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

Explain why it is 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

Explain why the lid is held with 2 pieces of tape and not sealed completely.

Answer 4

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

Question 5

Explain why a paper disc with water / no antimicrobial agent is used.

Answer 5

• Act as a control.
• Ensuring antimicrobial prevented growth, not paper disc.

Question 6

Explain why petri dishes are incubated upside down.

Answer 6

Condensation drips onto lid rather than surface of agar.

Question 7

Explain how zones of inhibitions can be measured if irregular.

Answer 7

Repeat readings in different positions, calculate a mean.

Question 8

Explain why a higher antimicrobial concentration isn’t used.

Answer 8

More bacteria killed so clear zones may overlap.

Question 9

Explain why bacteria should be
incubated at 25°C or less in a school laboratory.

Answer 9

Below human body temperature so prevent growth of pathogens.

Question 10

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

Answer 10

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 11

Explain the presence and absence of clear zones.

Answer 11

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.