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Negative Stain Lecture Notes

May 19, 2024

Negative Stain (Activity 3-6)

Purpose

  • Visualize bacterial cells.
  • Determine morphology, arrangement, and accurate cell size.

Key Concepts

Morphology

  • Refers to the shape of bacteria (e.g., rod, spiral, spherical).

Arrangement

  • How bacterial colonies are arranged when grown.
  • **Examples: **
    • Isolated cells
    • Pairs
    • Chains
    • Clusters

Heat Fix Step in Gram Staining

  • Purpose: Denature proteins so bacteria adhere to the slide and are killed.
  • Process: Pass the slide through the flame three times.

Why No Heat Fix in Negative Stain?

  • Heat can cause cells to shrink, leading to inaccurate cell size measurement.
  • Heat can damage fragile cells, like spirochetes.

Negative Stain Characteristics

  • Background is stained, cells appear clear.

Charge and Chemistry

  • Negative stain is acidic, carries a negative charge.
  • Repelled by negatively charged cells, hence staining the background.

Examples of Negative Stains

  • Congo Red
  • Nigrosin (black)
  • India Ink
  • Eosin

Bacterial Morphology Types

  • Cocci: Spherical or round.
  • Bacillus/Bacilli: Rod-shaped.
  • Spirillum: Rigid spirals.
  • Spirochete: Flexible spirals.
  • Vibrio: Curved rods.

Bacterial Arrangement Types

  • Strepto: Chains (e.g., Streptococcus).
  • Staphylo: Grape-like clusters (e.g., Staphylococcus).
  • Diplo: Pairs (e.g., Diplococcus).

Medically Important Bacteria

Treponema pallidum (causes Syphilis)

  • Signs: Canker sores, rash, late-stage involves internal organs.
  • Transmission: Sexual contact.
  • Treatment: Antibiotics (Penicillin).

Borrelia burgdorferi (causes Lyme Disease)

  • Transmission: Tick bites.
  • Signs: Bullseye rash, flu-like symptoms, neurological symptoms.
  • Treatment: Antibiotics.

Negative Stain Procedure

  1. Label slide (Name, Date, 'Negative Stain').
  2. Apply half-drop of Congo red onto the slide.
  3. Swab gum line with a sterile toothpick.
  4. Mix sample with Congo red.
  5. Use another slide to smear the dye across the slide.
  6. Allow slide to air dry.
  7. View under the microscope.

Observations

  • Background stained, bacteria clear.
  • Oral spirochetes or Staphylococcus epidermidis can be visualized.

Conclusion

  • Negative stain helps in observing bacteria without affecting fragile cells and provides accurate morphology and arrangement visualization.

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