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Saddleback BIO 3B - The Change in Growth Inhibition of Escherichia coli

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The Change in Growth Inhibition of Escherichia coli Across Generations in Conditions ofVarying pH Michael Harvey, Nika KusmierzDepartment of Biological Sciences, Saddleback CollegeMission Viejo, CA 92692.Abstract When environmental conditions change, members of a population that possess favorable traitstend to reproduce more than those that do not. This leads to evolution by means of naturalselection. A paper chad of high or low pH was added to a petri dish swabbed with E. coli. After a24 hour incubation period, an area of growth inhibition was observed around the chad. Then aportion of bacteria was scraped out from within the zone of inhibition and placed in nutrientbroth to be recultured in agar. After the a new sample petri dish was made from the broth, thissample then became trial 2. Group 1 consisted of 13 of these samples at low pH, group 2 was thecontrol consisting of 13 samples at a pH of 7, and group 3 consisted of 13 samples at high pH. Itwas hypothesized that bacteria will show less growth inhibition in the second and third trials thanthe first. The average diameter of growth inhibtion in group 1 of trial 1 was 0 cm, while trial 2was also 0 cm, and trial 3 was 0.87 cm. The average diameter of growth inhibition in in group 2of trial 1 was 0 cm, while trial 2 was also 0 cm, and trial 3 was 0.66 cm. All trials of the group 2had an average diameter of inhibition of 0 cm. There was a larger diameter of growth inhibitionin trial 3 than trial 2 of group 1 (p = 6.34 x 10-10, 1-tailed, paired t-test). There was a largerdiameter of inhibition in group 3 of trial 3 than trial 2 (p = 2.26 x 10-6, 1-tailed, paired t-test).There was no difference in growth inhibition of group 3 of trial 3 than group 1 (p = 0.07564, 2-tailed, unpaired t-test). The hypothesis was neither supported or refuted because not enough trialswere conducted.Introduction Organisms must adapt to changing conditions. Adaptation through natural selection is thereason for the incredible biodiversity on Earth. One such environmental condition is the acidityof the surrounding environment (Simon 1983). Prokaryotic cells are adapted for living in aparticular range of acidity. Escherichia coli is a bacteria found in the digestive tract of animals. Itserves as a model organism in scientific experiements (Cohen 1997). If the acidity becomes toohigh or low, those with a genetic advantage will reproduce and those without will die off off(Witkin, 1976). The pH range for survivability of E. coli is 4.9 to 9, while ideal pH is 6-7 (Small,1994). This study simulated the effects of natural selection on a population by studying thedifference in growth inhibition of E. coli from one trial to the next. A previous study found thatthe fitness of the experimental populations, as measured by the rate at which each populationgrew, increased rapidly for the first 5,000 trials and more slowly for the next 15,000 trials whencompared to the ancestral population (Cooper 2000). It was hypothesized that the second trialwould have a smaller diameter of inhibition than the first, and the third trial smaller than thesecond in all experimental groups. This is in support of the theory of natural selection; that the E.coli cells that lack the ability to survive in acidic medium will die off, and those that can survive1will take their place, increasing the frequency of a particular allele in a population (Thauer1997).This study has practical applications in biotechnology, where bacteria can be modifiedthrough natural selection to serve a purpose. One such purpose may be environmental wastecleanup. Bacteria can be bred to digest a certain organic comound, thus detoxifying a particulararea of the compound (Witkin 1976). A further application would be having an accuratemathematical formula that can model the rate of natural selection given the variables of time acidcontent (Hauke 2003). Further inquiries can determine the genetic makeup of a population at aspecific time based on the percentage of bacteria with an advantageous gene (Dahlia 1999).Materials and Methods A sample of E. coli was spread over agar in a petri dish. Two paper chads were then added toeach petri dish containing E. coli, and then these samples were allowed to incubate for 24 hours.Group 1 was the low pH group, group 2 was the control, and group 3 was the high pH group.Each group consisted of 13 samples for a total of 39 samples per trial. Group 1 had chads with apH of 5 added, group 2 had chads with a pH of 7 added, group 3 had chads with a pH of 9added. These pH’s were chosen because they are the outer limits in which E. coli can grow(Cohen 1997). Hydrochloric acid or sodium hydroxide was added to water to alter the pH, andwhen the proper pH was verified using pH paper. Using a micropipette, 0.05 mL of acid or basesolution was added to each chad. After 24 hours, the diameter of growth inhibition for eachsample was measured. A loop was then used to scrape out a portion of bacteria inside the zone ofinhibition. Inside the area of inhibition is where you find the highest concentration of bacteriawith resistant genes (Luria 1943). Each portion of bacteria was then placed in nutrient broth andallowed to incubate for 48 hours for a total of 39 broths. Forty-eight hours is required to achieveadequate proliferation in 50 mL of moderate nutrient broth (Kaeberlein 2002). After 48 hours, 0.5 mL of broth solution containing E. coli was added to a petri dishcontaining agar and spread evenly using a glass rod. Two chads were then added to thesesamples. Group 1 from the previous trial had chads with a pH of 4 added this time. Group 2 hadchads with a pH of 7 added again. This time, group 3 had chads with a pH of 10 added. Thesegroups now became trial 2. After 24 hours of incubation, the diameter of growth inhibition wasmeasured in each sample. Then a portion of bacteria was scraped out of each sample from insidethe zone of inhibition and placed in a separate nutrient broth solution for a total of 39 broths.These broths were allowed to incubate for 48 hours. Then 0.5 mL of broth solution was placed in a petri dish containing agar and spread aroundevenly using a glass rod. Then 2 chads were added to each petri dish. This time, group 1 hadchads with a pH of 3 added, group 2 had chads with a pH of 7 added, group 3 had chads with apH of 11 added. These samples were allowed to


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Saddleback BIO 3B - The Change in Growth Inhibition of Escherichia coli

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