Safety:
Methylated spirits is toxic and highly flammable. Therefore great care should be taken to prevent contact with any naked flame or other incandescent material.
Aseptic technique should be used at all times to prevent contamination of agar jelly or bacteria. All used Petri dishes should be autoclaved to destroy any living organisms and to sterilise the dishes. Aseptic technique is used to prevent microbial contamination. In this case we shall be using flaming sterilisation to prevent aerosols (airborne bacterium). This technique involves flaming the neck of all the opened bottles, just after opening and just before re-sealing. The work surfaces must be disinfected before and after use in order o prevent contamination and infection.
When taping the lid of the Petri dish shut, do not seal all of the way around the dish. Doing so might stimulate the growth of anaerobically respiring bacteria which can be harmful. Use 4 short strips to seal the dish in 4 places.
Variables:
Dependant variables
This is the variable which we shall be measuring. In this case it is the diameter of the zone of inhibition and therefore the effectiveness of the plant extract in question; Mint or Garlic.
Independent variables
These are the variables which we will be changing during the experiment. It is advisable to change only one variable at a time to maintain accuracy. In this experiment the independent variable is the plant matter with the antibacterial properties; Mint and Garlic.
Controlled variables
These are the variables that will be kept at constant for all experiments. There are several in this experiment:
- The temperature at which all Petri dishes are incubated at. Reduced or elevated temperatures have a negative effect on the growth of bacteria, too high and they are killed and too low and their growth is slowed. To prevent inaccuracies due to temperature difference, all Petri dishes should be incubated at the same temperature.
- the bacteria sample used for each Petri dish. Different samples of E.coli might have slightly different resistances to any antibacterial chemicals. This would lead to differing size zones of inhibition.
Method:
- Disinfect all work surfaces with virkon and set up a Bunsen burner near to the area in which work will be taking place. The purpose of the Bunsen burner is to flame the necks of all bottles opened and closed and to use convection currents to carry aerosols up and away from the work area.
- Place a bottle of agar jelly into the water bath and leave to melt.
- Divide the dish into 2 halves using the marker pen and write your name, the bacteria being used and the date.
- Once the jelly has melted, remove from bath and dry the bottle.
- Draw 5cm of E.coli into a sterile pipette, flaming the neck of the bottle before and after drawing E.coli.
- Grip the top of the agar jelly with the little finger and palm and twist the bottle itself to unscrew the top, flame the neck and squirt E.coli into the agar jelly, flame again then seal bottle.
- Agitate mixture by shaking agar-bacteria mixture.
- Partially open the Petri dish and pour the mixture into the dish, flaming before and after pouring. (Place empty bottle in virkon disinfectant with top off.)
- Gently swirl the Petri dish to ensure even covering of dish with agar-bacteria mixture.
- Using pestle and mortar, crush 4 cloves of garlic into a puree and add a few small drops of Methylated spirits into the mortar and mix. (repeat for mint)
- Place several filter paper discs into the garlic and mint purees and soak for 15 minutes.
- Remove discs using sterilised forceps and place 1 mint disc into one half of the dish and a garlic disc into the other half of the dish.(once again only partially opening the Petri dish)
- Seal Petri dish as shown below.
Reliability:
This experiment will only be accurate and therefore valid if aseptic techniques are used. This means flaming all bottles after unsealing and before sealing, autoclaving all materials before use and disinfecting all work surfaces before use.
When opening the Petri dish, use only a small opening and open near toa Bunsen burner. The purpose of the Bunsen burner is to carry away aerosols and to keep contaminations airborne, away from the experiment. If contamination is believed to have occurred, the experiment should be restarted to eliminate the risk of false results.
There is going to be some degree of error caused by the concentration of antibacterial matter on the filter paper discs; some may have absorbed more antibacterial matter than others which will provide a larger zone of inhibition.
Results:
Discussion:
From the results shown on the table and graph on previous pages, the mint had no anti-bacterial effect whatsoever. The garlic on the other hand had a more profound effect, both of which support my hypothesis; “Garlic will have the most effective antibacterial properties due to the presence of Allicin. Mint should in theory have little or no effect on the bacteria used in this experiment.”
The average zone of inhibition in mm for mint was 0, for garlic it was 17.3mm. this is conclusive evidence that garlic contains a chemical, or mixture of chemicals which posses antibacterial properties.
Recent scientific studies also back up my evidence for garlic and the role of Allicin, “In the studies, the scientists revealed and characterized a molecular mechanism by which Allicin blocks certain groups of enzymes. Allicin, created when garlic cloves are crushed, protects the plant from soil parasites and fungi and is also responsible for garlic's pungent smell.
“A natural weapon against infection, the research reported in Antimicrobial Agents and Chemotherapy revealed Allicin disables dysentery-causing amoebas by blocking two groups of enzymes, cysteine proteinases and alcohol dehydrogenases.”(Courtesy of )
There was a light degree of variation between the results, this may have been caused by contamination by a more resilient bacteria or, more likely, due to the concentration of antibacterial matter on the filter paper discs. Contamination due to another bacterium could be identified by any colonies forming on the agar jelly as the technique used here would create a “lawn” of bacteria spread evenly over the agar jelly. In order to overcome this in future experiments, all discs should be of equal size, equal depth in the paste and in the paste for an equal length of time, this is all that could be done to overcome this factor as it is very difficult to regulate.
Another factor that may have affected the results is contamination. If a more resilient bacterium is present in the mixture, the zone of inhibition would have been reduced. To overcome this in future experiments, more stringent aseptic techniques would need o be implemented to protect against contamination, or at least reduce it.
From the result, it is evident that mint is totally ineffective against infection and therefore useless in antibacterial toothpaste. The only service it could provide is the slight anaesthetic properties and its taste. Garlic on the other hand would be very effective in antibacterial toothpaste if a little pungent smelling. Allicin would be effective in toothpaste and the overpowering smell of garlic should not be present.
