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IntroductionAntimicrobial resistance is one of the most serious threats in the world today. As pathogens have been adapting to become resistant to various classes of antibiotics, infections from increasingly resistant bacteria become more common. As the available treatments and drugs becomes less able to effectively fight off infectious diseases, a additional threat is created for patients already left vulnerable for an assortment of different reasons-including surgery or dialysis- to contract a secondary infection. When the first round of antimicrobial treatments is limited in effectiveness due to this growing resistance among bacteria species, healthcare providers have to resort to the use of antibiotics that are more toxic to the patient’s health. This results in longer hospital stays and delayed recoveries. New strains of antibiotic resistant species can cross international borders and are a global issue. Each year, two million people in the United States acquire serious infections due to resistant bacteria that are immune to one or more antibiotics developed to stop that strain. More than 23,00 people die annually as a direct result of a drug resistant pathogen, with an additional 250,000 people who die from a health complication resulting from an infection caused by a antibiotic resistant microbial. The potent treatment eliminates the non-resistant bacteria cells, resulting in a lack of competition for the mutant antibiotic resistant bacteria which allows it to multiply more quickly- making copies of the resistant genes, creating the resistant strains and reducing the effectivity of the drug. Because bacterial cells reproduce so rapidly, some reproducing every 20 minutes, more mutations are possible, as well as more plasmids (A genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium or protozoan.) are going be activated in a shorter period of time creating a high speed evolution. As a result, the search for other methods to fight bacterial infections, other than by using antibiotics, is given so much importance in today’s modern science explorations. It is crucial to utilize different methods to combat bacteria in order to prevent creating a strong resistance and the development of “superbugs” ( a term used to describe microorganisms that are resistant to multiple antibiotics). By utilizing antimicrobial agents, or substances promoting bacterial cell death, to stop resistant strains spread and growth in the first place, the prevalence of harmful bacterial infections decreases. There are thousands of different variances of substances used to ward off bacterial growth and improve health that have high ranges of effectiveness as documented by the International Databank of Infectious Diseases.  In this extended essay, the Antimicrobial agents being focused on are hydrogen peroxide and copper sulfate solutions.Hydrogen peroxide (H2O2) shows a wide efficacy in combating virus’, yeasts, and bacteria. It is commonly used to clean of surfaces and medical tools. It is also commonly used to clean scrapes and wounds. Hydrogen peroxide is a better alternative for the environment then the other commonly used antimicrobial agents such as chlorine-based bleaches, because it is derived from water and oxygen and is categorized as safe by the Food and Drug Administration. Hydrogen peroxide has one more oxygen molecule than water meaning it acts as an oxidizer. This excess oxygen allows hydrogen peroxide to act as an oxidizer when it meets a bacterial cell’s membrane, causing the membrane to be “oxidized” or rust, leaving gaps in the cell’s lipid bilayer and as a result leading to cell death. Hydrogen peroxide can affect human cells too, if a high concentration is used and left on a wound for an extended period of time, however a standard household bottle is usually a 3% solution combined with a weak acid or water, leaving it harmless to human cells. There is some debate on hydrogen peroxides efficacy as an antimicrobial agent, as it is more effective towards gram-positive bacteria (Bacteria that retain the color of the crystal violet stain in the Gram stain. This is a characteristic of bacteria that have a cell wall composed of a thick layer of peptidoglycan), and some bacteria, such as certain strains of staphylococcus, are able to catalyse the molecules and effectively dilute the hydrogen peroxide leaving it less effective. However, hydrogen peroxide can also be a bacteriostatic agent, meaning it inhibits bacteria from reproducing and evolving to become more resistant.  This is because as mentioned previously, hydrogen peroxide has the ability to oxidize bacterial cells leaving them unable to reproduce through binary fission (a form of asexual reproduction commonly used by prokaryotes) and therefore inhibits the bacterial population to grow and further mutate. From this, the progression of infection and resistant strains are avoided.  Copper sulfate (CuSO4) is produced through treating copper with a high concentration of hot sulfuric acid. Copper, is a naturally antimicrobial material. It’s effectiveness has long been observed throughout history, as ancient civilizations such as Egypt found that carrying water in a container made from an copper alloy resulted in less algae or mold growth. Low quantities of copper are necessary for human and bacterial health, but in high quantities copper ions can cause critical damage to bacterial cells. This phenomenon has just begun to be studied, and scientists are just starting to form theories as to how copper has this effect on bacterial cells. One accepted explanation is that copper may cause the cell membrane of bacterial cells to weaken and rupture by interfering with the transmembrane potential, which is a differentiation of the voltage within the cell and outside the cell. It is possible that when copper interacts with a bacterial cell it short circuits this electrical current resulting in a weakened membrane prone to forming holes. These holes in the membrane may also be caused when a copper ion hits the bacterial cell membrane in the presence of oxygen creating oxidative damage. Because the cells outer envelope has been ruptured, copper ions now are able to flood inside of cell, putting many of the bacterial cell’s organelles and main functions at risk. Scientists hypothesize that copper bonds to the cells enzymes resulting in the enzymes being unable to fit into the active sites of their corresponding substrates and therefore halting the processes of metabolism within the cell. This leaves the bacterial cell unable to break down or transport nutrients ultimately resulting in cell death. The molecular mechanisms that create the antibacterial properties in copper are just beginning to be understood and have started being used to create antimicrobial copper alloy touch surfaces (Replacing frequently touched surfaces with antimicrobial materials to prevent the growth and spread of pathogens.) to defend against various microorganisms affecting public health.  The bacteria that will be used to test effectiveness of the 2 antimicrobial agents are Escherichia coli, a harmless and safe strand called k-12 will be used. Escherichia coli, often abbreviated to E.coli, is a gram negative, anaerobic (A microorganism that can live and grow with or without molecular oxygen.) coliform bacterium. It is categorized in the genus Escherichia. In nature, it is found in the lower intestine of endotherms such as humans. Most strains of E.coli are harmless, but certain strains can result in food poisoning. It has the ability, as most bacteria, to transfer copies of its DNA through transduction, resulting in the spread of its genetic information horizontally through a culture of the bacteria. E.coli, like most other prokaryotic bacteria, contain extra genetic material in the form of plasmids, which can provide genetic information that promotes mutation when the bacterium is met with different external threats. These mutations can quickly be passed down generations of E.coli, allowing the bacteria to evolve to withstand the original threat, while the other unmutated E.coli and other bacteria that acted as competition are killed off. This leaves a prime environment for cell reproduction, and therefore cultivates a new population of the evolved/resistant E.coli.Research Question “To what extent is 0.88 M copper sulfate more effective as an antimicrobial agent against strain k-12 Escherichia coli grown on nutrient agar over a 24/48 hour time period compared to 0.88 M hydrogen peroxide?”Hypothesis”Hydrogen peroxide will be more effective at controlling the growth of Escherichia coli than copper sulfate, and therefore result in larger zones of inhibition.”Null Hypothesis”There will be no difference between the effectiveness of Hydrogen Peroxide and Copper Sulfate as antimicrobial agents against strain k-12 Escherichia coli, and any differences in zones of inhibition measurements will be due to chance.”Methods and MaterialsApparatus List-  goggles- medical latex gloves- disposable micropipette tips (for p1,000 and p20 sizes)- micropipettes (p1,000, p20)- standard household disinfecting bleach- 25 plated with nutrient agar (purchased pre-made from Carolina Biological)- incubator- 3 empty petri dishes- 75 blank, sterile, antibiotic sensitivity discs (Disksare from Carolina Biological ¼” in diameter)- K-12 strain Escherichia coli living broth, (2 test tubes with 5 mL of liquid each)- 4 sterile plate spreaders – 100 mL of 0.88 M Hydrogen Peroxide (3%)-100 mL of 0.88 M Copper Sulfate – 4 sterile forceps – biohazard bag – ruler- permanent markerVariablesVariableIndependentAntimicrobial substance used- copper sulfate or hydrogen peroxideDependentDiameter (mm) of zone of inhibition ControleSection of dish tested with no disc, section of dish tested with blank disc Constants/Controlled VariablesLab environment, the amount of time the plates were places in the incubator, and microlitres of antimicrobial agent used for each disc.Preliminary Method : Day #1Obtain/put on safety goggles and gloves.Bleach and wipe down lab area where the experiment will be conducted.Set up biohazard bag.Obtain a p1000 micropipette and set it’s measurement for 350 microliters(l).Set out the 25 petri dishes with nutrient agar.Label the petri dishes 1-25 on their side so that the number is visible without lifting the dish.Using a permanent marker, on the bottom of the petri dish split the dish into 4 equal sections labeled as shown in figure 1. Place all petri dishes face up with lid still in place.Obtain the test tubes of  K-12 strain Escherichia coli living broth.Lift the cover off of an individual petri dish, then using the p1000 micropipette, dispense 350 microlitres of  the broth in the center of the nutrient agar.Using a sterile spreader, evenly distribute the broth across the surface of the dish, then place the cover back on.Repeat steps 10 and 11 on each petri dish until all bacterial lawns are prepared. Stop after every fifth plate and place the dishes upside down with the broth into the incubator at 37°C in order to have as little as possible condensation inside the dishes.Once all lawns are prepared and in the incubator, dispose of any used spreaders into the biohazard bag.Re-bleach and sanitize work space.On one empty petri dish, use sterile tweezers to lay out 25 blank antibiotic sensitivity discs.Set p20 micropipette to 20 microlitres.Use the micropipette to place 20 microlitres of the 0.88 M Copper Sulfate onto each disc.Once completed, thoroughly wash forceps, and place cover on the petri dish containing the completed discs.Label the petri dish as containing the discs with Copper Sulfate. Replace the tip of the p20 micropipette.On a new empty petri dish, use sterile forceps to lay out 25 blank antibiotic sensitivity discs.On each disk, use the micropipette to place 20 microlitres of 0.88 M Hydrogen peroxide onto each disc.Once completed, thoroughly wash forceps, and place cover on the petri dish containing the completed discs.Label the petri dish as containing the discs with Hydrogen peroxide.On the last empty petri dish, use sterile forceps to lay out 25 blank antibiotic sensitivity discs.Place lid on petri dish, and label the petri dish to be containing the blank discs that will be used as controls.Place all three petri dishes containing the discs in a safe place away from contamination to dry.Let the 25 petri dishes incubate for 24 hours so that the Escherichia coli living broth may form lawns.Let the prepared antimicrobial discs sit for 24 hours at room temperature to let the given antimicrobial liquid have time to be fully absorbed by discs. Day #2Obtain/put on safety goggles and gloves.After 24 hours has passed, remove all petri dishes from the incubator, and check to make sure an even lawn of Escherichia coli has grown.Lay all the discs down on a clean and sterile lab surface upside-down to prevent the condensation that collects on the lid of the petri dish from dripping onto the lawns.Obtain the petri dish containing the Copper Sulfate antimicrobial discs.Using sterile forceps, place a disc infused with Copper Sulfate firmly onto the designated labeled section of each created bacterial lawn of the 25 petri-dishes.Obtain the petri dish containing the Hydrogen Peroxide antimicrobial discs.Using sterile forceps, place a disc infused with Hydrogen Peroxide firmly onto the designated labeled section of each created bacterial lawn of the 25 petri-dishes.Lastly, obtain the petri dish containing the blank antimicrobial discs.Using sterile forceps, place a disc firmly onto the designated labeled section of each created bacterial lawn of the 25 petri-dishes.Once all discs are in place on the 25 petri dishes, place all the petri dishes back into the incubator upside-down at 37°C.Dispose of any unneeded materials, and use bleach to disinfect the surfaces used for the experiment.Day #3Obtain/put on safety goggles and gloves.After the plates have incubated for 24 hours with the discs, remove all plates and place them upside-down on a sterilized lab surface.Set p20 micropipette to 12 microlitres.Using the micropipette, put 12 more microliters of the 0.88 M Copper Sulfate onto each disc already containing copper sulfate in it’s designated section on each of the 25 discs.Change the micropipette tip.Using the micropipette, put 12 more microliters of the 0.88 M Hydrogen Peroxide onto each disc already containing hydrogen peroxide in it’s designated section on each of the 25 discs.Place all discs back into the incubator upside-down set to 37°C for an additional 24 hours.Dispose of any unneeded materials, and use bleach to disinfect the surfaces used for the experiment.Day #4Obtain/put on safety goggles and gloves.After the 24 hour incubation period, remove all petri dishes and place them upside-down on a sterilized lab surface.Using a ruler, measure the zone of inhibition as directed by figure 3, of each antimicrobial disc to the nearest millimeter (+/-1mm), for all of the 25 plates and record, separately recording the zone measurements for each section of the petri dish (see chart 1).After all data is collected, pour a 10% disinfecting bleach solution into all 25 petri dishes and into any open tubes of Escherichia coli broth and let them soak with bleach for 10 minutes to kill off the bacterial colonies.Dispose of bleach from petri dishes and test tubes and place the dishes and tubes into a biohazard bag. If any sealed, unused  Escherichia coli broth remains, make sure they are labelled, and place into a refrigerator, used for scientific purposes, to save.  Clean lab surfaces and ruler with bleach, then rinse with water.Chart 1: Resulting Zones of Inhibition from Control Variables, Copper Sulfate and Hydrogen PeroxideChart 2: Mean Zones of Inhibition and Standard Deviation of Measured Zones of Inhibition for Copper Sulfate and Hydrogen Peroxide                                                                                                                      Antimicrobial Agent’s Zones being Measured Mean Zone of Inhibition/mm (+/-0.5)Standard Deviation of Measured Zones of Inhibition/mm (+/-0.5)Copper Sulfate (CuSO4) 0.88 molarity10.12.3Hydrogen Peroxide (H2O2) 0.88 molarity9.41.9Graph 1: Graphed Mean Zones of Inhibition and Standard Deviation of Measured Zones Inhibition Compared Between Copper Sulfate and Hydrogen PeroxideT-TestIn order to statistically test whether copper sulfate’s greater average mean of a zone of inhibition compared to hydrogen peroxide is significant, a two tailed t-test for two independent samples was carried out to investigate whether there is a significant difference. T-test formula: Degrees of freedom= N1+  N2 – 1 = 49tcalc = 1.349tcrit(p = 0.05) = 2.01Because the t test value tcrit = 2.01 is greater than the critical value tcalc = 1.349 at p= 0.05, The Null Hypothesis should be accepted (see page 5). This is because the t-test indicates that the  probability that the average mean of zones of inhibition for copper sulfate are larger than the average mean of zones of inhibition for hydrogen peroxide are just due to chance is greater than the acceptable probability that would indicate its differences are most likely caused by efficacy.  The original hypothesis that hydrogen peroxide will be more effective at controlling the growth of Escherichia coli than copper sulfate, and therefore result in larger zones of inhibition was not supported in this experiment. It appeared that copper sulfate resulted in a larger zone of inhibition judging by the means, but this was not supported by the t-test analysis. Therefore, it can be concluded that 0.88 M copper sulfate and 0.88 M hydrogen peroxide are equally effective antimicrobial agents against strain k-12 Escherichia coli grown on nutrient agar over a 24/48 hour time period when comparing resulting zones of inhibition. Evaluation Hydrogen peroxide is seen in the scientific community to be more efficient as a bacteriostatic agent, meaning hydrogen peroxide is able to inhibit and control the reproduction of bacteria. Thus hydrogen peroxide is best used to inhibit culture growth, but is found less effective when used directly as an antimicrobial agent to kill off the bacterial colonies such as Staphylococcus aureus and other forms of Escherichia coli . In contrast to hydrogen peroxide, copper alone as a metal and it’s alloys have been studied and shown to eliminate over 99.9% of various E.coli strains as already existing colonies after just 2 hours of exposure. Results like this were not observed in the experiment. However, copper sulfate was used instead of a copper alloy as it provided for the use of the antimicrobial disks. The use of copper sulfate, which was a more practical approach given the equipment limitations and time constraints of the highschool lab setting, may have lowered the results as it is a solution of impure copper.  Therefore, the effectiveness of the hydrogen peroxide may have been higher if it was compared against copper sulfates ability to prevent the growth of k-12 strain of Escherichia coli as opposed to directly killing existing colonies. Such a comparison would be interesting to investigate further. After re-examining the procedure post experiment, it was noted that the procedure of the experiment could be altered to provide more accurate results, this would mean having lower standard deviation values so that the data collected would be more precise with all the measured zones of inhibition being closer to the mean values.Some recommended procedural changes are as follows:Increasing the initial amount of each antimicrobial solution used on the sensitivity discs. The initial 20 microlitres of solution proved to be too weak, meaning not concentrated enough, to show fast and visible results in terms of zones of inhibition, resulting in the need for and additional 12 microlitres of the antimicrobial solutions to be injected into the discs, which provided a high enough concentration of antimicrobial agent to lead to the cell death of Escherichia coli  and thus produce results . Testing out different higher molarities of copper sulfate and hydrogen peroxide, as only 0.88 molarity solutions were used. This could lead to more varied results in terms of zones of inhibitions, and a more effectively distinguish whether copper sulfate or hydrogen peroxide is more efficient as an antimicrobial agent against k-12 Escherichia coli.Expand the number of petri dishes used. This would increase the data pool and lead to more accurate and decisive results while decreasing the effects that any possible error could have made on the data.

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