MBIO 1010 Lecture 3

• Robert Koch

  • Studied anthrax - responsible for epidemics in livestock
  • He isolated bacteria from the carcass of a diseased animal - Bacillus anthracis
  • Injected healthy animals with the bacterium
  • animals became ill with anthrax
  • Re-isolated B. anthracis from the test subject and showed that it was identical
  • Established a set of criteria for relating a specific microbe to a disease
• Koch's postulates

  1. suspected pathogen must be present in all cases of the disease and absent from healthy animals
  2. the suspected pathogen must be grown in a pure culture
  3. cells from a pure culture of the suspected pathogen must cause disease in a healthy animal
  4. the suspected pathogen must be reisolated and shown to be the same as the original
• Koch's postulates don't work for all microbes because some microbes only affect humans
• Koch contributed to the realization that solid media provided a simple way to obtain pure cultures

  Broth medium solidified with agar
• broth = liquid
• agar is a solidifying agent
• koch had tried to use gelatin instead of agar but it served as food for the bacteria
• Agar starts to solidify at ~43 degrees celcius and melts at ~97 degrees celcius
• agar cannot be degraded by most microorganisms and is a polysaccharide derived from marine algae
• A typical petri plate = nutrient broth medium + 1.5% agar
• It is common to incubate microbes at 37 degrees celcius because our bodies are 37 degress and we have microbes that live on us
• A petri plate has no agar in them
• Peptone in agar is a good source of protein
• Beef extract in agar is a good source of nitrogen, carbon and sodium chloride
• NaCl in agar is there for osmolarity
• The Streak Plate technique

  One edge of a plate is inoculated with a concentrated sample of bacteria, sample is diluted by streaking it across the surface of the plate to deposit individual cells on the plate (separate from other cells), plate is incubated, individual cells grow to form colonies
• Colony

  a mass of cells that (ideally) arose from a single cell, can be used to create a pure culture
• Goal of the streak plate technique

  To deposit individual cells on the plate (separate from other cells)

  Morphology look the same

  streak plate for pure culture
• Spread plate and pour plate

  Sample is diluted before plating, diluted sample can be spread over the surface of the plate with a sterile spreader - separate cells grow into colonies on the surface of the plate

  it can be mixed with molten agar (~45 degrees Celsius)

  colonies form embedded inside the plate

  used for counting/enumeration
• Titre - how much is there
• Spread and pour plates allow you to calculate the concentration of bacteria in a population (bacterial titre)
• titre = # colonies / (volume) (dilution)

  titre is expressed in cfu/ml

  cfu = colony forming unit
• Serial dilution has to be long enough to reach a countable sample
• We normally count plates with between 30-300 colonies

  less than 30 - not statistically significant

  more than 300 - colonies grow into each other - inaccurate counts
• When we have more than one countable plate

  • calculate titre from each and then take the average
• TNTC = too numerous to count

  NEVER WRITE TFTC = to few to count because this is not true, you can count them they are just not statistically significant to count them