Introduction Bacteria Escherichia coli or E. coli is a Gram-negative1 rod-shaped bacteria commonly found in lower intestines of endotherms.

Most strains are harmless but some may cause serious food poisoning in humans. Harmless strains are part of the normal flora of the gut, benefits their host by producing vitamin K2 and preventing the establishment of pathogenic bacteria within the intestine2. The ability for E. coli to survive for brief periods outside the body makes them an ideal indicator organism to test samples for fecal contamination. Bacillus subtilis, known also as hay bacillus or grass bacillus is Gram-positive, catalase-positive bacteria commonly found in soil. It is a member of the genus Bacillus. B subtilis is rod-shaped and can form a tough, protective endospore thus allowing the organism to tolerate extreme environmental conditions. B. subtilis can reproduce by binary fission or producing a single endospore that is resistant to environmental factors. The endospore is formed at times of nutritional stress, allowing the organism to persist in the environment until conditions become favorable3. Serratia sp. is a genus of Gram-negative, facultatively anaerobic , rod-shaped bacteria of the Enterobacteriaceae family. Members of this genus produce characteristic red pigment, prodigiosin and can be distinguished from other members of the family by its unique production of enzymes DNase, lipase and gelatinase. Serratia sp. have been responsible for about 2% of nosocomial infections4 of the bloodstream, lower respiratory tract, urinary tract, surgical wounds, and skin and soft tissues in patients5.

Antimicrobial Properties Plants are susceptible to infection by bacteria and fungi. An antimicrobial is a substance that inhibits or destroys the growth of certain bacteria. Several plants are known to, or thought to have this antimicrobial property to repel attacks by microorganisms. It is a general term that includes antibiotics, antivirals, antifungals and antiprotozoa.

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bacteria that do not retain crystal violet dye in the Gram staining protocol Howard C. Berg (2004) E. coli in motion 3 http://www.rense.com/general4/bac.htm 4 infections that are a result of treatment in a hospital or a healthcare service unit 5 http://emedicine.medscape.com/article/228495-overview

Black Pepper The chemicals in black pepper responsible for pungency are Capsaicinoids which has two principles, capsaicin and dihydrocapsaicin. These two compounds represent between 80-95 % of the total Capsaicinoids. Studies have shown that these two compounds have antimicrobial properties against infection by pathogens6.

Garlic The chemical present in garlic is allicin. Allicin is the most powerful medicinal compound derived from garlic and has antibiotic properties. Allicin does not occur naturally in garlic, it is produced when garlic is chopped or crushed. The finer the chopping and the more intensive the crushing, more allicin is produced thus the medicinal effect is stronger. As a natural antibiotic, allicin does not build up a resistance to it compared to modern antibiotics. Cooking and aging can destroy the allicin7. Turmeric Turmeric has been used as anti-inflammatory and anti-arthritic. The main active ingredient in turmeric, curcumin is shown to have many therapeutic effects. It protects against free radical damage as it is a strong antioxidant. Turmeric has been investigated for its cholagogous influence on the secretion of bile, pancreatic, and gastric juices. It also has anticarcinogenic and antimutagenic properties8. Ginger Ginger has strong antibacterial and antifungal properties. In vitro studies have shown that active constituents of ginger inhibit multiplication of colon bacteria. These bacteria ferment undigested carbohydrates causing flatulence. This can be counteracted with ginger. It inhibits the growth of some bacteria. Ginger eases intestinal infections, especially related with digestive problems. Ginger can eliminate harmful bacteria such as E.coli especially in children9. Cinnamon Cinnamon is one of the ancient medicines. It was used for treating rheumatism and other inflammations. Its mild anti-inflammatory, anti-spasmodic, and anti-clotting properties are believed to be due to its content of cinnamaldehyde. The antimicrobial properties of cinnamon are thought to be due to eugenol and cinnamaldehyde10.

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http://themedicinalpepper.com/ http://www.garlic-central.com/allicin-chemistry.html 8 http://www.healthdiaries.com/eatthis/20-health-benefits-of-turmeric.html 9 http://www.academicjournals.org/ajmr/PDF/pdf2011/18Feb/Poeloengan.pdf 10 http://www.covenofthegoddess.com/herbproperties.htm

Hypothesis Different plants have different antimicrobial properties. Variables Manipulated variable: Type of plant used. Responding variable: Diameter of bacterial growth inhibition Constant variable: pH, incubation period, temperature. Apparatus Sterile petri dishes, mortar and pestle, sterile micropipette, paper discs, sterile forceps, tape, marker pen, incubator set at 25C, Bunsen burner Materials Plant material (ginger, black pepper, turmeric, cinnamon powder), denatured alcohol, sterile nutrient agar, bacterial broth (E. coli, Bacillus subtillis, Serratia sp.) Method 1. Obtain the plant extract (ginger) by crushing 3g of plant material with 10cm3 of industrial denatured alcohol and shake it from time to time for 10 minutes. 2. Repeat to obtain extracts of black pepper, turmeric and cinnamon powder. 3. Open the Schott bottle containing the agar solution and flame the neck of the bottle. 4. Pour the agar into a petri dish. The agar will start to solidify at about 42C. Take care not to let it cool too much. 5. Pipette 1cm3 of bacterial broth (E. coli) into the petri dish containing the molten agar using aseptic technique. The lid of the Petri dish should only be lifted enough to allow entry of the pipette. 6. Gently push the Petri dish back and forth, N-S, NE-SW and NW-SE to mix the bacteria with the agar and allow the agar to set. 7. Repeat steps 3 to 6 to obtain agar containing Bacillus subtillus and Serratia sp. 8. Dip a piece of sterile paper disc into the plant extract and let it dry for 10 minutes. 9. Prepare 3 paper discs dipped in alcohol. This will act as a control 10. Use sterile forceps to place the test discs onto the bacterial plate together with the control. Mark the underside of the Petri dish to distinguish between the different discs. 11. Close the Petri dish and tape it. 12. Incubate the plates for 24 hours at 25C 13. Observe the plates without opening them. Measure the diameter of the area where bacterial growth is inhibited.

Safety Measures and Risk Assessments Handle the bacteria with care. Gloves should be worn when handling bacteria to minimize skin contact with the bacteria. The neck of the Schott bottle and the bijou bottle containing bacteria must be heated first before it is poured out. This is to reduce the risk of contamination and to maintain a warm air current around the mouth of the bijou bottle. Ensure that all apparatus used is sterile and apply aseptic techniques throughout the experiment to avoid contamination. Wash your hands thoroughly with soap and water after completing the practical. Industrial denatured alcohol is harmful and highly flammable. It should not be used while naked flames are in use for example during the preparation and pouring of agar plates. Do not open the Petri dishes containing growing microorganisms. Only dispose of the used Petri dishes after they have been autoclaved. After closing the Petri dish, do not tape all around the dish as this can lead to the growth of anaerobic bacteria, some of which may be harmful and also exclude oxygen needed for the growth of bacteria in the agar. Make sure that all labels are at the bottom on the base of the plate. It is important that the plates are used for the investigation on the same day as the agar has set.

Results Extract Bacteria E. coli Bacillus subtilis Serratia sp. 0.5 0.5 0.5 0.5 Diameter (cm) 0.5 0.5 0.5 0.5 0.5 0.5 Black pepper Turmeric Cinnamon powder Ginger Alcohol (control)

0.5

0.5

0.5

0.5

0.5

A table of results obtained- The diameter of bacterial inhibition against with different plant extracts and in different bacterial culture.

Discussion The bacteria used are E. coli, Bacillus subtilis and Serratia sp. These bacteria have different resistance against antimicrobial properties of plant and through this experiment; it is possible to determine which one of the three has a stronger resistance towards microbial agents. Four different plant extracts are used to investigate their antimicrobial properties. This is to compare between the plants as well as with the control. The control used in this experiment is alcohol as alcohol is a positive control. Positive controls confirm that the procedure is competent in observing the effect and therefore minimizing false negatives. The plates are used for the investigation on the same day as the agar has set, otherwise once the bacteria have started to grow they will be unaffected by the microbial agent. The results obtained shows that different plant extracts do not affect the diameter of bacterial inhibition. The result also does not show any difference between the different bacteria species used. This reading is obtained by measuring the diameter of the paper disc containing the plant extract. This result does not coincide with the theories that plants have antimicrobial properties as plants are susceptible to infection by bacteria and fungi and several plants- mainly spices- are known to destroy or inhibit the growth of certain bacteria. The results should show different readings where perhaps one of the plant extract used has better antimicrobial properties. Our experiment should also use garlic as garlic is well-known for its antimicrobial properties. If garlic were to be used, there would be a visible clear area where bacterial growth is inhibited. Apart from that, the different plant extracts would react differently with different bacteria. As each species has different resistance, some bacteria may be resistant to the antimicrobial properties of the plant extract. If garlic were to be used, there would be a larger diameter in the Petri dish containing E. coli and a slightly smaller one in the Petri dish with Bacillus subtilis as B. subtilis is slightly more resistant than E. coli. Lastly, there would be no clear zone in the Petri dish with Serratia sp because Serratia sp is very resistant against the antimicrobial properties of even garlic. Even so, there should be results obtained with the ginger extract. This is because ginger has known antimicrobial properties against E.coli. Hence, there should be a clear zone around the paper disc with garlic extracts in the Petri dish containing E. coli. It could be said that this experiment was conducted wrongly and hence, no proper results could be obtained. Conclusion Different plants do not have different antimicrobial properties. The hypothesis is not accepted.

Limitation and Source of Error Among the sources or error that could be identified when conducting this experiment is that the bacteria was not properly mixed in the agar. This caused the bacteria to be more concentrated to one side rather than spread around evenly in the agar. Apart from that, there might be instances where the agar solidified before we put in the bacteria. This would cause the bacteria to be unable to mix evenly in the agar and thus concentrated to one corner only. The antimicrobial property of the plant is unable to be seen if there is no bacteria present in the vicinity of the paper disc with the plant extract. Suggestions for Improvement The bacteria and agar should be properly mixed together. The bacteria should be added when the agar has not solidified. This is to ensure the bacteria are evenly mixed in the agar. Thus when the bacteria grows, there would be a visible clear zone around the cloudy areas where the bacteria managed to thrive. Further Works This experiment could be repeated with different plant extracts such as mint, lemon and tea tree oil. Apart from that, different strains of bacteria could be used to determine its resistance towards antimicrobial properties of plants. The experiment would still require the same methods. Reference Howard C. Berg (2004) E. coli in motion http://www.rense.com/general4/bac.htm http://emedicine.medscape.com/article/228495-overview http://themedicinalpepper.com/ http://www.garlic-central.com/allicin-chemistry.html http://www.healthdiaries.com/eatthis/20-health-benefits-of-turmeric.html http://www.academicjournals.org/ajmr/PDF/pdf2011/18Feb/Poeloengan.pdf http://www.covenofthegoddess.com/herbproperties.htm