Required Practical 6

• Explain examples of aseptic techniques that could be used:

  • Wash hands with soap and disinfect surfaces -> kill microbes and prevents contamination
  • Sterilise pipette / spreader / boil agar growth medium -> kill microbes and prevents contamination
  • Flame neck of bottle of bacteria -> kill microbes and prevents contamination
  • Bunsen burner close -> upward current of air draws air-borne microbes away to prevent contamination
  • Lift lid of petri dish slightly and away from you -> prevent entry of microbes / contamination
• Describe a method to investigate the effect of antimicrobial substances on microbial growth:

  1. Prepare area using aseptic techniques
  2. Use a sterile pipette to transfer bacteria from broth to agar plate using aseptic techniques
  3. Use a sterile spreader to evenly spread bacteria over agar plate
  4. Use sterile forceps to place same size discs that have been soaked in different types / concentrations of antimicrobials for same length of time onto agar plate
  5. Lightly tape lid onto plate, invert and incubate at 25° for 48 hours
  6. Measure diameter of inhibition zone around each disc and calculate area
• Why is it important to maintain a pure culture of bacteria?

  • Bacteria may outcompete bacteria being investigated
  • Or could be harmful to humans / pathogenic
• Why hold lid with two pieces of tape instead of sealing completely?

  • Allows oxygen in to prevent growth of anaerobic bacteria
  • Which are more likely to be pathogenic / harmful to humans
• Why use a paper disc with water / no antimicrobial agent?

  • Acts as a control
  • Ensuring antimicrobial prevented growth - not paper disc
• Why incubate upside down?

  Condensation drips onto lid rather than surface of agar
• What if inhibition zones are irregular?

  Repeat readings in different positions, calculate a mean
• Why not use higher antimicrobial concentration?

  More bacteria killed so clear zones may overlap
• Why incubate at 25 or less?

  Below human body temp to prevent growth of pathogens
• Describe how data about the effect of antimicrobial substances can be presented as a graph:

  • Categorical data = Bar chart
  • Continuous data = Line graph
• Explain the presence and absence of clear zones:

  1)Clear zones -> antimicrobial diffuses out of disc into agar, killing / inhibiting growth of bacteria

  • Larger clear zones -> more bacteria killed -> more effective antimicrobial

  2)No clear zones -> if antibiotic used, bacteria may be resistant or antibiotic may not be effective against that specific bacteria