Gram Stain Quiz

Gram Stain

• Gram stain was developed by Christian Gram in 1884 to stain bacteria in tissues.
• Gram-positive bacteria stain purple, while Gram-negative bacteria stain pink.
• The differences in staining are due to the structural differences in their cell walls.
• Gram-positive bacteria have a thick peptidoglycan layer and large amounts of teichoic acid.
• Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane made of phospholipids, lipopolysaccharides, lipoproteins, and proteins.
• Gram-negative bacteria are decolorized by alcohol.
• Gram-positive bacteria retain the initial stain (crystal violet) after decolorization.
• The counterstain (safranin) gives Gram-negative bacteria a pink/red appearance.

Staining

• Staining is used to enhance contrast in microscopic images.
• Stains are classified based on their charge:
  • Basic stains have a positive charge and are used to stain negatively charged molecules like bacterial cell surfaces.
  • Acidic stains have a negative charge and are used to stain positively charged molecules like bacterial capsules.
  • Neutral stains have both charges.
• Stains are also classified based on their function:
  • Simple staining uses one dye and does not differentiate between bacteria.
  • Differential staining uses more than one dye and differentiates between bacteria based on their cell wall structure (e.g., Gram stain).
  • Special staining uses more than one dye and visualizes specific structures like capsules or spores.

Preparing Smears

• For liquid media:
  • Sterilize a loop with a Bunsen flame.
  • Cool the loop.
  • Withdraw a loopful of broth culture.
  • Spread the broth culture evenly on a clean slide to form a thin film.
  • Sterilize the loop.
  • Allow the smear to air dry.
  • Fix the smear by passing it through a Bunsen flame 3-4 times.
  • Allow the slide to cool before staining.
• For solid media:
  • Sterilize a loop with a Bunsen flame.
  • Cool the loop.
  • Place a loopful of clean water on a clean slide.
  • Sterilize the loop again.
  • Transfer a small portion of bacterial growth to the water on the slide.
  • Mix the growth with water thoroughly and spread evenly on the slide.
  • Allow the smear to air dry.
  • Fix the smear by passing it through a Bunsen flame 3-4 times.
  • Allow the slide to cool before staining.

Simple Staining

• This procedure uses a single basic dye like crystal violet, methylene blue, or safranin to stain bacteria.
• Bacteria will simply take on the color of the dye.

Differential Staining

• This procedure uses more than one dye solution.
• The dyes are added in multiple steps according to the procedure.
• Gram staining is used to stain bacteria.
• It helps differentiate between Gram-positive and Gram-negative bacteria.