Erron Jones 1

Erron Jones
Unknowns: 12 & 61
Unknown 12: Enterococcus faecalis
Unknown 61: Proteus mirabilis

Introduction
Enterococcus faecalis is a gram-positive bacterium that belongs to the Enterococcaceae family, and has
a cocci morphology, as well as a diplo or tetrad grouping of cocci cells. While E. faecalis, previously known in
microbiology as Streptococcus faecalis, is not very detrimental to people with healthy immune systems, it can
be quite disastrous in people with comprised immune systems, or other underlying health defects. Commonly
living in the mouth and vagina of the human body, E. faecalis can spread to other parts of the body due to
contamination during surgery or contact between areas of the body that do not normally have contact with each
other (Kayaoglu and Ørstavik, 2004), otherwise known as a nosocomial infection, or health-care associated
infection (Kau et al., Apr. 2005), which in terms of E. faecalis most commonly results in an urinary tract
infection. Due to their normal microbiota conditions, they can be resistant to high levels of salt, heat, and
acidity. Some of the complications that can occur with high infections of E. faecalis can include meningitis,
sepsis, and endocarditis. It can also affect dental health, as it can cause “acute periradicular abscesses” in the
gums in major infections (Rôças et al., 2004). E. faecalis can even be found to live in and around the roots of
the teeth with no access to nutrients for long periods of time, which aids their survivability in a person’s
immune system. These infections are also known to be more difficult to treat than most other infections, as E.
faecalis tends to be resistant to many drugs, some of which are known to be “last resort” gram-positive bacteria
drugs, such as vancomycin (Kau et al., Apr. 2005).
Proteus mirabilis is a gram-negative bacterium that belongs to the Enterobacteriaceae family. P.
mirabilis has a bacillus, or rod-shaped morphology, and is not normally grouped up together, and tends to stick
to being a single cell. One of its more notable virulence factors is its’ possession of urease and swarming
motility, making this bacterium an excellent pathogen at causing urinary tract infections, especially associated
with catheters (Armbruster et al., Feb. 2018). During infection, P. mirabilis can also use hemolysin to
essentially “cut” red blood cells. Interestingly, when displayed on a blood agar plate, P. mirabilis uses its
swarming motility factor to show a pattern on a blood agar plate that can be like that of a tree trunk, which will
be shown later. This “swarming” pattern is referred to as a Dienes line formation, and although little is known
about this formation, or P. mirabilis’s ability to create such an interesting structure, it has been concluded that
the swarms left behind in the smaller Dienes line formations have smaller, round shaped cells that also have less
viability than a normal P. mirabilis microorganism (Budding et al., Feb. 2007).
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Results and Discussion
The first part of researching and testing what
the bacteria in the unknowns involved creating a
dichotomous key for both gram-positive and gram-
negative bacteria that were used and tested on in lab.
As there were both a gram-positive bacterium and a
gram-negative bacteria chosen in this specific
unknown lab, both dichotomous keys were used to
test these bacteria. In addition to making the
dichotomous keys, the bacteria had to be tested for
whether they were gram-positive or gram-negative.
Figure 2a Figure 1b
This process, called gram staining, showed that
Unknown 12 was gram-positive (Figure 1a), and that
Unknown 61 was gram-negative (Figure 1b).
In the gram-positive dichotomous key, using blood agar to
test the kind of hemolysis Unknown 12 was capable of was by far
the most valuable initial test, as it provided three different
outcomes from the test, as well as a great general spread of the
bacterium among the results. The three different results that could
be yielded by this test were beta hemolysis, or complete
hemolysis, alpha hemolysis, or partial hemolysis, and gamma
hemolysis, or no hemolysis capabilities. After inoculating a blood
agar sample with Unknown 12, the result yielded a bacterium
with no capabilities of performing hemolysis, otherwise known
as a gamma result (Figure 2).
The next test that was performed to determine the species
of Unknown 12 was a motility test, using SIM. This test is an Figure 2
excellent test to use in the lab, as it can give you three
results all in one. It tests for sulfur production, indole
production, and if the organism is motile or not. In this
test, not only was motility test negative, but the indole
test and sulfur production all came back negative as
well (Figure 3).
The next test used in identifying Unknown 12
was the grouping of the bacteria found in the gram
stain. Most of the formations found in the bacteria
Figure 4a
sample were grouped up in pairs, or tetrads, and were cocci in shape
(Figure 4a), narrowing down our Unknown to two different bacterias;
Micrococcus luteus, and Enterococcus faecalis.
Figure 5 Figure 3
The last test performed in ordetr to differentiate between the final two
options was a lactose test, which was undertaken by using a Kligler’s iron agar innoculation. This test, much
like the SIM test, can provide many different results and insight into what the Unknown sample could be. The
results include positive or negative results in lactose fermentation, as well as positive or negative results in
glucose fermentation. The test also, like the SIM test, tests for sulfur production, but Kligler’s iron agar can also
test for gas production, which is usually characterized by gas bubbles forming on the medium, or large cracks in
and throughout the medium. Fortunately, the test showed that the sample was both lactose and glucose positive,
as well as negative for gas production, and as also shown in the SIM test earlier, negative for sulfide production
(Figure 5).
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One test that was not considered an extremely certain test in the gram-
positive dichotomous key was grouping of the bacteria in sample. To combat
this, as many bacteria did appear to form small chains throughout the gram-
stain procedure, an extra test was performed in order to confirm whether or
not Unknown 12 was E. faecalis, or Streptococcus bovis. S. bovis is
considered to be not salt-resilient, unlike E. faecalis. A salt broth can test
whether a bacteria can survive in salty conditions or not by yielding a
positive (foggy) growth or negative (clear) growth result. After innoculating a
6.5% NaCl broth, the result came back with a positive growth result (Figure Figure 4b
4b).
For Unknown 61, finding an initial test a little tougher, due to the amount of tests that
would yield the same results for many of the gram-negative species that were available in lab.
For the initial test, the best option chosen was a sulfide, or H2S production test. This test, much
like the tests used in Unknown 12’s identification, can be carried out by a wide variety of
different mediums. The medium chosen for this particular test was SIM, as it yields a great
sulfide production result compared to most other sulfide production tests. The result yielded a
high amount of sulfide production, which also indicated motility, as sulfide was present
throughout the entire medium (Figure 6). This result will also be referred to later on as well in
the tryptone/indole test.
The next test that was utilized was the sheep blood agar
test. Unlike the hemolysis portion of the test for the gram-positive
bacteria, blood agar can be used with gram-negative bacteria to
yield two different results. The results are either a gamma
hemolysis, or no hemolysis result, or a swarming result, which
resembles that of a tree trunk on the blood agar, as it forms a very
uniwue Dienes line formation very particular to the Proteus
genus. Unfortunately, the blood agar in the lab was damaged, so
this result had to come from the professor (Figure 7). This test led
to the conclusion that the sample could only either be Proteus Figure 7
Figure 6
mirabilis, or Proteus vulgaris.
The last test performed for Unknown 61 involved differentiating between P. mirabilis
and P. vulgaris. The only test that these two bacteria species differentiated in results with
certainty is the tryptone/indole test. For this test, two results were used in the conclusion of P.
mirabilis identification. One result derived from the initial SIM test used for this identification,
and the other involved the use of a tryptone broth test, both to use for tryptone or indole
production. At the beginning of innoculating the SIM test, five drops of Kovac’s reagent was
added, as this can be used to indicate indole production within the medium. When innoculating
the tryptone broth test, five drops of Kovac’s reagent is added as well to test for tryptone
production. The results of these tests can yield a red band at the top of the medium, indicating a
positive result, or no band, indicating a negative result. Both tests, for tryptone and indole, came
back negative (Figure 6, Figure 8). Figure 8
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(Red stars indicated paths taken in dichotomous keys.)
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Conclusion
After creating and following dichotomous keys made for both the gram-positive and gram-negative
bacteria, I have concluded that Unknown 12 is Enterococcus faecalis, and unknown 61 is Proteus mirabilis. I
did not have any issues with results, or inoculations on any certain tests, however some of the tests’ results did
seem to be more inconclusive than others, such as the %6.5 NaCl broth, and the indole test in the SIM media.
Nevertheless, I do believe with large certainty that these are the bacteria found within Unknowns 12 and 61.
One issue that unfortunately arose from this experiment was the SBA, or sheep blood agar medias going bad. It
would have been very interesting to see a swarming pattern from P. mirabilis myself, as I did not get to have a
good look at a swarming result in person during the time in the lab.
One very interesting thing to note about the samples that I had, specifically about my Unknown 12, was
the strange color I was getting in many of my inoculations involving Unknown 12. After inoculating the culture
medias, which were the medias that were used to inoculate all tests after the first day, the culture media started
to take on an orangish-gold color, a very similar characteristic of the very popular Staphylococcus aureus. On
top of that, the bacteria that appeared on the SBA media showed characteristics of gamma hemolysis, like that
of E. faecalis, but the bacteria on top of the plate, which is most of the time cream-colored or white, also
contained a yellowish hue. Still, I ruled S. aureus out, as the sample that was inoculated onto the SBA media
showed no signs of any hemolysis capabilities. One reason for this interesting result may be the effect of
contamination of the media, possibly from my hands, or another outside source. Still, the results from all medias
pointed towards E. faecalis being the identifiable bacteria within the sample.
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Citations

Armbruster, Charles E., et al. “Pathogenesis of Proteus Mirabilis Infection.” EcoSal Plus, U.S. National Library
of Medicine, 8 Feb. 2018, https://pubmed.ncbi.nlm.nih.gov/29424333/. Accessed May 1, 2023

Budding, A.E., et al. “The Dienes Phenomenon: Competition and Territoriality in Swarming Proteus Mirabilis.”
Journal of Bacteriology, U.S. National Library of Medicine, June 2009,
https://pubmed.ncbi.nlm.nih.gov/19251852/. Accessed May 1, 2023

Kau, Andrew L, et al. “Enterococcus Faecalis Tropism for the Kidneys in the Urinary Tract of C57BL/6J
MICE.” Infection and Immunity, U.S. National Library of Medicine, Apr. 2005,
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087416/. Accessed May 1, 2023

Kayaoglu, Güven, and Dag Ørstavik. “Virulence Factors of Enterococcus Faecalis: Relationship to Endodontic
Disease.” Sage Journals Pub, Sage Journals, Sept. 2004,
https://journals.sagepub.com/doi/full/10.1177/154411130401500506. Accessed May 1, 2023.

Rôças, Isabella N., et al. “Association of Enterococcus Faecalis with Different Forms of Periradicular
Diseases.” Journal of Endodontics, Elsevier, 16 Dec. 2005,
https://www.sciencedirect.com/science/article/abs/pii/S0099239905601070. Accessed May 1, 2023.