEN MODIFIED
  PLEASE WRITE DOWN THE CORRECTED TIMES.

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