Gram staining

• • Gram positive = stain purple
  • Gram negative = stain pink
• • Gram positive - thick, no outer membrane
  • Gram negative - thin, has outer membrane (lipopolysaccharide)
• How gram staining works

  • add crystal violet
  • add grams iodine
  • alcohol wash
  • add safranin
• Crystal violet

  • It is a dye that binds to the peptidoglycan of both gram negative and gram positive cells.
• Grams iodine

  • Fixes the crystal violet to the peptidoglycan   
• Alcohol wash

  • Dissolves the outer membrane and therefore exposes thin cell wall and therefore removes bound dye to gram negative bacteria
• Safranin

  • Also binds to peptidoglycan and therefore provides a pink colour to the gram negative bacteria making them easier to count
• Antibiotics

  • Gram positive bacteria are susceptible (can affect) to some antibiotics, such as penicillin, that can damage the peptidoglycan layer
  • This is because these bacteria have no outer (lipopolysaccharide) membrane and so penicillin can enter the bacteria. Once inside the cell, penicillin works
• Antibiotics pt2

  • Prevent cross-linking of protein chains in peptidoglycan
  • This disrupts cell wall synthesis
  • So new bacteria grow abnormally
  • Because they cannot maintain their rigidity
  • So they are more susceptible to bursting
  • The outcome is that fewer bacteria successfully replicate to produce new bacteria