RP6 - use of aseptic techniques to investigate effect of antimicrobial substances on microbial growth

Question 1

Examples of aseptic techniques

Answer 1

• Wash hands with soap and disinfect surfaces -> kill unwanted bacteria and prevent contamination
• Sterilise pipette/spreader/boil agar -> kill unwanted bacteria and prevent contamination
• Flame neck of bottle of bacteria -> kill unwanted bacteria and prevent contamination
• Bunsen burner close -> upward current of hot air draws airborne bacteria away to prevent contamination
• Lift lid of petri dish slightly/minimise opening -> prevent entry of unwanted bacteria

Question 2

Method to investigate effect of antimicrobial substances on microbial growth

Answer 2

• Prepare area using aseptic techniques
• Use sterile pipette to transfer bacteria from broth to agar plate using aseptic techniques
• Use sterile spreader to evenly spread bacteria over agar plate (flooding)
• Use sterile forceps to place same size discs soaked in different types/concentrations of antimicrobials for same length of time on agar plate
• Lightly tape lid, invert, incubate at 25 C for 48 hours
• Measure diameter of inhibition zone and calculate area

Question 3

Why is it important to maintain a pure culture of bacteria

Answer 3

Bacteria can outcompete bacteria being investigated -> could be harmful/pathogenic

Question 4

Why is lid loosely taped

Answer 4

Allows oxygen to prevent growth of anaerobic bacteria which is harmful/pathogenic

Question 5

Why is a paper disc with water/no antimicrobial agent used

Answer 5

Control disc - ensures antimicrobial is what prevented growth, not the disc

Question 6

Why are petri dishes incubated upside down

Answer 6

Condensation drips onto lid rather than agar

Question 7

Why is a higher antimicrobial concentration not used

Answer 7

More bacteria killed so clear zones may overlap

Question 8

Why should bacteria be incubated at 25 C or less in a school lab

Answer 8

Below human body temperature to prevent growth of pathogens

Question 9

How should zones of inhibition be measured if irregular

Answer 9

Repeat in different positions and calculate a mean

Question 10

Explain the presence and absence of clear zones

Answer 10

• Clear zones -> antimicrobial diffused out of disc onto agar - inhibits growth of bacteria
  -> larger clear zones means more bacteria killed so more effective antimicrobial
• No clear zones - bacteria may be resistant or antibiotic may not be effective