Lab Two

• Refraction

  change of light waves direction as they enter new environment
• Oil increases the resolution of the sample because oil has the same refractive index as the glass in the slide.
• Oil is used with --- objective.

  100x
• What is the general ocular magnification?

  10x
• Better resolution coincides with

  • the shorter the wavelength of light
  • the higher the numerical aperture (ability of the lens to gather light)
• Gram staining technique

  • distinguishes two types of bacteria based on cell wall structure
  • designed by christian gram in 1884
• Gram-positive bacteria

  • composed of many layer of peptidoglycan and teichoic acid
• Gram-negative bacteria

  • no teichoic acid
  • thin layer of peptidoglycan
  • additional outer membrane with lipopolysaccharide (LPS)
• Lipopolysaccharide (LPS)


  composed of: Lipid A (endotoxin) and O polysaccharide
• Gram-Negative stains...

  orange-red from safranin
• Gram-Positive stains...

  blue-purple from crystal violet and iodine
• Crystal Violet (primary stain)

  basic dye: positively charged

  cytoplasm: negatively charged

  dye attracted to cytoplasm, crystal violet stains cytoplasm purple
• Iodine (mordant)


  forms complex with crystal violet, decreasing solubility of dye within cell

  all cells become blue-purple
• Mordant


  substance that increases affinity of cellular components for a dye
• Decolorizer (alcohol wash)


  In g+: dissolves lipid in membranes and allow crystal violet iodine complex to leak out of cytoplasm --> becomes colorless

  In g-: dissolves lipid in membranes but large peptidoglycan content traps dye - iodine complex within cytoplasm --> remain blue-purple
• Safranin (counterstain)

  imparts contrasting color to colorless gram-negative --> turns orange-red