L1 - Innoculation

• Freezing stops bacterial growth
• Refrigeration (<5°C) slows bacterial growth down
• Bacteria grow rapidly at around room temperature and above, more slowly at higher temperatures
• Most bacteria are killed by boiling (high temperatures above 120°C)
• How to innoculate plates using aseptic technique (Part 1):

  1. Disinfect the desk and work next to Bunsen burner for uplift (sterile convection current)
  2. Wire loop is sterilised by 'flaming' in Bunsen flame. The heat will kill any microorganisms
  3. Place the innoculating loop into the sample
  4. Lift petri dish at 45° angle and sweep the loop across the surface of the agar, being careful not to dig in
• How to innoculate plates using aseptic technique (Part 2):

  5. Seal it with sellotape in a cross to allow oxygen to diffuse in (if totally sealed anaerobic pathogens will grow

  6. Plates are incubated at 25°C for 24-48 hours in school laboratories to encourage growth of the culture without growing pathogens

  7. Plates and equipment be autoclaved after use for safety reasons
• Aseptic Technique:

  • Disinfecting desk/work area with disinfectant spray
  • Sterilising equipment by autoclaving before or flaming during experiment
  • Boiling the agar jelly before using it (then allowing it to cool)
  • Only lifting the petri dish at 45° angle (reduces chance of other bacteria getting onto the plate)
  • Taping the lid shut in a cross pattern allows oxygen which reduces the chance of growing pathogenic bacteria. It also prevents the lid from coming off and letting bacteria/fungi in which didn't come from your sample
• One colony grows from one single bacterium
• Colonies are a high number of bacteria that have grown together
• If there are many colonies on a plate some may overlap. This is known as clumping and you cannot accurately estimate the number of colony forming units if clumping has occurred