Module 4. Core practical 6

Question 1

Explain examples of aseptic techniques that could be used

Answer 1

● 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

Question 2

Describe a method to investigate the effect of antimicrobial substances (eg. 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 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 (at equal distances)
5. Lightly tape lid onto plate (not fully sealed), 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 use a paper disc with water / no antimicrobial agent?

Answer 5

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

Question 6

Why incubate upside down?

Answer 6

● Condensation drips onto lid rather than surface of agar

Question 7

What if inhibition zones are irregular?

Answer 7

● Repeat readings in different positions, calculate a mean

Question 8

Why not use higher antimicrobial conc.?

Answer 8

● More bacteria killed so clear zones may overlap

Question 9

Why incubate at 25°C or less?

Answer 9

● Below human body temperature to 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 / mm ³)
● 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

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