THE EFFECT OF AMPICILIN ON

FUTURE GENERATIONS OF
ESCHERICHIA COLI
Imo Okojie
Lab Partners: Emma Kitko and Rose Ried
BIOL 220W lab sec. 001
Dr. Cornell
Submitted on 4/13/23
Abstract
The theory of evolution by natural selection has been happening since the beginning of time,

but only with artificial selection can we really understand this evolutionary process. This paper

represents the theory of artificial selection. Escherichia coli was exposed to the antibiotic,

ampicillin, for 4 generations. Over these 4 generations, we wanted to see a trend; an increase in

the amount of ampicillin-resistant E. coli. Conducted in a lab at Penn State Altoona, 8 petri

dishes were gathered; four being labeled as our selection group and the rest as our control

group. Using various lab and sterile techniques, the Zone of Inhibition or ZOI, was measured

from each petri dish over 4 generations (weeks). After the data was collected and averaged out

with the class, the control group showed no significant change in its length of ZOI from the 1st

generation to the 4th. The selection group, however, showed a significant change in its length of

ZOI from the 1st to the 4th generation. This supports our hypothesis, showing that if E. coli is

exposed to the ampicillin over a number of generations, then the number of ampicillin-resistant

E. coli would increase. Altogether, these observations look to support the theory of artificial

selection, and how it can be used to cultivate generations of organisms with a higher fitness

than their previous ancestors.

Introduction
Artificial selection experiments have been a practice and technique used for many years. These

experiments, in which strong selective forces are applied to laboratory or field populations over

a greatly curtailed evolutionary timescale, have been and continue to be an important source of

information on the genetics of quantitative characters and their effects on fitness (Hill et all.,
1992). Charles Darwin, an English naturalist, was the one who first developed the idea of

evolution through natural selection. Natural selection is the fundamental process in the

evolution of life and is a process that acts on all organisms, moreover, it can take longer than a

lifetime to study this evolutionary change (Williams, H. T. P., & Lenton, T. M., 1970). In order to

study the theory of natural selection, artificial selection studies are conducted. Artificial

selection is a human-directed evolution, and it has become an important tool for biologist

because of the information it provides on the given strength and form of what a selection can

accomplish in terms of phenotypic change and how quickly this change can occur (Conner,

2003). These artificial selection studies are performed on model organisms, such as Mus

musculus (house mouse) or Escherichia coli (bacterium). These model organisms are perfect

because of their short generation times and how easy they are to maintain, but also because of

their small size (Mostert-O’Neill et all.,1994-1996) .

Over the span of 4 weeks, artificial selection was conducted on the model organism, E. coli. The

purpose of this experiment was to assess the response of E. coli when it was exposed to the

antibiotic, ampicillin, and how well the generations of the selected E. coli grew in the presence

of that antibiotic (BIOL 220W, 2023). If the organism, E. coli, were to be exposed to the

ampicillin over time, then the number of bacteria resistant to the antibiotic would increase.

In order to test this hypothesis, the Disk Diffusion method was used to measure the generations

of the selected E. coli. Eight replicate lines of bacteria were gathered, with four being labeled as

our “Selection”, which contained the bacteria that exhibited the ability to survive in the

presence of antibiotic. The other four were labeled as “Control”, which represented the

colonies that did not exhibit the phenotype to grow in the presence of an antibiotic. This
method resulted in Zones of Inhibition (ZOI), which are clear, circular areas on the Petri dish

where no bacteria colonies have grown. These zones were measured and used to calculate how

much of the bacteria, that was resistant to the antibiotic, grew. After the experiment, I predict

that the Zones of Inhibition, for the selection treatment group, will become shorter over time,

resulting in the idea that more ampicillin resistant bacteria were produced over subsequent

generations.

Methods

This experiment was conducted in a laboratory setting located in Altoona, Pennsylvania. The

Petri dishes used were Muller-Hinton agar plates, which were kept at a temperature of 37

Degrees Celsius throughout the entire experiment. In order to perform this experiment, basic

sterile techniques were used with clean hands and the Disk Diffused Method was used to assess

the response of the selection groups to the ampicillin. During the 1st week of this lab, the

mixture of E. coli was made by adding 2.0 mL of each of the five bacterial strains to a sterile test

tube, using a sterile transfer pipet. In order to prepare the Petri dish, it was inoculated with 200

μL of the E. coli mixed population. Once the dish was inoculated, the bacteria was spread across

the dish using a “hockey stick” spreader. This spreader was sterilized by dipping it into a beaker

containing ethanol and flaming the stick. After sterilize the hockey stick, it was allowed to dry

for about 20 seconds before using it to spread the 200 μL of the E. coli across the Petri dish.

Once the E. coli was spread evenly across the Petri dish, 4 disk that contained 10 pg of

ampicillin were placed on the plate. Four selection plates were replicated this way, but only one

was use as the “selection” plate.

After 24 hours, a metric ruler was used to measure the ZOI that had the widest diameter, but

that was also the darkest portion visible. This is where the 4 replicates of the selection group

and control group were taken. For the selection group, a flame-sterilized inoculation loop was

used to select bacteria from the very edge of the ZOI. For the control group, a flame-sterilized

inoculation loop was used to select bacteria from the very edge of the of the Petri dish. These

groups were then incubated at 37 °C for 20 hours, and then placed in to a refrigerator (4 °C).

This was repeated for weeks 2 and 3, but for week 4, only the ZOIs were measured from the

inoculated plates.

Results

The ZOIs measured in the control group showed no significant difference from week 1 to week

4, instead displaying a constant trend (See Figure 1). The ZOIs measured in the selection group

showed a decreasing trend throughout the 4 weeks of data (See Figure 1). In the week 4 class

data, the average length of the ZOIs in the control line is higher than the average length of the

ZOIs in the selection line (See Figure 2). These results are further supported by our Analysis of

Variance (See Table 1, p-value = 0.002, F-value = 16.074).

Table 1. Analysis of Variance test results from the Week 4 Class Data.

Treatment Average ZOI SD DF F-value p-value

line (sig)

Control 21.53 1.319 11.00 16.07 0.002

Selection 18.63 1.186

Figure 1. This figure shows the Zones of Inhibitions between the Control and Selection Groups that were recorded over the span
of 4 weeks.
Figure 2. This figure represents the average length of the Zones of Inhibition of Week 4 only. The average was found using the
class data.

Discussion

In this experiment, we were able to use artificial selection to see if future generations of

the organism E. coli, could become more resistant to ampicillin by exposing previous

generations to this antibiotic. By first looking Figure 1, we can see that over the 4-week span of

our two treatment groups, the Zones of Inhibition for the Selection group became smaller with

each week. By the end of experiment, the Zone of Inhibition for the Selection group was around

18 or 19 millimeters. At the start of our experiment, the Zone of Inhibition for the Selection

group was around 22 or 23 millimeters. For our control group, the Zones of Inhibition did not

see significant change from the week 1 to week 4. The Zone of Inhibition for the Control Group

started at 22 or 23 millimeters, but by the end of the experiment, the Zone of inhibition showed
an increase to 24 millimeters. This increase may have been caused by insufficient sterile

technique or bacteria contamination during the process. By looking at just the results from

Figure 1, it looks like our hypothesis was correct, but just in case of possible error, the week 4

results of our data were taken and averaged with the week 4 results of the class data. These

averages can be found in Figure 2, where the week 4 averages of each treatment group are

compared. For the Control group, the average length was around 23 millimeters. For the

Selection group, the average length was around 18 millimeters. The results from Figure 2

support the data collected in Figure 1, indicating that there was no significant error that

plagued the results of the experiment. These results are further supported by the results found

in the ANOVA test (Table 1, p-value= 0.002, F-value= 16.072), indicating that there was a

significant different between the length of ZOIs in the control and selection groups. For this

experiment, our hypothesis stated that if the organism, E. coli, were to be exposed to the

antibiotic, ampicillin over generations, then the number of bacteria resistant to this antibiotic

would increase with each generation. But does this prove that natural selection is beneficial? As

scientist, our aim is never to prove anything. In hindsight, it could be a good thing that these

future generations of E. coli are resistant to ampicillin, but does that possibly make these

generations vulnerable to other antibiotics that were not tested in this experiment. Still, with

the results presented in this experiment, it’s safe to infer that our hypothesis was correct. In

the future, I hope to experiment with more types of organisms and also different antibiotics in

order to gain more knowledge about the theory of natural selection.

References

Conner, J. K. (2003). Artificial Selection: A Powerful Tool for Ecologist. Ecology, Ecological Society

of America. Volume 84, Issue 7. 1650-1660

Hill, W. G. & Caballero, A. (1992) Artificial Selection Experiments. Annual Review of Ecology and

Systematics, Volume 23

BIOL 220W (2023), Artificial Selection for Ampicillin Resistance in Escherichia coli

Williams, H. T. P., & Lenton, T. M. (1970). Artificial ecosystem selection for evolutionary

optimization, Advances in artificial Life (9th ed,)

Mostert-O’Neill, M. M., Tate, H., Reynolds, M. S., Mphahlele, M. M., Berg, G., Verryn, S. D.,

Acosta, J. J., Borevitz, J. O., Myburg, A. A. (2022) Genemoic consequences of artificial selection

during early domestication of a wood fibre crop. New Phytologist Foundation, Volume 235,

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