Gram Staining & Classifying Bacteria

• Differential staining, specifically Gram staining, is covered in this laboratory session
• This is week #4 of the BT233 General Microbiology Laboratory

Classification of Bacteria

• The initial step in classifying bacteria involves distinguishing between Gram-positive, Gram-negative, and acid-fast bacteria
• Further classification requires a specific key for each bacterial group, differing from others
• Accurate bacterial classification relies on the correctness of the initial classification

Methods to Classify Bacteria

• Three methods help confidently classify bacteria into Gram-positive and Gram-negative categories:
  • Selective media
  • 10% KOH
  • Gram-staining

Selective Media

• EMB (Eosin Methylene Blue), TSI (Triple Sugar Iron) agar, MacConkey agar, and Bismuth sulfite agar are examples of selective media
• These media types are selective for Gram-negative bacteria, so Gram-positive bacteria cannot grow on them
• If an unknown bacterium grows on these media after inoculation and incubation, it's likely 95% Gram-negative
• If the bacterium doesn't grow, it's likely Gram-positive

10% KOH

• Bacterial cell walls are divided based on main composition:
  • Peptidoglycan: Found in Gram-positive bacteria
  • Lipopolysaccharide: Found in Gram-negative bacteria
  • Mycolic Acid: Found in bacteria considered Gram-positive (Acid-Fast bacteria)

10% KOH Test Principle

• 10% KOH dissolves Lipopolysaccharide, but not Peptidoglycan or Mycolic Acid
• A saturated Lipopolysaccharide mixture becomes viscous (mucoid)
• This method distinguishes Gram-negative bacteria from others, but cannot differentiate between Gram-positive and bacteria considered Gram-positive

Materials

• Slides
• Loop
• Gram-positive bacteria example, Bacillus, and Gram-negative bacteria example, E. coli
• 10% KOH

Gram Staining

• It is used to classify bacteria into three main groups:
  • Gram-positive (+ve)
  • Gram-negative (-ve)
  • Considered as Gram-positive

Requirements for Gram Staining

• Bacteria for Gram staining should be no older than 24 hours
• For motile bacteria, use the fresher center of colonies

Materials for Gram Staining

• Bacterial culture of E. coli and Bacillus
• Slides
• Sterile distilled water
• Filter paper
• Loop
• Crystal violet or Methylene blue stains
• Iodine
• Safranin
• Ethanol 95% or Absolute 100%
• Compound Microscope

Gram Staining Procedure

• Smear preparation involves creating three complete smears up to heat fixation:
  • Smear for Bacillus
  • Smear for E. coli
  • A mixed smear of Bacillus and E. coli

Gram Staining Principle

• Staining bacteria depends on:
  • Charge of bacteria
  • Charge of stains
  • Cell wall permeability
    • Peptidoglycan and Lipopolysaccharides are stain permeable
    • Mycolic acid is stain impermeable
  • Bacterial cell wall structure

Gram Staining outcomes

• Classifies bacteria into:
  • Gram-positive: Retain primary stain color (violet)
  • Gram-negative: Accept counter stain color (pink)
  • Bacteria considered gram-positive: Remain colorless

Gram Staining Details

• It helps determine both the shape and arrangement of bacterial cells
  • Bacilli, Cocci, Spirilli, Coccobacilli, and Vibrio

Important Note

• Acid-fast bacteria will appear colorless after gram staining
• This is because their cell wall is mainly composed of Mycolic acid, which prevents stain entry
• Special staining protocols are needed

Troubleshooting Gram Staining

• If primary stain and counterstain are switched:
  • Gram-negative bacteria become violet after decolorizing and adding crystal violet
  • Gram-positive bacteria retain safranin (pink) but appear violet due to crystal violet masking the pink color
• Differentiation between Gram-positive and Gram-negative bacteria is not possible

Gram Staining Pure Culture Concerns

• If a pure culture displays mixed staining patterns, the culture might be older than 24 hours
• The bacteria is likely gram-positive
• Decline or stationary phase bacteria stain Pink
• Bacteria in lag or log phase stain violet

Fresh Culture

• Fresh cultures are vital for accurate Gram staining (less than 24 hours old)
• Older bacteria have fragile cell walls that may break down, leading to protoplast formation
• Without cell walls, cells accept the final stain (Safranin), and Gram-positive bacteria may appear Gram-negative

Issues & Troubleshooting

• Gram Reaction is hard to determine if the following occurs:

  • Over Decolorization occurs when more alcohol is added than needed
  • Low decolorization happens when a low amount of alcohol is added than needed

• The following can occur

  • Either the bacteria is gram positive and this side is stained correctly
  • The bacteria is gram negative and the side is stained correctly