OCR AS Biology

Revision Notes

1.2.2 Practical: Aseptic Techniques

Practical: Aseptic Techniques

  • When investigating the effect of antimicrobial substances on microbial growth it is essential that aseptic (sterile) techniques are used
  • Aseptic techniques ensure the microbes being investigated don’t escape or become contaminated with another unwanted, and possibly pathogenic, microbe
    • This is especially important in preventing the accidental culture of human pathogens
  • Aseptic techniques include:
    • Washing hands thoroughly to disinfect them
    • No food or drink allowed in the lab
    • Disinfecting work surfaces with disinfectant or alcohol
    • All apparatus, glassware and collecting loops must be fully sterilised before use
    • The culture medium (either a culture broth or an agar plate) must be sterilised
    • Using flamed loops or sterile swabs when transferring cultures to avoid collecting microbes in the atmosphere
    • Flaming culture bottlenecks to prevent contamination
    • Not allowing the growth of microorganisms at body temperature
    • Only removing petri dish lids when necessary
    • Sterilising or disposing of all used equipment promptly once they are no longer needed

Example of a practical using aseptic techniques: testing for bacterial antibiotic resistance using the disc diffusion method

  • The disc diffusion method is commonly used to test for antibiotic resistance in bacteria
    • It allows for multiple antibiotics to be tested at once

Apparatus

  • Sterile agar plates
    • The agar can be made sterile by boiling
  • Diluted bacterial broth with a concentration of 1 x 108 CFU mm-3
    • Colony-forming unit (CFU): a live bacterial cell that is able to divide and form a colony on the agar plate
  • Multiple different antibiotic solutions of a standard concentration
  • Paper disks
  • Pipettes
  • Spreaders
  • Bunsen burner
  • Gloves
  • Goggles
  • Incubator

Method

  • Pre-soak paper discs in the different antibiotic solutions
    • The different antibiotic solutions need to be the same concentration so that the effects of the different antibiotics can be compared
  • Spread a sample of the diluted bacterial broth onto the surface of the sterile agar plate
  • Lightly press the paper discs onto the surface of the agar
    • Make sure the discs are evenly distributed in the plate
    • They should not be touching the edges of the plate or any other discs
  • Keep the agar plate in the incubator overnight
    • The incubator maintains an optimum temperature for bacterial growth
  • Remove the agar plate from the incubator and examine the results with the petri dish lid on

Results

  • Overnight the antibiotics will diffuse outwards from each paper disk so that a gradient of antibiotic forms. The antibiotic is most concentrated where the paper disk is located
  • If the bacteria being investigated is vulnerable to an antibiotic then a clear area will be visible around the disc
    • There are no bacteria present in the clear area
  • The clear area will end when the concentration of antibiotic reaches a level that the bacteria are no longer susceptible to
  • More effective antibiotics require a lower concentration to kill bacteria and so they will produce larger clear zones
  • If a bacteria is completely resistant to an antibiotic then there will be no clear zone around that particular paper disk

Microbial Growth Agar, downloadable AS & A Level Biology revision notes

Image showing the bacterial growth on an agar plate following a disc diffusion experiment. The most effective antibiotics produce the largest clear zones while. The antibiotics that the bacteria are resistant to produce no clear zone.

Exam Tip

It is expected that you will be able to suggest aseptic techniques that should be used for specific experiments. Make sure to learn a few of the ones above so that you can get those marks!

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