Practical: Aseptic Technique

Section 1 of 8Why aseptic technique?

Two aims

• Two aims To stop the cultured microbes escaping into the lab and to stop unwanted microbes contaminating the culture.

Safe temperature

• Safe temperature Cultures in schools are not grown at body temperature, so that microbes which can infect humans do not thrive.

Section 2 of 8Techniques to know

Technique	Reason
Wash hands; no food or drink	Prevents transfer of microbes
Disinfect surfaces	Kills microbes on the bench
Flame loops and bottle necks; use sterile swabs	Kills microbes on equipment
Autoclave glassware and agar	Sterilises with high-pressure steam
Lit Bunsen burner nearby	Rising warm air carries contaminants away from the agar
Lift petri dish lids only when needed	Limits entry of airborne microbes
Wear gloves and goggles	Protects the worker
Sterilise or dispose of used equipment	Prevents release of the culture

Section 3 of 8Disc diffusion method

1

Soak paper discs in different antibiotic solutions of the same concentration so the comparison is fair.

2

Spread diluted bacterial broth over the surface of a sterile agar plate with a sterile spreader.

3

Lightly press the discs onto the agar, spaced apart and away from the edge of the plate.

4

Incubate the plate overnight at a suitable temperature.

5

Examine the plate with the lid on and measure each clear zone.

Section 4 of 8Interpreting results

1Diffusion

• Diffusion Antibiotic spreads out from each disc, most concentrated at the disc.

2Clear zone

• Clear zone Where bacteria are susceptible, no bacteria grow around the disc.

3Bigger zone

• Bigger zone A more effective antibiotic kills bacteria at lower concentrations, so the zone is larger.

4No zone

• No zone The bacteria are resistant to that antibiotic.

Section 5 of 8Variables and calculation of zone size

Variables

• Variables The independent variable is the type of antibiotic;
  • the dependent variable is the diameter of the clear zone.
• Control variables include antibiotic concentration, disc size, volume of bacteria, agar depth, incubation time and temperature.

Area of the zone

• Area of the zone The clear zone is circular, so its area is πr², where r is half the diameter measured in mm.

Example

• Example A zone of diameter 16 mm has r = 8 mm and area = π × 8² ≈ 201 mm².

Section 6 of 8Safety and ethics

Working safely with microbes

Seal the plate

• Seal the plate Tape the lid with two or three short pieces and never seal it all the way round, so oxygen can enter.

Do not open after incubation

• Do not open after incubation The plate may contain harmful bacteria, so the lid stays on and the plate is sterilised in an autoclave after use.

Sterile spreaders

• Sterile spreaders Use a sterile spreader or loop, and flame the neck of the culture bottle each time it is opened.

Hands and surfaces

• Hands and surfaces Wash your hands and disinfect the bench both before and after the practical.

Section 7 of 8Key terms

Term	Meaning
Aseptic technique	Methods that prevent unwanted microbes from contaminating a culture or escaping.
Agar	A jelly containing nutrients on which microorganisms are grown.
Autoclave	A machine that sterilises using high-pressure steam.
Antibiotic resistance	The ability of bacteria to survive an antibiotic.
Clear zone	The area around an antibiotic disc where bacterial growth is prevented.
Incubate	To keep cultures at a controlled temperature so that microorganisms can grow.

Section 8 of 8Exam technique

1

Using different concentrations of antibiotic and then comparing the clear zones.

2

Taping the lid of the petri dish fully shut, which could allow anaerobic bacteria to grow - tape only in a few places.

3

Incubating at 37 °C in school, rather than around 25 °C.

4

Opening the lid to examine the plate after incubation.