Bio Lab Report 2

Duncan Ackerman
Partners: Morgan Keegan, Cait Miller

Page #: N/A
Chicken Farm Lab Report
Sec. 911
12 December 2013

Introduction:

Antibiotics are used in large scale farming in order to prevent bacterial
contamination of the livestock, which will prevent large-scale loss of livestock and
reduce the risk of people becoming ill from the consumption of said livestock. The
use of antibiotics has become an increasingly important part of successful livestock
farming. Some bacteria have become resistant to certain antibiotics, like Kanamycin,
which will be examined in this report (Cyr et. al. 2010). Bacteria have genetic
material contained outside their nuclei called plasmids, which can assist in their
ability to be resistant to antibiotics (Cyr et. al. 2010). Plasmids promoting antibiotic
resistance can be transferred from one bacteria cell to another at a fast pace,
resulting in large populations of resistant bacteria. Kanamycin resistance can lead to
massive bacterial contamination resulting in the chickens dying and meat
contamination, which can cause human harm upon consumption. It is imperative
that this problem be addressed for the safety of all who consume products made
from these contaminated chickens. In this particular case, the resistant bacteria
have arisen in 3 different farming locations: Luckys Eggs, Tuckers Cluckers, and
Mias Eggcellent Eggs. In order to prevent future contaminations, the genes must be
identified to determine the source of the outbreaks (Cyr et. al. 2010). This can be
done by examining the three known genes that can lead to Kanamycin resistance. If
the source of the contamination results from a single gene, then it can be concluded
that the source of the resistance has come from the same source, most likely the
chicken food. To determine the source of the contamination, the resistant bacteria in
question must be isolated and counted (Cyr et. al 2010). Then, gel electrophoresis
must be used to determine which type of plasmid caused the infection. If the same
plasmid is found to have caused each of the three infections, then it can be
concluded that the contamination source is the same for the three farms (Cyr et. al.
2010). This particular report was done with specific focus on the Luckys Eggs farm.
The question that the experiment set out to answer is Which of the three gene
known to cause Kanamycin resistance was responsible for the contamination in
each farm and is there a common source of resistance for each of the three farms in
question?

Methods:

The first step in the procedure was to obtain liquid cultures of antibiotic resistant
bacteria from each of the three chicken farms in question. Serial dilutions were then
taken for each of the samples and placed on agar plates with and without the
antibiotic Kanamycin at factors of 10
-2
, 10
-4
, and 10
-6
ul. This was useful in order to
make the bacteria colonies more visible and easily countable. If the bacteria were
able to survive in the Kanamycin, then they were determined to be resistant to the
antibiotic and those that died were determined not to be resistant. A PCR reaction
was run to allow the genes in question to multiply to a level that can be viewed.
After the PCR was run, the appearance of each plate was observed. Plates with
countable colonies were then selected and counted. The percentage of Kanamycin
resistant bacteria was then calculated. Gel electrophoresis was then performed on
the resistant bacteria genes with a control lane and a DNA ladder was used as a size
marker. The first electrophoresis well contained the DNA ladder, the others
contained our samples (colonies 1,2, and 3) in wells 2,3, and 4 respectively, as well
as the control genes (wells 4,5, and 6) for the three types of Kanamycin resistant
plasmids (A, B, and C). The size of the resistant genes were then compared from
each of the three farms and the controls to determine which gene caused the
resistance and whether or not this gene was common to each farm.

Results:

Luckys Eggs:
Dilution Factor K Plate Non-K Plate
10
2
83 Lawn
10
4
7 2045
10
6
0 116
Freq:
N/A
0.03
0

K Resistance Frequency in Luckys Eggs: 7:2045 = .03%

Class Frequencies:

Luckys Eggs 1 .03%
Tuckers Cluckers 1.11%
Mias Eggcellent Eggs .154%
Lucky Eggs 2 3.9%

Electrophoresis Set Up:
Lane/Well Sample Contents
1 DNA Ladder Molecular weight
standard
2 PCR sample 1 Plasmid DNA from
colony 1
colony 2
colony 3
5 PCR sample 4 Standard of plasmid
A
B
C

Picture of Electrophoresis Result

The experimental group Luckys Eggs, which was tested in this particular report
lined up with the control for plasmid B.

Discussion:

The gene from Luckys Eggs #1 corresponds to the B plasmid. The plasmids from
each of the other three farms did not match each other (no specific info given about
the other electrophoresis results). According to the results from the experiment it
can be concluded that the contamination was not due to the same gene. Since the
electrophoresis results matched different genes for each of the three farms, the
source of the contamination could not have been the same for all of them. The
overall frequency of the kanamycin resistant bacteria in the chicken farm cultures
was less than 5%. Based on these results, it is recommended that farms: Divert eggs
to pasteurization facility until contamination levels drop to less than 1% for 8
weeks; monitor the farm weekly; identify the sources of contamination; and change
the antibiotic regime. Some source of experimental error could have been improper
measurement and counting of the bacterial colonies as well as calculation errors for
frequencies.

References:

Cyr, R., 2002. Title of the tutorial being cited. In, Biology 110: Basic
concepts and biodiverity course website. Department of Biology, The
Pennsylvania State University. http://www.bio.psu.edu/

Cyr, R., Hass C., Woodward D., and Ward A., 2010. Using Genes for
Antibiotic Resistance to Trace Source(s) of Bacterial Contamination. In,
Biology 110: Basic concepts and biodiverity course website. Department of
Biology, The Pennsylvania State University. http://www.bio.psu.edu/

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Duncan Ackerman

Partners: Morgan Keegan, Cait Miller

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