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
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
Examiner 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!