Required practical 6 -Use of aseptic techniques to investigate the effect of antimicrobial substances on microbial growth

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 25oC 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

● 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 25oC or less in a school laboratory

Answer 9

● Below human body temp to prevent growth of pathogens

Question 10

Explain the presence and absence of clear zones

Answer 10

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

Question 11

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

Answer 11

● Categorical data → bar chart, eg.
○ X axis type of antimicrobial
○ Y axis area of zone of inhibition / mm3

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