Lab Exam 1

• Lab Box Contents

  loop

  spreader

  bunsen burner attachment

  fuel canister

  pen

  tweezers

  striker

  lens paper
• Pipettes

  P20

  P200

  P1000
• P20

  tens

  ones

  tenths
• P200


  hundreds

  tens

  ones
• P1000

  thousands

  hundreds

  tens
• TSA

  Tryptic Soy Agar
• Streak plate isolation


  Purpose: isolate single colony from liquid culture using metal loop
• Two ways to measure bacterial concentrations

  Optical density

  Viable counts
• Optical density


  absorbance of 600 nm

  measures concentration of dead and living cells
• Viable counts


  measures concentration of living cells only
• Viable counts procedure

  1. dilution
  2. plate with spreader
  3. compare numbers after 30-37C
• Dilution equation

  Dilution = V stock / V total
• Better resolution =

  shorter wavelength of light

  higher numerical aperture
• Oil

  increases resolution of sample because oil has same refractive index as glass
• Refraction

  change of light waves direction as they enter new environment
• Gram staining


  differentiates between two types of cell structure

  designed by christian gram in 1884
• Gram positive bacteria

  cell wall = thick peptidoglycan

  has teichoic acid

  forms spores, no UV effect

  stains blue-purple
• Gram negative bacteria

  cell wall = thin peptidoglycan

  outer layer with lipopolysaccharide (LPS)

  stains orange-red
• LPS

  Lipid A (endotoxin)

  O polysaccharide
• Gram staining procedure

  crystal violet

  iodine

  alcohol wash

  safranin
• Crystal violet: primary

  basic dye: positive charge

  cytoplasm: negative charge

  dye bind to cytoplasm

  all cells: purple
• Iodine (mordant)


  form complex with crystal violet

  decrease solubility of dye

  all cells: blue purple
• Mordant


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


  dissolves lipid in membranes

  gram pos: crystal violet iodine trapped in peptidoglycan: blue purple

  gram neg: crystal violet iodine leaks out: colorless
• Safranin (counterstain)


  contrasting color to gram negative bacteria: orange red
• UV light

  wavelength 100 - 400 nm

  265 nm: DNA absorbs UV light energy

  induces formation of thymine dimers
• UV light effectiveness

  direct exposure

  doesn't go through thick things

  not effective against all spores
• Thymine dimers


  cause localized distortion of DNA helix: interferes with DNA replication adn transcription
• SASP

  small acid-soluble spore protein
• How spores resist UV light

  SASP binds to DNA: untwists double helix

  structural change makes spore DNA resistant to UV light
• Pasteurized


  high heat for short period of time
• 3 goals of pasteurization

  destroy all pathogens

  reduce concentration of other microbes: increase time needed for spoilage

  save flavor
• Potential sources of contamination in raw milk


  cow udder

  skin

  feces

  milking machinery
• Raw milk process

  wash teats

  sanitize machinery

  milk the cow

  strain milk

  chill milk rapidly

  store in cold
• California law for raw milk

  not greater than 10 coliform bacteria/mL and not greater than 15,000 total bacteria/mL
• Coliform

  found in animal guts

  rod shaped

  gram negative

  ferment lactose with production of acid and gas
• MacConkey agar analysis

  selective: inhibits growth of gram positive

  differential: lactose fermenting & non fermenting
• MacConkey agar interpretation

  Non lactose:

  no lactose, use peptone

  forms ammonia (raise pH)

  colorless / yellow

  Lactose:

  forms acid

  lowers pH below 6.8

  pink
• Enterotube II / EnteroPluri Test


  used for rapid interpretation of unknown enteric bacteria

  12 compartments

  15 biochemical tests
• Enterobacteriaceae family


  found in digestive tract of animals

  gram negative

  anaerobic rods