Inoculation

• How does freezing affect bacterial growth?

  STOPS bacterial growth
• How does refrigeration affect bacterial growth?

  slows bacterial growth down (<5 degrees)
• Bacteria grow rapidly around room temperature and above
• Bacteria grow slower at higher temperatures
• Most bacteria are killed by boiling (120+)
• Aseptic = free from contamination of microorganisms
• Pathogens grow at human body temperature
• Pathogen = a disease-causing microorganisms
• Method for prac:

  1. Disinfect desk + work next to Bunsen burner for updraft
  2. Sterilise wire loop by flaming in Bunsen burner
  3. Place inoculating loop into sample
  4. Lift Petri dish at 45 degree angle and sweep loop across surface of agar
  5. Seal with sellotape in a cross to allow oxygen to diffuse in
  6. Incubate plates at 25 degrees for 24-48 hours
  7. Autoclave plates and equipment after use
• What does flaming do?

  heat kills any microorganisms
• updraft has?

  sterile convection currents
• why do allow oxygen to diffuse in to Petri dish?

  as otherwise sealed anaerobic pathogens will grow
• Why do we incubate at 25 degrees for 24-48 hours?

  To encourage growth of culture without growing pathogens
• Aseptic techniques:

  • Disinfect desk/working area with disinfectant spray
  • Sterilise equipment by autoclaving/flaming
  • Boiling agar jelly before using (cool first)
  • Lift Petri dish at 45 degrees
  • Taping lid shut in a criss cross pattern
• Why do we only lift lid at 45 degrees?

  reduces chance of other bacteria getting onto plate
• Why do we seal with a criss-cross?

  • Allows oxygen to diffuse in -> prevents growth of pathogens
  • Prevents lid coming off and letting in bacteri/fungi that didn’t come from sample
• What is agar?

  a nutrient jelly used to grow cultures
• Plating = growing a culture of bacteria on a sterile Petri dish
• inoculating loop = a sterile loop used to transfer bacteria onto an agar plate
• One single bacterium -> one colony
• Colonies = high number of bacteria that have grown together
• What don’t we want when counting colonies?

  clumping
• Clumping = colonies overlap
• Why is clumping bad?

  cannot accurately estimate number of colony forming units