Required 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 (as above)
2. Use a sterile pipette to transfer bacteria from broth to agar plate using aseptic techniques (as above)
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 25oC for 48 hours
6. Measure diameter of inhibition zone around each disc and calculate area using πr^2

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 25C or less?

Answer 9

● Below human body temp 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 / 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

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