UNIVERSITI TEKNOLOGI MARA INTERNATIONAL EDUCATION CENTRE (INTEC)

BIOLOGY LABORATORY REPORT A LEVEL PROGRAMME 2014 17 MARCH 2014

NAME: MAISARAH BINTI ZAINUDDIN IC NUMBER: 950201-14-5724 GROUP: 11SC6 SID NUMBER: 1311171707 TITLE: THE ANTIMICROBIAL PROPERTIES OF PLANTS

1.0

TITLE
The Antimicrobial Properties of Plants

2.0

OBJECTIVES
To identify the plant that has the greatest antimicrobial properties To study the effects of microbial properties in plants

3.0

INTRODUCTION
Escherichia coli Escherichia coli which is commonly abbreviated as E. coli is a Gram-negative, facultative anaerobic and is a rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms. There are many different types of E. coli where some E. coli strains live in the intestine quite harmlessly while some serotypes can cause serious food poisoning in their hosts, and are occasionally responsible for product recalls due to food contamination. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2 and preventing colonization of the intestine with pathogenic bacteria.i

Image 1: Scanning electron micrograph of Escherichia coli, grown in culture and adhered to a cover slip.

The commonest infection caused by E. coli is infection of the urinary tract, the organism normally spreading from the gut to the urinary tract. E. coli is also the commonest cause of cystitis (infection of the bladder), and in a minority of patients the infection may spread up the urinary tract to the kidneys, causing pyelonephritis.

Otherwise healthy patients in the community may develop cystitis, and patients in hospital who have catheters, or tubes, placed in the urethra and bladder are also at risk. E. coli is also present in the bacteria that cause intra-abdominal infections following leakage from the gut into the abdomen, as for example with a ruptured appendix or following traumatic injury to the abdomen.ii

Image 2: Disease caused by E. coli

E. coli bacteria may also cause infections in the intestine. Diarrhoeal infections (intestinal) are caused by a group of E. coli known as 'enter virulent' (harmful to the intestines). Overspill from the primary infection sites to the bloodstream may cause blood poisoning (E. coli bacteraemia). In rare instances, E. coli may cause meningitis in very young children. Turmeric Turmeric (Curcuma longa) is a rhizomatous herbaceous perennial plant of the ginger family, Zingiberaceae. India is a significant producer of turmeric which has regional names based on language and country. It is also native to tropical Tamil nadu, in southeast India, and needs temperatures between 20 C and 30 C (68 F and 86 F) and a considerable amount of annual rainfall to thrive. Plants are gathered annually for their rhizomes, and propagated from some of those rhizomes in the following season. Turmeric has a peppery, warm and bitter flavour and a mild fragrance slightly reminiscent of orange and ginger, and while it is best known as one of the ingredients used to make curry, it also gives ballpark mustard its bright yellow colour. It also has a tough brown skin and a deep orange flesh.

Image 3: Turmeric

Curcumin is also a powerful antioxidant. Antioxidants scavenge molecules in the body known as free radicals, which damage cell membranes, tamper with DNA, and even cause cell death. Antioxidants can fight free radicals and may reduce or even help prevent some of the damage they cause. In addition, curcumin lowers the levels of two enzymes in the body that cause inflammation. It also stops platelets from clumping together to form blood clots.iii Its anti-inflammatory also makes it able to cure many other diseases such as flatulence, jaundice, menstrual difficulties, bloody urine, haemorrhage, toothache, bruises, chest pain, and colic.iv Fennel

Image 4: Fennel

Fennel (Foeniculum vulgare) is a plant species in the genus Foeniculum (treated as the sole species in the genus by most botanists). It is a member of the family Apiaceae (formerly the Umbelliferae). It is a hardy, perennial, umbelliferous herb, with yellow flowers and feathery leaves. It is indigenous to the shores of the Mediterranean but has become widely naturalized in many parts of the world, especially on dry soils near the sea-coast and on riverbanks. It is a highly aromatic and flavorful herb with culinary and

medicinal uses and, along with the similar-tasting anise, is one of the primary ingredients of absinthe. Fennel is used for various digestive problems including heartburn, intestinal gas, bloating, loss of appetite, and colic in infants. It is also used for upper respiratory tract infections, coughs, bronchitis, cholera, backache, bedwetting, and visual problems. Some women use fennel for increasing the flow of breast milk, promoting menstruation, easing the birthing process, and increasing sex drive. Fennel powder is used as a poultice for snakebites. In foods and beverages, fennel oil is used as a flavouring agent. In other manufacturing processes, fennel oil is used as a flavouring agent in certain laxatives, and as a fragrance component in soaps and cosmetics. Cinnamon 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. While Cinnamomum verum is sometimes considered to be "true cinnamon", most cinnamon in international commerce is derived from related species, which are also referred to as "cassia" to distinguish them from "true cinnamon".

Image 5: Cinnamon

Numerous studies show that cinnamon regulates blood sugar, making it a great choice for diabetics and hypoglycaemic alike. It also reduces LDL cholesterol levels. LDL is also known as the harmful cholesterol. Reducing it may help reduce the risk of cardiovascular disease. Besides, it has natural anti-infectious compounds. In studies, cinnamon has been effective against ulcer-causing H. pylori bacteria and other pathogens.v

 Mint Mint, the well known mouth and breath freshener that is scientifically known as Mentha, has more than two dozen species and hundreds of varieties. It is an herb that has been used for hundreds of years for its remarkable medicinal properties. The market is full of products like tooth paste, chewing gum, breath fresheners, candy and inhalers which have mint as their base element. Most of us are familiar with the refreshing application of mint, but it has far more to offer than that.

Image 6: Mint

Mint is a great appetizer or palate cleanser, and it promotes digestion. It also soothes stomachs in cases of indigestion or inflammation. When you feel sick to your stomach, drinking a cup of mint tea can give you relief. Also, if you are someone who travels long distances via plane or boat, the menthol oil derived from mint can be very soothing for nausea and related motion sickness. viSome more, the strong aroma of mint is very effective in clearing up congestion of the nose, throat, bronchi and lungs, which gives relief for respiratory disorders that often result from asthma and the common cold. As mint cools and soothes the throat, nose and other respiratory channels, it relieves the irritation which causes chronic coughing. This is the main reason why so many balms are based on mint.vii Ginger Ginger or ginger root is the rhizome of the plant Zingiber officinale, consumed as a delicacy, medicine, or spice. It lends its name to its genus and family (Zingiberaceae). Other notable members of this plant family are turmeric, cardamom, and galangal. The distantly related dicots in the Asarum genus have the common name wild ginger because of their similar taste. Ginger cultivation began in South Asia and has since spread to East Africa and the Caribbean.

Image 7: Ginger

According to the National Library of Medicine, part of the NIH (National Institutes of Health), ginger is widely used throughout the world for treating loss of appetite, nausea and vomiting after surgery, nausea resulting from cancer treatment, flatulence, stomach upset, colic, morning sickness and motion sickness. Some people find ginger helps them with the symptoms of upper respiratory tract infection, bronchitis, cough, menstrual cramps, and arthritis and muscle pain. In some parts of the world, ginger juice is applied to the skin to treat burns. Ginger is also used as flavouring by the food and drinks industry, as a spice and flavouring in cooking, and for fragrance in soaps and cosmetics.viii Garlic Allium sativum, commonly known as garlic, is a species in the onion genus, Allium. Its close relatives include the onion, shallot, leek, chive and rakkyo. With a history of human use of over 7,000 years, garlic 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. It was known to Ancient Egyptians, and has been used for both culinary and medicinal purposes.

Image 8: Garlic

Garlic is used for many conditions related to the heart and blood system. These conditions include high blood pressure, high cholesterol, coronary heart disease, heart attack, and atherosclerosis. Garlic actually may be effective in slowing the development of atherosclerosis as well and seems to be able to modestly reduce blood pressure. Some people use garlic to prevent colon cancer, rectal cancer, stomach cancer, breast cancer, prostate cancer, lung cancer and might as well cure diabetes, osteoarthritis, hay fever, high blood pressure late in pregnancy (pre-eclampsia), cold and flu. It is also used for building the immune system, preventing tick bites, and preventing and treating bacterial and fungal infections. Other uses include treatment of fever, coughs, headache, stomach ache, sinus congestion, gout, rheumatism, haemorrhoids, asthma and bronchitis, shortness of breath, low blood pressure, low blood sugar, high blood sugar, and snakebites. It is also used for fighting stress and fatigue, and maintaining healthy liver function.ix

4.0

PROBLEM STATEMENT
Different plant extracts have different antimicrobial properties. This experiment is conducted to determine which ingredient disc that has the biggest clear zone around the disc. This is because; having big clear zone indicates that the plant extract has better ability to inhibit the bacterial growth around it thus, proving that the plant has greater antimicrobial properties.

5.0

HYPOTHESIS
Garlic has the greatest antimicrobial properties compared to other plant extracts which is turmeric, fennel, cinnamon, mint and ginger.

6.0

VARIABLES
Use different plant extracts namely turmeric, fennel, cinnamon, mint, ginger and garlic. Measure and record the diameter of the clear zone after 2 days. prepared y diluting 3g of plant extract in 10cm3 of distilled water. Temperature at Keep the petri dish in the incubator with temperature

Manipulated Variable: Type of plant extract used Responding Variable: Diameter of clear zone Constant Variable:

Concentration of For every plant extract, 30% solution concentration was plant extract

which the petri 60C. dish was kept Volume and type For every petri dish, 200l of E. coli was poured into of culture used bacterial them. being

7.0

APPARATUS
Sterile petri dish, micropipette with sterile tips, test tubes, mortar and pestle, sterile forceps, cellophane tape, marker pen, Bunsen burner, laminar flow chamber, weighing machine, incubator, small measuring cylinders, metre rule and small beakers.

8.0

MATERIALS
Agar warmed at 60C, ethanol, distilled water, plant extracts (turmeric, fennel, cinnamon, mint, ginger and garlic) and E. coli kept in sterile bottles.

9.0

TECHNIQUES
Measure and record the diameter of the clear zone by using metre rule after the ingredient disc was placed into agar with E. coli mix with it after 2 days.

10.0

PROCEDURES

Preparation of the plant extract solution 1. Six types of plant extracts were chosen namely turmeric, fennel, cinnamon, mint, ginger and garlic. 2. Garlic, fennel, mint, ginger and garlic were pounded by 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 was done by dissolving 3g of each extract in 10ml of distilled water. 4. 3g of each plant extract was weighed by using weighing balance while 10ml of distilled water was measured by using small size of measuring cylinder. 5. Both substances (3g of plant extract and 10ml of distilled water) were mixed thoroughly in a small beaker respectively.

Image 9: Plant Extracts

 Preparation of the agar plates 1. Hand must first be washed wished ethanol before preparing the agar plates. 2. Two sterile petri dishes were labelled with 1, 2,3,4,5,6,7,8 and 9. Each of the number represented different type of plant extracts as shown below: 1 2 3 4 5 6 7 8 Turmeric Fennel Cinnamon Distilled Water Mint Ginger Garlic Distilled Water

Table 1: Plant Extracts and Its Respective Quadrant

Image 10: Quadrants of the Petri Dishes

3. Agar solution which had been kept in incubator of 60C earlier was poured into both of the sterile petri dishes until half-filled (at least covered the whole surface of the petri dishes). 4. Starting from this procedure, everything must be conducted in the laminar flow chamber to ensure that the surrounding air was also well-sterilised. 5. Using aseptic technique, 200l of the E. coli broth was transferred in sterile condition inside a laminar flow chamber. 6. Using last finger, the bottle containing E. coli broth was opened. The mouth of bijou bottle was then flamed with Bunsen burner to kill any pathogens that may present.

7. The E. coli 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. 8. The mouth of bottle flamed again before closed to avoid contamination. 9. The E. coli broth was dispensed into the Petri dish (lid of Petri dish only lifted enough to allow entry of the pipette tip) and closed quickly by putting the lid onto the Petri dish to avoid contact with air too long. 10. Step 5-8 were repeated on another petri dish. 11. Both of the petri dishes were moved sideways slowly to allow the E. coli broth to be scattered in the agar solution evenly. 12. The petri dishes then were allowed to solidify within 10 minutes. 13. Once it had been solidified, placed one paper disc into each beaker containing the plant extracts earlier by using a pair of sterilised forceps. 14. All of the paper discs that have been immersed in the respective plant extracts were then placed onto the solidified agar at their respective quadrant in the petri dish. 15. The Petri dishes were closed and cellophane tape was used to tape it sideways to prevent growth of anaerobic bacteria and enable entering of oxygen for the bacteria to respire. 16. The Petri dish containing agar and paper discs with plant extracts were then inverted and placed into incubator and left for two days to enable bacterial growth. 17. Observations were done on the next day and the area of clear zone (area of inhibition of bacterial growth) was calculated.

11.0 RESULTS
Type of Plant Extract Turmeric Fennel Cinnamon Distilled Water Mint Ginger Garlic Distilled Water Diameter of Bacterial Growth Inhibition Zone (cm) 1 0.8 1.0 0.7 0.6 1.0 1.1 2.0 0.6 2 0.8 1.3 0.8 0.6 1.4 1.1 2.4 0.6 Average 0.80 1.15 0.75 0.60 1.20 1.10 2.2 0.60

Petri Dish

Quarter

1 A 2 3 4 5 B 6 7 8

Table 2: Diameter of Bacterial Growth Inhibition for every Plant Extracts

Graph of Diameter of Bacterial Growth Inhibition Zone against Types of Plant Extract
2.5

2.2

Diameter of Bacterial Growth Inhibition Zone (cm)

1.5

1.2 1.15 1.1 1 0.8 0.75 0.6 0.5

0 Turmeric Fennel Cinnamon Mint Ginger Garlic Control Type of Plant Extract

Graph 1: Graph of Diameter of Bacterial Growth Inhibition Zone against Different Types of Plant Extracts

12.0

DISCUSSION
Table 2 above shows the area of clear zones (inhibition zone) surrounding the paper

discs that were soaked into respective plant extracts. This data will be analysed by measuring the diameter of the clear zone instead of the cloudy zone after at least 24 hours being kept in incubator of 25C. Image below shows the difference between the clear zone and the cloudy zone:

Image 11: Inhibition Zone

The clear region is an indication of the effective inhibition of microbial growth by the antimicrobial agent. This method is preferable since it is quite fast and inexpensive relative to other laboratory tests for antimicrobial activity. Based on this table, every plant extracts show different diameter of the clear zone thus proving that different type of plants has different antimicrobial properties. At the end of this experiment, garlic has the longest diameter followed by mint, fennel, ginger, turmeric and cinnamon. On the other hands, paper discs in Quadrant 4 and 8 have been immersed in distilled water to act as a point of comparison (controlled variable) to the others that have been immersed in plant extracts. Theorically, ginger should have longer diameter compared to mint and fennel however, it does not turn out that way when at the end, mint and fennel are having larger diameter than ginger. This is due to a few limitations. During the preparation of mixing the ginger powder into distilled water, it does not seem to mix well with the denatured alcohol and formation of clump can be observed. This might overall affect the concentration of the plant extracts in the distilled water thus causing errors in diameter of the clear zone.

13.0

FURTHER STUDIES
Another experiment can be carried out using different type of bacteria which can be

staphylococcus aureus. Different bacteria may have different effect towards the antimicrobial actions. All of this depends on its resistance towards the chemical it encounter.

14.0 SAFETY PRECAUTION

In order to avoid and prevent any kind of accidents or injuries from happening while conducting this experiment, there are a few common safety precautions that everyone must follow. First and foremost, wearing a lab coat and a pair of suitable and closed shoes are the very first things everyone must do once entering the laboratory. This is to protect our clothes and exposed skin from spillage of any chemical substances or bacteria culture. Wearing hand gloves is also very crucial since we were handling bacteria throughout this experiment. However, wearing hand gloves alone does not protective enough so we still have to rinse and wash our hands with clean water before and after conducting the experiment. These are all very important to protect ourselves from getting any kind of infections due to the presence of microorganisms and bacteria. Besides, all of the glass wares that being used in this experiment must be handled with full care since they are very fragile. All of the samples and apparatus must also be placed back to their original position. Since we are dealing with bacteria throughout this experiment, all of the apparatus used must be sterilised. The sterilising process can be done by heating the apparatus for a while at the flame of the Bunsen burner such as the forceps and the mouth of the bottle that stored E. coli broth. The tip of the micropipette also must be sterilised by using a steriliser machine (since it cannot be heated or else the tip will melt) and must be changed when we are using other solutions. Almost all of the procedures must be conducted in the laminar flow chamber because in this chamber, the surrounding air is well-sterilised so contaminations of the agar solution or the plant extracts are unlikely to occur. If there is no laminar flow chamber, we can use flame to sterilise the air by lighting up Bunsen burner and allow it to be lighted up a few minutes before conducting the experiment. For the agar solution, since it can easily solidified once it does not exposed to heat, it is advisable to use it immediately after it has been taken out from the vortex machine. This is to avoid the agar from being solidified too fast even before the bacteria mere mixed and also to prevent wastage of the agar solution. Once the E. coli broth had been successfully placed

into the agar solution in the petri dish, the bacteria and the agar solution must be mixed well as this will affect bacterial growth across the agar. 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. After done all of the procedures, both of the petri dish must be placed in incubator for at least 24 hours. The temperature of the incubator must be kept constant which is at 25C because at this point only E. coli that able to grow. However, both of the petri dish cannot be kept at 37C because at this temperature, other harmful bacteria are also able to grow as well as E. coli. By keeping the temperature at 25c, it is not only protecting us from any other harmful bacteria but also important in accuracy of the results.

15.0

CONCLUSION
Theoretically, each and every plant has its own strength of antimicrobial property in

order to fight against bacterial infection. From this experiment, it can be concluded that garlic has the highest antimicrobial property toward E. coli followed by cinnamon powder, ginger and mint. Thus, the hypothesis is accepted.

16.0
i

REFERENCES

http://www.mayoclinic.org/diseases-conditions/e-coli/basics/definition/con-20032105 Public Health England at

ii

http://www.hpa.org.uk/Topics/InfectiousDiseases/InfectionsAZ/EscherichiaColi/GeneralInformation/
iii

Turmeric | University of Maryland Medical Center

http://umm.edu/health/medical/altmed/herb/turmeric#ixzz2vwZhrO3Y
iv v vi vii

http://www.whfoods.com/genpage.php?tname=foodspice&dbid=78 http://www.care2.com/greenliving/10-surprising-health-benefits-of-cinnamon.html http://www.organicfacts.net/health-benefits/herbs-and-spices/health-benefits-of-mint.html http://timesofindia.indiatimes.com/life-style/health-fitness/diet/Health-benefits-of-mint-

leaves/articleshow/10452719.cms
viii ix

http://www.nlm.nih.gov/medlineplus/druginfo/natural/961.html

http://www.nlm.nih.gov/medlineplus/druginfo/natural/300.html