Microbiology

• Obligate Anaerobe - cell that cannot metabolise in the presence of oxygen

  
  

  Facultative Anaerobe - cell that can metabolise in presence of oxygen and without, usually prefers aerobic

  
  

  Obligate Aerobe - cell that can only metabolise in the presence of oxygen
• 1 - mesosome

  2 - capsule

  3 - plasma membrane

  4 - peptidoglycan cell wall

  5 - cytoplasm

  6 - plasmid DNA

  7 - 70s ribosomes

  8 - flagellum

  9 - free circular DNA

  
• Coccus - spherical shaped bacteria

  Bacillus - rod shaped bacteria

  Spirillum - spiral shaped bacteria
• Gram Stain method:

  1. Apply crystal violet (purple dye)
  2. Apply Grams Iodine (covalently bonds crystal violet to peptidoglycan)
  3. Alcohol wash - dissolves lipopolysaccharide layer
  4. Apply Safranin (pink counter-stain)
• Colour of bacteria at each step:

  1:

  Gram positive - purple

  Gram negative - purple

  2:

  Gram positive - purple

  Gram negative - purple

  3:

  Gram positive - purple

  Gram negative - colourless

  4:

  Gram positive - purple

  Gram negative - pink
• Gram negative bacteria contain a lipopolysaccharide layer on the cell wall whereas gram positive bacteria have a fully peptidoglycan cell wall
• Which test tube contains which type of bacteria?

  1 - Obligate aerobes

  2 - Facultative anaerobes

  3 - Obligate anaerobes

  
• Aseptic techniques have 2 main purposes:

  1. Preventing contamination of pure culture by microbes from the environment
  2. Preventing contamination of the environment by cultures being grown
• Preventing contamination of pure culture:

  • Sterilise all media and equipment (such as inoculating loops) before use to prevent initial contamination
  • Handle cultures carefully, flame the neck of culture bottles before opening and closing
  • Use a bunsen burner to create a convection current
  • Disinfect work benches before hand - such as with 3% lysol
• • For glassware and metalware, the method of sterilisation is autoclaving
  • For plasticware, the method of sterilisation is gamma irradiation
• In autoclave, items should be:

  • sealed in an autoclave bag
  • heated to 121 degrees in steam
  • under high pressure

  for 15 minutes
• Total cell count - count of number of living and dead cells in a bacterial sample

  Total viable count - count of number of living cells in a known volume of liquid medium
• A method of a total viable count is using a serial dilution
• Serial dilution:

  1. place 9cm3 of distilled water into five sterile test tubes using sterile pipette
  2. place 1cm3 of the original bacterial culture sample in the first tube and mix (10^-1 dilution)
  3. transfer 1cm3 of that dilution into the next tube and mix, repeat this for the remaining tubes
  4. transfer 1cm3 of each diluted sample onto a sterile nutrient agar plate
  5. seal and incubate plates
  6. look for the dilution that shows distinct colonies without merging and count the number of distinct colonies
  7. count number of colonies and scale back up to find number of bacteria in original sample