Required Practical 6

Question 1

Explain examples of aseptic techniques that could be used.

Answer 1

1. Wash hands with soap/disinfect surfaces.
   
   • Kill microbes/prevent contamination.
2. Sterilise pipette/spreader/boil agar growth medium.
   
   • Kill microbes/prevent contamination.
3. Flame neck of bottle of bacteria.
   
   • Kill microbes/prevent contamination.
4. Bunsen burner close.
   
   • Upward current of air draws air-born microbes away to prevent contamination.
5. Lift lid of petri dish slightly/minimise opening.
   
   • Prevent entry of microbes/contamination.

Question 2

Describe a method to investigate the effect of antimicrobial substances (e.g. antibiotics, disinfectants, antiseptics) on microbial growth.

Answer 2

1. Prepare area using aseptic techniques.
2. Use a sterile pipette to transfer bacteria from broth to agar plate using aseptic technique.
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 at equal distance.
5. Lightly tape lid onto place, invert and incubate at 25°C for 48 hours.
6. Measure diameter of inhibition zone around each disc and calculate area using πr².

Question 3

Why is it important to maintain a pure culture of bacteria?

Answer 3

• Bacteria may outcompete bacteria being investigated.
• Or could be harmful to humans/pathogenic.

Question 4

Why hold lid with 2 pieces of tape instead of sealing it completely?

Answer 4

• Allows oxygen in preventing growth of anaerobic bacteria.
• Which are more likely to be pathogenic/harmful to humans.

Question 5

Why incubate upside down?

Answer 5

Condensation drips onto lid rather than surface of agar.

Question 6

What if inhibition zones are irregular?

Answer 6

Repeat readings in different positions, calculate a mean.

Question 7

Why not use higher antimicrobial concentration?

Answer 7

More bacteria killed so clear zones may overlap.

Question 8

Why incubate at 25°C or less?

Answer 8

Below human body temperature so prevent growth of pathogens.

Question 9

Describe how data about the effect of antimicrobial substances can be presented as a graph.

Answer 9

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.

Question 10

Explain the presence and absence of clear zones.

Answer 10

CLEAR ZONES

1. Antimicrobial diffuses out of disc into agar, killing/inhibiting growth of bacteria.
   
   • Larger clear zones, more bacterial killed, more effective antimicrobial.

NO CLEAR ZONES

2. If antibiotic used, bacteria may be resistant or antibiotic may not be effective against that specific bacteria.