Gram stain and AFB

• Introduction to Simple Stain:

  • Bacteria have no color, so staining is necessary for microscopic examination
  • Staining allows observation of bacteria's shape, size, and arrangement
  • Steps for stained bacterial smear preparation include placing a bacterial colony on a glass slide, fixing it with heat, and applying a basic stain with a positive charge
• Direct Smears from Swab:

  • Roll the swab across the slide to avoid destruction of cellular entities
• Smears from Cultures (Subculture Smears):

  • Spread a thin film of bacteria on a clean glass slide
  • Place a loopful of culture in the square without spreading unless growth is heavy
• Gram Stain Introduction:

  • Developed by Hans Christian Gram in 1882
  • Important staining technique in microbiology for identifying bacteria
  • Crystal violet primary stain distinguishes bacteria as Gram-positive (purple) or Gram-negative (red)
• Gram Stain Procedure:

  1. Apply crystal violet stain, rinse excess

  2. Add iodine solution, rinse

  3. Decolorize with a few drops of decolorizer, rinse

  4. Counterstain with basic fuchsin, rinse

  5. Air dry or blot the slide
• Introduction to Acid-Fast Bacilli (AFB) Stain:

  • Acid-fastness is a feature of Mycobacteria due to waxy mycolic acids
  • Acid-fast organisms retain primary dye, while non-acid fast organisms take up counterstain
  • Sample collection for AFB staining involves early morning sputum
• Preparation of Sputum Smear:

  • Transfer sputum to a slide, make an oval smear, air dry, and fix with heat
  • Disinfect tools and practice aseptic technique
• AFB Stain Procedure:

  1. Flood slide with carbol fuchsin, steam

  2. Wash off excess stain

  3. Decolorize with acid alcohol, counterstain with methylene blue

  4. Wash off excess stain, air or blot dry, examine under a microscope
• Microscopy for AFB Stain:

  • Read the slide from left to right and then from right to left
  • Record results: Acid-fast organisms appear red, non-acid-fast organisms appear blue