Required practical 6 Flashcards
Use aseptic techniques to investigate the effect of antimicrobial substances on microbial growth
Why are aseptic techniques used
- To avoid contamination of the sample from the outside substances such as microorganisms
- This is important to get reliable and repeatable data
What equipment is needed for this practifcal
- Bunsen burner
- Disinfectant
- Soap
- Cotton wool
- Sterilised forceps
- Sterilised spreader
- Inoculating loop
- 2x petri dish with agar in E.coli
- 4x antibiotic discs
Describe the method for this practical
- Carry out aseptic techniques detailed above.
- 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). - Spread bacteria evenly over plate using a sterile plastic spreader.
- Use sterile forceps to place a multi disc antibiotic ring on the plate. Ring
should only be moved by holding the centre, NOT the arms. - 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. - Sterilise equipment used to handle bacteria and disinfect work surfaces.
What should you do after the dissection
- Measure the diameter of the inhibition zone (clear circle) for each antibiotic.
DO NOT remove the lid from the agar plate. - Work out the area of the inhibition zone using the formula: A = (πd^2)/4
where d is the diameter
What are the hazards associated with this practical
- Disinfectant
- Biohazard
- Naked flame
What are the risks associated with the disinfectant
Flammable
What are the risks associated with the biohazard
Contamination and infection
What are the risks associated with the naked flame
Fire hazard and could cause burns.
What are the precautions for the disinfectant
Keep away from naked flame.
What are the precautions for the biohazard
- Use disinfectant
- Wash hands with soap after dissection
- Do not incubate at human body temperature
- Do not open agar plate post incubation
What are the precautions for the naked flame
- Keep away from flammable materials
- Tie up long hair
- Wear goggles
What should the graph look like for this practical
● Plot a bar chart of the area of the inhibition zone against antibiotic.
● Graph could include range bars to show the uncertainty from the ruler used
when measuring the diameter.
What is the conclusion for this practical
● If there is a larger inhibition zone around the antibiotic, it has killed more
bacteria. Therefore, the larger the inhibition zone, the better the antibiotic works.
● Some antibiotics will have no/very little inhibition zone. This shows that the
bacteria are resistant to this antibiotic and are not killed by it
What are aseptic techniques
Working in sterile conditions to prevent contamination and infection of others.
What aseptic techniques should be done pre-inoculation
- Sterilise all equipment to kill microbes
- Sterilise work surfaces with disinfectant to kill microbes
- Wash hands with soap to remove any microbes
What aseptic techniques should be done whilst inoculating
- Work near a bunsen burner (convection currents continually sterilise the air)
- Only open the petri lid slightly and only when necessary
What aseptic techniques should be done post-inoculation
- Sterilise all equipment to kill microbes
- Sterilise work surfaces with disinfectant to kill microbes
- Wash hands with soap to remove any microbes
