Effectiveness of Bacterial Control Agents By Megan Messer Abstract In this modern age, many methods of bacterial control are

available, including antiseptics and disinfectant cleaners, antibiotic drugs, and ultraviolet light sources, and deciding which method to use can be difficult. The best way to decide on a method is to see how well it inhibits the growth of different bacterial species, which is why we experimented with several household cleaners, antibiotic drugs, and ultraviolet light on several gram positive and gram negative bacteria. The cleaners and antibiotics were tested by placing saturated dots of each chemical on petri dishes of several species of bacteria and observing their growth patterns. The ultraviolet light was tested by exposing a gram positive and gram negative species for varying exposure times. The cleaners Fabuloso, Pinesol, and Lysol were fairly successful against different species of bacteria due to the chemicals they contained, and Listerine was unsuccessful due to its low alcohol concentration. The broad spectrum drugs tetracycline, chloramphenicol, and streptomycin were more successful than penicillin, which is narrow spectrum. Penicillin and ampicillin were also unsuccessful due to penicillin resistant genes. The ultraviolet light affected the gram negative species more severely than it affected the gram positive species, due to the spore forming characteristics of Bacillus species. Introduction Ever since people have been aware that bacteria cause infections and diseases, we have been devising ways to eliminate or reduce the number of bacteria on surfaces or within the body. Chemicals are the weapon of choice against bacteria in the home, as included in home cleaners and personal hygiene products. Disinfectants are intended to target vegetative microbes on objects such as kitchen and bathroom surfaces, whereas antiseptics are used on body surfaces (Talaro and Chess 2012). In this experiment, we tested the effectiveness of several household disinfectants and antiseptics, to see if they actually eliminated 99.9% of germs as many products marketed today seem to claim. Chemicals to eliminate bacteria are also important in medical settings, because treatments are needed to cure the many infections and diseases caused by harmful bacteria. Antimicrobial drugs can be derived naturally from the metabolic products from microorganisms that inhibit other microorganisms, which are called antibiotics. They can also be obtained from natural sources and then chemically modified (semisynthetic drugs), or synthesized totally in a laboratory without the use of natural sources (synthetic drugs) (Talaro and Chess 2012). In commercial production settings such as food packaging and drug manufacturing, ultraviolet light is used to eliminate bacteria on surfaces used to prevent contamination to their products (Zeuthen 2003). For this experiment, the antiseptic Listerine was used, as well as the disinfectants Lysol, Fabuloso and Pinesol. The bactericidal actions of household cleaners depend on the chemicals

they contain. Lysol contains phenolic compounds, which disrupt bacterial cell walls and membranes and damage proteins. Even in low concentrations, they inactivate enzymes necessary for the bacteria to function (Talaro and Chess 2012). Listerine contains alcohol as its active ingredient, which works against bacteria by disrupting the lipids in their cell membranes. However, alcohol must account for at least 50% of the cleaners concentration to be effective, and Listerine only contains 21.6% alcohol (Listerine Ingredients 2012). Fabuloso contains a surfactant called sodium dodecyl benzene sulfonate. Surfactants work in cleaners by lowering the surface tension between two unlike substances, making it easier to remove them during cleaning (Talaro and Chess 2012). Pinesol contains 8-10% pine oil, which is a phenolic compound like the one in Lysol. It also contains isopropyl alcohol (rubbing alcohol), which works by disrupting bacterial cell membranes (Talaro and Chess 2012). In this experiment, the effectiveness of common antibiotic drugs was tested. Antibiotics target many different structural characteristics of bacteria, and can be specific to certain groups of bacteria (narrow spectrum) or effective against many different types of bacteria (broad spectrum), depending upon its mode of action (Talaro and Chess 2012). Penicillins structure has a beta lactam group that interferes with proteins that make the bacterial cell wall, making the bacteria vulnerable to lysis. Penicillin has a narrow spectrum and is most effective against gram positive bacteria, because its external cell wall is more easily penetrated by the penicillin (Ophardt 2003). Ampicillin is a modified penicillin drug that has a side chain allowing it to pass through the outer membrane layer of gram negative bacteria, making it a broad spectrum drug (Talaro and Chess 2012). Chloramphenicols structure allows it to interfere with protein synthesis by blocking peptide bond formation within the bacterial cell, making it a broad spectrum drug. Streptomycin is part of the aminoglycoside antibiotic group. Aminoglycosides contain one or more amino sugars and a six carbon ring called aminocyclitol, a substance which is naturally produced by the fungi Streptomyces and Micromonospora, or synthetically produced. This substance works by binding to a bacterias ribosomal subunit and blocking protein synthesis, making it a broad spectrum drug (Todar 2012). Tetracycline also targets protein synthesis by attaching to bacterial ribosomes, making it another broad spectrum drug (Ophardt 2003). The use of ultraviolet (UV) light is a powerful way to cut down the number of bacteria on surfaces due to the damage it causes to the bacteria cells. When a bacterial cell is first exposed to UV light, its DNA is damaged, because the rays cause the pyrimidines, cytosine and thymine, that are adjacent to each other on a DNA strand to form abnormal bonds with each other, resulting in an incorrect DNA strand (Olson 2011). This causes the cell to stop growing because its instructions to make proteins, the DNA strand, is now incorrect. UV light also cause highly reactive free radical compounds to form, which inhibit vital cellular processes by binding to RNA, DNA, and proteins (Talaro and Chess 2012). It is hypothesized that due to the chemical differences in the household cleaners, they will have varying effectiveness against different species of bacteria. For example, alcohol must be in a

certain concentration to be effective, and Lysol and Pinesol both contain phenolics, so they are likely to react similarly against the bacterial species. In the antibiotic effectiveness experiment, it is hypothesized that the broad spectrum drugs ampicillin, chloramphenicol, streptomycin, and tetracycline will be effective against more species than the narrow spectrum penicillin drugs. Also, penicillin resistant genes are extremely common, so the drugs penicillin and its relative ampicillin are unlikely to be effective. For the ultraviolet light experiment, it is hypothesized that the harmful effects of ultraviolet rays will cause progressively worse damage to the bacteria the longer they are exposed. Materials and Methods The antiseptics and disinfectants were tested by first obtaining samples of Staphylococcus epidermidis, Enterobacter aerogenes, Staphylococcus aureus, Proteus vulgaris, Bacillus cereus, Eschericia coli, and Micrococcus luteus. Sterile swabs were used to apply each species onto a separate petri dish, using straight lines in several directions, taking care to cover the entire surface of the dish with an equal amount of bacteria. Next, disks of paper were saturated with Lysol, Listerine, Fabuloso, Pinesol, and laboratory table cleaner. Forceps were passed through a Bunsen burner flame and then used to place a dot of each chemical on each petri dish for a total of six plates each with five dots of chemicals. The petri dishes were labeled to identify the location of each disinfectant. The petri dishes were incubated for one week, and then observed for changes. For the antibiotics experiment, the bacterial species Bacillus cereus, Proteus vulgaris, Eschericia coli, Enterobacter aerogenes, Staphylococcus aureus, and Staphylococcus epidermidis were used. Several types of antibiotics were used, including penicillin, chloramphenicol, streptomycin, ampicillin, and tetracycline. The procedure for this experiment began with using a sterile swab to sweep each bacterial species all over the surface of its own petri dish, taking care to cover the entire surface, making a total of six plates. Then, using forceps sterilized by Bunsen burner flame, dots containing each antibiotic were placed onto each dish, making a total of 5 different antibiotics per plate. The plates were labeled with the first letter of each antibiotic name to identify their locations. These plates were then incubated for one week, and then observed for changes. For the ultraviolet radiation experiment, the gram positive species Bacillus cereus and the gram negative species Serratia marcescens were used. A sterile swab was used to cover the entire surface of five petri dishes for each species, for a total of ten petri dishes, five for each species. For each species, the five petri dishes were labeled lid, 30 seconds, 60 seconds, 90 seconds, and no lid. The dishes labeled lid had a complete lid covering, and the 30, 60, and 90 second dishes had partial lids with letters cut out of them. The dishes labeled no lid had no lid covering. The dishes were then placed under an ultraviolet light source, with 170 millimeters distance between the light source and the dish. A timer was set, and the dishes labeled 30 seconds were removed after 30 seconds, the dishes labeled 60 seconds were removed after 60 seconds, and the dishes

labeled 90 seconds were removed after 90 seconds. The lid and no lid plates were also removed after 90 seconds. Immediately after being removed from under the ultraviolet light source, the plates were covered with tin foil. These plates were incubated for one week and then observed for changes. Results Resistant <10mm All species to Listerine (0) E. aerogenes to all chemicals(0) P. vulgaris to all chemicals(0) E. coli to Lysol (0) M. luteus to Fabuloso (0) Intermediate 10-14mm S. aureus to Pinesol M. luteus to Lysol Susceptible >14mm S. epidermis to Fabuloso S. epidermis to Lysol S. aureus to Fabuloso S. aureus to Lysol B. cereus to Pinesol B. cereus to Lysol B. cereus to Fabuloso M. luteus to Pinesol E. coli to Fabuloso E. coli to Pinesol

Table 1 Chart of the results of the disinfectant and antiseptic test.

Resistant<13mm All species to penicillin (0) S. aureus to ampicillin B. cereus to ampicillin S. epidermidis to all drugs(0) E. aerogenes to tetracycline E. aerogenes to ampicillin (0) P. vulgaris to ampicillin (0)

Intermediate 14-16mm S. aureus to streptomycin P. vulgaris to tetracycline

Susceptible >17mm S. aureus to chloramphenicol S. aureus to tetracycline B. cereus to streptomycin B. cereus to chloramphenicol B. cereus to tetracycline E. coli to ampicillin E. coli to tetracycline E. coli to streptomycin E. coli to chloramphenicol P. vulgaris to streptomycin P. vulgaris to chloramphenicol E. aerogenes to streptomycin E. aerogenes to chloramphenicol

Table 2 Chart of the results of the antibiotic sensitivity test.

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To measure to results of the disinfectants and antibiotics experiments, the diameter in millimeters was taken for each clear zone of inhibition around each dot of disinfectant or antibiotic (Table 1, Table 2, Figures 1-5). The S. epidermidis plate had many white colonies on its surface (Figure 4). For the ultraviolet radiation test, the amount of colonies present was noted. The B. cereus plate with a lid had abundant growth (Figure 11). On the region exposed to ultraviolet radiation for the 30 second B. cereus plate, there were lots of colonies, but less growth than the regions that were not exposed to the light (Figure 12). The region exposed to ultraviolet radiation on the 60 second plate had fewer colonies than the 30 second plate, and the 90 second plate had even fewer colonies than the 60 second plate (Figures 13,14). For the 60 and 90 second plates, the regions not exposed to light also had abundant growth (Figures 13,14) . The plate with no lid had only four colonies (Figure 15). The S. marcescens plate with a lid had abundant growth (Figure 6). The regions exposed to ultraviolet radiation on the 30, 60, and 90 second plates had no growth, and the regions not exposed to ultraviolet radiation had abundant growth (Figures 7,8,9). The S. marcescens plate with no lid also had no growth (Figure 10). Discussion The measurements of the zone of inhibition gave us an idea of how effective each chemical agent of control was. Zones that were less than 10 mm were considered resistant, zones 10 to 14 mm were considered intermediate, and zones 14 or greater were considered susceptible to the chemical agent. The cleaner with the best results was Fabuloso, which produced susceptible zones of inhibition for four species of bacteria, S. epidermidis, S. aureus, B. cereus, and E. coli, which suggests that the surfactant action of the sodium dodecyl benzene sulfonate was effective (Table 1). Both Lysol and Pinesol produced three susceptible zones of inhibition and one zone of intermediate inhibition. S. epidermidis, S. aureus, and B. cereus were susceptible to Lysol, and M. luteus was intermediate. B. cereus, E. coli, and M. luteus were susceptible to Pinesol, and S. aureus was intermediate (Table 1). Bacteria reacted similarly to Pinesol and Lysol, because they both contain phenolic compounds, and these results support the hypothesis that both phenoliccontaining cleaners will react similarly. Phenolic compounds kill bacteria by damaging their cell membranes and enzyme systems (Talaro and Chess 2012). All species were resistant to the antiseptic Listerine. This was expected because Listerine contains only 21.6% alcohol, and alcohol is only effective against bacteria by disrupting lipids in their cell membrane if it is at least 50% of the concentration (Talaro and Chess 2012). These results support the hypothesis that Listerine will not be effective due to its low alcohol concentration. P. vulgaris and E. aerogenes did not have any zones of inhibition, which means that they are left behind when these cleaners are used. Both P. vulgaris and E. aerogenes are gram negative, so they were likely resistant to the chemicals due to their protective outer membrane that acts as a

barrier to the intake of disinfectant chemicals ("Factors Affecting the Efficacy of Disinfection and Sterilization." 2008). For the antibiotics experiment, species that had zones of inhibition of 13 mm or under were considered resistant, species that had zones of 14-16 mm were considered intermediately susceptible, and species with zones 17 or greater were considered susceptible to the antibiotic. Penicillin has been in use for a while, so it is not surprising that no species showed susceptibility to it, because evolution has favored the survival of bacteria with penicillin resistant genes (Talaro and Chess 2012). A close relative of penicillin, ampicillin, was also not successful in inhibiting the growth of 5 species of bacteria out of 6 that we tested. Ampicillin is more broad spectrum than penicillin, because it has a side chain that facilitates its transport across gram negative outer membrane layers. However, ampicillin is still affected by penicillin resistance genes (Talaro and Chess 2012). This data supports the hypothesis that penicillin will be less effective due to its narrow spectrum of action and the prevalence of penicillin resistance genes. Tetracycline, chloramphenicol, and streptomycin were all very successful in inhibiting bacterial growth, as indicated by their large diameters of zones of inhibition. Streptomycin intermediately inhibited the growth of S. aureus, and the species P. vulgaris, E. aerogenes, and E. coli were considered susceptible. Streptomycin inhibits bacterial protein synthesis by binding to their ribosomes (Talaro and Chess 2012). It was expected that streptomycin would be more effective against gram negative bacteria, because the drug passes through the thin outer membrane and inner peptidoglycan layers easier than through the thick peptidoglycan of gram positive bacterial cell walls (Ophardt 2003). Tetracycline intermediately inhibited the growth of P. vulgaris, and the species S. aureus, B. cereus, and E. coli were considered susceptible. Tetracycline is a broad spectrum drug that targets the bacterial cells protein synthesis process by attaching to the ribosomes, which is why it was effective against both gram positive and gram negative bacteria (Ophardt 2003). Tetracycline has been used regularly for decades; so many bacteria have developed tetracycline resistance, which explains why tetracycline did not inhibit the growth of all species (Todar 2012). Chloramphenicol was the most successful, inhibiting the growth of the five susceptible species S. aureus, B. cereus, E. coli, P. vulgaris, and E. aerogenes. Chloramphenicol interferes with protein synthesis by blocking peptide bond formation. Chloramphenicol is not used regularly to treat medical infections, because it has the potential to harm human bone marrow cells causing aplastic anemia (Ophardt 2003). Since the drug is not used often, many species of bacteria have not developed resistance, as supported by this experiments data. Tetracycline, chloramphenicol, and streptomycin are all broad spectrum drugs, so it is expected that they were the most effective in inhibiting the growth of bacteria.

The plate containing S. epidermidis was likely contaminated, because there were white, raised circular colonies of bacteria that did not occur on any other lab groups plate. The plate was likely contaminated by improper practice of aseptic technique, such as an uncovered sneeze. The plate showed no growth inhibition by any of the antibiotics, but since the identity of the contaminant is not known, results are not conclusive to support whether it was the S. epidermidis or the contaminant that was resistant. Ultraviolet light causes damage to cells by mutating their DNA and creating harmful free radicals. For the ultraviolet light experiment, the gram positive B. cereus species showed more resistance to light damage than the gram negative S. marcescens, as observed by the visible growth of colonies of B. cereus following incubation. Bacillus species such as B. cereus have the ability to form extremely resilient endospores with many-layered protective coats when subjected to harsh environmental conditions such as desiccation by light, explaining why B. cereus was more resistant to the light damage than S. marcescens (Talaro and Chess 2012). The areas of the plate that were covered with cardboard or a lid showed growth, because ultraviolet rays are weak and the bacteria must be exposed directly to be vulnerable to damage (Olson 2011). For the B. cereus colonies, the number of colonies was progressively smaller according to the length of time they were exposed to the ultraviolet light. The plate exposed for 30 seconds had the most growth, the plate exposed for 60 seconds had less growth, and the plate exposed for 90 seconds had the least amount of growth. These results support the hypothesis that a longer exposure time to ultraviolet light will produce more damage. It can also be explained by the fact that bacterial cells have mechanisms to repair damage due to mutations in their DNA, but if the cells are exposed to ultraviolet light for longer time periods they are unable to keep up with the accumulating damage (Olson 2011). However, S. marcescens showed consistent damage for each of the 30, 60, and 90 seconds of exposure, but S. marcescens also did not have the spore forming capabilities like B. cereus. This experiment was useful in determining the effectiveness of household cleaners, antibiotic drugs, and ultraviolet light. Bacterial control is a very popular practice, and it is important to be aware of what agents are actually effective. Thorough knowledge of bacterial control methods are very important to health professionals, scientists, and even home owners, so they can utilize the best methods possible to prevent infection from contaminated surfaces or treat infections to improve their quality of life and the lives of others.

References "Factors Affecting the Efficacy of Disinfection and Sterilization." CDC, 2008. Web. 6 Aug 2012. <cdc.gov>. "Listerine Ingredients." Listerine. Johnson and Johnson, 2012. Web. 6 Aug 2012. <listerine.com/products>. Olson, Andrew. "Death Rays: What Duration of Ultraviolet Radiation Kills Bacteria." Science Buddies. N.p., 2011. Web. 6 Aug 2012. <sciencebuddies.org>. Ophardt, Charles. "Introduction to Drug Action:Antibiotics." Virtual Chembook . Elmhurst College, 2003. Web. 6 Aug 2012. <elmhurst.edu>. Talaro, Kathleen, and Barry Chess. Foundations in Microbiology. 9th. New York : McGraw Hill, 2012. Print. Todar, Kenneth. "Antimicrobial Agents in the Treatment of Infectious Disease." Todar's Online Textbook of Bacteriology. N.p., 2012. Web. 6 Aug 2012. <textbookofbacteriology.net>. Zeuthen, Peter. Food Preservation Techniques. First edition. Cambridge: Woodhead Publishing Limited, 2003. eBook.