AQA A Level Biology

Revision Notes

4.4.6 Investigating Microbial Growth

Required Practical: Affecting Microbial Growth

  • When investigating the effect of antimicrobial substances on microbial growth it is essential that aseptic techniques are used
  • Aseptic techniques ensure the microbes being investigated don’t escape or become contaminated with another unwanted, and possibly pathogenic, microbe
  • Aseptic techniques include:
    • Washing hands thoroughly
    • No food or drink allowed in the lab
    • Disinfecting work surfaces with disinfectant or alcohol
    • Not allowing the growth of microorganisms at body temperature
    • Using flamed loops or sterile swabs when transferring cultures
    • Wearing gloves and goggles
    • Flaming culture bottlenecks to prevent contamination
    • Sterilising or disposing of all used equipment
    • Washing hands thoroughly
    • Having a lit bunsen burner in the room
    • Only removing petri dish lids when necessary

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.

The minimum inhibitory concentration (MIC)

  • When antibiotics are used to treat bacterial infections, the dosage used is carefully controlled
  • The minimum inhibitory concentration (MIC) is the lowest concentration of a substance that will inhibit the growth of a microorganism

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!

Author: Lára

Lára graduated from Oxford University in Biological Sciences and has now been a science tutor working in the UK for several years. Lára has a particular interest in the area of infectious disease and epidemiology, and enjoys creating original educational materials that develop confidence and facilitate learning.
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