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. (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 petriPetri 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 petriPetri 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).
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).
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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 petriPetri dish. Two paper chads were then
added to each petriPetri dish containing E. coli, and then these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 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 theThe
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 petriPetri 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 aA 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 petriPetri dish containing agar and spread
around evenly using a glass rod. Then 2 Two chads were added to each petriPetri dish. This
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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).
 CENTER GRAPH
Figure 1: (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)  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
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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, theThe 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, theThe 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 dodoes 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.
References 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 HardyWeinberg Disequilibrium at a Marker Locus. Genetics 63: 1531-1540.
Hauke, L. 2003. Advances in Refolding Proteins of Escherichia coli. Current Biology 9: 497501. (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)
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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.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current
state of knowledge.
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Overall, the paper is fairly well written and clear. This is a rough draft however and needs revision. Sentence
structure needs some editing in order to avoid being repetitive. The investigators have organized their paper well
and content flows. Weaknesses include repetitive sentence structure and grammatical errors throughout. Also,
there are some awkwardly worded sentences that should be revised. The graph explained the data well, but error
bars must be explained.
Technical Criticism
Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical errors, and
minor textual problems. It's not the reviewer's job to copy Edit the paper, mark the manuscript.
Grammar errors are present. Typos are also present. Petri dish is capitalized (this mistake occurred often
throughout the paper). There is a little confusion when explaining the groups and trials. Consider revising.
Grammar errors were crossed out, highlighted and fixed in red font.
This paper was a final version
This paper was a rough draft
Recommendation
 This paper should be published as is
 This paper should be published with revision
 This paper should not be published
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