Practical 6

• Explain examples of aseptic techniques that could be used

  ● Wash hands with soap / disinfect surfaces → kill microbes / prevent contamination

  ● Sterilise pipette / spreader / boil agar growth medium → kill microbes / prevent contamination

  ● Flame neck of bottle of bacteria → kill microbes / prevent contamination

  ● Bunsen burner close → upward current of air draws air-borne microbes away to prevent contamination

  ● Lift lid of petri dish slightly / minimise opening → 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 diff concs of antimicrobials for same time, onto agar plate

  5. Lightly tape lid onto plate invert + incubate at 25oC for 48 hours

  6. Measure diameter of inhibition zone around each disc and calculate area using πr
• 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 2 pieces of tape instead of sealing it completely?

  ● Allows oxygen in preventing growth of anaerobic bacteria

  ● Which are more likely to be pathogenic / harmful to humans
• Why use a paper disc with water / no antimicrobial agent?

  ● Act 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 conc.?

  More bacteria killed so clear zones may overlap
• Why incubate at 25oC 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 (X axis type of antimicrobial, Y axis area of zone of inhibition / mm3)

  ● Continuous data → line graph joined by a line of best fit (X axis concentration of antibiotic / μgmL-1, Y axis area of zone of inhibition / mm3)
• 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