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Bacterial Unknowns Identification
Anthony Ngo
TA: Yevgeniy Marusenko
Monday, Wednesday, 3:40
10 April, 2011
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Abstract
In this project, a broth that contained a mixture of two unknown bacteria was given. The
objective of the experiment was to isolate the two different bacteria from the mixture, grow them
in pure culture, and perform several specific tests to identify the two bacteria. In order to isolate
the bacteria, the streak plate was used. Many other methods that helped identify the bacteria were
used. These included the gram stain, endospore stain, motility test, the catalase test, nitrate
reduction test, coagulase test, IMViC, and several others. After all of these tests were performed,
the identity of the two bacteria was determined. One of the bacteria was found to be the gram
positive coccus Kocuria Rosea. The other bacteria was identified as Serratia liquefaciens.
Introduction
A mixture contains two or more organisms. To determine the identity of these organisms,
they must first be isolated. This can be done by using the streak plate. Steven Woeste who is an
assistant professor in the Department of Basic Biomedical Sciences, Schull College of Podiatric
Medicine, believes that the plate streak is important and that “To master the technique is to be
able to produce pure cultures on demand, and it is the foundation of modern microbiology. (4)
The isolated bacteria can then be grown in pure cultures using the Mac and CNA plates. The
CNA plate inhibits the growth of gram negative bacteria and the Mac plate inhibits the growth of
gram positive bacteria. With the availability of many tests that determine the properties and
characteristics of bacteria, the identities of the bacteria can be observed. Thus, if a mixture of
two bacteria can be isolated, grown in culture and their properties observed, then the identities of
the two bacteria can be determined.
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Materials and Methods
In order to properly conduct all of the experiments and tests for this project, several
materials were required. In addition to all of the materials mentioned in each of the required
sections from the lab manual, a Bunsen burner, several inoculating loops, a striker, test tubes,
and a test tube rack were necessary. The procedures from the sections from the lab manual
Microbiology Labaratory Theory & Application by Michael J. Leboffe were used to perform the
experiments. To isolate the mixture of organisms, section 1.4 Streak Plate Methods of Isolation
was used. The organisms were grown in pure culture and other methods were used to help
identify them. The procedures for these were sections 3.9 Capsule stain, 3.10 Endospore stain,
2.7 Fluid thioglycollate medium, and 5.20 SIM medium. Lastly, the tests from the flow charts 713 from section 7.7 and 7-17 from section 7.8 were used to identify the gram negative and gram
positive bacteria respectively (3). All tests were performed aseptically.
Results
This table shows the results for the early tests that were performed to help identify the unknown
bacteria
Gram staining
Endospore staining
Capsule staining
Fluid Thioglycollate Medium
(FTM)
SIM test
Gram +
Coccus
Absence of endospores
Absence of capsules
Strict anaerobe
Gram Rod
Absence of endospores
Absence of capsules
Facultative anaerobe
No motility
Some motility is observed,
sulfur is produced
The results show that gram staining both the gram positive and gram negative bacteria led
to no results. After the endospore and capsule staining were performed, it was determined that
both the gram positive and gram negative bacteria lacked endospores and capsules. The FTM test
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for gram positive was determined to be a strict anaerobe. For gram negative, the result was a
facultative anaerobe. The SIM test resulted in no motility for the gram positive bacteria and some
motility and sulfur production for the gram negative bacteria.
Gram Positive Cocci
Glucose Oxidation-Fermentation
Test
Oxidation
Fermentation
Nitrate Reduction
+
Coagulase Tube Test
-
-
+
Nitrate Reduction
+
Kocuria rosea
Micrococcus luteus
-
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus saprophyticus
Flow chart for identification of gram positive cocci. Red arrows indicate path followed.
To determine the identity of the gram positive bacteria, the above flow chart was used
from the lab manual. The glucose oxidation-fermentation test was performed, which resulted in
green and yellow colors. This indicated the presence of oxidation. The nitrate was reduction was
then tested, which ended with a positive result. Thus, the identity of the gram positive bacteria
was K. rosea.
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Gram Negative
IMViC
(Indole/Methyl red/Voges-Proskauer/Citrate)
-
+
+
+
Sucrose Fermentation
+
-
Gelatinase
Urease
-
+
-
+
Xylose fermentation
-
+
Klebsiella pneumoniae
Hafnia alvei
Serratia marcescens
Proteus mirabilis
Serratia liquefaciens
Flow chart for identification of gram negative bacteria. Red arrows indicate path followed.
In order to determine the unknown negative bacteria, the first test performed was the
IMViC test. This test consisted of the indole, methyl red, voges-proskauer and citric tests. The
results for these tests were negative, positive, positive, and positive respectively and thus, led to
the use of the flow chart 7-13 from section 7.8 from the lab manual. The sucrose fermentation
test was then performed and had positive results. The gelatinase and xylose fermentation tests
also had positive results. Thus, the identity of the gram negative bacteria was determined to be S.
liquefaciens.
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Discussion
Following the flow chart, the identities of the two bacteria were determined. The gram
negative bacteria was identified as S. liquefaciens and the gram positive bacteria was K. rosea.
The results from the gram staining test were difficult to read. However, the gram negative
bacteria was rod shaped and the gram positive bacteria was coccus. These results comply with
the actual shapes of the bacteria. Furthermore, S. liquefaciens are facultative anaerobes and are
motile and these characteristics match with the results from the FTM and SIM tests. K. rosea are
not motile and the SIM test performed also confirms this. K. rosea are found in freshwater, and
soilwater. Additionally, they may cause infections in patients who have a low immune system. In
patients with catheter infections and where K. rosea are unresponsive to medical treatment,
patients must be treated by catheter removal (1) S. liquefaciens can cause infections in the
iatrogenic bloodstream (2). They can also infect the respiratory tract within humans and they are
naturally found in water reservoirs. Thus, these bacteria can have great impacts on human lives
and this is why it is important to study them and to be able to identify and observe them.
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Works Cited
1. Altuntas, F. 2004. Catheter-related bacteremia due to Kocuria rosea in a patient undergoing
peripheral blood stem cell transplantation. BMC Infectious Diseases. 4:62.
2. Chuang,T. Aortic valve infective endocarditis caused by Serratia liquefaciens. Journal of
Infection. 54:161-163.
3. Leboffe, M.J. Pierce, B.E. Microbiology Laboratory Theory & Application. Morton
Publishing
Company.
4. Woeste ,S. 2011. A Template for the Streak Plate Isolation Technique for Laboratory
Classrooms. Journal of Biological Education.30:1