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The Inhibitory Affect on Growth of E.docx

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Biology (Biological Sciences)
Richard Moses

Gillian Goobie Tuesday, February 10, 2009 The InhibitoryAffect on Growth of E. coli and M. luteus due to the ChemicalAgents Scope, Mr. Clean and Mercury Introduction: The domain bacteria is comprised of prokaryotes, which are unicellular organisms that do not contain membrane bound organelles or nuclei and can function alone or in colonies. Prokaryotes are essential to all ecosystems and to all life we encounter.As some bacteria are harmful, chemical agents have been developed to retard the growth of and kill these bacteria. Specific chemical agents often target bacteria based on their cell wall composition. Gram-positive bacteria have a cell wall comprised of a thick peptidoglycan layer, whereas Gram-negative bacteria have a lipopolysaccharide outer cell wall above a thin peptidoglycan layer. This outer wall in Gram-negative bacteria often makes it less susceptible to some chemical agents (Campbell et al. 2008). In this experiment, the effect of the chemical agents Scope, Mr. Clean and Mercury on the growth of the bacteria, Escherichia coli and Micrococcus luteus, is examined. Through examining the zones of inhibition in both species in response to exposure to the chemical agents, the relative survival of the two species can be analyzed. E. coli is a Gram-negative bacteria that is present in the human digestive tract, but may also cause illnesses such as traveler’s diarrhea and urinary tract infections (Biology 108 lab manual, 2008). M. luteus is a Gram-positive bacteria that is commonly present on human skin and can cause meat to spoil (Biology 108 lab manual, 2008). The purpose of this experiment is to test the effect of the three chemical agents on these bacteria, providing insight on their action in bacterial inhibition. This study is relevant because bacterial infections are a common risk for humans, and in order to stay safe, we must come up with effective methods in preventing contamination. Through testing the effectiveness of various chemical agents, the best antiseptic and disinfecting methods can be determined, thus helping to prevent the spread of infections. If M. luteus and E. Coli are exposed to Mr. Clean, Scope and Mercury, the growth of these bacterial species will be inhibited in varying amounts. Mercury will likely be the most effective anti- microbial agent against both the Gram-positive M. luetus and the Gram-negative E. coli because in high concentrations the Hg will poison both types of bacterial cells. In both bacteria the mercury metal salt will react with proteins in the cell membrane leading to insoluble heavy metal protein salts, which will cause the poisoned bacteria to clump together in a heavy mass in which they cannot survive (Rahn, 1945). Mr. Clean’s main active ingredient is NaOH, which is effective in killing Gram-negative bacteria such as E. coli, but should be less effective in killing Gram-positive bacteria such as M. luteus. NaOH causes the denaturation of integral membrane proteins and the saponification of membrane lipids, but the thick peptidoglycan cell wall of Gram-positive bacteria should keep the cells intact, whereas gram-negative bacteria are more likely to lyse due to their thin peptidoglycan layer (Amoroso et al. 1994). Mr. Clean will likely be less effective than Mercury in both cases. Scope contains more than one anti-microbial agent and thus will likely be effective against both bacteria.Alcohol is able to penetrate the cell wall of bacteria and disrupt hydrogen bonding of cell membrane proteins, causing the formation of new hydrogen bonds with the alcohol (Campbell et al. 2008).According to the Biology 107 Laboratory Manual (2008), alcohol dissolves the lipopolysaccharide layer of Gram-negative bacteria, which suggests that E. coli are more susceptible to the alcohol than M. luteus. Sodium benzoate and benzoic acid are two other chemicals in Scope that are effective in killing Gram + bacteria (Phillips and Walker, 2007). Thus Scope will affect both E. coli and M. luteus. It will likely be more effective in inhibiting bacterial growth than Mr. Clean due to its various chemical agents. Scope will likely be less effective in inhibiting growth than mercury because scope is handled by humans, suggesting it is not very toxic, whereas mercury is a very poisonous substance that people avoid handling because of its high toxicity. Materials and Methods: The Biology 108 lab manual (2008) states that using sterile technique, three PCAplates are inoculated with M. luteus and three with E. coli. There is a control plate for each bacterial species that has not been impregnated by a chemical agent. The other plates have one of each disk impregnated completely by the chemical agent solutions placed in one of three sections on each experimental PCA plate. There are two experimental plates for each species to incorporate replication, ensuring viable results.After preparation the cultures are allowed to cultivate and diameters of zones of inhibition are recorded in mm around the disks of each plate the next week. Results: It is seen from the results summarized in Table 1 that the three chemical agents tested inhibited the growth of both types of bacteria in varying amounts. In looking at the control groups for M. luteus and E. coli, there is no zone of inhibition when no chemical agent has been used. However, when a chemical agent has been used, there are varying zones of inhibition depending on the species and the agent. The respective zones of inhibition represent the amount that a specific chemical agent inhibits bacterial growth, and by measuring the diameter of each zone of inhibition, the growth prevention caused by each chemical agent can be quantified. It was found that the Mr. Clean treatment resulted in the smallest zones of inhibition for both species. There was a slightly larger zone of inhibition of M. luteus (with 7.17mm inhibition) than E. coli (with 6.67mm inhibition) with Mr. Clean. Scope resulted in the second largest zones of inhibition, inhibiting the growth of M. luteus (with 15.5mm inhibition) more than E. coli (with 10.5mm inhibition). Mercury resulted in the largest zones of inhibition for both species with E. coli having a slightly larger zone of inhibition (29.5mm) than M. luteus (26.0mm). Table 1: The average diameters of zones of inhibition as induced by three chemical agents on 10mm diameter disks placed in PCAagar plates on the growth of E. coli and M. luteus. Diameters of Zones of Inhibition (mm) Mercury Mr. Clean Scope Control Organism Escherichia Coli 29.5 6.67 10.5 0 Micrococcus luteus 26.0 7.17 15.5 0 Discussion: As was seen in the control tests, there were no zones of inhibition, demonstrating that the PCA medium in which the bacteria were grown did not inhibit the growth of either bacterial species. It also demonstrates that the paper disks, which were impregnated with chemical agents in the experimental plates, did not inhibit bacterial growth in the absence of chemical agents. As was predicted in the hypothesis, mercury was the most effective chemical agent in inhibiting the bacterial growth of both species due to its extreme toxicity to most living organisms. The mercury was, however, slightly more effective in inhibiting the growth of E. coli than it was in inhibiting M. luteus. The diameter of the zone of inhibition on E. coli was 29.5mm, suggesting that mercury had a very strong inhibiting effect on the bacteria. The slightly smaller diameter of 26.0mm for M. luteus still suggests a very strong inhibiting effect. Mercury is an effective chemical agent because it interacts with the cell membranes and dissolves into the bacterial cytoplasm, interrupting cell function by forming heavy, insoluble metal protein salts (Rahn,1945).Apossible reason for E. coli being slightly more inhibited by the mercury than M. luteus might be because of interaction of the mercury with proteins, lipoproteins and perhaps lipopolysaccharides and phospholipids of the outer cell wall (Kaiser, 2008). In Gram-negative bacteria the mercury would immediately come in contact with proteins of the outer membrane to which it could combine and inhibit the cell faster and more effectively than it would Gram-positive bacteria. In Gram-positive bacteria, the mercury has to pass through the thick peptidoglycan layer in order to get to the cell membrane or through the cell wall to begin the process of interaction with and thus inhibition of the proteins. Also as was predicted, Scope was the second most effective inhibitor of both species of bacteria due to the large amount of various disinfectants it contains. Scope was significantly more effective in inhibiting the growth of M. luteus, with a zone of inhibition of 15.5mm, than it was in inhibiting E. coli, with a zone of inhibition of 10.5mm. Scope contains
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