MICROPARA LAB

• Bacteria


  Colorless in nature, difficult to examine in a light microscope
• Motile bacteria


  Going in a definite direction, non-motile bacteria appear to move due to Brownian movement
• Staining


  Coloring agent used to make bacteria visible

  
• Different dyes are used as not all bacteria exhibit the same morphological properties
• Bacterial morphology can be used for classification
• Staining techniques


  • Enhance contrast of images formed by compound microscope
  • Used to examine tissues
• Stain


  Substance that adheres to a cell giving it color
• Chromophore group


  Chemical group that imparts color to benzene
• Auxochrome group


  Chemical compound that conveys ionization property of chromogen and binds to fibers or tissues
• Direct staining


  Organism is stained, background is left unstained

  
• Direct stains


  • Methylene blue, crystal violet, basic fuchsin
• Negative staining


  Background is stained, organism is left unaltered
• Negative stains


  • Nigrosine, Indian ink
• Bacterial smear preparation


  1. Write organism and staining procedure on slide

  2. Flame sterilize inoculating loop and cool

  3. Fish out colonies from culture medium

  4. Spread inoculum on slide

  5. Air dry

  6. Heat fix bacteria

  
• Simple staining


  Single basic dye to highlight entire microorganism

  
• Simple staining procedure


  1. Create bacterial smear slide

  2. Apply crystal violet, safranin or methylene blue for 30-60 seconds

  3. Wash with distilled water and air dry

  4. Observe under oil immersion

  
• Differential staining


  Uses more than one chemical stain to differentiate microorganisms or cellular components

  
• Gram staining


  • Crystal violet (primary stain), iodine (mordant), 95% ethanol (decolorizer), safranin (counterstain)
  • Gram positive retain primary stain, gram negative retain counterstain
• Cell wall staining


  Demonstrates bacterial cell wall

  
• Chance's method for cell wall staining

  1. Apply 0.5% new fuchsin for 3 minutes

  2. Remove excess stain (no washing)

  3. Apply 0.5% congo red for 4 minutes

  4. Gentle wash with water and air dry

  5. Observe under oil immersion

  
• Mechanism of Chance's method

  New fuchsin stains cell wall and cytoplasm, congo red removes new fuchsin from cytoplasm but not cell wall
• Acid-fast staining


  Differentiates acid-fast and non-acid-fast bacteria, binds strongly to bacteria with waxy cell walls

  
• Acid-fast bacteria


  • Mycobacterium tuberculosis, Mycobacterium leprae, Nocardia
• Ziehl-Neelsen method (acid-fast staining)


  • Apply carbol fuchsin, heat, decolorize with acid-alcohol, counterstain with methylene blue
  • Acid-fast bacteria retain pink/red, non-acid-fast appear blue
• Spore staining


  Demonstrates bacterial endospores

  
• Schaeffer-Fulton method for spore staining

  1. Primary stain is malachite green, stains both vegetative cells and endospores

  2. Vegetative cells appear pink, endospores appear green

  
• Bacterial endospore


  Metabolically inactive, dormant or resting structure, highly resistant
• Non-acid-fast cells


  Appear blue after the counterstain is applied
• Acid-fast bacteria


  Still red after the counterstain is applied
• Spore staining


  • Demonstrates a special part of the organism

  
• Schaeffer-Fulton method


  • Most used spore staining method
  • Similar procedure with acid fast but the stain is different
  • Demonstrates spore in bacteria as well as free spores

  
• Bacterial genera that produce endospores

  • Bacillus spp
  • Clostridium spp
• Vegetative cells


  • Pink
  • Actively dividing cells (cells that don't have a spore)
• Endospores


  • Green
  • Metabolically inactive (dormant or resting structure of bacteria)
  • Highly resistant structure produced by bacteria as a defensive strategy against unfavorable environment
• Primary stain (malachite green)

  • Stains both vegetative cells and endospores
  • Heat is applied to help the primary stain penetrate the endospore

  
• Water


  Decolorizer which removes the malachite green from the vegetative cell but not the endospore
• Safranin


  Counterstain any cells which have been decolorized
• Spore staining procedure


  1. Prepare smear

  2. Flood smear with malachite green, steam slide for 5 mins

  3. Remove paper, rinse with water

  4. Apply safranin counterstain for 1 min

  5. Wash with water, air dry, observe under OIO

  
• Capsule


  • Synthesized in the cytoplasm of the cell and secreted to the outside of cell where it surrounds the bacterium
  • Most capsulated bacteria are made up of polysaccharide (sugar) layer, some are made up of polypeptide and glycoprotein
  • Capsules are non-ionic (neither basic nor acidic stain will adhere to the surface)
• Capsule staining


  Uses acidic and basic dyes to stain the background and bacterial cell respectively, leaving the capsule unstained