Core Practical 9: Antimicrobial Properties of Plants

antimicrobial meaning

ability to kill or prevent the growth of micro-organisms

apparatus

• Broth containing bacterial culture and nutrients

• Agar plate

• Pipette

• Plastic spreader

• Plant tissue

• Pestle and mortar

• Ethanol

• Funnel 

• Glass beaker

• Filter paper

• Forceps

• Stopwatch

• Incubator

method

1. Prior to this practical, bacteria would have been grown in a mixture of distilled water and nutrients, along with a specific bacterial culture

   • This mixture is called a broth

2. Transfer some of the bacteria from the broth onto an agar plate (which is a petri dish filled with agar jelly that will serve as a growth medium for the bacteria) using a sterile pipette

3. Make sure the bacteria is evenly spread out by using a sterile plastic spreader

   • Open the lid of the of the agar plate as little as possible when doing this to avoid contaminating the plate with other fungi or bacteria present in the surrounding air

   • Place the lid back on top of the agar plate immediately afterward to prevent contamination

4. To prepare the plant extracts, plant tissue must be dried and ground finely

5. This should be soaked in ethanol to extract the antimicrobial substances, after which it should be filtered

6. Equal sized discs cut from sterile absorbent paper should be dipped in the plant extract using sterile forceps

7. Leave the discs in the extract for the same amount of time to ensure that they absorb a similar amount of the plant extract

   • The disc that will serve as the control will only be dipped in ethanol

8. Space the discs out evenly on the agar plate, before taping the lid on, inverting the plate and incubating it at 25°C

   • This temperature will ensure good bacterial growth without stimulating the growth of human pathogens

9. Incubate for 24 to 48 hours

ms method

-bacteria would have been grown in a mixture of distilled water and nutrients, along with a specific bacterial culture

• This mixture is called a broth

• • filter paper discs soaked in plant extract (placed on agar) / plant extract placed in well (cut in agar) / plant extract added to broth (1

• control disc size/ extract volume/ temperature (1)

• flaming/ dish lid/ use autoclave/safe temeprature (<32 C)

• • incubate for suitable time (1) eg 24 hours

• • clear zone around {disc / well} {measured / scored / turbidity measured (1)

analysis

• The area around each disc where bacteria cannot grow is known as the clear zone

• The larger the clear zone, the more effective the antimicrobial properties of that plant extract was

• The size of the clear zone can be determined by measuring the diameter or by calculating the area (area = πr²)

• Repeat the experiment at least three times and calculate the mean of the results

why aseptic techniques important

• These techniques are important to use in order to prevent the bacterial cultures on the agar plate from being contaminated by other micro-organisms or human pathogens from outside

• Contamination will have a negative impact on the growth of the bacteria under investigation

aseptic techniques used

• Keep windows and doors closed to prevent air movement

• Disinfect surfaces and utensils regularly to prevent contamination

• Ensure that you use sterile equipment and discard afterwards (especially plastic instruments)

• Work near a Bunsen flame when transferring bacteria to ensure that microbes in the air are drawn away by rising hot air (convection current)

• For the same reason as above, hold the flame close to the neck of the glass container of the broth every time it's opened or closed 

why work should be carried out in from of lit bunsen on yellow flame

• flame creates a convection current above the bench, preventing contamination of any microorganisms in the air

why hot agar jelly is poured into a sterilised petri dish then agar left to cool and set?

• the petri dish and culture medium are heated to a high temp to kill any potential microorganisms that could contaminate the experiment

why an inoculating loop is passed through a hot flame before it is used to transfer bacteria to the culture medium?

• any microorganisms on the loop are killed to prevent contamination

why petri dish should only be opened as little as possible at the side facing the bunsen burner?

• this decreases the risk of microorganisms contaminating the dish

why the lid of the petri dish should be secured with tape at intervals around the dish and stored upside down

• this prevents drops of condensation (another source of contamination) from dripping onto the surface of the agar

why the cultures should not be incubated above 25 degrees ceclius in school laboratory

• this restricts the growth of harmful pathogens which are more likely to grow at higher temepratures

why one paper disk on bacterial agar plate is soak din ethanol not plant extract

This is to ensure that any differences in bacterial growth observed can be attributed to the antimicrobial properties of the plant extracts used and not some other factor (such as the paper discs themselves or the presence of ethanol, for example)

why agar plates kept at 5 degrees celcius, before they were incubated?

to allow chemicals in extract to diffuse into agar/to stop growth of bacteria

describe control disk

• filter paper disk

• soaked in solvent/water

for fruit experiment, what other factors other than antimicrobial effect could effect are of zone of inhibiiton?

• solubility/conc/volume of extract/solvent used

• size of molecules in extract

• rate of diffusion of extract into agar

explain aseptic techniques used when transferring bacteria form one culture to another

• flame {loop / spreader / neck of culture bottle} to {kill the bacteria / sterilise it / disinfect it} (1)

• have a Bunsen lit to move microorganisms in the air away (1) • lift (receiving) {Petri dish / culture bottle} lid partially to minimise entry of bacteria (1)

• use disinfectant on workplace to kill bacteria (1)

how to measure zone of inhibition accurately

• use of squared paper (1)

• detail of what to do with paper to get area (1) eg treatment of prt squares

OR

• measure {diameter / radius} {using callipers / in more than one direction} (1)

• detail of calculation of area from this (1) pi R2

factors that affect rate of diffusion of the antimicrobial molecules

• size (of antimicrobial molecule) (1)

• {solubility (of antimicrobial molecule) / properties of agar} (1) • concentration (of antimicrobial molecules) (1)

• (incubation) temperature (1)