RP6 - Use of aseptic techniques to investigate the effect of antimicrobial substances on microbial growth.

Explain examples of aseptic techniques that could be used (5)

1. Wash hands with soap and disinfect surfaces → kill microbes and prevent contamination
2. Sterilise pipette / spreader / boil agar growth medium → kill microbes and prevent contamination
3. Flame neck of bottle of bacteria → kill microbes and prevent contamination
4. Keep Bunsen burner close → upward current of air draws air-borne microbes away to prevent contamination
5. Lift lid of petri dish slightly / minimise opening → prevent entry of microbes and contamination

Describe a method to investigate the effect of antimicrobial substances (eg. antibiotics, disinfectants, antiseptics) on microbial growth

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

Why is it important to maintain a pure culture of bacteria?

● Bacteria may outcompete bacteria being investigated
● Or could be harmful to humans / pathogenic

Why hold lid with 2 pieces of tape instead of sealing it completely?

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

Why use a paper disc with water / no antimicrobial agent?

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

Why incubate upside down?

● Condensation drips onto lid rather than surface of agar

What if inhibition zones are irregular?

● Repeat readings in different positions, calculate a mean

Why not use higher antimicrobial conc.?

● More bacteria killed so clear zones may overlap

Why incubate at 25C or less?

● Below human body temp to prevent growth of pathogens

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

● 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 )

Explain the presence of clear zones

1. Clear zones → antimicrobial diffuses out of disc into agar, killing / inhibiting growth of bacteria
● the larger the clear zones → the more bacteria killed → more effective antimicrobial

Explain the absence of clear zones

2. No clear zones → if antibiotic used, bacteria may be resistant or antibiotic may not be effective against that specific bacteria