(9) Antimicrobial Properties of Plants
& (15) Effects of Antibiotics on Bacteria
Independent Variable: Plant extract or Antibiotic
Dependent Variable: Size of zone of inhibition around disc
Control Variables:
• Concentration of plant material or antibiotic
• Volume of plant material or antibiotic
• Lawn of bacteria on petri dish
Aseptic Technique:
• Wipe down surfaces with antibacterial cleaner both before and after experiment
• Use Bunsen burner in work space so that convection currents draw microbes away from culture
• Flame wire hoop before using to transfer bacteria
• Flame neck of any bottles before use to prevent any bacteria entering the vessel (air moves out so
unwanted organisms don’t move in)
• Keep all vessels containing bacteria open for the minimum amount of time
• Close windows and doors to limit air currents
Method:
1. Use a sterile pipette to transfer the bacteria from the broth to the agar plate
2. Spread the bacteria over the plate using a sterile plastic spreader
3. (9) Dry and grind each plant and soak in ethanol
This will extract the antimicrobial substances, as they are soluble in ethanol
4. Use sterile forceps to dip equally sized discs of sterile absorbent paper in the plant extract or
antibiotic
Leave each disc in the extract for the same amount of time, so they will absorb the same volume of
liquid
5. Place the discs on the agar plate, ensuring they are equally spaced out
6. Lightly tap the lid on, invert and incubate at around 25°C for 24-48 hours
This temperature is high enough for the bacteria to grow, but low enough to prevent the growth of
unwanted human pathogens
7. Measure the diameter of the clear zone around each disc
Control Test: Use one disc with no plant extract
Data:
• Half the diameter to find the radius of each clear zone
• Calculate the area using the formula A = πr2
Conclusion:
• The size of the clear area shows how well the antimicrobial plant extract or antibiotic is working
• The larger the zone of inhibition, the more effective the antimicrobial plant extract or antibiotic is
against the bacteria
Validity:
• There will be inconsistencies when adding plant extract or antibiotic to the paper discs
• There will may be contamination of equipment
• Unwanted microbes may grow on the agar plat due to bad aseptic techniques
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