# Core Practical 9

Investigate the antimicrobial properties of plants, including aseptic techniques for the safe handling of bacteria
OBJECTIVES
● To successfully compare the effect of plants on microbial growth
● To understand the safety issues of microbiological work and how to apply good aseptic techniques
SAFETY
● Wear eye protection.
● The microorganisms are a potential biological hazard.
● Wash your hands with soap and water before and after the activity.
● Wear gloves when handling mint and garlic.
● Avoid skin contact with the disinfectant.
● Do not decant ethanol near a naked flame and stopper the bottle after use.
● Use aseptic techniques when transferring the bacteria to the Petri dishes.

● Only open the Petri dish slightly when adding the discs with forceps (not fingers) and, once it is closed with some strips of tape, do not open it again.
● Disinfect the bench before and after working. Leave the disinfectant on the bench for about 10 minutes.
● If the agar plate shows signs of contaminating growth, do not open the plate. A technician will destroy it by autoclaving.
● Use ratios, fractions and percentages.
● Calculate the circumferences, surface areas and volumes of regular shapes.
● Plot two variables from experimental or other data.
● Use logarithms in relation to quantities that range over several orders of magnitude.
● Substitute numerical values into algebraic equations using appropriate units for physical quantities.
MATHS SKILLS
● Use ratios, fractions and percentages.
● Calculate the circumferences, surface areas and volumes of regular shapes.
● Plot two variables from experimental or other data.
● Use logarithms in relation to quantities that range over several orders of magnitude.
● Substitute numerical values into algebraic equations using appropriate units for physical quantities.
EQUIPMENT
● eye protection
● bench spray of disinfectant
● paper towels or cloth
● agar plate seeded with bacteria
● sterile agar plate
● marker pen
● incubator
● forceps
● pestle and mortar
● mint
● garlic
● ethanol or denatured alcohol
● small filter paper discs
● 50 cm3 beaker

PROCEDURE

DAY 1
1. Wash your hands with soap and water and disinfect your bench area, leaving it to soak for 10 minutes before wiping it down.
2. Place a piece of garlic in the mortar and grind it into a paste with the pestle. Add 10 cm3 of alcohol to the garlic paste.
3. Use the forceps to pick up a paper disc and place it in the mortar to soak up the garlic and alcohol solution.
4. Use the forceps to place the disc on the sterile agar plate to dry.
5. Use a marker pen to mark four quarters on the bottom of the agar plate that has been seeded with bacteria; label one garlic, one mint and two control.
6. Open the lid of the Petri dish containing the bacteria-seeded agar. Open it away from yourself and only open it slightly. Use the forceps to place the dry disc in the centre of the quarter labelled garlic.
7. Close the Petri dish.
8. Clean the pestle and mortar, then repeat steps 2–7 with the mint.
9. Add a small amount of alcohol to the small beaker.
10. Using clean forceps, add two paper discs to the beaker. Remove them and place them on the sterile agar plate dish to dry.
11. Open the lid of the seeded Petri dish, making sure you open it away from yourself and only open it slightly. Add one disc to each of the quarters labelled control.
12. Place one small piece of tape on each side of the Petri dish lid to hold it down. Do not tape it all the way around.
13. Invert the plate and incubate at a temperature no higher than 30 °C for 24 hours; alternatively, leave at room temperature for 48 hours.
DAY 2
14. Use a ruler to measure the diameter of the clear zone around each disc. (It’s also possible to measure the area of the clear zone using squared paper.) Record your results in a suitable
table.
15. Clear away all the equipment you have used. Petri dishes should be returned for sterilisation. Wash your hands and disinfect surfaces before leaving the laboratory.
ANALYSIS OF RESULTS
1. Collate the results from the whole class. Calculate the mean area of the clear zone for each treatment.
2. Carry out some research to find out about one other plant material that can be used for its antimicrobial properties.

LEARNING TIP
● Most microorganisms require a good source of carbon and nitrogen as well as specific minerals. These nutrients can be supplied in a solid form. The solid medium used in the Petri dishes in this investigation was nutrient agar (agar with some nutrients added), but many bacteria will not grow effectively on this simple medium. Agar is a jelly extracted from seaweed.