Gram staining

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Gram staining
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Types of staining

• Simple
• Bacteria stained to contrast environment
• Differential
• Bacteria differing chemically react differently
  to staining

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Gram stain

• Differential
• Gram positive purple

• Gram negative pink

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Step 1
Fixation Add 1 drop of deionized water Burn
bacteria to slide
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Step 1

• Primary stain
• Basic dye crystal violet
• 30 seconds

• Rinse (DI water) 5 seconds

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Step 2

• Mordant
• Iodine
• Helps dye interact with bacteria
• Dye tightly bond to cell
• 60 seconds

• Rinse 5 seconds

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Step 3

• Decolorizer
• Ethanol
• Gram () bacteria retain crystal violent-iodine
  dye when decolorized
• Gram (-) bacteria lose crystal violent-iodine dye

Rinse 5 seconds
20 seconds
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Cell Wall

• Peptidoglycan
• Polymer of cell wall
• Gram ()
• 20 to 80 nm thick
• Gram (-)
• 2 to 7 nm thick
• Lipopolysaccharide (fats sugar) Proteins

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Step 4

• Counterstain
• Safranin
• Purple color of Gram () bacteria not altered
• Colorless Gram (-) bacteria turn pink

Rinse 5 seconds
70 seconds
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Gram () Deep purple Gram (-) pinkish

• Heat Affix Bacteria to slide 1 drop of H2O
• Crystal violet
• Stain 30 seconds
• Iodine
• Stain 60 seconds
• Ethanol
• Stain 20 seconds
• Safranin
• Stain 70 seconds

E. Coli M. Luteus B. megasterium R. Rurum S.
aurantaca S. Marcescens

• Rinse 5 seconds
• Rinse 5 seconds
• Rinse 5 seconds
• Rinse 5 seconds