The Change in Growth Inhibition of Escherichia coli Across Generations in Conditions of Varying pH
Michael Harvey and Nika Kusmierz
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692. ß(no period)
Abstract
When environmental conditions change, members of a population that possess favorable traits tend to reproduce more than those that do not. This leads to evolution by means of natural selection. A paper chad of high or low pH was added to a petriàPetri dish swabbed with E. coli. After a 24 hour incubation period, an area of growth inhibition was observed around the chad. Then a A portion of bacteria was scraped out from within the zone of inhibition and placed in a nutrient broth to be recultured in agar. After the a new sample petriàPetri dish was made (prepared?) from the broth, this sample then became trial 2. (This sentence sounds awkward. Consider revising). Group 1 consisted of 13 of these samples at low pH, group 2 was the control consisting of 13 samples at a pH of 7, and group 3 consisted of 13 samples at high pH. It was (past tense verb) hypothesized that bacteria will (future tense verb, change to would) show less growth inhibition in the second and third trials than the first. The average diameter of growth inhibtion in group 1 of trial 1 was 0 cm, while trial 2 was also 0 cm, and trial 3 was 0.87 cm. The average diameter of growth inhibition in in group 2 of trial 1 was 0 cm,(there was an extra space here on original copy) while trial 2 was also 0 cm, and trial 3 was 0.66 cm. All trials of the group 2 had an average diameter of inhibition of 0 cm. There was a larger diameter of growth inhibition in trial 3 than trial 2 of group 1 (p= 6.34 x 10-10, 1-tailed, paired t-test (one-tailed, paired t-test)). There was a larger diameter of inhibition in group 3 of trial 3 than trial 2 (p= 2.26 x 10-6, 1(one)-tailed, paired t-test). There was no difference in growth inhibition of group 3 of trial 3 than group 1 (p= 0.07564, 2two-tailed, unpaired t-test). The hypothesis was neither supported or (nor) refuted because not enough trials were conducted. (Consider revising this statement. Investigators should avoid statements like “not enough trials were conducted” because it seems as if the investigators did not complete their study. Simply mention that the hypothesis was neither accepted nor rejected).
(Abstract should be a MAXIMUM of 225 words. This abstract is over 300 words).
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Introduction
Organisms must adapt to changing conditions. Adaptation through natural selection is the reason for the incredible biodiversity on Earth. One such environmental condition is the acidity of the surrounding environment (Simon, 1983). Prokaryotic cells are adapted for living in a particular range of acidity. (awkward transition) Escherichia coli is a bacteria found in the digestive tract of animals. It serves as a model organism in scientific experiements (Cohen, 1997). (Why does E. coli serve as a model organism in scientific experiments?) If the acidity becomes too high or low, those with a genetic advantage will reproduce (more than those without an advantage) 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 the difference in growth inhibition of E. coli from one trial to the next. A previous study found that the fitness of the experimental populations, as measured by the rate at which each population grew, increased rapidly for the first 5,000 trials and more slowly for the next 15,000 trials when compared to the ancestral population (Cooper, 2000). It was hypothesized that the second trial would have a smaller diameter of inhibition than the first, and the third trial smaller than the second 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 survive will take their place, increasing the frequency of a particular allele in a population (Thauer, 1997).
This study has practical applications in biotechnology, where bacteria can be modified through natural selection to serve a purpose. One such purpose may be environmental waste cleanup. Bacteria can be bred to digest a certain organic comound, thus detoxifying a particular area of the compound (Witkin, 1976). A further application would be having an accurate mathematical formula that can model the rate of natural selection given the variables of time acid content (Hauke, 2003). Further inquiries can determine the genetic makeup of a population at a specific 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 petriàPetri dish. Two paper chads were then added to each petriàPetri dish containing E. coli, and then theseàThese 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 a pH of 5 added, group 2 had chads with a pH of 7 added, and group 3 had chads with a pH of 9 added. These pH’s were chosen because they are the outer limits in which E. coli can grow (Cohen, 1997). (Probably doesn’t belong in materials and methods). Hydrochloric acid or sodium hydroxide was added to water to alter the pH, and when theàThe proper pH was verified using pH paper. Using a micropipette, 0.05 mL of acid or base solution was added to each chad. After 24 hours, the diameter of growth inhibition for each sample was measured. (how?) A loop was then used to scrape out a portion of bacteria inside the zone of inhibition. Inside the area of inhibition is where you find the highest concentration of bacteria with resistant genes (Luria, 1943). Each portion of bacteria was then placed in nutrient broth and allowed to incubate for 48 hours for a total of 39 broths. Forty-eight hours is required to achieve adequate proliferation in 50 mL of moderate nutrient broth (Kaeberlein, 2002). (Last sentence seems unnecessary)
After 48 hours, 0.5 mL of broth solution containing E. coli was added to a petriàPetri dish containing agar and spread evenly using a glass rod. Two chads were then added to these samples. Group 1 from the previous trial had chads with a pH of 4 added this time. Group 2 had chads with a pH of 7 added again. This time, group 3 had chads with a pH of 10 added. These groups now became trial 2. After 24 hours of incubation, the diameter of growth inhibition was measured in each sample. (how?) Then aàA portion of bacteria was then scraped out of each sample from inside the 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 petriàPetri dish containing agar and spread around evenly using a glass rod. Then 2 Two chads were added to each petriàPetri dish. This time, group 1 had chads with a pH of 3 added, group 2 had chads with a pH of 7 added and group 3 had chads with a pH of 11 added. These samples were allowed to incubate for 24 hours at which time the diameter of each zone of inhibition was measured. The area of inhibition of both the control and experimental samples of E. coli were then statistically compared. (How did you compare the data statistically? Include the test you ran upon them and also include how you transferred your data. Ex: Data were transferred to MS Excel (Microsoft Corporation, Redmond, Washington).
Results
There was no growth inhibition in all groups for trial 1 and 2. Trial 3 showed inhibition. The average diameter of inhibition in group 1 of trial 3 was 0.87 cm. There was no growth inhibition for group 2 of trial 3. The average diameter of inhibition of group 3 in trial 3 was 0.66 cm (Figure 1).
ß CENTER GRAPH
Figure 1: ß(should be a period) There was greater inhibition in group 1 for trial 3 than trial 2 (p= 6.34 x 10-10, 1one-tailed, paired t-test). There was greater inhibition for trial 3 than trial 2 in group 3 (p= 2.26 x 10-6, 1one-tailed, paired t-test). There was no difference between groups 3 and 1 in trial 3 (p= 0.07564, 2two-tailed, unpaired t-test). Group 1 is the low pH group, group 2 is the control group, and group 3 is the high pH group. (Need to add what error bars represent. Ex: “Error bars represent the average diameter ± SEM”).
Discussion
In our this study, no inhbition was observed in trial 1 and 2. (This has been mentioned many times. Getting a little repetitive). This may be because (A possible explanation for these results may be because) the pH of the chads added to the bacteria was not far enough from the ideal growth conditions. The data collected here differs from what another study has found. (Awkward sentence) à the next sentence repeats Another study found that growth of E. coli is significantly reduced by small changes in pH. The same study (repeating “another study” and “study” a lot here) found that natural selection occurs rapidly in early trials and then begins to slow in later trials (Small, 1994). Our study differs from this study because our study (too much “study”!) has observed that large shifts in pH are needed to inhibit growth of E. coli. A reason for this difference may be due to subtle differences in experimental technique. For example, E. coli has many strains, and it is very difficult to obtain the same strain of E. coli as used in previous experiences. Therefore, theàThe sample used in this experiment may have an unusually high resisitance to acidic and basic conditions. A study found that genetic makeup of a strain of bacteria can vary widely from another strain (Cohen, 1997). The particular strain of E. coli used in this experiment turned out to be unusually resillient to varying pH.
No conclusion can be drawn about the change in growth inhibition due to natural selection because growth inhibition was only observed in the final trial. At least two more trials would be needed to be carried out in order to observe the effects of natural selection. For this particular strain, chads with a pH of 2 and 12 must be used to observe growth inhibition. Not enough data was collected in this experiment to support or reject the hypothesis that there would be less growth inhibition in subsequent trials.
In future studies, very acidic and basic conditions are needed to properly observe growth inhibition. Clearly, theàThe conditions in this experiment were not hostile enough to induce inhibition. There was not a significant difference between group 1 and 3 in trial 3. This asserts (suggests) that E. coli doàdoes not tend to favor acidic conditions over basic conditions or basic conditions over acidic conditions, but rather tolerates both equally well.
There was greater inhibition in trial 3 than trial 2 for groups 1 and 3. This is likely due to the pH being lower for group 1 in trial 3 than in trial 2. The lower pH was more hostile to the bacteria. The bacteria could not survive as well when a pH of 3 was added compared to when a pH of 4 added. Similarly, when a pH of 11 was added in group 3 of trial 3, there was a greater inhibition than in group 3 of trial 2 when a pH of 10 was added.
Although the effects of natural selection were not observed, insight into the lethal limits of acid and base content was gained from this experiment. Through further experimentation, more information can be gained on the theory of evolution.
Referencesà Literature Cited
Cohen, S. 1997. Nonchromosomal Antiobiotic Resistance in Bacteria. Nature 390: 232-34.
Cooper, V. and Lenski, R. 2000. The Population Genetics of Ecological Specialization in
Evolving Escherichia coli populations. Nature 407: 736-739.
Dahlia, M. and Weir, S. 1999. Detecting Marker Disease Association by Testing for Hardy-
Weinberg Disequilibrium at a Marker Locus. Genetics 63: 1531-1540.
Hauke, L. 2003. Advances in Refolding Proteins of Escherichia coli. Current Biology 9: 497-501. (An extra spacing at the beginning of this)
Kaeberlein, T. and Lewis, K. 2002. Isolating “Uncultivable” Microorganisms in Pure Culture in a Simulated Natural Environment. Science 296: 1127-1129. (An extra spacing at the beginning of this)
Luria, S. E. 1943. Mutations of Bacteria from Virus Sensitivity to Virus Resistance. Genetics 6: 491-511.
Simon, R. 1983. A Broad Host Range Mobilization System in Gram Negative Bacteria. Nature Biotechnology 1: 784-791.
Small, P. 1994. Acid and Base Resistance in Escherichia coli. J Bacteriol 176: 1769-1770.
Thauer, H. R. 1997. Energy Conservation in Chemotrophic Anaerobic Bacteria. Microbiology 41: 47-49.
Witkin, E. 1976. Ultraviolet Mutagenesis and Inducible DNA Repair in Escherichia coli. Bacteriological Reviews: 869-898.
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s):Michael Harvey and Nika Kusmierz
Title: The Change in Growth Inhibition of Escherichia coli Across Generations in Conditions of Varying pH
Summary
The study looked at simulating the effects of natural selection over time in Escherichia coli. Bacteria were cultured in pH ranges of low, medium and high. Using a metal rod, bacteria was swabbed from the zone of inhibition and placed on new agar plates with pH’s of low, medium and high. The proposed hypothesis was that E coli. would show less growth inhibition in the second and third trials than in the primary trial. The idea behind this was that the E coli. that survived the varying pH’s in the previous trials would be genetically advanced than others that died off. This would create less growth inhibition in future generations of the cultured bacteria due to natural selection. The results, however, showed no difference in growth inhibition in group 1 and 2 trials. But, there was a greater growth inhibition in trial 1 and 2 than in trial 3.