Gram staining

• Gram staining- Stage 1

  • The bacteria are placed on a microscope slide and crystal violet stain is applied to the sample.
  • The result - he sample will stain purple regardless of whether they are Gram positive or Gram negative but the dye is not fixed and you cannot tell the difference between the two types of bacteria.
• Gram staining- stage 2

  • Gram’s iodine solution (iodine with potassium iodide) is added.
  • The result - he stain is now fixed so it should stay in the cell walls it has managed to reach without being washed out.
• Gram staining- Stage 3

  • Sample is washed with ethanol or acetate. This removes excess crystal violet stain but not where it has stained bacterial cell walls – these will remain purple.
  • The result- Gram positive bacteria will remain purple. Gram negative bacteria will have their dye washed out because it did not dye the cell wall due to the capsule protecting the cell wall. Gram negative bacteria will lose their purple colour.
• Gram staining- Stage 4

  • Finally, the pink dye safranin is added to the sample before a cover slip is applied and the sample is viewed under the light microscope.
  • The result-The safranin dye will stain both Gram negative and Gram positive bacteria. However, it will only be visible on Gram negative bacteria which had no stain. The Gram positive bacteria will still be seen as purple because the crystal violet overshadows the pink colour of the safranin.
• What is the difference in wall thickness between Gram positive and Gram negative bacteria?

  Gram positive bacteria have thicker walls
• What structure do Gram negative bacteria have that Gram positive bacteria lack?

  A capsule outside the cell wall
• Why was crystal violet able to stain Gram positive bacteria?

  Because of their thicker cell wall
• Why was crystal violet unable to stain Gram negative bacteria?

  It couldn't reach the cell wall due to the protective wall
• What happens to crystal violet stain in Gram negative bacteria when washed with acetate/ethanol?

  It gets washed off
• How do many antibiotics work against bacteria?

  By disrupting the cell wall
• What happens to bacteria when their cell wall is disrupted by antibiotics?

  They absorb water and eventually burst
• Why are Gram negative bacteria more resistant to antibiotics compared to Gram positive bacteria?

  The outer capsule protects their cell wall
• What is the role of the outer capsule in Gram negative bacteria?

  It protects the cell wall from antibiotics