BIOLOGY LAB REPORT TITLE PREPARED BY I/C NUMBER STUDENT ID GROUP LAB PARTNER LECTURERS NAME PRACTICAL DATE

SUBMISSION DATE : THE EFFECTS OF ANTIMICROBIAL PROPERTIES IN PLANTS : : : : : : : :

Abstract Different plant has different reaction towards bacteria, thus exhibit different degree of antimicrobial property towards bacteria. In this experiment, plant extract was prepared by diluting it in denatured alcohol and was left to soaked up by the paper discs. These are then take using a pair of sterilize forceps and pressed onto an agar which has been prepared with bacteria (E.coli and B.subtilis). The area of inhibition of bacterial growth (or the clear zone) around the paper disc was calculated and recorded.

Introduction 1. Escherichia coli (1) Escherichia coli or well known as E.coli is a Gram-negative, rod-shaped bacterium that commonly found in the lower intestine of endotherms. Most E.coli strains are harmless ; they produce vitamin K2 and prevent formation of pathogenic bacteria in intestines. Some of E.coli strains may cause serious food poisoning and result in fatal death. These cells able to survive outside the body of endotherms for a limited amount of time, which makes them ideal indicator organisms to test environmental samples for fecal contamination. E.coli strain is a sub-group that has unique characteristics that distinguish it from other strains. These differences are often detectable only at the molecular level; however, they may result in changes to the physiology or lifecycle of the bacterium. Because of its long history of laboratory culture and ease of manipulation, E. coli also plays an important role in modern biological engineering and industrial microbiology. Modified E. coli cells have been used in vaccine development, bioremediation, and production of immobilised enzymes.

Figure 1 : E.coli Bacterium (2)

Figure 2 : Disease caused by E.coli (3)

2. Bacillus subtilis (4) Bacillus subtilis is a Gram-positive, catalase-positive bacterium that commonly found in soil. It is rodshaped, and has the ability to form tough, protective endospore which enables this organism to tolerate extreme environmental condition. B. subtilis has proven highly amenable to genetic manipulation, and has become widely adopted as a model organism for laboratory studies, especially of sporulation, which is a simplified example of cellular differentiation. It is also heavily flagellated, enabling B. subtilis to move quickly in liquids. This organism also often used as the Gram-positive equivalent of Escherichia coli, an extensively studied Gram-negative bacterium. Enzymes produced by B. subtilis are widely used as additives in laundry detergents, commercial production of the Japanese food natto, as well as the similar Korean food cheonggukjang, as a natural fungicidal activity, as an immune stimulatory agent to aid treatment of gastrointestinal and urinary tract diseases and help in speeding corm growth and increasing stigma biomass yield.

Figure 3: Reproductive cycle of B.subtilis (5)

Figure 4 :B.subtilis (6)

3. Garlic (7) Garlic or its scientific name Allivum Sativum is native to central Asia and has long been a staple in the Mediterranean region, as well as a frequent seasoning in Asia, Africa, and Europe. Garlic is easy to grow and can be grown year-round in mild climates. While sexual propagation of garlic is indeed possible, nearly all of the garlic in cultivation is propagated asexually, by planting individual cloves in the ground. Garlic plants can be grown close together, leaving enough room for the bulbs to mature, and are easily grown in containers of sufficient depth. When selecting garlic for planting, it is important to pick large heads from which to separate cloves. Garlic plants prefer to grow in a soil with a high organic material content, but are capable of growing in a wide range of soil conditions and pH levels. Garlic is widely used around the world for its pungent flavor as a seasoning or condiment. Other parts of the garlic plant are also edible. The leaves and flowers (bulbils) on the head are sometimes eaten. They are milder in flavor than the bulbs, and are most often consumed while immature and still tender. Inedible or rarely eaten parts of the garlic plant include the "skin" and root cluster. The papery, protective layers of "skin" over various parts of the plant are generally discarded during preparation for most culinary uses, though in Korea immature whole heads are sometimes prepared with the tender skins intact. The root cluster attached to the basal plate of the bulb is the only part not typically considered palatable in any form.

Figure 5 : Garlic (8)

Figure 6: Structure of Garlic(9)

4. Cinnamon(10) Cinnamon is a spice obtained from the inner bark of several trees from the genus Cinnamomum that is used in both sweet and savoury foods. It is principally employed in cookery as a condiment and flavoring material. It is used in the preparation of chocolate, many dessert recipes, savory dishes of chicken and lamb and many more. It is also used in sambar powder which is an Indian cuisine giving it a rich aroma and tastes unique. Cinnamon also has been used as an insect repellent, and it is very effective in killing mosquito larvae. Through studies, Cinnamon found to be the most effective extracts against HIV-1 and HIV-2. Cinnamon's essential oils also qualify it as an "anti-microbial" food. In laboratory tests, growth of yeasts that were resistant to the commonly used anti-fungal medication fluconazole was often stopped by cinnamon extracts. Cinnamon's antimicrobial properties are so effective that recent research demonstrates this spice can be used as an alternative to traditional food preservatives. In a study, published in the August 2003 issue of the International Journal of Food Microbiology, the addition of just a few drops of cinnamon essential oil to 100 ml (approximately 3 ounces) of carrot broth, which was then refrigerated, inhibited the growth of the food borne pathogenic Bacillus cereus for at least 60 days. When the broth was refrigerated without the addition of cinnamon oil, the pathogenic B. cereus flourished despite the cold temperature. In addition, researchers noted that the addition of cinnamon not only acted as an effective preservative but improved the flavor of the broth. (11)

Figure 7 : Cinnamon powder(12)

5. Ginger(13) Ginger or ginger root is the rhizome of the plant Zingiber officinale, consumed as a delicacy, medicine, or spice. Ginger produces a hot, fragrant kitchen spice. Young ginger rhizomes are juicy and fleshy with a very mild taste and they are often pickled in vinegar or sherry as a snack or just cooked as an ingredient in many dishes. They can also be steeped in boiling water to make ginger tea. Ginger can also be made into candy. The traditional medical form of ginger historically was classified as a stimulant and carminative and used frequently for dyspepsia, gastroparesis, slow motility symptoms, constipation, and colic. It was also frequently employed to disguise the taste of medicines. Some studies indicate ginger may provide short-term relief of pregnancy-related nausea and vomiting. Studies are inconclusive about effects for other forms of nausea or in treating pain from rheumatoid arthritis, osteoarthritis, or joint and muscle injury. Side effects, mostly associated with powdered ginger, are gas, bloating, heartburn, and nausea.

Figure 8 : Ginger plant (14)

6. Mint (15) Mint is a popular Mediterranean herb that is known for its cool, breath freshening properties. As such, it is a favorite flavor found in toothpastes, chewing gums, mouthwashes, breath sprays, candies and even inhalers. Mint is also widely used in cooking where its cooling properties are used to temper hot dishes and perk up soups. Mint is widely known to have great therapeutic properties that can be used to treat a number of ailments. Mint provides relief from flatulence, helps prevent food poisoning, and treats indigestion. Mint leaves have a strong and pleasant aroma which activate the saliva glands and the digestive glands to secret enzymes that promote digestion. It is also anti-spasmodic and relieves stomach discomfort coming from indigestion and inflammation. Mint is also used to provide relief from sore throat and cough and is the main ingredient in inhalants, rubs, cough medicine, and throat lozenges. Mint juice also often used in skin cleansers to help fight skin infections and pimples. It is also effective in treating insect bites by mosquitoes, honey-bees, hornets, wasps, gnats and others as it soothes and cools the affected areas. Mint is thought to remove excess body heat by inducing sweating in a person suffering from high fever. It also heals and soothes burns, cleans the mouth, prevents tooth decay, boosts libido and stamina, and relaxes the body.

Figure 9: Mint(16)

Objective To investigate the effects of antimicrobial properties in plants. Problem Statement Which plant has the greatest antimicrobial property? Hypothesis Different plant extract has different antimicrobial property. The ingredient disc that has the biggest clear zone around the disc shows that it is able to inhibit bacterial growth near it thus, has the highest antimicrobial properties. It is predicted that garlic has the greatest antimicrobial property than ginger, mint and cinnamon powder.

Apparatus Sterile paper discs, micropipette with sterile tips, test tubes, mortar and pestle, sterile Petri dish, sterile forceps, tape, marker pen, Bunsen burner, vortex machine, laminar flow chamber, weighing machine, small measuring cylinder, small beakers , cellophane tape.

Materials Agar warmed at 60C, denatured alcohol, plant material (garlic, ginger, mint and cinnamon powder), distilled water, Escherichia coli and Bacillus subtilis bacterial broth in sterile bijou bottles.

Variable : Types of Variables Manipulated Variable: Type of bacteria and plant material used Use different types of bacterial cultures Ways to control the variables

(Esherichia coli and Bacillus subtilis) on different type of plant extracts namely garlic, ginger, mint and cinnamon powder.

Responding Variables:

Area of the white zone

The radius clear zone calculated was measured using a ruler after 24 hours and by using formula area = r2, area of bacterial inhibition was calculated.

Fixed Variables: Volume of bacterial broth used About 200L of bacterial broth was used in all samples. Temperature at which agar was kept Liquidified agar was kept at 60C and kept back into incubator after used. Concentration of plant extract For each plant extract, 30% solution

concentration was prepared by diluting the 0.3g of plant extract in 1ml of denatured alcohol

Procedure a) Preparing the agar plates 1. Hands were washed using disinfectant before the experiment carried out. 2. 2 sterile Petri dishes were labeled with respective name of bacteria and numbers were written according to the paper discs (immersed with plant extract) on the back of Petri dish. 3. Using aseptic technique, 200L of the bacterial broth (Escherichia coli and Bacillus subtilis) was transferred in sterile condition inside a laminar flow chamber (condition inside laminar flow chamber are very much sterile, clean and free from bacteria). 4. Using last finger, the bijou bottle containing bacterial broth was opened. The mouth of bijou bottle was then flamed with Bunsen burner to kill any pathogens that may present. 5. The bacterial broth was then pipette using micropipette that has been calibrated to 200L. The knob of the micropipette was gently pressed until the first pressure exerted felt and released inside the bijou bottle (in the middle of the bacterial broth solution) so that no air bubble will be formed. 6. The mouth of bijou bottle flamed again before closed to avoid contamination. 7. The bacterial broth was dispensed into a Petri dish (lid of Petri dish only lifted enough to allow entry of the pipette tip) according to their label and closed quickly by putting the lid onto the Petri dish to avoid contact with air too long. 8. The tip of micropipette was ejected into the beaker of disinfectant and a new sterile one was used for another time. 9. Step 2 to 7 was repeated for each bacterium (Escherichia coli and Bacillus subtilis). 10. The agar solution that was kept at 60C to maintain its liquid form was also prepared using aseptic technique. 11. Using hand glove, the bottle containing the agar was taken out from vortex machine, opened and the agar was poured into the Petri dishes until enough to cover the bacteria (around 18-20 ml). 12. Both bacterial broth and agar was then gently mixed together by swirling the Petri dish in all direction. This has to be done to ensure thorough mixing of both components so that the bacteria will grow at a similar rate at all areas of the agar.

b) Preparation of plant extract 1. Four types of plant extracts were chosen namely garlic, ginger, mint and cinnamon powder. 2. Garlic, ginger and mint were pounded using mortar and pestle while the cinnamon powder was readily prepared in powder form. 3. The plant extract had to be prepared in a 30% solution. This is done by dissolving 0.3g of each extract in 1ml of denatured alcohol. 4. 0.3g of each extract was measured using a weighing machine while 1ml of denatured alcohol was measured using small measuring cylinder. Both substances were then mixed in a small beaker respectively. 5. Two sterile paper discs were then placed in each beaker containing plant extract using a pair of sterilized forceps. Then, they were taken out and left to dry on another Petri dish inside the laminar flow chamber. 6. After all the paper discs have been prepared, they were then placed onto the prepared agar plates according to their respective numbers using a pair of sterilized forceps (different forceps used for each solution to prevent mixture of plant extracts). The lid of Petri dishes were only lifted enough to place the paper discs. 7. The paper discs were pressed gently on the solidified agar to prevent them from fall of when the Petri dish is inverted. Paper discs that were soaked in only denatured alcohol were also placed on the agar (act as control). 8. The Petri dishes were closed and cellophane tape was used to tape it in side ways to prevent growth of anaerobic bacteria and enable entering of oxygen for the bacteria to respire. 9. The Petri dish containing agar and paper discs with plant extracts were then inverted and placed into incubator and left for 24 hours to enable bacterial growth. Observations were done on the next day and the area of clear zone (area of inhibition of bacterial growth) was calculated.

Safety precaution In order to avoid any accident or injury during the experiment in laboratory, the precautionary steps should be taken and applied. Wearing lab coat and a pair of suitable shoes are compulsory when conducting an experiment in the lab at all times to protect the skin and clothing from spillage of any chemical substance or bacteria culture. Hands need to be thoroughly washed before and after performing the experiment. This is to avoid ourselves from getting infected from any of the microorganism. Since only bare hands were used in handling the bacteria, thus it is important to maintain a minimal contact, such as using only the last finger in opening the bijou bottle. Furthermore, the glassware such as beaker should be handled with full care because they are fragile. The apparatus such as forceps is also sterilized to prevent infection of microorganism. After using all samples and apparatus at the end of experiment, they should be discarded properly and returned back to their places to avoid injuries and unnecessary accidents that may result fatal results.

Risk Assessment The tip of the micropipette has to be changed each time different bacteria used to avoid contamination. All sterile apparatus such as sterile forceps were used to avoid contaminations. The preparation of agar and placing the paper discs that were soaked in plant extracts were carried out in laminar flow chamber to avoid contamination too. Hot agar is used while it is still in its liquid form and it is advisable to use it immediately after it has been taken out from the vortex machine. This is to avoid the agar from solidifying too fast even before the bacteria were mixed which may affect the result later. The agar is put inside the vortex machine as soon as it is finish used to prevent agar solidification that may contribute to wastage. After placing the bacteria in the Petri dishes (done at fast pace) to avoid long exposure to open air, the agar solution and bacteria needs to be mixed thoroughly as this will affect bacterial growth across the agar.

Results Type of plant extract used Type of Bacteria Bacillus subtilis Radius r

Alcohol (control) -

Garlic

Ginger

Mint

Cinnamon powder

1.00 3.142 X (1.00)

0.90 3.142 X (0.90) 2.5450

0.70 3.142 X (0.70) 1.5396

1.00 3.142 X (1.00) 3.1420

Area of inhibition of bacterial growth (cm) Escherichia coli Radius r

No change

3.1420

0.90 3.142 X (0.90)

0.90 3.142 X (0.90) 2.5450

0.70 3.142 X (0.70) 1.5396

0.80 3.142 X (0.80) 2.0109

Area of inhibition of bacterial growth (cm)

No change

2.5450

Table 1 : Area of bacterial growth respective to the plant extracts

Data Analysis

Table 1 above show the area of clear zones surrounding the paper discs that were soaked into respective plant extracts. This data will be analysed according to each type of bacteria namely Escherichia coli (E.coli) and Bacillus subtilis. Both bacteria have shown the same results with denatured alcohol that act as a control. For plant extracts, different results were obtained. Both bacteria has reacted with all of the plant extracts. For Bacillus subtilis area of clear zone is bigger in garlic (3.142cm) which is similar with cinnamon powder shows that both garlic and cinnamon powder have the highest antibacterial activity than ginger and mint towards Bacillus subtilis. While ginger has a clear zone at about 2.5450 cm and mint has 1.5396 cm, thus it can be said that ginger has higher antimicrobial property than mint. Overall it can be concluded that the plant extract that exhibit highest antimicrobial property is garlic, cinnamon powder, ginger and lastly mint. Escherichia coli also has reacted with all four plants extracts. Inhibition area of garlic and ginger (2.5450cm) is the highest among the all plant extract, followed by cinnamon powder (2.0109cm) and mint (1.5396cm). Thus, it can be concluded that garlic and ginger has the highest antibacterial property against Escherichia coli, followed by cinnamon powder and mint. It can be seen that the area of clear zone in both mint and ginger Petri dishes were the same even though they contain different microbial broth. For cinnamon powder and garlic, it can be seen that area of clear zone is bigger in Petri dish containing Bacillus subtilis than in E.coli.; showing that B.subtilis has the greatest microbial properties towards plant extracts.

DISCUSSION From the result obtained, it can be seen that different plant extract exhibit different antimicrobial properties. According to the hypothesis, highest antimicrobial activity is exhibited by garlic, followed by ginger, cinnamon powder and mint.

Both bacteria showed that garlic has the greatest antimicrobial properties with average area of 2.8435 cm. This is followed by cinnamon powder by having average area of 2.5765 cm. For ginger, it has average clear zone area at about 2.5450 cm, followed by mint having an average area of 1.5396 cm. These results were having some limitations as some sources of error can be seen during preparation of extracts. During the preparation of mixing the cinnamon powder into denatured alcohol, it doesnt seem to mix well with the denatured alcohol and formation of clump can be observed. As for garlic, ginger and mint, since they were pounded, some extract may be lost during transfer and when soaked in denatured alcohol, it seems not to mix well too. This might overall affect the concentration of these plant extracts in the denatured alcohol.

In both bacterial culture, mint and ginger show same strength of antimicrobial properties.. This may be due to limitation in preparation as told earlier. The average inhibition diameter in B.subtilis is much bigger (5.1843 cm) compared to E.coli (2.160cm) in all the plant extract samples. This is because B.subtilis is the positive Gram bacteria which are easily treated with antibiotics. On the other hand, E.Coli is the gram negative bacterium which is hardly treated with antibiotics. The lipopolysaccharides produce antitoxin in the E.Coli which makes the inhibition diameter is much smaller compare to B.subtilis.

Limitations There are several limitations that have been identified throughout this experiment. The whole preparation wasnt done inside the laminar flow chamber except for some crucial steps may cause contamination by bacteria in air thus, contributing towards reduction in result accuracy. Bacterial culture that was prepared left for more than 24 hours. This bacterial culture actually should sub cultured after 24 hours to prevent the concentration of bacterial culture become too concentrated. And since the bacteria left for too long, the bacteria may enter lag phace or stationary phase which will affect the growth of bacteria in agar. During preparation of plant extract, some ingredients seem to not mix well with denatured alcohol. Cinnamon powder form clumps while the part of onion that was used to soak in alcohol was merely its epidermis rather than inside parts. This could reduce the volume of chemical from the plant ingredients that could possibly mix well with alcohol. The paper discs that were used werent sterile properly and some of the apparatus such as forceps were not in sterile condition which later affects the result. Forceps should be sterilized by dipping them in alcohol and burn it in Bunsen burners flame. But due to safety of the students, this step wasnt carried out, thus act as limiting factor for the result.

Sources of errors Several sources of error in this experiment were identified and steps were taken to minimize these errors to make the result more accurate. After the whole reparation or while waiting for plant extracts to be ready, the Petri dish with bacterial broth and agar solution were kept inside laminar flow chamber instead of being left outside. This is to avoid contamination of bacteria that may present in air. When the paper discs were placed onto the agar, the lid of Petri dish was only lift up enough to let the forceps to place the discs. This is to, prevent contamination of other bacteria. The Petri dish may open when it is inverted before placed into incubator. This was prevented by taping the sides of Petri dish. The Petri dishes were not taped all round as it will encourage growth of anaerobic bacteria, some which may be harmful and also exclude oxygen needed for growth of bacteria in the agar.

Conclusion Theoretically, each and every plant has its own strength of antimicrobial property in order to fight against bacterial infection. Same goes to the bacteria. Different bacteria may have different effect towards the antimicrobial actions .All of this depend on its resistance towards the chemical it encounter. From this experiment, it can be concluded that garlic has the highest antimicrobial property toward both E.coli and Bacillus subtilis followed by cinnamon powder,ginger and mint. Thus, the hypothesis is accepted.

Further Investigation Another experiment can be carried out using different type of bacteria. It is also possible to use organisms from other kingdom to test their responses towards the antimicrobial activity of the same or different plants.

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