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Darian Schube

Section # 6
Unknown # 84

Introduction:
In general exercises are done to practice a plan or procedure. The point of the exercise is
to get practice, gain knowledge and show understanding on how to classify and isolate a
bacterium microbe through a series of tests and procedures followed. The bacteria utilized to
conduct the exercise were Staphylococcus epidermidis, Proteus vulgaris, Staphylococcus aureus,
Bacillus cereus, Escherichia coli, Citrobacter freundii, Proteus mirabilis, Enterobacter
aerogenes, Providentia stuartii, Bacillus subtilis, and Enterococcus faecalis.

Bacteria are

ubiquitous and play an important role to everyday life especially in humans and in the medical
field. Bacteria are important to humans for several reasons. For example, Bacteria in the
digestive tract help to break down food that is ingested by humans. In other areas of the body
bacteria prevent other more toxic bacteria from taking up living accommodations. According to
Robert Bauman, author of Microbiology with Diseases by Body System, bacteria have several
different kinds of relationships with their hosts. One of those relationships is a commensal
relationship. In other words bacteria live on or within humans and neither benefit them or harm
them. Another relationship is mutualism were both benefit (411). Without bacteria humans would
become sick and would have problems with the basic processes of life. Also bacteria can have
beneficial factors or negative factors to the food and beverage industry. A negative factor is that
some bacteria are responsible for the decomposition of food and for the food borne illnesses that
make people sick; however, there are beneficial factors to the bacteria in food. According to the
Encyclopedia Britannica, bacteria are responsible for the making of dairy products other than
milk. Bacteria break down the components of milk and help convert them into items like ice
cream, cheese and yogurt (9). This enables human to eat these products. Not only do bacteria
play a role in humans and in food but they also play a role in the medical field. Since bacteria are

ubiquitous and can cause parasitism they play a big role in the medical field (Bauman 411).
Bacteria may help humans like digesting food and expelling wastes or by creating food to eat but
bacteria are also what makes humans sick and can cause death. Bacteria that are not naturally
found in the body can cause harm to humans when introduced. In other words they become
pathogens. Pathogens can spread causing high health risks. For example, the Black Plague swept
through Europe severely diminishing the population. This can be a concern to the health
sciences. Another example is bacteria that cause gangrene when introduced to the body and if left
untreated can cause appendages to be lost or in severe cases cause death. It is important for
health professionals to identify what microbes cause such problems and to isolate them for
treatment. By isolating a bacteria species it can be identified. It is important to properly identify a
bacterial species to ensure the safety of the immune compromised patient, the health care
professionals, and people around the patient who are not infected but exposed. When a bacterium
is properly identified the right medicine/ treatment can be given. If the bacteria are not properly
identified then the safety of others could be compromised and the treatment could damage
healthy cells in the patient, making them sicker.
To isolate bacterial microbes for treatment health professionals must classify them first.
When classifying a microbe the ideal goal is to get it down to the species. In order to classify a
microbe to a species a series of test are utilized. A common way to start is to look at the microbe
under a microscope. A series of other test can be preformed to further classify the microbe.
Staining, morphology and interaction with other cells and microbes can be utilized to further
narrow down the classification (Bauman 117). The series of test preformed are biochemical tests.
Biochemical tests have advantages and disadvantages. Some advantages are that these types of
tests can help to identify the species of bacteria by helping to distinguish between types, these

tests can identify specific properties of a given microbe like if it is Gram positive or Gram
negative which is told by Gram staining, and these tests can also tell what environment a
microbe flourishes in, for example if it is an anaerobe or it lives in an acidic environment or not.
Some disadvantages to biochemical tests can be the lack of what they can do for example
biochemical tests that are PR-broths can only test for one sugar at time, tests can be time
consuming for the different ones and for incubation time, and results can come out different than
expected. For example, growth can occur on a medium when it was not supposed to.
Materials and Methods:

Biochemical tests follow a procedure to ensure safety and maximum accuracy. Aseptic
techniques were utilized throughout the procedure to ensure safety and accuracy. Stated by Chess
aseptic techniques utilized to inoculate tubes, plates and slants follow simple and main steps.
Disinfectant to wipe surface before and after all procedures are done. A test tube rack to prevent
breaks and spills. Inoculating loops and needles employed for proper transference. A Bunsen
burner instrumented for proper sterilization of the loops and needles before and after inoculation.
Hand washing to prevent transference (30-32). For plates the lid is usually opened at a tilt to
prevent the spread of pathogens through the air. The loop is run through the burner before and
after use and a standard streak is used. Same procedure is followed for tubes except they are run
through the burner before and after inoculation and then recapped and usually swirling of the
loop was the technique for liquid medium or a needle to stab non liquid mediums. Slants are run
through the burner to heat fix bacteria. Water used to rinse slant between steps of staining.
Bibulous paper was utilized to remove water at end of the procedure. For non-staining slants the
loop was heated before and after procedure and inoculation. All materials were disposed of
properly in biohazard bags to prevent contamination.
The first method utilized to classify the unknown was a simple stain called a Gram Stain.
This was chosen as the first test because of the differential properties between Gram negative and
Gram positive bacteria. The crystal violet stain allows for this to happen. The iodine seals in the
stain. Ethyl alcohol breaks down lipids of Gram negative to allow for the other stain to take
place. Safranin is utilized to give Gram negative bacteria their color. In the end Gram Positive
bacteria come out purple in color and Gram negative come out with a pink color to them. Gram
Stain is also good at telling morphology of bacteria. The second test that was utilized was a
catalase test. Catalase test is a differential test between aerobic organisms and anaerobic

organisms. Hydrogen peroxide is the differentiator between the two types. Hydrogen Peroxide is
added to the organism on a slant and if catalase is present then it will bubble releasing oxygen
gas into the sir making the organism aerobic. This test just takes a matter of a couple seconds to
complete. In other words no incubation is needed. The next tests to be done were done at the
same time. They were a SIM test, a PR-Mannitol broth, and a Citrate test. The SIM test was also
a differential test like the others because it told if the unknown was motile, indole or produced
sulfur. The indicator for indole was Kovacs. The other indicators were a color change for sulfur
or spreading of growth for motility. A positive for indole would be a red ring at the top, for sulfur
would be a black color change and for motility it would be more than one skinny straight line.
Incubation time was forty-eight hours. The PR-Mannitol broth was utilized to differentiate
between mannitol fermenting organisms. Incubation time was forty-eight hours. A positive result
would have been a change of yellow in color and a negative would have stayed pink. The
indicator is phenol red. The citrate test incubated for forty-eight hours. A positive result is a
change in color to blue. A negative would be a green color. Bromthymol blue is the indicator to
differentiate between Gram positive bacteria. It tests for the production of ammonia. The final
two test used were Urease test and a Blood Agar test. Urease test was incubated twenty-four
hours. It is a differential media to separate species. It tests for ammonia production. If ammonia
is produce the medium turns pink making it a positive result. Phenol red acts as the indicator.
Blood agar is very unique because it only differentiates between the types of hemolysis and
whether or not it is produced. The active indicator is hemolysin. There are three possible results
for this test to be positive. A beta result would be a white growth all over the media and a clear
color in the agar. An alpha appearance would be a bluish green change in agar. A gamma would

just have clear growth with no change in color to the agar. It was incubated for twenty-eight
hours.
Results:
Potential

Gra

Morpholog

PR-

Catalas

Citrat

Unknown

Mannito

Staphylococcu

Stain
+

Agar

l
Cocci

s
Aureus
Staphylococcu

Cocci

yellow

pale

alpha

pink
+

Hot

gamma

Pink

epidermidis
Enterococcus

Urea Blood

pink
+

Cocci

faecalis
Source: Brenner, Don J. ., Noel R. . Krieg, James T. . Staley, and George
M. . Garrity. Bergey's Manual of Systematic Bacteriology. New York:
Springer, 2005. Print.
The results for the Gram stain were purple and seemed to be in an irregular cluster format
( Chess 51). The morphology of the unknown was a circular or cocci shape (Chess 51). The
Catalase test produced a positive result with lots of bubbles. The PR-Mannitol was a negative
result. The citrate test was a negative result but seemed to be cloudy on the top. The Urea test
was a negative result with absolutely no color change. The Blood Agar was a positive result with
lots of clear growth and no color change to agar.

Discussion:
The first test conducted on the unknown was Gram Stain and morphology. This was to
differentiate between Gram positive and Gram negative bacteria and to tell the shape of the
bacteria to differentiate between Gram positive if that were the case. When the stain was
complete the work was examined with the microscope and the species appeared to be purple. So
after that conclusion Morphology was looked at as shown in the dichotomous key above. The
bacteria had a cocci shape to it. That narrowed the choices down to three bacteria
Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis. From the finding
in the Bergeys manual the next step decided was a catalase test since it did not take that long for
results. Since Enterococcus faecalis has a negative result, which is shown in the table above a
positive result would narrow the bacteria down to two species. The results came back with lots of
bubbles meaning a positive so the next test was conducted. A PR-Mannitol broth was utilized
because as shown in the charts above there are clear results of a color change. When no color
change occurred it narrowed the two remaining choices down to one. Staphylococcus
epidermidis was the assumed bacteria but a conformation test was needed. The next test
conducted was a citrate test based on the results from the chart above. The test came back
negative with no color change(pictured left). This led to suspicion if a test was done wrong. This
lead to a urea test to be done for sure since the info on the citrate test was a little sketchy. A urea
test would have a positive for Staphylococcus period. The test was conducted twice. Both times
the test came out negative(pictured right). This led to a bafflement. It had to be Staphylococcus
because of the catalase test. As one last try and because other doing a similar project where
having trouble with the urea test all the way around a Blood Agar test was decided. The test was

done with extreme caution to ensure accuracy. The test came back confirming previous
assumptions of Staphylococcus epidermidis. This ended the testing.
The results prove that the unknown bacterium is Staphylococcus epidermidis. The color
of the bacteria came out to be stained purple making it a Gram positive. The
Morphology came out to be cocci making it a circular bacterium. The Catalase
test came back with lots of bubbles meaning that it was a aerobic organism. The
PR-Mannitol came back negative (pictured at right), which means that the
organism could not ferment mannitol. The Citrate and Urea test both came back
negative meaning that ammonia could not be
made(Chess 169,173). This could be due to not enough
incubation time or improper inoculation techniques. The
Blood agar came out with growth but no color changes to
the agar this means that it was a gamma hemolysis
reaction(pictureed to the left). In other words it could not
produce hemolysis.
Staphylococcus epidermidis tends to be abundant in the skin and finds it
very

suitable for growth and reproduction (Bauman 447,558). The species

Staphylococcus can cause disease within the human body thanks to the slime layers that
prevent phagocytes and leukocytes from attacking it. One disease that it can cause is
Folliculitis, which is basically an infection of the hair follicle. The are becomes read and
swollen and even pus filled. Most doctors would classify it as an acne bump.
Staphylococcus epidermidis can be related to other skin conditions as well. Another more serious
illness that it can cause is Endocarditis. This is a disease that effects the endocardium on the

heart. Symptoms may be fever, murmurs, fatigue and trouble breathing. Clots, strokes and heart
failure can occur if treatment is not received (Bauman 636-637). In general Staphylococcus
epidermidis is a part of the normal microbes on the skin because it is present all the time and
seems to not be a parasite unless opportunity presents itself (Bauman 411). This is proof that it is
relevant to human health because it could become an opportunistic pathogen. This also proves
that it is important to medicine in the way to which we decided to attack such infections
especially since it is present in abundance everywhere.

Literature Review:
bacteria. Encyclopedia Britannica. Encyclopedia Britannica Online. Encyclopedia Britannica
Inc.2013.Web.27Mar.2013
http://www.britannica.com/EBchecked/topic/48203/bacteria/27230/Bacteria-in-medicine.
Bauman, R. Cosby, C. Fulks, J. Lammert, J. Machunis-Masuoka, E. Montgomery, J. Berriman,
L. Seibel, K. et. al. 2012. Microbiology with Diseases by Body System. Pearson Education
Inc. San Francisco, California.
Brenner, Don J. ., Noel R. . Krieg, James T. . Staley, and George M. . Garrity.
Bergey's Manual of Systematic Bacteriology. New York: Springer, 2005.
Print.
Chess, B. 2009. Selected Material from Laboratory Applications in Microbiology: A Case Study
Approach. The McGraw-Hill Companies Inc. United States of America.

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