Fellenz

1
Taylor Fellenz
Bio 110 Written Assignment #2
Jeffrey Hall
December 2, 2015

Beef Farm Lab Report

Introduction:
Genes for antibiotic resistance can be traced to bacterial contamination in beef
cattle. Bacterial antibiotic resistant genes are often carried on plasmids. Bacterial
plasmids are circular DNA molecules in bacteria. Some plasmids even make their hosts
immune to toxins. The most common known plasmid gene is an antibiotic resistant gene.
Antibiotics are produced by organisms like fungi for example and that is why it we have
the capacity to resist them. The use of antibiotics has increased the frequency of antibiotic
resistance in populations of bacteria. Today cattle feeders and growers add antibiotics to
the cattles food to increase their growth. Tetracycline is a common antibiotic used by
cattle feeders to increase growth.
Reports of the stomach flu have been linked to raw uncooked meats and have led
the FDA to look into the sources of the contamination. Scientists have learned that E.
coli carries plasmids containing tetracycline resistant genes. E. coli is one of the three
most important pathogens that spread through food and water afflicting people
worldwide. Recently, E. coli is listed as the most serious foodborne infection because of
its high risks (Liao, et. al., 2015). These contaminated meats come from three different
cattle farms. The three different genes responsible for tetracycline resistance genes each

Fellenz
2
code for a different protein. Bacteria that contain these genes are resistant to tetracycline.
Scientists have found that the different tetracycline resistant genes are different sizes and
are independent from each other. Being able to identify a certain gene can help trace the
sources of a particular bacterial strain. Since bacteria is so small, reproduce quickly and
grow to high concentrations in a sample, the class performed serial dilutions to count the
bacteria. Serial dilution is easy and gives accurate approximations of the number of
bacteria on a sample. The main point to serial dilution is to moderate the sample where
individual colonies appear and can be easily counted.
PCR is a DNA polymerase that intensifies large amounts of specific regions of
DNA. There are several types of DNA polymerases. Taq polymerase is a technique often
used for PCR. Taq allows for the temperature to function under heating and cooling
cycles. Three cycles of PCR are denaturation, annealing, and elongation. During
denaturation DNA is separated into two strains. The annealing step involves the
temperature to be lowered so the primers can bind to other sequences on two separated
strands. During Elongation two new strands of DNA are synthesized.
To separate different sized DNA fragments, determine the size, and compare it to
the size of the actual genes the technique of agarose gel electrophoresis is used. By
applying 160 volts the DNA sample moves through the gel toward the positive electrode.
Once The PCR product is exposed to the electric field it will migrate to the positive
electrode because the phosphate groups on DNA have a negative charge. Since the gel
contains the DNA binding dye it will be easier to see the fragments.
Contaminated meat have been linked to three farms, Bovines R Us, Rebels of
Renovo, and Jumping Steer Farm. For this experiment the class is trying to find the

Fellenz
3
source of the outbreak and make recommendations on steps that people can take to
prevent more outbreaks. The class will also be looking for any shared sources to the
bacteria. For example, shared feed producer, supplier of materials, or if the bacteria is
different in each farm. To find this information the class will perform, serial dilutions,
use PCR, and gel electrophoresis. Is the bacterial contamination at these three farms due
to the same strain of bacteria or different strains? What is the frequency of tetracycline
resistant bacteria in the cattle farm cultures? These are some of the research questions
that will be used to help make recommendations after the investigation. The bacterial
contamination among the three farms will not have the same strains of the tetracycline
bacteria but different strains.

Methods:
In experiment part A the class prepared serial dilutions of bacterial cultures on
plates with and without tetracycline. Each table received a code letter; this code letter
represented which cattle farm the sample came from. Then each pair received three
tetracycline plates labeled K (with tetracycline) and three unlabeled plates (no
tetracycline). On the top of the plate each group member wrote their names, the date,
section number, and the code letter of the sample and label one of each 10-2, 10-4, and
10-6. Next, each group added 990l of water into each microtube. Then they took the
microtube and mixed it using a gentle flicking motion. Each person removed 10 l from
the microtube and used this at the starting sample to make the three serial dilutions. In
the next step each person in the group lined up all of their petri dishes in order of dilution.
Then used a pipette to add 100-l samples of bacteria from the microtube to each petri

Fellenz
4
dish. To distribute the bacteria evenly the class used sterile beads. Each group started
with the 10-6 plate. Then used the same beads on the 10-4 and 10-2 plates. The plates were
then sealed and taken to an incubator for 24 hours and then to a cold storage room until
the following week so each group could count the bacteria present on the plates (Burpee,
et. al., 2015).
In Experiment Part B, each group used PCR to amplify specific genes for
tetracycline resistance. Each table was given six tubes of the PCR reaction mix. The
tubes were color-coded. Three tubes (orange, blue, and yellow) had primers and the other
three (red, green, and pink) had a control plasmid. Then the group identified and
numbered three antibiotic-resistant colonies growing on plates labeled T. Next the
group took a small sample of colony #1 and put it into the orange tube. The process was
repeated twice. The group put colony #2 into the blue tube and colony #3 into the yellow
tube. Red, green, and pink tubes were positive controls. The six tubes were then put into
the PCR machine. While the PCR machine was running each table completed experiment
part C. In experiment part C each group counted and recorded the bacterial cultures
growing on the plates with and without tetracycline (Burpee, et. al., 2015).
The next part in the procedure was to identify genes for tetracycline resistant
present in the bacterial sample. The first step in experiment part D was to prepare and
pour the gel. First the group made sure the gel apparatus was put together. Then one
person in the group weighed the agarose and placed it into a flask. TAE buffer was
measured next and put into the flask. The agarose and the buffer were gently mixed and
then microwaved for 35 seconds. The gel cooled for 2 minutes and 1 l of ethidium
bromide dye was added. In the second procedure the gel was casted. The dams on the gel

Fellenz
5
rig were put snugly into the slots and the gel was poured into the gel tray. The gel
hardened in 10 minutes and the dams were removed so the TAE buffer can be poured in
the gel electrophoresis unit. In the third procedure the gel was prepared to put in the gel
electrophoresis. 5 l of the PCR DNA ladder was loaded into the first well and 2 l of the
loading dye were added to the six tubes containing the samples.15 l from the six colored
tubes are loaded into the next six wells. The electrodes was then turned on and put on
160 volts. Once the tracking dye migrated half way the gel was removed and put into the
UV light box to photograph the gel (Burpee, et. al., 2015).
During Experiment Part E each group calculated their frequency of tetracycline
resistance in the beef farms. To calculate the frequency of the tetracycline resistance in
the bacterial population of the three beef farms the equation
of bacterial colonies with tetracycline
of bacterial colonies without tetracycline

X 100 was utilized.

Results:
Table 1. Numbers of bacterial colonies on plates with and without tetracycline

Treatment

Dilutions
-2

-4

10

10

10-6

Volume Plated

100 l

100 l

100 l

Tetracycline

680

29

No Tetracycline

Lawn

1755

93

Table 2. Summary of Tetracycline Resistance in Beef Farms

Fellenz
6

Tetracycline Resistance Gene

Frequency
of
Resistance

Beef Farm
1
Bovines
R Us

Rebels of
Renovo

Jumping
Steer Farm

Comments

X
X
X

1.23%

600 Base
Pairs

6.85%

500 Base
Pairs

0.035%

300 Base
Pairs

Cattle feed supplier and growers add antibiotics to cattle feed to increase growth.
When using antibiotics populations of bacteria resistant occur to whatever antibiotic that
is being used. The most common form of antibiotic used to heighten growth is
tetracycline. Tetracycline interferes with bacterial growth by interacting with bacterial
ribosomes, inhibiting, and protein synthesis because it interferes with the translation stage
of protein synthesis (Burpee, et. al., 2015).

Fellenz
7

Farm A: Bovines R Us

Farm B: Rebels of Renovo

Farm C: Jumping Steer

Sample Calculation for Antibiotic Resistance Percentage:

B=N/D
B=

29
x 100=1.65
1755

Discussion:
After counting the number of colonies in the bacteria my group found that the
plates with no tetracycline had more colonies. For the dilution 10-2 , the plate with
tetracycline had 680 colonies and the plate with no tetracycline had a lawn. A lawn is a
high concentration of bacteria that is uncountable. The dilution 10-4 had 29 colonies on
the tetracycline plate and 1755 on the plate with no tetracycline. The dilution 10-6 had no
colonies on the tetracycline plate and 93 colonies on the plate with no tetracycline. The

Fellenz
8
frequency of resistance in beef farm A (Bovines R Us) was 1.23%, in beef farm B
(Rebels of Renovo) the frequency was 6.85%, and in beef farm C (Jumping Steer Farm)
the frequency was 0.035%. These frequencies are important in making recommendations
for the CDC on how to handle the bacterial contaminations at each cattle farm. Farm C
(Jumping Steer Farm) can change their antibiotic regime by identifying sources of
contamination, monitor farm weekly, and divert meat to pasteurization facility until
contamination levels have been <1% for 8 weeks (Burpee, et. al., 2015). Farm A
(Bovines R Us) and Farm B (Rebels of Renovo) can change their antibiotic regime,
identify sources of contamination, monitor farm weekly, identify and treat individuals
infected, and destroy all the meat until levels have been <1% for 8 weeks (Burpee, et. al.,
2015).
After putting the gels in the UV light box each group could see the size of their
tetracycline resistance gene. Farm A had 600 base pairs, Farm B had 500 base pairs, and
Farm C had 300 base pairs. After learning this information each group could tell which
tetracycline gene they had. Each group had a different tetracycline resistance gene. This
information determined that my hypothesis was correct. The farms did not have the same
strains but different strains. There were no unexpected findings. Future research that
can be done is to study if there is a reduction in E. coli in cattle would that prevent
humans being infected with E. coli (Katayama, et. al., 2011).

Fellenz
9

References:
Burpee, D., Cyr, R., Hass, C., Ikis, D., Richter, K., Ward, A. and D. Woodward, eds. A
Laboratory Manual for Biology 110 Biology: Basic Concepts and Biodiversity.
2015. Department of Biology, The Pennsylvania State University Park, PA.

Katayama, S., Kusukawa, M., Murakami, M., Sasaki, Y., Tsujiyama, Y. and Y. Yamada.
Prevalence and characterization of Shiga toxin-producingEscherichia coli O157
and O26 in beef farms. Science Direct. 11 Jan. 2011. Web. 1 Dec. 2015.

Liao, Y., Liu, F., Liu, H., Wei, J., Xing, D. and X. Zhou, Multiplex detection and
genotyping of pathogenic bacteria on paper-based biosensor with a novel
universal primer mediated asymmetric PCR. Science Direct. 15 July 2015. Web.
1 Dec. 2015.