HOW TO INVESTIGATE THE ANTIMICROBIAL PROPERTIES OF PLANTS

by mugo

Plants are susceptible to infection by bacteria and fungi and therefore they find ways of killing or repelling them. A plant with this property is antimicrobial plant. These chemicals will either kill them or inhibit their growth.

An antimicrobial plant produces chemicals that either kill or inhibit the growth of bacteria and fungi that cause infections to these plants. The method:

Sterile agar plates mixed with bacteria are prepared. Aseptic techniques (methods of avoiding or minimising contamination) are observed. Discs containing the chemical from a plant are placed on the agar. Incubate the plates for 24 hours at 250C (not 370C as it can encourage growth of harmful bacteria that can affect humans). Bacterial growth on a plate looks cloudy. A clear area (inhibition zone) where the bacteria have been inhibited or killed should surround each of the discs.

The measurements of the effectiveness of the chemicals from plants would include: i. ii. iii. Use a ruler and measure the diameter of the cleared area, especially if they are circular areas. If the diameter varies, measure the widest points. For more precise measurements, the area of the clear zone would have to be determined.

PROCEDURE

1) 2)

Agar plates seeded with suitable bacteria are prepared. Obtain a plant extract by crushing 3 g of plant material with 10 cm3 of industrial denatured alcohol, and shake it from time to time for 10 minutes. The advantage of using alcohol instead of water is that it is likely to kill any bacteria that might otherwise contaminate the extract.

3) 4) 5) 6) 7) 8)
9)

Pipette 0.1 cm3 of extract onto a sterile paper discs. Let the paper discs dry for 10 minutes on open sterile Petri dishes. Pipette 0.1 cm3 of distilled water onto a disc as a suitable control. Use sterile forceps to place the discs onto the bacterial plate together with the suitable control per plate. Three test discs and a control can be placed on a single Petri dish. Incubate the plates for 24 hours at 25c. Observe the plates without opening them and determine the area or diameter of the inhibition zone. Repeat the above procedure with different plants to determine the most effective chemical. POURING AGAR PLATES

Procedure:

1) 2) 3) 4)

15 cm3 of sterile nutrient agar is added into a culture bottle. Agar liquefies (melts) at 97oC hence the bottle is placed in a hot water bath to liquefy it. If the bottle has a screw cap it is loosened to allow air to escape. Remove the bottle using a cloth because it is very hot to avoid burning. Allow the agar to cool to about 50c, a temperature:

5.

At which you can handle the bottle and which will reduce the amount of condensation when the agar is poured into the Petri dish. The agar will start to solidify (set) at about 42c. Take care not to let it cool too much or it will

set as you pour it into the Petri dish.

6.
agar plate.

Pour the 15cm of molten agar into the sterile petri dish and gently swirl to ensure uniform

distribution on molten agar in the petri dish and place the lid to minimise contamination. This allows it to solidify. This is

7.

Pipette 1 cm of bacterial broth into a sterile agar plate using aseptic technique. The lid of the

Petri dish should only be lifted enough at an angle to allow entry of the pipette to reduce contamination. Place the used pipette into the beaker of disinfectant.

5) Please note: it is important that the plates are used for the investigation on the same day as the agar has set, otherwise
once the bacteria have started to grow they will be unaffected by the antimicrobial agent.