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4.4.6Genetics, Variation and Interdependence

Required Practical- Aseptic Techniques — AQA A Level Biology 7402 Revision Notes & Topical Questions

5 min read 9 parts Lesson 26 of 46 1 quiz

📚 AQA A Level Biology 7402 Hub / Topical Revision
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4.4.6 Required Practical- Aseptic Techniques — AQA A Level Biology 7402 notes: Describe aseptic techniques and how they are used in the preparation and investigation of microbial cultures, including the effect of antimicrobial substances.Microorganisms are everywhere: in the air,…

  • prevent contamination
  • Disinfect the work surface
  • Sterilise equipment
  • Work near a Bunsen flame
  • Minimise exposure
  • Seal the Petri dish

Section 1 of 9Why aseptic technique?

Microorganisms are everywherein the air, on skin, on benches and on equipment.

Prevent contamination
When bacteria are being cultured, the aim is to prevent contamination of the culture with unwanted microbes,
  • and to protect the worker and the environment from the culture,
  • which may be pathogenic. Aseptic technique is a set of procedures to achieve both aims.
In the AQA practical, it is used to investigate the effect of antimicrobial substances (antibiotics, disinfectants, plant extracts) on bacterial growth.

Section 2 of 9Key aseptic procedures

1Disinfect the work surface
Disinfect the work surface and wash hands before and after work.
2Sterilise equipment
Sterilise equipment glassware and agar are sterilised in an autoclave (steam at 121 degrees C under pressure) before use; inoculating loops are heated in a Bunsen flame until red hot and then allowed to cool near the flame without touching anything.
3Work near a Bunsen flame
Work near a Bunsen flame the rising hot air creates an upward current, which carries airborne microbes away from the work area.
4Minimise exposure
Minimise exposure open lids of Petri dishes only as far as needed, and for a short time.
  • Flame the neck of bottles when removing and replacing the lid.
5Seal the Petri dish
Seal the Petri dish with two or four pieces of adhesive tape (not all round) so that air can enter,
  • which prevents anaerobic conditions where harmful anaerobic pathogens might grow, and so that the lid cannot be removed accidentally.
6Incubate upside down
Incubate upside down so that condensation does not drip on the agar surface.
7Incubate at 25 degrees C or lower
Incubate at 25 degrees C or lower in school: this temperature is below the human body temperature,
  • which reduces the chance of growing human pathogens.
8Disposal
Disposal sterilise the cultures in an autoclave before disposal, and disinfect surfaces.

Section 3 of 9Investigating antimicrobial substances

Lawn
Using aseptic technique, spread a standard volume of a bacterial culture over the surface of nutrient agar with a sterile spreader to produce a lawn.
Place sterile paper discs soaked in different antibiotics (or concentrations of one substance) on the agar surface using sterile forceps.
  • Include a control disc soaked in sterile water.
Seal and incubate the plates upside down at 25 degrees C for 24 to 48 hours.
Zones of inhibition
Observe the zones of inhibition, the clear areas around the discs where bacteria have not grown.
Measure the diameter of each zone (preferably twice, at right angles) and calculate the area using pi x r2.

A larger zone of inhibition shows that the substance is more effective at inhibiting growth. The control confirms that the paper and the solvent do not affect the growth.

Section 4 of 9Variables, errors and evaluation

Variables to control include the species and the volume and density of the bacterial culture, the volume and concentration of the antimicrobial on each disc, the type and depth of agar, the incubation temperature and the time.

Variables, errors and evaluation
The independent variable is the substance or the concentration being tested,
  • and the dependent variable is the diameter or area of the zone of inhibition.
Repeat each treatment on several plates and calculate a mean.

Limitations include the diffusion of different substances through the agar at different rates, so a smaller zone may be due to slow diffusion, not weaker action. Zones may overlap if discs are too close, and the edges of a zone can be hard to define. Safety points include risk assessment, avoiding opening the plates after incubation, and washing hands.

Section 5 of 9Links

This practical links to bacterial growth and the log scale, to antibiotic resistance and selection, to the structure of bacterial cell walls, and to the practical skills of drawing conclusions from data. In exam questions on the practical, state why each precaution is used, as the mark is usually for the reason: 'heating the loop kills the bacteria on it, so that the culture is not contaminated'.

Section 6 of 9Safety and ethical points

Cultures grown in schools use non-pathogenic strains only, and plates are never opened after incubation.

1
Spills are treated with disinfectant, and contaminated materials are autoclaved.
2
Never culture microbes from the body or the environment unless this is approved, since an unknown culture might contain a pathogen.
3
A risk assessment lists hazards (such as a hot flame, ethanol and the culture itself), the possible harm and the precautions.
4
Keep hair tied back, wear eye protection, and keep ethanol away from flames.
5
If a bacterial colony appears that has an unusual shape or colour, it may be a contaminant,
  • and the result of that plate should be treated with caution or excluded and noted in the evaluation.

Section 7 of 9Exam-style questions

  1. Explain why the lid of the Petri dish is taped at only two or four points and why the dish is incubated upside down. [3]3 marks

  2. A student placed discs of antibiotics A and B on a lawn of bacteria. The zone of inhibition around A had a diameter of 14 mm and B had a diameter of 20 mm. Calculate the area of each zone, and suggest which is the more effective. [3]3 marks

  3. Describe how an inoculating loop is sterilised and why this is necessary. [2]2 marks

Section 8 of 9Key terms

TermMeaning
Aseptic techniqueProcedures that prevent contamination of cultures and spread of microbes.
AutoclaveA device that sterilises equipment using high-pressure steam.
LawnA continuous layer of bacterial growth on agar.
Zone of inhibitionA clear area around an antimicrobial where bacteria have not grown.
Nutrient agarA jelly containing nutrients on which bacteria are grown.
InoculateTo introduce microorganisms into a culture medium.
SterileFree from all living microorganisms.
AntimicrobialA substance that kills or stops the growth of microorganisms.

Section 9 of 9Exam technique

1
Taping all the way round the lid of the Petri dish, which could cause anaerobic growth.
2
Saying that the flame kills bacteria in the air. It creates an updraught that carries them away.
3
Forgetting the control disc.
4
Measuring the zone radius when the question asks for the diameter, or vice versa, when calculating the area.
5
Incubating at 37 degrees C in a school laboratory, where it would encourage human pathogens.

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