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Abstract
With few new antibiotics being created, doctors are running out of ways to treat
antibiotic-resistant pathogens. One remedy scientists are considering is bacteriophages, viruses
that kill bacteria. Conventionally-raised cows are treated with hormones to grow larger and
produce more milk. They may end up in the milk; when drunk they might affect gut bacteria.
Does conventional milk make E. coli bacteria less susceptible to infection by bacteriophage T4r
than organic milk does?
If E. coli grown in organic milk are more susceptible to T4r than E. coli grown in
conventional milk, there will be more plaques on the bacteria culture that was grown in organic
milk possibly because the organic cows are not given extra hormones. Bacteriophage, milk, and
E. coli were added to gels which were poured onto plates and incubated for 24 hours.
The results support my hypothesis. There were more plaques (1075) on the plate with
organic milk than on the plate with conventional milk (777), which suggests that organic milk
causes more susceptibility than conventional does. The presence of more plaques on the milk
plates than the control also suggests there is a substance in milk overall that increases
susceptibility.
The difference between the conventional plaque count and the organic was minimal
(298). Also, only one trial was done. A statistical analysis must be done to determine whether
this is a substantial difference. If a milk substance makes E. coli susceptible, then perhaps it can
be isolated and used to make bacteriophage treatments more effective.



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Introduction
Could viruses replace antibiotics as cures for pathogenic infections? Could milk make
viruses more effective weapons against pathogens? Could the way milk is farmed, organic or
conventional, make a difference? My project question is, Does conventional milk make E. coli
less susceptible to infection by bacteriophage T4r than organic milk does? I chose this topic
because I am fascinated by both bacteria and viruses. Also, I want to learn more about the value
of conventional and organic foods. A 2012 Stanford study sparked my interest because it
suggested that there is no nutritional benefit to organic food over conventional. Last year, I
compared the amount of antibiotic resistance in the bacteria found on organic versus
conventional chicken. As many organic milk brands claim to have no added hormone, I
wondered whether this confers an advantage. Bacteriophages are viruses that infect and kill
specific species of bacteria. Because of this property, they can be used like antibiotics, and in the
future they may be used in this fashion because of the mounting numbers of antibiotic-resistant
bacteria like MRSA. I want to know whether a possible difference in the amount of hormones in
milk can influence the susceptibility of a bacterium like E. coli to bacteriophage infection. This
question integrates my multiple interests and some of the techniques I learned in my previous
research project.
The independent variable in my experiment is the type of milk. I will vary it by adding
organic milk to one experimental culture broth and conventional milk to another. The dependent
variable is the amount of bacteria killed by the bacteriophage, measured by the number of
plaques formed on the bacterial culture grown on Tryptone agar. My control treatments are (1)
the bacteria cultured without bacteriophage or milk and (2) the bacteria cultured with
bacteriophage but without milk. All of the cultures will be grown on the same type and quantity
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of agar. The species and amount of bacteria, species and amount of bacteriophage, the amount of
milk, and the temperature will all be held constant.
It might be beneficial to know whether one type of milk can be more harmful than the
other. Since antibiotics are no longer as effective as they used to be, bacteriophages may be a
good alternative way to kill pathogens. This possibility is being researched. If hormones in milk
influence the susceptibility of E. coli to viral infection, we may have to safeguard against
creating a strain of E. coli that cannot be killed by antibiotics or bacteriophages.
Background Research
Fantastic Phages
Viruses can infect a variety of hosts; bacteriophages are viruses that infect bacteria. One
type of bacteriophage is T4, which infects the E. coli bacterium. T4 is part of the T even phage
family, which includes other phages like T2. A mutant form of T4 called T4r is used in this
experiment. T4 is a lytic phage because it lyses and kills its host. It does this by inserting its
genetic material into the host cell to reproduce. The infected bacterium is used as a replicator for
the virus. After creating many copies, the cell is lysed, releasing the new viruses. T4 has a unique
body made of proteins that allow it to accomplish this task. The head of a T-phage is called a
capsid. It encloses the double-stranded DNA, the genetic material. The tail consists of a
contractile tail sheath, six tail fibers, and a base plate. The fibers are used to attach the
bacteriophage to its host. It is important to understand the basic structure of the T4r phage
particle since I am interested in studying its ability to infect E. coli.
"Bacteriophage Fact Sheet." Wisconsin Institutes for Discovery. Morgridge Institute for Research
Outreach Experiences, n.d. Web. 10 Apr. 2014.
<http%3A%2F%2Fdiscovery.wisc.edu%2Fmedia.acux%2F98e1c6ba-bceb-45d3-90a4-
1af754693e73>.
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Terrific T4
T4 is one of the most complex virus particles known, and it is studied very frequently. It
is more sophisticated than many other viruses in that it can inhibit lysis, or put off reproduction
of itself and the killing of its host, to make the fullest use of hosts. Forty percent of T4s genetic
material is devoted to the synthesis and assembly of its virus particle. Most of the time, when
lysis occurs there are already 100 to 150 phage particles in the cell. Since many strains of
pathogenic bacteria are resistant to many antibiotics, some people are looking to viruses as a
disease treatment replacement for antibiotics. The possibility that viruses may someday be used
instead of antibiotics to cure bacterial infections is basic to my hypothesis.
Miller, Eric S., Elizabeth Kutter, Gisela Mosig, Fumio Arisaka, Takashi Kunisawa, and Wolfgang
Rger. "Bacteriophage T4 Genome." PMC. National Center for Biotechnology Information, Mar.
2003. Web. 10 Apr. 2014. <www.ncbi.nlm.nih.gov/pmc/articles/PMC150520/>.

T4 Treatment
T4 and other such viruses could be used to deliver vaccines, to detect malevolent
bacteria, to screen antibodies, or to do gene therapy. They could even be used to protect crops
and fish from infections. Sadly, little research has been done on these uses. The only countries
doing much research are Russia and Georgia. Phages were used during World War II by both the
Russians and Germans. Some scientists think there need to be more in vivo tests. Fourteen
thousand people in the US die each year from multiple-resistant bacteria acquired in hospitals.
MRSA (multiple-resistant Staphylococcus aureus) can be carried by individuals to infect less
healthy people because it is an opportunistic bacterium. With no antibiotic solutions for MRSA,
phages may be patients only hope.
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Researchers at Kenyon College suggest that a cocktail of phages would be most useful
against MRSA. Phage cocktails would be applied directly to a wound to avoid interaction with
the immune system. Theoretically, a phage could be developed for any bacterium. Resistance to
a bacteriophage is not very concerning, as a replacement would only take a few days to develop
and phages evolve as quickly as bacteria. Phages are very cheap to produce. Unfortunately, an
exact diagnosis of the type of pathogen would be necessary in order to choose the right phage to
destroy it.
There are several advantages to viral treatment. Phages are quick; they do not cause
collateral damage; they are not toxic like antibiotics; and, once all the target bacteria are killed,
they die too. The immune system can get in the way and the spleen can absorb the phages too
early. For this reason, some scientists believe that a harmless species of bacteria should deliver
the phage to its pathogenic target. Most of the time, multiple doses would not be required since
viruses can reproduce on their own; although when treatment is early, multiple doses may be
superior. This information is important because it explains why some doctors believe the future
of antipathogenic medicine is bacteriophages.
"Phage Therapy." Microbewiki. Kenyon College, n.d. Web. 10 Apr. 2014.
<http://microbewiki.kenyon.edu/index.php/Phage_Therapy>.

Hormone Hunger
Currently no genetically engineered animals are approved by the FDA, but genetically
engineered salmon from AquaBounty are currently being reviewed. They would be engineered to
produce hormones year round so to grow larger. However, there are lots of genetically
engineered fruits and vegetables. Recombinant bovine growth hormone (rBGH) was approved by
the FDA in 1993. When given to cows it makes them produce more milk. Many people fear that
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insulin-like growth factor (IGF), another hormone, might act like human growth hormone in
dangerous ways. rBGH-treated cows produce milk that has up to 10 times more IGF than milk
from other cows. It is disputed, but a few studies suggest that lots of IGF in the blood can cause
people to be around 50% more likely to get cancer. That amount of IGF in one quart of milk is
still only about 1.05% the amount of IGF used by the human body each day. It is possible that
milk in general may influence the human body to produce more IGF. Still another hormone
found in cow milk, estrogen, is a hormone used to fatten up cattle. Cows often have an implant in
an ear that gives them hormone. Another concern about hormones in food is that they may cause
puberty to occur earlier. The amount in estrogen in a three-ounce serving of beef treated with it is
only 0.00025% the amount of estrogen in a woman and 0.001% the amount in a man. This is
important to my study because it suggests the effects added hormones may cause.
Brovall, Sandra. "Hormones In Food: Should You Worry?" The Huffington Post.
TheHuffingtonPost.com, 31 Jan. 2011. Web. 10 Apr. 2014.
<http://www.huffingtonpost.com/2011/01/31/hormones-in-food-should-y_n_815385.html>.
Good Genes
Bacteriophages were involved in several important experiments. Martha Chase and
Alfred Hershey used bacteriophages in the 1950s to prove that DNA was the genetic material of
a cell. They watched the viral DNA enter the bacterium, and when it replicated the virus they
knew that it was the necessary particle of the virus for its reproduction. DHerelle took bacteria
from recovering dysentery patients and filtered them through porcelain. When the bacteria died,
he added new bacteria. When those died, he knew that it must be something smaller than
bacteria, bacteriophages, that was killing the bacteria. One study treated bacterial lung infections
in mice with bacteriophages. The genetics of soil, vegetation, and the ocean all are affected by
bacteriophages. Not all phages kill their hosts; sometimes they change cells and offer new genes
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to them through conjugation. Viruses in the bacteria of aphids contain a gene that protects the
aphids from wasps. There are phages used to protect tomatoes. There are no phage therapies for
humans approved by the FDA, but in France, Russia, and Poland there are nonprescription phage
cocktails. There are 1031 known viruses worldwide. Nonessential genetic material is called a
moron. Twenty-five septillion, or 25,000,000,000,000,000,000,000,000 replications of viral
genomes occur each second, and one in a thousand of those is a mutation. This information is
important because it demonstrates the possibility that viruses may be helpful.
Kirby, Breeann, and Jeremy J. Barr. "Going Viral." The Scientist. LabX Media, 13 Sept. 2013. Web. 10
Apr. 2014. <http://www.the-scientist.com/?articles.view/articleNo/37208/title/Going-Viral/>.

Hypothesis
If E. coli bacteria grown in organic milk are more susceptible to the T4r bacteriophage
than E. coli grown in conventional milk, then there will be more plaques on the bacteria culture
that was grown in organic milk possibly because the cows that produce it are not given extra
hormones.
Materials
10 Petri Dishes
Hard Tryptone Agar
E. coli on Agar Slant
Tryptone Broth
10 Soft Agar Gels
Organic Milk
Conventional Milk
5 mLs T4r in Broth
Safety Goggles and Gloves
Water Bath
25 Sterile Pipettes
Incubator




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Procedure
24 Hours Before:
Preparation
1. Pour Tryptone broth into the tube with the E. coli on slant, agitate, and incubate at room
temperature.
2. Pour solid agar plates.

Day of Experiment:
Preparing the Cultures
3. Heat soft gels.
4. Put them in 48C water bath.
5. Add 0.2 mL E. coli to gels 1, 2, 4, 5, 7, and 8.
6. Add 0.2 mL Organic Milk to gels 3, 4, 5, and 9.
7. Add 0.2 mL Conventional Milk to gels 6, 7, 8, and 10.

Serial Dilution of Bacteriophage Stock
8. Take 1 mL of T4r (original stock) using a sterile pipette.
9. Put in 9 mL of broth (now diluted by 10 to the -1
st
power).
10. Cover and invert.
11. Repeat steps 8 10 by taking 1 mL of the previous diluted broth until dilution 10 to the -6
th

is reached.
Adding T4r Bacteriophage to E. coli Gel Cultures
12. Add 1 mL of T4r (10 to the -6
th
) each to gels 2, 5, 8, 9, and 10.
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13. Invert each soft gel 3 times gently and pour each onto its respective plate. Swirl.
14. Incubate plates at 37C for 24 hours.
15. Take pictures of all the plates and import them to Paint. Count the plaques on each plate.
Summary of Treatments
Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Plate 7 Plate 8 Plate 9 Plate 10
E. coli E. coli E. coli E. coli E. coli E. coli
O milk O milk O milk O milk
C milk C milk C milk C milk
T4r T4r T4r T4r T4r
Data
Trial 1: For this trial the dilution of T4r I used was too potent, so the E. coli were wiped out on
all plates but my controls that had no virus.
Dilution Mini Experiment: For this trial I tested various dilutions to see which would be best
(the ideal plate has about 300 plaques). I discovered that dilution 10 to the -6
th
was best.
Trial 2: The E. coli culture was very weak, so there were no lawns in this trial.
Trial 3: This was my final trial, giving me the results elaborated upon below.

Plate
Number
1 2 3 4 5 6 7 8 9 10
Ingredients E. coli E. coli
T4r
O milk E. coli
O milk
E. coli
O milk
T4r
C milk E. coli
C milk
E. coli
C milk
T4r
O milk
T4r
C milk
T4r
Number of
Plaques
0 270 0 0 1075 0 0 777 0 0



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(Above is a picture of my counting method using Microsoft Paint.)
Graph
The graph below shows the impact of different types of milk on the virulence of T4r on
E. coli. The data support my hypothesis that organic milk (compared to conventional milk)
causes E. coli to be more susceptible to viral infection. There were more plaques on the plates to
which milk had been added. It seems as though something in milk (both organic and
conventional) increases the virulence of T4r on E. coli, but there is less of that substance in
conventional milk.
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Conclusion
If E. coli bacteria grown in organic milk are more susceptible to the T4 bacteriophage
than E. coli grown in conventional milk, then there will be more plaques on the bacteria culture
that was grown in organic milk possibly because the cows that produce it are not given extra
hormones. My hypothesis was supported. There were more plaques on the E. coli lawn from the
culture grown with organic milk. However, both the cultures grown with milk had more plaques
than the plain control E. coli culture. This suggests that there is something in milk that increases
T4rs virulence or E. colis sensitivity. My data cannot confirm or deny that it is the hormone
that makes the difference.
Discussion
The plaques were very difficult to count. I imported the pictures to Microsoft Paint, and I
put a red dot in the center of each plaque I counted. There were some large areas of inhibited
growth, and I counted these as many plaques. Because I had to estimate the number of normally-
shaped plaques that would have fit in that space, they might have slightly upset my count. It
might have been beneficial if I had measured the entire area of that zone, and equated it to a
number of plaques mathematically, rather than placing red dots on it as evenly spaced as I could.
Recommendations
My results suggest that milk increases the sensitivity of E. coli, but that conventional
milk does not have as large an impact on this as organic milk. I would like to discover what there
is in milk that causes this effect. I would need to isolate different constituents of milk, and then
repeat this experiment with each of the isolated substances. However, first I would like to repeat
this same experiment. I only had the opportunity to do one trial, so I would like to gather more
data.
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Applications
As more and more bacteria become resistant to an increasing number of antibiotics, we
will need alternative therapies. Bacteriophage viruses may be an important replacement. They
only target specific species, they do not infect human tissues, they reproduce on their own, and
they die when all of their target bacteria are destroyed. Drinking milk may enhance the therapy.
My results suggest that a substance in milk makes E. coli more susceptible to T4r. If that
substance is isolated and it has the same effect with other viruses and other bacteria, then it could
increase the potential effectiveness of phage treatments tremendously.
Works Cited
"AquaAdvantage Fish." AquaBounty Technologies. AquaBounty.com, n.d. Web. 10 Apr. 2014.
<http://www.aquabounty.com/products/aquadvantage-295.aspx>.
"Bacteriophage Fact Sheet." Wisconsin Institutes for Discovery. Morgridge Institute for Research
Outreach Experiences, n.d. Web. 10 Apr. 2014.
<http%3A%2F%2Fdiscovery.wisc.edu%2Fmedia.acux%2F98e1c6ba-bceb-45d3-90a4-
1af754693e73>.
Brovall, Sandra. "Hormones In Food: Should You Worry?" The Huffington Post.
TheHuffingtonPost.com, 31 Jan. 2011. Web. 10 Apr. 2014.
<http://www.huffingtonpost.com/2011/01/31/hormones-in-food-should-y_n_815385.html>.
Carolina Biological Supply Company. Plaque Assay Plating Set Instructions. N.p.: Carolina Biological
Supply, 1999. Print.
Carolina Biological Supply Company. Techniques for Studying Bacteriophages of Escherichia Coli
Reference Manual. N.p.: Carolina Biological Supply, n.d. Print.
Chang, Kenneth. "NEWS ANALYSIS; Parsing of Data Led to Mixed Messages on Organic Food's
Value." The New York Times [New York] 16 Oct. 2012, Science Sec.: N. Pag. Print.
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Kirby, Breeann, and Jeremy J. Barr. "Going Viral." The Scientist. LabX Media, 13 Sept. 2013. Web. 10
Apr. 2014. <http://www.the-scientist.com/?articles.view/articleNo/37208/title/Going-Viral/>.
Miller, Eric S., Elizabeth Kutter, Gisela Mosig, Fumio Arisaka, Takashi Kunisawa, and Wolfgang
Rger. "Bacteriophage T4 Genome." PMC. National Center for Biotechnology Information, Mar.
2003. Web. 10 Apr. 2014. <www.ncbi.nlm.nih.gov/pmc/articles/PMC150520/>.
"Phage Therapy." Microbewiki. Kenyon College, n.d. Web. 10 Apr. 2014.
<http://microbewiki.kenyon.edu/index.php/Phage_Therapy>.


(Above are all of my plates from my final trial.)