Bacterial Unknown Report (150 pts)
Biol 251 and 351 students will identify the genus of one bacterium using morphological,
biochemical, physiological characteristics and analysis of a 16S rRNA sequence. The term
paper will emphasize the logical sequence used to identify the unknown bacterium from a list of
15 possible candidates. The Unknown Table Worksheets will be turned in with the term paper.
Due Date
Biol 351 Microbiology XXXX
Late Penalty
10 pts will be deducted for each day that the paper is submitted after the deadline.
Cover Page
A cover page is mandatory and must include the following information:
Student Name
Lab section number
Lab TA's name
Unknown number
The cover page is not included in the page limit. Papers with an incomplete or missing
coversheet will be returned to the student. Late penalty applies.
Organization of the Term Paper
The unknown term paper will consist of 5 sections and all sections must be present to receive
full credit. Each section of the paper should be introduced with the appropriate heading or
subheading in bold type. The maximum length of the term paper is 20 pages double spaced.
The APA format will be used for citing in-text references and students may review examples at
the UNLV Writing Center website (http://writingcenter.unlv.edu/writing/apa.html).
Section I – Introduction and Definition of a Bacterial Species (20 pts)
This section will provide a general overview of the methodology employed to identify a bacterial
unknown. Students should specifically mention the importance of aseptic technique,
microscopic examination, the use of differential media, and the importance of using positive and
negative controls for each test.
In their own words, students must provide an explanation of how bacterial species are
defined. Students must clearly state how the concept of a bacterial species differs from
that of the biological species concept used with animal and plant species.
All references must be cited.
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Section II – MATERIALS AND METHODS (40 pts, 4 pts/test)
This section will contain a concise summation of the microscopic examination, 5 tests not
including the 16S rRNA blast that were central to the identification of the bacterial genus, and
the results of the 16S rRNA sequence analysis. The name of each test should be listed as a
subheading in bold text, followed by a description of that test, and the results for the unknown.
Results for positive and negative controls for each test must be reported. Tests with negative
results should be reported if they were important in the identification of the unknown. The
results of the 16S rRNA sequence analysis should identify the top three matches from a
BLASTn search and a clear description of the significance of the E values and the raw scores.
Examples from past student unknown term papers follow.
Microscopic Examination
"Unknown 09 is rod shaped bacterium. The simple stain procedure with
methylene blue allows students to clearly see the shape and arrangement of the
bacterial cells. Methylene blue is a basic cationic stain and is attracted to the
negative charge of bacterial cells.
Cultures of Staphylococcus (clusters of
cocci), Streptococcus (chains of cocci), Bacillus (rod) and Escherichia (rod) were
used as controls."
Mannitol Salts Agar
"Unknown 79 was streaked for single colonies onto a mannitol salts agar plate
and incubated at room temperature. The recipe for mannitol salts agar plates
consists of beef extract, peptone, sodium chloride (7.5%) and phenol red. This
high concentration of salt inhibits most Gram negative and positive bacteria, but
does not inhibit Streptococcus. Mannitol cannot be used as a carbon source by
all bacteria and only species capable of fermenting this sugar are able to grow.
The pH indicator, phenol red turns yellow as an indication of acid production due
to the fermentation of mannitol. Unknown 79 grew as yellow colonies with a
halo on mannitol salts agar. Staphyloccocus aureus was used as a positive
control and grew as single yellow colonies with halos. The negative control, E.
coli did not grow as was anticipated."
16S rRNA Sequence Analysis
"The sequence of the 16S rRNA gene for unknown 43 was submitted to the National
Center for Biotechnology Information (NCBI) database. The sequence was pasted into
the BLAST program for nucleotides and the best three matches were Burkholderia
fungorum, Rhizobium radiobacter, and Xanthomonas sp. The E-value is a measure of
the number of alignments between the submitted… Burkholderia fungorum had an E
value of 10-5 and this suggests… E-values are calculated based upon… Raw scores
are a measure of… The raw score for Burkholderia fungorum and Rhizobium
radiobacter suggest that…"
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Section III – RESULTS and DISCUSSION (40 pts)
This section will consist of a flow chart illustrating the key tests that led to the identification of the
bacterial genus. The flow chart must contain the tests described in Section II. Flow charts can
be hand drawn, but must be legible. The flow chart must also demonstrate how closely related
genera were eliminated.
Unknown 24
Gram negative, rod, non-spore former,
oxidase negative, facultative anaerobe,
glucose fermenter
Unknown 24
Students must discuss the process of elimination that led to the identification of their unknown.
“A Gram negative, rod shaped, oxidase negative bacterium that is a facultative
anaerobe and able to ferment glucose fits the description of the
Enterobacteriaceae (Bergey’s Manual, 2001). However, there are more than 20
genera within this family of bacteria (Brock, 2009). The motility agar test was
performed and unknown 54 was found to be highly motile.
The motility test
eliminated the genera Klebsiella and Shigella. A positive citrate utilization test
(bright blue) eliminated Esherichia, Proteus, Providencia and Morganella. A
negative litmus milk test (lactose catabolism) eliminated Citrobacter and
Enterobacter. A positive methyl red test further eliminated Salmonella as a
possible choice. The logical conclusion is that unknown 54 is a member of the
genus Erwinia. The results of the BLASTn search were consistent/not consistent
with the with the results presented in the flow chart."
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Included in Discussion Inconsistent Results (10 pts)
This section will contain an explanation for any test results that were inconsistent with the
identification of the bacterial genus.
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"All preliminary test results suggested that unknown 46 is E. coli. However, the
result of the Gram stain test indicated that unknown 46 was a Gram positive
coccus and this result was inconsistent with the initial identification. This
inconsistent result could have been caused by…"
"The best E-value from the BLASTn 16S rRNA sequence analysis was for
Pseudomonas fluorescens. This result was not consistent since unknown 13
was demonstrated to be a facultative anaerobe and P. fluorescens is an obligate
aerobe. These results are not consistent and a likely explanation is…"
Part IV – CONCLUSION
Naming the Organism and Background Information (10 pts)
All students must clearly state the identity of their unknown. In addition, an explanation of the
derivation of the genus and species name must be included. Discuss any particular host,
location, physiological, genetic and historical fact that was used to describe the genus.
"Unknown 12 is Escherichia coli. The genus Esherichia was named after Dr.
Theodor Escherich, the physician that first described this bacterium. Escherichia
was originally isolated from a child with diarrhea in 1885 (Brock, 2009). The
species, coli is derived from the Latin for colon. Escherichia coli is a common
commensal and probably inhabits all mammalian digestive tracts (The
Prokaryotes, 2006)."
This section must also briefly summarize the medical, industrial or historical significance of the
unknown. Background information is available in Bergey's Manual and other sources.
"Mycobacterium tuberculosis is the bacterium that causes tuberculosis.
Tuberculosis is a serious public health problem in many parts of the world. The
World Health Organization estimates that 8 million people are exposed to this
bacterium and 2 million develop active tuberculosis each year (Brock, 2009)."
(1 page maximum)
References Cited
Reference information is mandatory. Each reference must include the names of all authors (in
the same sequence in which they appear in the publication), the article, journal or book title,
volume number, page numbers, and year of publication. Website addresses should be use to
identify sources or documents that are only available electronically.
Visit the UNLV Writing Center website for specific examples of how to properly cite a source
(http://writingcenter.unlv.edu/writing/apa.html).
Wikipedia is not an acceptable reference.
There will be a maximum deduction of 10 pts for improper reference citations.
Additional Guidelines
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Proofread the term paper
Proper grammar, correct spelling and accurate punctuation are all important and make grading
easier. 25 pts will be deducted from term papers that are that are difficult to read due to a
lack of proofreading. The UNLV Writing Center is available to review and comment on
student term papers (http://writingcenter.unlv.edu/owl/).
Greek Letters, Special Symbols, Superscripts and Subscripts
Pay attention to the use of special symbols including, Greek letters (), degree signs (°C),
and fractions (½). Subscripts (CO2) and superscripts (Cu2+) should be used with molecular
formulas (C6H12O6) and chemical equations (2H2O2 → 2 H2O + O2).
Do not use an ampersand (&) in place of the word "and".
Scientific Names
All scientific names consist of a genus and species. The genus is capitalized and the species is
written in lower case. Scientific names should always be italicized.
Escherichia coli
Staphylococcus epidermidis
The genus and species for every organism should be spelled out when the organism is
mentioned for the first time. A name can be abbreviated by using the first letter of the genus
and the full name of the species in subsequent entries.
E. coli
S. epidermidis
Abbreviations for species, both singular (sp.) and plural (spp.) can be used with the genus.
Escherichia sp.
(Indicates an unknown species within this genus)
Staphyloccus spp.
(Refers to several species)
Headings and Subheadings
Identify each section of the paper with either an appropriate heading or subheading in bold type.
Grammatical Person
Scientific papers are written in the third person. The pronouns I (singular) and we (plural) are
used in first person writing, as in a letter or an email to a friend. The pronoun you, the second
person is used to identify the individual being addressed. He, she, it, and they are pronouns
used in third person writing.
Do not use the first person as exemplified below.
"I performed the Gram stain, anaerobic growth and oxidase tests and they were all
negative."
However, do use the third person.
"the results of the Gram stain, anaerobic growth and oxidase tests indicated that
unknown 35 was…"
Formatting
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Margins in all directions must be one inch
Font size of 11
Standard business font: Arial, Times New Roman, Palatino or Georgia
Text must be double-spaced
Pages must be numbered consecutively
Papers not complying with formatting requirements will be returned to the student. Late penalty
applies.
In-text Citations
References to any sources (textbook, lab manual, Bergey's Manual, scientific journal article,
newspaper or periodical, website) that were employed in any portion of the term paper must be
properly cited. The UNLV Writing Center (http://writingcenter.unlv.edu/writing/apa.html) has
specific examples of how to handle in-text citations.
Wikipedia is not an acceptable reference.
The UNLV Lied Library has many excellent resources on prokaryotes. Commonly used
references include, but are not limited to the following:
Bergey's Manual of Systematic Bacteriology
The Prokaryotes
Brock Biology of Microorganisms
Microbiology Laboratory Theory and Applications
Plagiarism
No form of plagiarism is acceptable and all instances of academic dishonesty will be reported to
the UNLV Office of Student Conduct. Students should familiarize themselves with the UNLV
Writing Center's description of plagiarism (http://writingcenter.unlv.edu/writing/plagiarism.html).
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Item
Formatting
Cover Page
Section I
Section II
Section III
Section IV
Section V
Grading Scheme
1 inch margins in all directions
Font size of 11
Standard business font
Doubled-spaced
Pages numbered consecutively
Papers not complying with formatting requirements will be
returned to the student. Late penalty applies.
Paper with an incomplete or missing coversheet will be returned
to the student. Late penalty applies.
20 pts
Overview of methodology
Aseptic technique
Microscopic examination
Differential media
Importance of using + and - controls
Definition of a procaryotic species
Explanation of how the concept of a procaryotic species differs from a
eukaryotic species
40 pts
Microscopic examination
8 other tests
Description of the test and the results for the unknown
Results for + and - controls
Identification of three best matches from BLASTn
Results and explanation of E-value and raw score for the 16S rRNA
sequence analysis
40 pts
Legible flow chart
Demonstrates how closely related genera were eliminated
Detailed discussion on the process of elimination
All tests from Section II mentioned
10 pts
Clearly stated the identity of their unknown
Explanation of the name (genus & species)
Background information: medical, industrial or historical significance
10 pts
Complete description of inconsistent results
Unknown Table
Worksheets
30 pts
References
10 pts will be deducted for improper citations
Proofreading
25 pts will be deducted from poorly proofread papers
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