TITLE Antimicrobial properties of plants.

INTRODUCTION Some plants may have antimicrobial properties as one of their own chemical substances that their have. Antimicrobial properties actually refer to a type of substances which help to inhibit the growth of microorganisms such as bacteria and fungi thus provide protection for plants. On the other hand, disinfectants are actually referring to the antimicrobial substances which are being applied on inanimate objects such as surface of the floor. The chemical interfere with biological process of microorganisms such as Escherichia coli (E. coli) in various ways, therefore, this experiment is designed in order to find the effect of different antimicrobial properties on the bacteria stated before. In this experiment several types of plants is being used one of it is ginger. Ginger is actually tuber plant which is being consumed by Malaysians as delicacy itself. The young parts of it is very juicy and fleshy therefore are often pickled in vinegar or sherry as a snack. Ginger has been found to effectively treat nausea caused by sea sickness, morning sickness and chemotherapy. It is also known as a digestive aid through increasing the production of digestive fluids and saliva. It also helps to relieve indigestion, gas pains, diarrhoea and stomach cramping. There are others conditions that it can treat which are mainly about digestion system. We also used cinnamon as one of the plants extract being used. It is mainly the inner bark of a tropical evergreen tree which can sub-divide into two main species. It is being valued due to it usage in preservative value for meats and medicinal use. It is also mainly used as spices in nowadays cuisines. There are a lot benefit comes from cinnamon. One of it is it helps boost the brain power. Other than that, it also helps in digestive aid and controlling blood glucose level. Cinnamon is universally prescribed as an astringent, stimulant carminative by most herbalists. Studies in Japan also show that it can kills fungi, bacteria and other microorganisms. These abilities of cinnamon are believe derived from various terpenoids found in the volatile oil of this herbs.

This is to name a few of the herb or plants being used in the experiment because we also used several other plants such as ginger, garlic, and turmeric to name a few. We test the antimicrobial properties of all these plants against Escherichia coli (E. coli) which is actually one of the most common disease-causing bacteria. It is negative bacillus with rod shape, and mostly occurs in huge numbers in lower part of intestines of endothermic vertebrae. Its strain are harmless however there are also the pathogenic ones in which they also can cause serious food poisoning and kidney damage. Usually the disease is spread through the mean of ingestion contaminated food. One of the most important apparatus that is being used in this experiment is micropipette. It helps to take small amount of solution that we want in which for this experiment is the bacteria. It has pipette teat which is the part of the disposable plastic tip and the one that come into contact with the solution. Each of the micropipette uses their own specific size tip. It is very handy and easy to use. We used it just like how the syringes are being used. For which the first things we need to do for this pipette is to attach the tip. After that by the ease of pushing a button to fill it with bacteria and then put it into a Petri dish. The tip is then discarded into special dustbin to prevent contamination from occurring.

PROBLEM STATEMENT How do different types of plants differ in their antimicrobial properties in inhibiting the growth of bacteria? OBJECTIVE To study the antimicrobial properties of different plants extract in inhibiting the growth of Escherichia coli (E. coli) HYPOTHESIS Different plants have different antimicrobial properties. NULL HYPOTHESIS Every plant has the same antimicrobial properties regardless of its species APPARATUS Bunsen burner, sterile forceps, sterile Petri dish with cover, micropipette, pipette teats, mortar and pestle MATERIALS Bacteria such as Staphylococcus aures (S. aures) or Escherichia coli (E. coli) in bottle, plant extract such as ginger, garlic, turmeric, cinnamon, cumin and mint, sterilized distilled water, industrial denatured alcohol, agar solution, label stickers, marker pen, ruler, sterile filter paper discs, hand soap, disinfectant spray VARIABLES Manipulated variable: Types of plant extract used Responding variable : Diameter of inhibition zone (mm) Fixed variables : Amount of bacteria used, incubation temperature, incubation period, Volume of sterilised nutrient agar solution , size of paper discs

PROCEDURE Part A:Petri dish culture preparation 1. Before preparing an agar plates seeded with suitable bacteria we must wash our hands and clean the table first. 2. In order to prepare the agar plates first of all collect a bottle or test tube containing 15 cm of sterile nutrient agar. 3. After that, the bottle or test tube containing the agar is put in a hot bath to liquefy the agar. If the bottle has a screw cap loosen it in order to allow for air to escape. 4. Remove the bottle once the agar has been liquefied. A cloth is used to do this. The agar is allowed to cool to about 50C, a suitable temperature to handle the bottle and can reduce the condensation of the agar when it is poured into a Petri dish. At about 42C the agar will start to solidify. Do not let it to cool too much or it will when being poured into Petri dish. 5. 1 cm of bacterial broth is pipette into a sterile Petri dish using antiseptic technique. The lid of the Petri dish is lifted enough to allow entry of the pipette bacteria. The pipette is placed into the beaker of disinfectant. 6. 15 cm of molten agar is then poured into the Petri dish and replace the lid. The plate is gently pushed back and forth, N-S, NE-SW, and NW-SE or according to the eight numbers direction to mix the bacteria with the agar and the agar is allowed to set. 7. The plates used for the investigation must be on the same day that the agar is being set, otherwise the growth of bacteria which have started readily will be unaffected by the antimicrobial agent. Part B : Obtaining plant extracts 1. A plant extract is obtained by crushing 3 g of plant material with 10 cm of industrial alcohol, and from time to time shake it for 10 minutes. Denature alcohol is being used rather than water because it readily kills any bacteria that may contaminate the extract when in contact. 2. 0.1 cm of extract is pipette onto a sterile 13 mm Whatman antibiotic assay paper disc (or discs cut from new filter paper using a hole puncture.

3. The paper discs are left to dry for 10 minutes on open sterile Petri dishes. 4. Steps 1 to 4 is repeat for other plants, making separate test discs for each extract. 5. The test discs are placed onto the bacterial together with the suitable control per plate by using a sterile forceps. Three test discs and a control can be placed on a single Petri dish. The different discs must be able to be distinguished by marking the underside of the Petri dish. Part C : Incubating the bacteria culture solution 1. The Petri dish is closed and tapes it. But do not tape all round the dish because this can lead to the growth of anaerobic bacteria, some of which can be harmful and also exclude oxygen needed for growth of bacteria in the agar. The name, the date, the bacteria, and plant used are recorded on the base of the plate. 2. Then we transfer all of the Petri dish into the incubator and place it upside down at a constant temperature of 37C for about 24 hours. 3. The working area is then cleaned with disinfectant and wiped it cleaned to avoid any contamination around the experimented area which can lead to infection. The hand must also be sanitized after the experiment. 4. After 24 hours which is the period of the incubation , the diameter of the inhibition zone is measured using a ruler and all the data obtained is recorded and tabulate.

RESULTS
Types of plant extracts E. coli Bacillus sp.

Diameter of the inhibition of bacterial growth (cm)

Disc 1

Disc 2

Disc 3 (Control)

Average

Disc 1

Disc 2

Disc 3 (Control)

Average

Garlic

2.5

1.3

0.0

1.9

0.9

1.0

0.0

1.0

Mint Ginger

0.9 0.9

0.7 0.8

0.0 0.0

0.8 0.9

0.7 0.7

0.6 0.6

0.0 0.0

0.7 0.7

Onion

0.9

0.8

0.0

0.9

0.8

0.7

0.0

0.8

Cloves

0.5

1.3

0.0

1.8

0.7

0.5

0.0

0.6

Cinnamon

1.0

1.9

0.0

1.5

0.8

0.9

0.0

0.9

DATA REPRESENTATION
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Garlic Mint Ginger Onion Cloves Cinnamon

E. coli Bacillus sp.

DISCUSSION The main purpose of this experiment is to investigate antimicrobial properties of plants towards bacteria in which in the experiment we used E.coli. This is mainly because it is the most well understood bacteria in the world in which it is used it a lot in other experiment throughout the world. Besides that it is also safer compared to any other bacteria which are much more harmful. Moreover it can be cultivated and easily grown if at optimum condition it can reach up to 20 minutes per cell cycle. Furthermore, a large number of E.coli can be grown in a small space therefore it does not take much of spaces to be used. This is also applicable to the Bacillus sp.in which we also used in the experiment. Generally from the results we obtained all of the microbial properties of plants have higher effect on E.coli rather than on bacillus sp. This is probably because E.coli has lower tolerant value towards the antimicrobial properties of those plants compared to bacillus. Therefore, more of E.coli is hindered to reproduce and is more prone to be killed by the extract of plants. However, it can be seen that from the results much of the bacillus bacteria has low inhibition zone area. This shows that they are much more tolerant and can withstand the action of antimicrobial substance. This is also depends on the type of antimicrobial substances secreted by the plants itself because different type of antimicrobial substances has different effect on different bacteria. From the results we also can see that different antimicrobial substances have different effect. However, in this experiment the inhibition zone is prone to flaws cause by different concentration used because we do not control the concentration of antimicrobial substances. In the results we can conclude that for both plants the strongest antimicrobial properties is derived from garlic as it has the biggest inhibition zones compared to the others. Therefore we can conclude that garlic antimicrobial properties are effective on both bacteria species. On the other hand, the weakest among all of the antimicrobial substances differ between the two species. For E.coli they can stand up to mint the most. This maybe because of E.coli is less prone to mint rather than any other antimicrobial properties of other species of plants. But baccilus can withstand eefect from clove rather than E.coli. Therefore, we can see that bacillus is more resistant to clove maybe due to certain biological factors.

In conclusion, different antimicrobial substances have different effects on different bacteria. This is maybe due to the different ways they interrupt the biological process of the bacteria. But these are not differ much between the bacillus and E.coli as they have about the same results even though there are still some different. Therefore, we must use different substances to fight different bacteria if any infection occurs. SOURCES OF ERRORS AND WAYS TO OVERCOME IT Sources of errors The diameter of the inhibition zone is not regular around the filter paper discs Antibiotic may spread out from the filter paper discs when it is being put into unsolidifies agar solution due to it movement Contamination or mixing of antibiotics may occur due to the forceps being use is the same. Therefore, there will be more than one antibiotics (from different plants)in one plates which will influence the inhibition zone due to different antimicrobial agent have different effects. The bacteria may be contaminated through air borne or due to exposure to air which contains pathogen The amount of the antimicrobial chemicals of the plants on the filter paper disc is different The filter papers are being placed close to each other which can cause overlapping of inhibition zone thus harder to make measurements. The bacteria is not widespread in the agar solution but concentrate on certain parts of it. Ways to overcome it Take several readings and find the average of all the readings for a specific inhibition zone by a filter paper Therefore the agar solution must be ensured to be solidified before putting the filter discs inside Thus in order to prevent such condition from occurs the forceps must be cleaned properly and use flame to sterilize it to prevent any biological material from coming into contact with the forceps. Besides that it also helps to denature the biological agent which comes into contact with the forceps. Lit up a Bunsen burner to heat the air surrounding the Petri dish to kill the airborne bacteria or any other contamination factors In order to reduce this kind of error the period of time the plates being dipped must be constant throughout the experiment. Therefore, the discs cant be placed too near with each other or with the edges of the Petri dish to avoid obstruction of the inhibition zone. The Petri dish with bacteria and agar is swirled gently in a figure of eight

Systemic error (due to faulty calibration of the instrument) Error The ruler use to measure the diameter has problem with the calibration like the end of it is not zero Calibration of micropipette is not accurate and is too small Ways to overcome it Measure the error cause by the faulty calibration and add It back to the results to obtained the accurate value Recalibrate the micropipette or take enough bacteria based on the others so that it is sufficient for the experiment

Random error (due to surrounding) Error The temperature fluctuate can cause the growth of bacteria to be hindered or cease The concentration of nutrients in the agar are too low or less Ways to overcome it Keep it in an incubator that has constant temperature throughout the experiment During agar preparation it is advised that the procedure is being done meticulously and according to the procedure so that the agar has sufficient nutrients Same amount of plant and denatured alcohol is used for all plant extract

Inconsistent concentration of plant extract

Increasing reliability of the experiment Problem The results just obtained once, and it is doubt to be unreliable There are extreme results being obtained Ways to overcome it Repeat the experiment twice to investigate the consistency Ignore the anomalous results and discarded it from the observation The time exposure of the agar to the environment is ensured to be minimized It is incubated for a fixed period of time and does not exceed the time limit

The agar is contaminated with others bacteria from surrounding Petri dish is over incubated thus resulting in the bacteria growth overcome antimicrobial plant extract Contamination may occur which outcompete The experiment is done under aseptic the bacteria condition Control variables can affect the experiment All of the control variable must be consistent to prevent any other factors from involving thus altering the results being obtained. Inhibition zone is hard to measure Count the area of inhibition rather than taking its diameter as it is much more accurate due to the inhibition zone is not 100% spherical.

FURTHER INVESTIGATION In this experiment we are investigating the effect of different antimicrobial properties on E.coli which is one of the most well known bacteria. Therefore, there are a lot of ways in order to extend our knowledge regarding these fields. One of the extent experiments that we can do is by trying the antimicrobial properties of different plants on other types or species of bacteria. This will provide with data which has more range and can be assumed to be more reliable due to wide number of species is being used. The experiment will have the same method except different species of bacteria will be in used. Other than that, we also can discovered what is the effect of varying concentration on the bacteria directly by soaking the plate in different concentration of the extract to see what is the best and optimum temperature for its to function. Other steps are just mere repetition of this experiment. RISK AND SAFETY PRECAUTION Risks Microbial contamination on the apparatus The hot agar solution may spilled on the experimenter and scorched it The biological waste may have spilled and come into contact with experimenter. Safety precaution All the apparatus such as Petri dish and forceps should be sterilised Handle with care when transferring the hot agar solution from the oven to the respective places All of those who are involved are ordered to wear lab coats and gloves to avoid any direct contact with the experimenter. The experimenter must also clean their hands with antiseptic wash to dispose any bacteria that landed on their hand during the experiment. Dispose all of the biological waste properly by discarding it into a specific container. When observing it make sure the temperature is not at 37C which is the optimum condition for the bacteria to grow. The incubator also can used a slightly lower temperature to prevent it from growing excessively Please take extra precaution when moving around the Bunsen burner to avoid any unnecessary contact Beware of the distant between the Bunsen burner and the experimenter

The biological waste can contaminate the surroundings or environment of the labs. The bacteria may reproduce rapidly and burst out or escape from the Petri dish and cause infection

The lit Bunsen burner can cause burn to skin when comes into contact

The apparatus used is subjected to fragile and cause harm to us

Therefore, the experiment need to be done according to the procedure and all the apparatus must be handled with extra care from the researcher.

CONCLUSION Different plants have different antimicrobial properties based on their own species. Hypothesis is accepted. BIBLIOGRAPHY 1. http://en.wikipedia.org/wiki/Ginger 2. http://en.wikipedia.org/wiki/Garlic 3. http://www.streetdirectory.com/travel_guide/119290/science/what_are_microp ipettes.html 4. A Pearson Company, Bacteria, Edexcel A2 Biology, 2008, pg 86-87 5. Clegg C.J., Introducing micro-organisms, Edexcel Biology, Hodder Education A2, London, UK, 2008, pg 70-72

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