Running Head: Bacterial Warfare
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The Effect of (Manipulated Variable) on (Responding Variable)
Student Name
Liberty Union High School
Friday, November 12
Teacher Name
Bacterial Warfare
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Table of Contents
1. Introduction…………………………………………………………………………………..2
2. Hypothesis……………………………………………………………..………………………8
3. Methods and Materials……………………………………………………….……….8
4. Results………………………………………………………..………………………………….10
5. Conclusion……………..……………………………………………………………………....12
6. Graphs…………………………………………………………………………………………….13
7. Pictures………………………………………………………………………………………….16
8. References Cited…………………………………………………………………………22
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Introduction
Year after year, cases of antibiotic resistant infections are increasing all over the
world. Recently, a wrestler in Ohio fell victim to one of the most prominent antibiotic
resistant infections, MRSA, or Methicillin Resistant Staphylococcus Aureus. Bacteria
from the mat or another person, may have entered his body through a scrape or cut. When
the cut came in contact with MRSA, an infection occurred. This study is a continuation of
a study performed last year. The first study tested the effectiveness of the antibacterial
agents, oregano, garlic, olive leaf extract, and Amoxicillin. Garlic proved to be most
effective at destroying Staphylococcus Epidermis and did not affect the Lactobacillus.
The current study was done using garlic, colloidal silver, thieves oil (combination of
lemon, cinnamon, and clove oil), and Amoxicillin. This study demonstrats how the
abtibacterial agents work and how effectively each one killed the Staphylococcus without
killing the beneficial bacteria in the body. The beneficial bacteria are necessary in the
body, because it helps fight infection by creating a non-acidic environment in which
pathogenic bacteria cannot live.
In 2011, improvements were made to more proficiently test antibacterial agents.
In 2010, there was a problem with mold growth in the Petri dishes in the experimentation.
Techniques were used to achieve a more sterile environment for the bacteria to grow. An
inoculating loop was created using a dowel rod and 25 gauge nickel chrome wire. It was
sterilized by holding it in a Bunsen burner flame, then obtaining the bacteria instead of
using cotton swabs. This study also used a refurbished, hospital-grade lab incubator
instead of a fish tank and heat lamp to grow bacteria. The incubator was calibrated and
monitored for an optimal, consistent temperature. Mannitol salt agar was used to grow
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certain strains of Staphylococcus but inhibit mold growth. It also identified the
Staphylococcus Aureus by forming a green ring around it. Petri dishes had grids (fiftytwo 1 cm2 squares) on the bottom for recording bacterial growth. Finally, this study tested
the effectiveness of additional alternative remedies such as colloidal silver and thieves’
oil.
The study of bacteria and treatments also bears historical significance. On
September 17, 1683, Antony Van Leeuwenhoek found bacteria in the plaque on his teeth.
He described them in a letter to the Royal Society of London, according to Lisa Yount in
her article, “Antony van Leeuwenhoek and his Little Animals,” Leuwenhoek describes
them as, “an unbelievably great company of living animalcules, a-swimming more
nimbly than any I had ever seen up to this time. The biggest sort bent their body into
curves in going forwards. Moreover, the other animalcules were in such enormous
numbers, that all the water seemed to be alive," (Yount, Lisa. “Fleming, Sir Alexander.”
A to Z of Biologists. Facts on File, Inc.).
“Almost 4,000 years ago,” says Rob McCaleb, from the website, Boosting
Immunity with Herbs, “the Chinese used herbs such as echinacea, astralagus, and garlic to
treat common maladies. (McCaleb, Rob. “Boosting Immunity,” Herb Research
Foundation). Recently, in a study to find alternatives to antibiotics, oregano and other
herb’s effectiveness, Harry G. Preus proved that oregano was effective at killing bacteria
in lab rats.
Because of the overuse of antibiotics, hand sanitizers, antibacterial cleaning
products, and antibiotics used in raising poultry and livestock, bacteria are mutating and
becoming immune to antibiotics. This is why there is a need for alternate remedies.
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Science Online states that these ‘superbugs’ are the culprits behind thousands of deaths in
hospitals each year. (Report: “Superbugs killed record number. Science Online”. Facts on
File, Inc.)
MRSA, Methicillin Resistant Staphylococcus Areus, is a harmful antibiotic
resistant strain of Staphylococcus Aureus. Staphylococcus Aureus lives naturally on the
skin of 20% of the world’s population. Strains of Staph, growing out of control, are the
culprit behind most skin infections. Symptoms of MRSA include skin rashes and boils,
but can ususally be treated with certain powerful antibiotics. (Michelle, Todd. “Superbad
Superbug.” MRSA Packs a Potent Punch”) In England, according to the British Office
for National Statistics, about 1,625 people died from the infection. (British Office for
National Statistics). A person can get MRSA almost anywhere. MRSA can only enter the
body through a break in the skin.
Swine Flu has been a recent viral pandemic all over the world. The mortality rate
from swine flu is .5% while seasonal influenza has a mortality rate of .77%. Why is that?
According to Dr. Mercola from Mercola.com, when the H1N1 weakens your body’s
defenses against infection, Staphylococcus Aureus can enter your body and cause
pneumonia, an infection in the lungs that leads to death. (Dr. Mercola. “Swine Flu, One
of the Most Massive Cover-ups in American History.”)
One type of bacteria used in this experiment is Staphylococcus Epidermis.
Staphylococcus Epidermis is a naturally occurring bacteria on the skin that is potentially
pathogenic. Staphylococcus Epidermis is very similar to Staphylococcus Aureus however,
it does not break down Mannitol compounds in Mannitol Salt agar and does not produce
the deadly toxins produced by Staphylococcus Aureus.
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Another antibiotic resistant infection that is at large today is C.Diff (Clostridium
Difficile). These bacteria live as part of one’s normal flora in the intestinal track. When
there is not a balance of normal flora or consisting of several beneficial bacteria, harmful
bacteria can take over. This can happen when a substance such as an antibiotic, steroids
or certain medications destroys all the beneficial bacteria, but do not kill the pathogenic
bacteria. The harmful bacteria then have nothing to hinder them from growing and taking
over. When doctors prescribed antibiotics for this infection, it eventually became
antibiotic resistant. C.Diff. kills over 6,424 people each year. It causes watery diarrhea,
nausea, and abdominal pain. C Diff. can be found in hospitals, nursing homes, childcares,
and adultcare facilities. In combination with probiotics, the only cure is an antibiotic
called Flagyl.
Penicillin, the first antibiotic to be created, was invented by Alexander Fleming in
1928, when he tested secretions from different molds to kill bacteria. Antibiotics are
chemicals used to kill or decrease the growth of bacteria. They also interfere with the cell
structure, thus destroying the bacteria. Antibiotics work one of two ways according to
Christian Nordqvist from Medical News.com. They can kill the bacteria directly causing
a bacteria’s cell wall to collapse, (a bactericidal). The second way causes the bacteria not
to multiply or to suffocate by disabling the bacteria’s ability to absorb oxygen by
blocking chemical receptors that allow the bacteria to reproduce, called a bacteriostatic.
This study used a bactericidal antibiotic called Amoxicillin. Some antibiotics are
made naturally, by growing certain types of bacteria or mold and using the substance
secreted to make antibiotics. Others are made synthetically (man-made), by using a
mixture of chemicals. There are various side effects from antibiotics. The following are
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just a few: diarrhea, nausea, abdominal pain, vomiting and dizziness. (Nordqvist,
Christain. “The New Chemistry.” Medilexicon International.)
Bacteria can become immune to antibiotics, causing bacterial resistant infections.
According to Stanley Maloy from San Diego State University, ((Maloy, Stanley.
"Horizontal Gene Transfer". San Diego State University), when bacteria are exposed to a
certain antibiotic, all the bacteria are killed off except the ones that have acquired the
genes to resist the antibiotic through horizontal gene transfer or through a natural
mutation. Horizontal gene transfer is the bacterias’ method of genetic variation. During
this process, genes are switched between two bacterial organisms. The result is antibiotic
resistant infection.
Garlic is an herb that belongs to the onion family. It has antibacterial properties
(kills bacteria), is antifungal (kills fungi), and is an antioxidant (helps combat against
diseases). Garlic works like an antibiotic without the side affects. It has even been used to
combat MRSA, according to Michelle Moore from Staph Infection Resources. The garlic
causes the cell wall of the bacterium to swell, causing the bacterium to burst. As a result,
the bacterium dies. Cloves of garlic contain an amino acid (building block of a protein)
called allicin. Allicin contains enzymes (substance causing a chemical reaction), which
react with the bacteria to break down their cell walls. Bacteria do not have the
opportunity to become immune to garlic. Garlic can be used in various forms, including
oil, extract, or a capsule. (Moore, Michelle. “Garlic, A Natural Treatment for MRSA
Infections.)
Thieves’ Oil is a type of herbal supplement that contains clove oil, lemon oil,
and cinnamon oil. Studies have been conducted by such facilities as the Cleveland Clinic
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for the oil’s effectiveness against hospital acquired infections. Each component of
Thieves Oil has been tested to test its effectiveness against certain types of bacteria such
as Staphylococcus. Both lemon oil and cinnamon oil were tested against Staphylococcus.
Lemon oil, the killing agent in Thieves Oil, killed the Staphylococcus in less than two
hours while cinnamon oil was shown to inhibit the growth of Staphylococcus. (Eaton,
Caleb. “Manage bacteria with a revolutionary set of products offering a multi-prong
approach...powerful external applications but safe enough to take internally.)
Colloidal silver is a negative silver ion (AG-) suspended in liquid, and it
transfers two electrons to the oxygen in the bacterial cell wall creating instability. Since
oxygen forms most of the chemical bonds in the cell wall, the cell wall breaks apart.
There are not many studies on colloidal silver, and the results are inconclusive (Gerstner,
Louis. "Colloidal silver." Memorial Sloan-Kettering Cancer Center). This study will
show if colloidal silver is effective as an alternative to antibiotics.
While sometimes medication is needed to cure infections, preventative measures
are also necessary. Most people use hand sanitizer; it kills most harmful bacteria, but it
also kills beneficial bacteria on the skin called Staphylococcus Epidermis. Overuse of
hand sanitizer can cause pathogenic bacteria to mutate. Because of improper hand
washing, 9,000 adults get food poisoning every day caused by an overgrowth of bad
bacteria growing in food. According to the U.S. Center for Disease Control, proper hand
washing techniques include lathering one’s hands with soap, scrubbing hands and under
the finger nails for 15-20 seconds, rinsing thoroughly with warm water, and drying one’s
hands completely.
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Proper wound care also prevents bacterial infections. First, clean the wound with
soap and water. Next, treat it with Neosporin or another antibiotic ointment. Lastly, cover
it with a bandage. If debris is embedded in the wound, a substance such as hydrogen
peroxide would be helpful in cleaning out the wound (Proper Hand Washing Procedure.
Lyn Distributing Personal Care Products).
Because of the lack of information on the effectiveness of herbs and alternative
remedies, this project will test whether antibiotics or alternative remedies are better at
killing Staphylococcus (harmful bacteria) and not killing the beneficial bacteria, such as
Lactobacillus.
Hypothesis
The hypothesis states that out of the four antibacterial agents that will be tested:
(garlic oil, colloidal silver, Thieves oil, and Amoxicillin), colloidal silver will work the
best at killing the Staphylococcus (potentially harmful bacteria) and not killing the
Lactobacillus (beneficial bacteria), which is necessary for fighting infections.
Methods and Materials
The materials used for this experiment were twenty prepared Mannitol Salt agar
plates and twenty prepared plates of LMRS agar, named for its inventors (de Man,
Rogosa and Sharpe). Both types of petri dishes had grids (52 1 cm2 squares) on the
bottom for easy recording of bacterial growth. Different types of agar were needed,
because certain types of bacteria require different types of nutrients and PH (acidity) to
grow. Lactobacillus requires an acidic enviroment and different nutrients, which the
LMRS agar provides.
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Mannitol Salt agar is a selective media that allows only species of Staphylcoccus
to grow, also identifying Staphylococcus Aureus by forming a greenish-yellow ring
around it. Some Staphylococcus was actually identified and treated. The antibacterial
agents used were Amoxicillin, colloidal silver, Thieves Oil, and garlic oil. The bacterium
used to inoculate the agar was Staphylococcus Epidermis which makes up normal human
skin flora. The beneficial bacterium used was a strain of Lactobacillus from probiotic
capsules (pulled apart and dissolved in sterile water). The mixture was applied using a
sterile inoculating loop to inoculate the LMRS and Mannitol Salt Petri dishes. For safety
measures, latex disposable gloves were used to keep the bacteria from potentially
spreading. A refurbished hospital-grade, lab incubator was used for optimal bacterial
growth. The incubator’s temperature was tested and monitored to maintain a constant
98.6° F
When this experiment was performed, five Petri dishes with Mannitol salt agar
were inoculated with Staphylococcus, and five LMRS dishes were infected with
Lactobacillus. After three to four days of growth in the incubator, the Petri dishes were
treated with Amoxicillin, garlic oil, Thieves Oil and colloidal silver by using a sterile
dropper and distilled, sterile water for mixing and applying antibiotics to bacteria. The
Petri dishes were covered in the selected bacterial agent using the sterile dropper. A
treated Petri dish from Lactobacillus was used as a control. The appearance of the
Staphylococcus was recorded and the bacteria were grown for two days. The entire
process was repeated for a second trial. Pictures were taken to record the growth and
destruction of the bacteria for comparison of the two trials.
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Results
During this experiment, the goal was to find out which of the following
antibacterial agents were most effective at killing the Staphylococcus and not killing the
Lactobacillus (responding variable): garlic oil, colloidal silver, Thieves oil, or
Amoxicillin, (manipulated variables). When the Staphylococcus was first grown, it
looked like tan, flat, shiny spots scattered throughout the Petri dish. The Lactobacillus
resembled tiny white, slightly raised, specks growing very closely together in colonies,
scattered throughout the entire Petri dish.
These antibacterial agents included garlic oil, colloidal silver, Thieves’ Oil and
the antibiotic Amoxicillin. Among these materials, colloidal silver was hypothesized to
be the one that worked the best. The following results were achieved during the
experiment.
Colloidal silver was the most effective at killing the Staphylococcus and leaving
the Lactobacillus unharmed. In the first trial, 29 out of 32 cm2 was left. In the second
trial, the colloidal silver killed 1 cm2 of the bacteria out of 8. Colloidal silver did not kill
any of the Lactobacillus, making it the most effective. The colonies that were left were
smaller in appearance. Colloidal silver killed an average total of 10.94% of the harmful
bacteria grown.
The second most effective was Thieves’ oil. Thieves oil fell third to antibiotic in
killing the Staphylococcus Epidermis and Staphylococcus Aureus and other harmful
bacteria, but, unlike the antibiotic, did not kill any of the Lactobacillus. During the first
trial, out of 33 cm2, 30 were left. In the second trial, 34 cm2 out of 35 was left. The
Thieves oil killed 5.97% of the Staphylococcus.
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Falling into third was the antibiotic. The antibiotic was second in killing the
Staphylococcus. Out of 52 cm2, 50 were left in the first trial. During the second trial, out
of 18 cm2, 16 cm2 were left. An averaged 6.51% of the Staphylococcus was killed. On the
other hand, the antibiotic also killed the Lactobacillus. The antibiotic killed 3 out of 34
cm2 in the first trial, 8 cm2 out 45 cm2 in the second trial, and 6 cm2 out 44 leaving 38
behind. The antibiotic killed 11.49% of the Lactobacillus.
Last are the results for garlic. In 2010, garlic placed first because the antibiotic
killed almost all the Lactobacillus (beneficial bacteria). In 2011, the garlic never killed
the Lactobacillus, but was not as effective as Thieves Oil or colloidal silver at killing
Staphylococcus. In the first trial, the garlic killed 2 cm2 out of 24 but did not kill any
Staphylococcus in the next trial. The garlic killed an average of 4.16% of the
Staphylococcus, making the garlic fall into last place.
Conclusion
Because of the overuse of antibiotics in the world today, causing antibioticresistant strains of bacteria like MRSA and C.Diff, doctors are looking for new
alternative ways to treat bacterial infections. When testing some of the most popular
alternative methods to treat bacterial infections, including garlic, Thieve’s Oil, colloidal
silver, and Amoxicillin, results proved that colloidal silver worked the best at killing the
Staphylococcus while leaving the Lactobacillus unharmed, supporting my hypothesis.
Colloidal silver binds to the oxygen in bacterial cell walls, causing the cell to
fall apart. This makes colloidal silver the most effective out of all the antibacterial agents.
Bacteria also cannot become immune to colloidal silver because of the way it destroys the
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bacteria. Because of this, colloidal silver could be used to treat patients with antibiotic
resistant infections and eventually may be used as an alternative to antibiotics.
If repeating this project in the future, the quality of the agar would be examined
more closely. During this study, the agar shrank and crystallized. It was also noted that
the original agar was inconsistent in thickness and some of the agar was very thin. As a
result, only two trials could be conducted for the Staphylococcus without controls. The
length of treatment time also had to be shortened. If treatment times could have been
longer, more substantial results might have been obtained. Due to expense and lack of
resources, additional trials could not be performed.
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