rp6 aspetic

Explain examples of aseptic techniques that could be used

● 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

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

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 the 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 25

oC 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 clear zones

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