LAB 5

• Staining


  Artificially coloring microorganisms with dye
• Procedures for staining

  1. Smear - thin layer of microorganisms over the slide

  2. Fixing microorganism - passing slide through a flame / covering slide with Methyl Alcohol for 1 minute
• Fixation


  • Fixing / killing microorganisms and attaching them to the slide
  • to preserve various parts of the microorganism in their natural state with minimal distortion
• Ways of fixation

  • Heat-fixing (5 seconds)
  • Air-drying
  • Treatment with alcohol solution
• Dye


  Compound consisting of benzene rings, the "aniline" dyes
• Functional groups of dye

  • Chromophore - gives a specific color to a compound
  • Auxochrome - salt forming properties responsible for transferring color of the dye
• Types of dye

  • Basic dye - dye with a positive charge (e.g. Crystal Violet, Safranin, Methylene Blue, Malachite Green)
  • Acidic dye - dye with negative charge (e.g. Eosin, Carbolfuchsin, Nigrosin, Congo Red, Acid Fuchsin)
• Simple staining


  Uses simple stain; one dye only
• Purpose of simple staining

  • To highlight the entire microorganism
  • To reveal shapes, sizes, and arrangements
• Differential staining


  Uses two or more dyes; type of staining that reacts differently with different microorganisms
• Purpose of differential staining

  • To contrast two or more organisms
• Gram staining


  Developed by Hans Christian Gram in 1884; classifies bacteria as Gram positive or Gram negative
• Gram staining reagents

  • Primary stain - Crystal Violet
  • Mordant - Gram's Iodine
  • Discolorizer - Acid Alcohol
  • Counterstain - Safranin
• Gram positive organisms


  • Mycobacterium
  • Clostridium
  • Bacillus
  • Corynebacterium
  • Lactobacillus
  • Erysipelothrix
• Gram negative organisms


  • Neisseria
  • Veilonella
  • Moraxella
  • Listeria
  • Nocardia
• Gram positive vs Gram negative differences

  • Gram positive - Thick peptidoglycan, 90% peptidoglycan, Teichoic acid
  • Gram negative - Thin peptidoglycan, 5-10% peptidoglycan, Outer membrane has lipids, lipopolysaccharides
• Acid fast staining


  • Ziehl-Neelsen stain
  • binds only to bacteria that have a waxy material (mycolic acid) in cell walls
  • classifies bacteria from acid fast to non-acid fast
  • used to identify bacteria in genus mycobacterium and nocardia spp
• Acid fast staining reagents

  • Primary stain - Carbolfuchsin (easily penetrates mycolic acid)
  • Decolorizer - Acid Alcohol
  • Secondary stain - Methylene Blue
• Acid fast vs non-acid fast staining

  • Acid fast - Primary: Red, Decolorizer: Red, Counterstain: Red
  • Non-acid fast - Primary: Red, Decolorizer: Colorless, Counterstain: Blue
• Negative staining


  • "Indirect staining"
  • preparing colorless bacteria against a colored background
  • reagents: Nigrosine or India Ink
• Special staining


  Used to color, isolate, identify, and study specific parts or structures of microorganisms (endospores & flagella) and to reveal the presence of capsules
• Endospore staining


  • Schaeffer-Fulton Endospore Stain
  • endospore is a special resistant, dormant structure formed within a cell that protects a bacterium from adverse environmental condition
  • identifies organisms like Bacillus and Clostridium
• Endospore staining reagents

  • Primary stain - Malachite Green (then heat —mordant)
  • Decolorizer - Water
  • Secondary stain - Safranin
• Capsule staining


  Stains the bacteria and the background leaving the capsule unstained
• Capsule staining reagents

  • Primary stain - Nigrosine, India Ink, Congo Red (either of the 3)
  • Mild decolorizer - Copper Sulfate (Blue Vitriol)
• Capsule staining - Welch Method


  • Capsular materials are soluble in water
  • Polymers of the capsule tend to be uncharged and as such they are not easily stained
  • Glycocalyx - made of polysaccharide, polypeptides, and both; sticky substance surrounding the bacterial cell wall
• Types of capsule

  • Capsule - slime layer, gelatinous covering; determines organism's virulence
  • Virulence - degree to which a pathogen can cause disease
• Capsule staining steps

  Congo Red, India Ink, Nigrosin

  Air dry (not heat)

  Acid alcohol (fixation)
• Examples of organisms with capsules

  • S. Pneumoniae
  • Cryptococcus neoformans
• Flagella staining


  Many bacteria are motile
• Types of flagella

  • Monotrichous - one tail on one side
  • Lopotrichous - many tail on one side
  • Amphitrichous - many tail on both sides
  • Peritrichous - tail on entire microbe
• Flagella staining steps

  Air dry bacteria on glass slide

  Flagella stains (tannic acid & potassium alum)

  Carbolfuchsin for 5 minutes

  Rinse and do not blot with absorbent paper
• Special stains for spirochetes

  • Giemsa Stain
  • Wright Stain
  • Silver Stain
  • Dark-Field Microscopy
• True motility


  Movement of microorganisms from one position to another in a fluid environment
• Brownian movement


  Not a true motility but rather is movement caused by the molecules in the liquid striking an object and causing the object to shake or bounce
• Materials for observing living microorganisms

  • Depression slide & cover slip
  • Petroleum jelly - to seal and reduce evaporation
  • Pasteur pipettes
  • Hay infusion - Hay macerated in water for 1-2 weeks, store in open jar either light or dark place
• Microorganisms in hay infusion

  • Protozoans
  • Algae
  • Bacteria
  • Algal-Like Protists
  • Animal-Like Protists