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UNIVERSITI TEKNOLOGI MARA INTERNATIONAL EDUCATION CENTRE (INTEC)

DATE OF EXPERIMENT TITLE :

19 AUGUST 2010.

THE ANTIMICROBIAL PROPERTIES OF PLANTS

Objective Hypothesis

: :

To investigate the antimicrobial properties of plants. Different plants have different antimicrobial properties. In this experiment, garlic has the strongest antimicrobial properties against both bacteria.

Variables :
Manipulated variable : Responding variable : Fixed variables : Types of plant extract used. The diameter of the inhibition zone. Surrounding temperatures, concentration of agar, the size of the antibiotic-impregnated paper discs.

Introduction :
Garlic
Garlic has been used as both food and medicine in many cultures for thousands of years, dating back to when the Egyptian pyramids were built. In early 18th -century France, gravediggers drank a concoction of crushed garlic in wine they believed would protect them from the plague that killed many people in Europe. More recently, during both World Wars I and II, soldiers were given garlic to prevent gangrene. Today garlic is used to help prevent heart disease, including atherosclerosis (plaque buildup in the arteries that can block the flow of blood and possibly lead to heart attack or stroke), high cholesterol, high blood pressure, and to improve the immune system. Garlic may also protect against cancer.While the science is not conclusive, research shows promise for garlic in the areas of cancer protection and heart-related risk factors for patients. Garlic is rich in antioxidants, which help destroy free radicals -- particles that can damage cell membranes, interact with genetic material, and possibly contribute to the aging process as well as the development of a number of conditions, including heart disease and cancer. Free radicals occur naturally in the body, but environmental toxins (including ultraviolet light, radiation, cigarette smoke, and air pollution) can also increase the number of these damaging particles. Antioxidants neutralize free radicals and may reduce or even help prevent some of the damage they cause over time. There are several types of garlic preparations. Most clinical studies have been performed on aged garlic extract (AGE) or enteric coated, dried garlic tablets.

Cumin
Cumin grows in most hot countries like India, China, North Africa, and the Americas. The cumin seeds should be lightly roasted before being used whole or ground to bring out the aroma. Cumin may also be pounded with other spices in mixtures such as

curry powder. Ground cumin must be kept airtight, to retain its pungency.Cumin seeds contain protein, fat, carbohydrates, fiber, calcium, iron and phosphorous. Cumin is used as a spice in Indian, Mexican, Portuguese, Eastern, Middle Eastern, and Spanish cookery. It is an important ingredient of most curry powders. Cumin seeds are used in many recipes to improve taste and flavor. Cumin stimulates the appetite. Cumin is believed to increase lactation and reduce nausea in pregnancy. In India cumin is given to new mothers in puddings and other dishes for increased lactation. Cumin is diuretic, stimulant, astringent, emmenagogic, and antispasmodic. It is helpful in dyspepsia diarrhoea and hoarseness. Cumin helps to cure colic pain. It relieves swelling of the body, especially of breast or testicles, if used in a poultice.

Cinnamon
Cinnamon is a small tree that is commonly found in South Asia and the Middle East region. The cinnamon that we typically purchase in our supermarkets is actually the bark of this tree, either sold as sticks or ground into a powder. The two most popular types of cinnamon are Ceylon and Cassia, each of which are derived from different trees. Cinnamon has extremely high anti-oxidant activity, and the oil of cinnamon has strong anti-bacterial and anti-fungal properties. Cinnamon is also a great source of manganese, fiber, iron, and calcium. As a result, it has been used as an effective home remedy for:

Reducing blood sugar levels and treating Type 2 Diabetes. Lowering cholesterol. Aiding digestion. Treating diarrhea. Curing the common cold. Reducing arthritis pain. Boosting memory and cognitive function.

Treating toothaches. Eliminating bad breath. Curing headaches and migraine pain. Some recent studies in Europe have shown that cinnamon derived from the Cassia

plant contains a toxic compound known as Coumarin. This compound is known to cause liver and kidney damage in high concentrations. Since the powdered cinnamon that we purchase in our supermarkets is typically derived from the Cassia plant, it is important to avoid consuming high doses of it. Since cinnamon has an anti-clotting effect on the blood, caution should also be exercised when it is used in combination with other blood-thinning products such as aspirin. Large quantities of cinnamon should also be avoided during pregnancy due to the possibility of adverse effects on the uterus. Anyone that is currently taking medication for controlling blood sugar levels or blood thinners should consult their doctor before using cinnamon as a regular supplement.

Material and apparatus

Two sterile Petri dishes, sterile forceps, plant materials (garlic cloves, cumin powder, cinnamon powder), beakers, sterile nutrient agar in a universal bottle, sterile pipette, beaker to receive used pipette, bacteria culture in bijou bottle (E.coli, and Staphylococcus sp.), pestle and mortar, industrial denatured alcohol, paper discs, Bunsen burner, permanent marker pen.

Procedure

1. Hands were washed thoroughly with handwash. 2. The bench was sprayed thoroughly with ethanol prepared by the lab assistant. It was left for a few minutes, then wiped with a paper towel. 3. One Petri dish was labeled on the base at the edge by using a marker pen with initials, the date and the type of bacterium it was inoculated with, which in this case, was E.coli. 4. The base of the Petri dish was then labeled at 4 different areas with G (Garlic), Ci (cinnamon), Cu (cumin), C (control, distilled water). 5. A micropipette was calibrated to 200 microlitres. 6. About 1cm3 of E.coli was piped from bijou bottle using a micropipette into a sterile Petri dish using aseptic technique. The lid of the Petri dish was only lifted enough to allow entry of the pipette. The used pipette was then placed into the beaker of disinfectant. 7. A bottle of liquefied sterile nutrient agar was obtained from the oven. 8. The neck of the agar bottle was heated with flame in order to create an upward lift of air (part of the aseptic technique) after loosening its screw cap. The molten agar was poured into a sterile Petri dish and the lid was replaced. The plate was then gently swirled in a motion shaped 8.

Figure 1 : The neck of the agar bottle was flamed.

9. The Petri dish was then left for 10 minutes for the molten agar to solidify. Meanwhile, the lid was left opened slightly to avoid the water vapour from dripping back into the Petri dish. 10. The garlic was crushed using pestle and mortal.

Figure 2 : Crushing garlic using pestle and mortar. 11. Two spatulas of cinnamon powder was dissolved in very little amount of water. This was done to cumin powder too. 12. The forceps were flamed and it was used to pick up a paper disc. One piece of sterilised paper disc was dipped into each extracts. 13. The paper discs were then placed on the different marked regions, respectively. 14. Step 3 to 13 were then repeated with Staphylococcus sp. replacing E.coli. 15. Both the Petri dishes were placed upside down inside an incubator for the growth of bacteria. They were left inside for a day. 16. The next day, the Petri dishes were taken out from the incubator and the diameter of each inhibition zone was measured using a ruler. Three measurements are taken and the average diameter was then calculated. 17. Hands were washed with handwash again before leaving the laboratory. 18. The readings were tabulated and one bar chart was plotted.

Results

Tabulation of data
Type of bacteria Escherichia coli Diameter of Inhibition Zones in Distilled Water / mm 0.0 (contamination observed) 0.0 0.0 0.0 Average Diamete r / mm 0.0

Staphylococc us aureus

0.0

0.0

0.0

Table 1 : Area of the inhibition zones in distilled water. Type of bacteria Escherichia coli Staphylococc us aureus Diameter of Inhibition Zones in Garlic / mm 16.9 24.0 17.0 24.0 17.1 24.0 Average Diamete r / mm 17.0 24.0

Table 2 : Area of the inhibition zones in garlic. Type of bacteria Escherichia coli Staphylococc us aureus Diameter of Inhibition Zones in Cumin/ mm 10.1 12.0 10.0 12.0 10.2 12.0 Average Diamete r / mm 10.0 12.0

Table 3 : Area of the inhibition zones in cumin.

Type of bacteria Escherichia coli Staphylococc us aureus

Diameter of Inhibition Zones in Cinnamon/ mm 6.0 8.2 6.0 8.1 6.1 8.1

Average Diamete r / mm 6.0 8.0

Table 4 : Area of the inhibition zones in cinnamon.

Interpretation of data

A e g d m te of in ib v ra e ia e rs h itionz e of on s d re t p n e tra ts iffe n la t x c


D m ia etersof inhibition z ones(m ) m 30 25 20 15 10 5 0
W at er (c on tr ol ) Ci nn am on Cu m in Ga rli c

E .coli S .aure us

P n e tra tsu ed la t x c s

Chart 1 : Diameters of inhibition zones against types of plant extracts used.

Discussion

In this experiment, we are to investigate the antimicrobial properties of plants. Three different plant extracts are used, namely garlic, cinnamon and cumin. These plants can only release their active chemicals or antibiotics when they are crushed or chopped into smaller pieces. Two types of bacteria are used to determine the strength of the antimicrobial effect of three plant extracts - Escherichia coli and Staphylococcus aureus. All the three extracts are tested on these bacteria. From the results obtained, we are able to compare the effectiveness of the antimicrobial properties of different plant extracts on these two types of bacteria. From the results obtained, it is observed that in both Petri dishes, there is no inhibition zone observed with the treatment of distilled water, which acts as control in this experiment. This is observed because there is no compound or substance in distilled water which can kill bacteria. On the other hand, the three plant extracts show antimicrobial properties towards E.coli as clear zones are noticed in the Petri dish. Garlic shows the greatest effectiveness in performing the task (17mm), followed by cumin (10mm), and cinnamon (6mm). This proves these plant extracts carry antimicrobial properties. Among these extracts, garlic has the strongest antimicrobial property which is followed by cumin and the weakest property is cinnamon. In the case of Staphylococcus sp., garlic shows the greatest effectiveness in performing its task (24mm), followed by cumin (12mm), and cinnamon powder (8mm). To sum up, we can see that Staphylococcus sp .is a more vulnerable food borne bacterium as it experiences bigger inhibition zones in all the cases. On the other hand, E.coli has stronger resistance to the antimicrobial properties of extracts used. Garlic shows the highest effectiveness in inhibiting the growth of as the greatest diameter of inhibition zone is recorded, whereas cinnamon is least effective in inhibiting the bacteria

growth. From this experiment, we can see some correlation in the usage of spices in preventing the growth of food borne bacteria, thereby reducing the risk of getting food poisoning. This is why most industrial processed food and products contain the active ingredients from plants, for example allicin from garlic as food perservatives and mint in toothpaste.

Evaluation

There are some precautions to be taken into account in this experiment. Firstly, as the microorganisms are a potential biological hazard, aseptic techniques have to be used when transferring the bacteria to the Petri dishes. Only sterilized apparatus can be used. Besides, the bench has to be cleaned with ethanol and hands have to be washed thoroughly to prevent bacteria infection. Other than that, the Petri dishes cannot be opened once they have been incubated. On the other hand, there are also some sources of errors which may affect the accuracy of the data obtained. For example, systemic error might occur if the pipette is calibrated wrongly. Besides that, random error, such as zero error might happen while taking the diameter of the inhibition zones. To minimize zero error, the eye position while taking readings must be directly proportional to the scale. Besides, while it is assumed that the inhibition zones are circle in shape, it might not be a complete circle. This might affect the results, but only a little, as the inhibition zones are generally circular. If it is not, square grids can be used to determine the total surface areas of the inhibition zones. Not forgetting also, there are some limitations in this experiment. As bacteria is everywhere, it is almost impossible for us to sterilize the workplace completely. Therefore, the Petri dishes might be exposed to contamination, which will in turn affect the results obtained. Next, the bacteria in the agar solution may have not spread evenly across the petri dish and more bacteria may gather at specific region in the petri dish than the other regions. This error would lead to the results recorded to be inaccurate for the concentration of the bacteria would affects the diameter of the clear zone. When the bacteria is in higher concentration at a specific zone than it should be, the clearing zone would be smaller in diameter than it should be as more of the antibiotic are used to kill the higher concentration of bacteria instead of spreading wider out.

All in all, the results obtained are valid because the trend of the curve obtained is explainable and supported by theories which are stated in the discussion. Besides that, the results are also reliable as the data obtained is almost the same as other practical groups and there are no apparent anomalies in the results and none of the sources of errors or limitations is enough to deem the results unreliable. A set of data is obtained from another practical group and it is presented in the table below: Average diameter of clear zone including paper disc (mm) Petri dish 1 Agar plate 2 extracts Escherichia coli Staphylococcus aureus Garlic 18.0 26.0 Ginger 16.0 18.0 Cumin powder 9.0 11.0 Turmeric 8.0 12.0 Mint 12.0 14.0 Cinnamon powder 5.0 8.0 Water(control) 0.0 0.0 Table 5 : Average diameter of inhibition zones in different extracts. The data from this practical group also shows that garlic is most effective against both bacteria, whereas cinnamon is least effective, just like the data obtained by our practical group. Hence, the results obtained by my practical group are reliable. Type of Plant

Conclusion

Different plants have different antimicrobial properties. In this experiment, garlic has the strongest antimicrobial properties in combating the invasion of food borne bacteria such as E.coli and S.aureus, followed by cumin and lastly cinnamon. Thus, it can be said that the method of using spices in preventing food poisoning (as food perservatives or additives) is a proper way to be done as this can greatly increase the qualities of the food we consumed.

Bibliographies :

1. Biology (Eighth Edition), Campbell, Reese, Pearson. (Retrieved on 21 August

2010)
2. http://www.helpwithcooking.com/herb-guide/mint.html (Retrieved on 22 August

2010)
3. http://www.bionewsonline.com/i/what_is_staphylococcus_aureus.htm (Retrieved

on 22 August 2010)
4. http://www.food-india.com/ingredients/i001_i025/i010.htm (Retrieved on 22

August 2010)
5. http://www.fatfreekitchen.com/spices/cumin.html (Retrieved on 22 August 2010) 6. http://www.whfoods.com/genpage.php?tname=foodspice&dbid=78 (Retrieved on

22 August 2010)

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