The use of aseptic techniques to investigate the effect of antimicrobial substances on microbial
growth.
Equipment:
o Agar plate seeded with bacteria (Ecoli)
o Plant material (garlic cloves and mint leaves)
o Pestle and mortar 10 cm3 industrial methylated spirits
o Pipette (sterile)
o Paper discs
o Sterile Petri dish
o Sterile forceps
o Tape
o Marker pen
o Incubator set at 25 °C
Aseptic Techniques:
o Wipe down surfaces with antibacterial cleaner, both before and after experiment.
o Use a Bunsen burner in the work space so that convection currents draw microbes away
from the culture.
o Flame the wire hoop before using it to transfer bacteria.
o Flame the neck of any bottles before using them to prevent any bacteria entering the vessel
(air moves out so unwanted organisms don’t move in).
o Keep all vessels containing bacteria open for the minimum amount of time
o Close windows and doors to limit air currents
Method:
1. Carry out aseptic techniques detailed above.
2. Use a sterile pipette or wire hoop to transfer bacteria from broth (distilled water, bacterial
culture, nutrients) to agar plate (petri dish containing agar jelly).
3. Gently spread bacteria evenly over plate using a sterile plastic spreader. (This is supposed to
be done gently as the agar might get damaged if you apply too much force onto it).
4. Use sterile forceps to place a multi disc antibiotic ring on the plate (garlic, mint, ethanol and
plain control). Rings should only be moved by holding the centre, not the arms.
5. Lightly tape a lid on, invert and incubate at 25°C for 48 hours. Do not tape around the entire
dish as this prevents oxygen entering and so promotes the growth of more harmful
anaerobic bacteria.
6. Sterilise equipment used to handle bacteria and disinfect work surfaces.
7. Incubate the plates for 24 hours at 25 °C.
8. After incubation examine the plate and try to identify the colonies which have not been able
to grow near the multidisc arm(s). These are called zone(s) of inhibition.
, 9. Turning the plate upside down and using a ruler measure the diameter of the zones of
inhibition. Calculate the area of the zone of inhibition using the formula Area of zone = πr2
se 3.14
10. Record your results in a suitable table.
Risk assessment:
Hazard Risk Safety In emergency Risk level
precautions
Keep away from Put out fire; seek
Disinfectant Flammable naked fire assistance Low
Use disinfectant;
wash hands with
soap after Low/medium
dissection; do not (depends on
Biohazard Contamination; incubate at Seek assistance likeliness of
Ecoli bacteria infection human body bacteria sample
used temperature; do used to cause
not open agar infection)
plate post
incubation
Keep away from Put out fire; seek
flammable assistance, run
Naked flame Fire hazard; burns materials; tie up burns under cold Low
long hair, wear water
goggles immediately
growth.
Equipment:
o Agar plate seeded with bacteria (Ecoli)
o Plant material (garlic cloves and mint leaves)
o Pestle and mortar 10 cm3 industrial methylated spirits
o Pipette (sterile)
o Paper discs
o Sterile Petri dish
o Sterile forceps
o Tape
o Marker pen
o Incubator set at 25 °C
Aseptic Techniques:
o Wipe down surfaces with antibacterial cleaner, both before and after experiment.
o Use a Bunsen burner in the work space so that convection currents draw microbes away
from the culture.
o Flame the wire hoop before using it to transfer bacteria.
o Flame the neck of any bottles before using them to prevent any bacteria entering the vessel
(air moves out so unwanted organisms don’t move in).
o Keep all vessels containing bacteria open for the minimum amount of time
o Close windows and doors to limit air currents
Method:
1. Carry out aseptic techniques detailed above.
2. Use a sterile pipette or wire hoop to transfer bacteria from broth (distilled water, bacterial
culture, nutrients) to agar plate (petri dish containing agar jelly).
3. Gently spread bacteria evenly over plate using a sterile plastic spreader. (This is supposed to
be done gently as the agar might get damaged if you apply too much force onto it).
4. Use sterile forceps to place a multi disc antibiotic ring on the plate (garlic, mint, ethanol and
plain control). Rings should only be moved by holding the centre, not the arms.
5. Lightly tape a lid on, invert and incubate at 25°C for 48 hours. Do not tape around the entire
dish as this prevents oxygen entering and so promotes the growth of more harmful
anaerobic bacteria.
6. Sterilise equipment used to handle bacteria and disinfect work surfaces.
7. Incubate the plates for 24 hours at 25 °C.
8. After incubation examine the plate and try to identify the colonies which have not been able
to grow near the multidisc arm(s). These are called zone(s) of inhibition.
, 9. Turning the plate upside down and using a ruler measure the diameter of the zones of
inhibition. Calculate the area of the zone of inhibition using the formula Area of zone = πr2
se 3.14
10. Record your results in a suitable table.
Risk assessment:
Hazard Risk Safety In emergency Risk level
precautions
Keep away from Put out fire; seek
Disinfectant Flammable naked fire assistance Low
Use disinfectant;
wash hands with
soap after Low/medium
dissection; do not (depends on
Biohazard Contamination; incubate at Seek assistance likeliness of
Ecoli bacteria infection human body bacteria sample
used temperature; do used to cause
not open agar infection)
plate post
incubation
Keep away from Put out fire; seek
flammable assistance, run
Naked flame Fire hazard; burns materials; tie up burns under cold Low
long hair, wear water
goggles immediately
