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By: Soh Yee Kiat (1P1), Justin Soh (1P1), Leow Shawn Tao (1A4), Sow Jeng Wei (1O2). Group ID: 1-010 A Category 1 Experimental Project (IS)

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Table of Contents Information About Chlorine Materials Used Information About Bacteria Variables Methodology Data Collection and Comparison of Results Conclusion

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Rationale Since 1920, chlorine water has been commonly used to disinfect water supplies and swimming pools. During this period of 92 years, these bacteria might have adapted to chlorine, thus scientists have recently doubted chlorines effectiveness in killing bacteria.

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What is CHLORINE? Chlorine is a rather abundant halogen with an atomic number of 17 and a symbol of Cl. It was first discovered by the Swedish scientist Carl Wilhelm Scheele.

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Uses and effects of CHLORINE It can be deadly even at an amount of 430 parts per million(ppm) and exposure of 0.2ppm already affects the human body. Overexposure to chlorine can cause temporary inflammation of the eyes and hyper-excretion of mucus. Chlorine water is commonly used as a disinfectant in swimming pools.

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HCI Pool Information InformationSize (metres)Chlorine Water Added (mass) Value50x25x1.4Approximately 1kg

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HYPOTHESIS Chlorine water is ineffective in killing common harmful bacteria.

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Materials Bacteria Equipment E.coli Agar Plates Sterile Swabs Microscope Chlorine Water (Collected from HCI swimming pool) Pipette Centrifuge Tubes Nutrient Agar Nutrient Broth Measuring Cylinder

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What about E.coli? It is a type of bacterium that is commonly found in the lower intestines of warm blooded organisms and is common cause of food poisoning in humans. It is generally transmitted through consumption of contaminated food. E.coli

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VARIABLES ControlledDependent I ndependent Variable Volume of Chlorine water used Colonies of bacteria left after the experiment Type of bacterium used Amount of bacteria used The time in which the chlorine water is collected Area of collection of water Volume of Silver Nitrate Used

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Content Water Collection Broth Preparation Agar Preparation Agar Spreading Overnight Culture Preparation Observations Methodology

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Methodology (Water Collection) 1. A measuring cylinder was used to collect 30ml of water at the timings 7.50am, 2.30pm and 5.00pm. 2. Water collection took place on the day before the bacteria were spread. The water was then placed in the labs refrigerator.

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Methodology (Broth) 1. Using a weighing machine (to 2 decimal places), we measured precisely 3.25 grams of nutrient broth and placed it in a 250ml sterilised bottle. 2. Using a measuring cylinder of maximum volume 500ml, we measured 250ml of de-ionized water and carefully poured the distilled water into the bottle before thoroughly and carefully stirring the mixture.

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Methodology (Broth) 3. After a clear, pale yellow solution was formed, the broth was placed in the lab refrigerator. This methodology was carried out a second time to prepare another 250ml of nutrient broth.

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Methodology (Agar) 1. Using a weighing machine (to 2 decimal places), we measured precisely 14 grams of nutrient agar before placing the agar inside a 500ml bottle. 2. Secondly, using a 1 litre measuring cylinder, we collected 500ml of de-ionized water and poured the water into the bottle before thoroughly mixing the mixture until a cloudy suspension was formed. This agar was then placed in the fridge for autoclaving the next day. 3. This procedure was repeated once for another 500ml bottle of agar.

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Methodology (Bacteria) 1. To prepare for the overnight culture, we borrowed a plate of E.coli from the SRC and using a bacteria streaking rod, we displaced some bacteria onto a spare agar dish of our own. 2. This procedure was carried out in the bio- safety cabinet to prevent contamination.

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Methodology (Agar Spreading) 1. Using the recently autoclaved agar, we displaced the agar onto Petri dishes to a height of about the agar plates. It was then left to solidify for around 15 minutes. 2. Then, water vapour found on the agar plates were wiped off with sterile wipes to prevent the water from interrupting with our experiment. All these steps were carried out in the biosafety cabinet.

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Methodology (Overnight Culture) 1. Using autoclaved nutrient broth prepared on the day before, we poured 10ml of nutrient broth into 4 centrifuge tubes. 2. Then, using a bacteria streaking rod, bacteria that was prepared a day earlier was scraped and placed into the broth in the centrifuge tubes.

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Methodology (Overnight Culture) 3. To minimise the risk of contamination, we flamed the streaking rod, the broth bottle and the centrifuge tubes both before and after usage. 4. The centrifuge tubes were placed in the shaking incubator for 1 day in preparation for dilution and spreading.

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Methodology (Dilution) 1. Using the overnight culture, we started with dilution. Firstly, our aim was to reach 1 x 10- and 1 x 10- of bacterial dilution. Using 6 centrifuge tubes, we filled each centrifuge tube with 9ml of autoclaved nutrient broth using a pipette gun and pipette. 2. Using a micropipette, we displaced 1ml of the bacterial broth from the overnight culture into a centrifuge tube labeled 1 x 10-.

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Methodology (Dilution) 3. By repeating step 2, we displaced 1ml of broth from the centrifuge tube containing 1 x 10- into a centrifuge tube labeled 1 x 10-, and continued this procedure with the tubes labeled 1 x 10-, 1 x 10- , 1 x 10- and 1 x 10- . 4. After this method of dilution had been carried out in the biosafety cabinet with proper flaming, we used the tubes with the smallest and second smallest concentration of bacteria for use in the bacterial spreading.

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Methodology (Bacterial Spreading) 1. Firstly, our agar plates were labeled as accordingly to the type of water that they will be filled with, the bacteria and the dilution used. Each type of water has 3 prototypes for triplication. Also, 3 agar plates with only bacteria and sterile water was set up. (E.g.: First plate of7.50am water with E.coli with 1 x 10- concentration:7.50am,E.coli, 1 x 10- ,1) (E.g.: Second Control with 1 x 10- : E.coli, Sterile Water, 1 x 10- ,2)

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Methodology (Bacterial Spreading) 2. Next, according to the labeling on our agar plates, we carried out our experiment in the bio- safety cabinets. Using micropipettes of the range 0.5-10 microlitres, we pipetted 10 microlitres of bacteria together with 10 microlitres of chlorine water of their respective timings. The controls were done last, with sterile water instead of chlorine water.

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Methodology (Bacterial Spreading) 3. Flaming was carried out after each use of the centrifuge tubes and we used a sterilised and flames bacteria spreader to spread the bacteria after each filling. 4. To let the bacteria set in appropriately, Parafilm was used to securely tape up the agar dishes and prevent contamination. The bacteria was then left in an incubator at 36.00 Celsius for 1 day.

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7.50am Water Water SampleNumber of colonies 7.50am, E.coli, 1 7.50am, E.coli, 2 7.50am, E.coli, 3 Control

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2.30pm Water Water SampleNumber of colonies 2.30pm, E.coli, 1 2.30pm, E.coli, 2 2.30pm, E.coli, 3 Control

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5.00pm Water Water SampleNumber of colonies 5.00pm, E.coli, 1 5.00pm, E.coli, 2 5.00pm, E.coli, 3 Control

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Silver Nanoparticles These are particles of mainly silver oxide ranging from 1 to 100 nanometres in length. They are scientifically proven to possess strong antibacterial properties.

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Problems 1/9 of people are allergic to the nickel present in silver nanoparticles. Scientists have conducted a test in which the silver nanoparticles have actually caused severe developmental defects in zebrafish. Overexposure might cause degeneration of body parts due to the strong antibacterial properties.

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Timeline MonthsFebMarchAprilMayJune Events:PlanningMentorship Approved PrelimsCollection Of Data Conduction Of 1 st Experiment Project Confirmation First Project Meeting First SRC Meeting PlanningConduction of 2 nd Experiment Group Formation PowerPoint Prelim Creation Final Experiment Conduction

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Timeline MonthJulyAugustSeptemberOctober EventFinalisation of Project Conduction of Silver Nanoparticles Experiments Full Completion of Project Grand Finals Semi-FinalsRepetition of S.Particles Experiments Finals Silver Nanoparticles Planning

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Bibliography http://www.medicalnewstoday.com/articles/68511.php http://natsci.edgewood.edu/wingra/wingra_bacteria.h tm http://natsci.edgewood.edu/wingra/wingra_bacteria.h tm http://www.cbc.ca/news/health/story/2009/07/02/f- ecoli-recall-food-safety.html http://www.cbc.ca/news/health/story/2009/07/02/f- ecoli-recall-food-safety.html http://www.sciencebuddies.org/science-fair- projects/project_ideas/MicroBio_p013.shtml http://www.sciencebuddies.org/science-fair- projects/project_ideas/MicroBio_p013.shtml http://proquest.umi.com.libproxy.nlb.gov.sg/pqdweb?i ndex=9&did=2037658811&SrchMode=1&sid=2&Fmt=6 &VInst=PROD&VType=PQD&RQT=309&VName=PQ D&TS=1332762445&clientId=13402 http://proquest.umi.com.libproxy.nlb.gov.sg/pqdweb?i ndex=9&did=2037658811&SrchMode=1&sid=2&Fmt=6 &VInst=PROD&VType=PQD&RQT=309&VName=PQ D&TS=1332762445&clientId=13402

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Bibliography(2) http://biofilmbook.hypertextbookshop.com Usborne Encyclopedia Of Science Usborne Mini-Encyclopedia: All About Chlorine Usborne Information- All About Bacteria http://www.charpan.com/antibacterial-silver- nanoparticles http://www.charpan.com/antibacterial-silver- nanoparticles http://www.nanowerk.com/spotlight/spotid=5966.ph p http://www.nanowerk.com/spotlight/spotid=5966.ph p