Louise Margaret Tomas
SCB260.7733
Professor John Bihn
Fall 2007 Session II
Unknown Microbe Number 16
The Process of Identification
Morphology:
On the first day, I started with Gram Staining by obtaining a sample from
the pure culture that was provided to me. The results that I observed by Gram
Staining was Gram Positive cocci. Next I performed a Negative Stain in order
to further more validate that I had cocci, since Negative Staining would yield an
accurate determination of the shape of the microbe by staining the background of
the microbe. In observation of my unknown using Negative Staining; I knew that
I indeed had a microbe with cocci.
With this information, I was able to eliminate a number of potiential
microbes and was then able to focus my attention towards the microbes that
were Gram + cocci which were:
Lactococcus lactis
Micrococcus luetus
Micrococcus roseus
Micrococcus varians
Streptococcus agalactiae
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus faecalis
Staphylococcus saprophyticus
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Mannitol Salt Agar
On the first day; I prepared a streak plate and it was incubated at 37˚C.
When I returned 3 days later to view my results; I observed that white colonies
had grown.
White colony formation helped me eliminate M. luteus from my list of
possible microbes, because on Mannitol Salt Agar, M. luteus grows yellow
colonies.
I was able to eliminate S. aureus; because this microbe produces acid in
MSA; thus producing a yellow colony as well.
S. epidermidis was noted to grow white colonies, so I noted that potential
microbe. In my mind, I was thinking my unknown was either S. epidermidis, or
perhaps a cousin of that microbe.
TSA
On the first day I also prepared 4 TSA petrie dishes. These Petri dishes
were incubated at 37 ˚C. When I return 3 days later, I planned on performing a
Catalase Test and an Oxidase Test.
Temperature Requirements:
Temperature requirements are used to determine optimum temperature
for growth of the unknown in a glucose broth. I used 3 glucose broth tubes to
determine the growth temperature of the unknown microorganism.
When I returned I was able to observe the following:
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Glucose broth at 4˚C  no growth
Glucose broth at 27˚C  some growth
Glucose broth at 37˚C  growth (large abundance)
The results of my temperature experiment lead me to believe that my
unknown is a Mesophile microbe; which is able to sustain life within between
20˚C-40˚C. Microbes within the Staphylococcus genus fall within this category.
Fermentation Testing:
I inoculated 2 tubes of sucrose and 2 tubes of glucose. A positive result
for fermentation would be a change of the color (original color is a red color 
to a yellow color) of the sucrose and glucose as well as if gas was produced, gas
bubble present within the tube inside the test tube.
All 4 of my test tubes came out positive; all 4 had the distinctive color
change, as well as the presence of the gas bubbles within the tubes of the test
tubes. Microbes within the genus Staphylococcus are supposed to yield a
positive result for fermentation.
Biochemistry Testing:
Catalase Test
During my Catalase test, I used one of the Petri dishes and dropped 5
drops of Hydrogen Peroxide onto the growth in the dish. A positive Catalase
result would yield bubbling of the area that the hydrogen peroxide was placed;
and this was the result I observed. I then performed the catalase test again to be
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extra sure on a second TSA Petri dish, and again I received a positive catalase
test result.
Oxidase Test
For my Oxidase Test I used another incubated TSA Petri dish and
dropped the oxidase reagent on the colonies. A positive Oxidase result should
yield a color change, and the new color observed should be pink within one
minute of added the oxidase reagent and then blue then black. I had a negative
oxidase result; meaning the color on my colonies did not change color at all with
the introduction of the oxidase reagent. I also repeated this test for a second
time on another unused inoculated incubated Petri dish, and again I had a
negative oxidase result.
With these results I was able to eliminate the potiental microbes within the
Micrococcus genus; because those microbes yield positive catalase and a
positive oxidase results. My unknown only tested positive for the catalase test.
I was also able to eliminate S. faecalis because that microbe yields a
negative catalase test result.
I also eliminated S. agalactiae because that microbe is supposed to yield
a negative catalase and negative oxidase test.
Thus far since I was able to eliminate those microbes, I was then left with
the following microbes that could potentially be my unknown:
S. epidermidis
S. saprophytricus
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Novobiocin Sensitivity Testing
By this time I with the results of my previous tests, all I needed to do was
differentiate between S. epidermidis and S. saprophyticus. By performing a
Novobiocin Sensitivity Test, I would be able to find out which Staphylococcus
species my unknown is.
To perform this test I used a sterile cotton swab to create a “lawn” on a
Meuller Hinton Agar with my unknown. I did this to 2 Meuller Hinton Agar Petri
dishes. I then placed 2 Novobiocin discs within each Petri dish and allowed it to
be incubated at 37˚C.
When I returned the results I received on both Mueller Hinton Agar Petri
dishes were negative, meaning there was no zone of inhibition; thus whatever
microbe I had was resistant to the anti-biotic Novobiocin.
With this final test, I was able to eliminate S. epidermidis, because this
microbe would have yielded sensitivity to Novobiocin (ie: a zone of inhibition
would have been observed around each of the 2 Novobiocin dishes deposited).
I was then left with S. saprophytricus as my possible unknown since this
microbe is resistant to Novobiocin.
Urease
Although my tests have been conclusive to my unknown being S.
saphrophytricus, I still wanted to perform one more test just to be little bit more
sure. My last test was to inoculate 2 test tubes of Urease and left it to be
incubated at 37˚C. When I returned, my test tubes had both yielded positive
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results. A positive result with this test is a color change of the Urease (yellow)
 to a fuchsia pink (bright pink).
S. saphrophytricus yields a positive result in this test; thus firming my
conclusion that my unknown microbe is indeed S. saphrophytricus.
Conclusion
The flowchart summarizes the results of the tests that determine the
morphological characteristic, physical requirements and biochemical/enzymatic
reactions that are necessary for isolating the identity of the unknown organism.
These tests can be thought of as a series of questions in sequence known as a
Dichotomous key that uses the process of elimination to identify an unknown
microorganism. The unknown that fits the test results as a criteria for identity,
was Staphylococcus saphrophytricus..
The cell morphology, gram stain, Mannitol Salt Agar colony growth,
catalase test, oxidase test, temperature test, Novobiocin Sensitivity Test helped
in differentiating Staphylococcus saphrophytricus from the other microbes that
could have been possible. The catalase and oxidase tests were very important
in eliminating many of the other potienal microbes (ie: I eliminated the entire
genus of Micrococcus with these results alone). The test that helped specify
between the last two microbes; was the Novobiocin Sensitivity test, since one
was resistant (Staphylococcus saphrophytricus) and the other was sensitive
(Staphylococcus epidermidis). I did one more test; the Urease Test; to just be
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sure that I indeed had S. saphrophytricus and when the result of that test came
back positive the microbe my unknown was is Staphylococcus saphrophytricus.
Positive (+)/ or Yes
Gram

Cocci

Catalase Test

Negative (-)/ or No

Oxidase Test
Fermentation

Fermentation with

Gas Produced
Growth on MSA
Novobiocin





Sensitive
Urease
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