Gram Staining

• Gram-positive bacteria - have thick cell wall made of peptidoglycan, which traps the crystal violet in the cytoplasm. The alcohol rise does not remove the crystal violet, which masks the red safranin dye.
• Gram-negative bacteria - have thinner layer of peptidoglycan, locatied in a layer between the plasma membrane and outer membrane. The crystal violet is easily rinsed from the cytoplasm, and the cell appears pink or red from the dye
• Gram stain - the most frequently used stain in a clinical microbiology laboratory and is usually the first step in identifying bacteria; categorize bacteria as either gram-positive (stains dark purple) or gram-negative (stains pink). In addition to the gram reaction, the morphology and configuration of the bacteria can be observed, i.e., gram-positive coccus in chains or gram-negative rod.
• Gram Staining Steps:

  1. Heat fix slide
  2. Crystal Violet (1 min), rinse
  3. Iodine (1 min), rinse
  4. Alcohol (5-10 s), rinse
  5. Safranin (1 min), rinse
  6. Blot dry, view under microscope
• .Slide not heat-fixed - smear will wash off
• Slide over heat-fixed - cellular morphology may be distorted
• Slide over-decolorized - gram-positive bacteria will appear gram-negative
• Slide under-decolorized - gram-negative bacteria will appear gram-positive
• Smear too thick - cells in very thick areas will not decolorize properly and gram negative bacteria will appear gram-positive
• Insufficient time for safranin counterstain - gram-negative bacteria may be very faint and difficult to see
• Staphylococcus aureus - gram positive coccus
• Escherichia coli - gram negative rod shape