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1. just follow Unknown Lab Report Instructions and rubric.2. I put two samples and and my notes in the class about #1 bacteria.3. My bacteria is #1 Bacillus subtilis.4. I have a flow chart, so you do not need draw a flow chart.
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General Microbiology Lab Report Grading Rubric
Title (5 pts total):
PointValue Points Earned
Student included a clear, brief title describing the particular
experiment performed and the conclusion reached.
5
Abstract/Summary (20 pts total):
A. Introductory sentence to put work in context
5
B. Summary of method(s) used-WITHOUT technical details-and statement of overall purpose
5
C. Summary of results
5
D. Summary of conclusions
5
Introduction (20 pts total):
A. The student used valid outside sources to place
experminent(s) performed in the context of a larger field
(e.g. explaining why unknown investigations or the various
forms of biotechnology are imporant and how they are used
in society).
5
B. The student used valid outside sources to introduce the
specific background information relevant to the
experiments that were conducted. Student included
background on specific techniques used (brief explanation
of why it is used by researchers, how it works, and why it
was used in this experiment). Clear, accurate, detailed and
developed .
10
C. The student provided a clear, accurate, detailed and
developed purpose statement for the lab completed.
5
Methods (20 pts total):
The student explained all procedures used in a clear,
accurate, detailed and developed manor. TO RECEIVE
CREDIT for this section: the procedures were explained in
the past tense and paragraph form. Student explained only
major points and noted how and why each procedure was
done. Student noted controls used, essential
equipment/reagents, type of data analysis, and why that
type of analysis was chosen.
Results (20 pts total):
20
A. The student Included properly formatted figures and/or
tables to represent the data. Each figures/table included
title and legend noting the general method that was used to
generate the data shown and the overall point readers
should glean from the figure/table. (Note: Figures and
tables should be self-explanatory so that the reader can
understand the content based on the legend, without
needing to refer to the text of the report.)
B. The student described his/her observations in a clear,
accurate, detailed and developed manor in PARAGRAPH
form. Student stated the relationships between different
variables, and referred the reader to figures/tables as
necessary. Student described key observations/trends
without discussing the implications/interpretation of these
observations.
Discussion (20 pts total):
A. The student restated the purpose of the experiment and
the significant results observed.
B. The student provided a clear, accurate, detailed, and
developed interpretation of the results. Student explained
what the data obtained imply or suggest (without claiming
that anything was proven). Student explained what the
data obtained in this experiment likely mean/suggest based
on the available background information (with outside
sources cited as necessary). Student noted whether or not
the data obtained support the initial hypothesis, and noted
possible explanations for any problems that occurred.
C. The student used outside sources to explain how the
results and interpretations presented fit into a larger
context (that was mentioned in the introduction). Student
noted how the work conducted fits in with what is already
known and/or how this type of work is used in society.
Properly formatted Work Cited and Textual Citations (10 pts total):
To receive full credit not only for this section but also for the
introduction and discussion sections, the student included
parenthetical citations in the body of the report, and also a
works cited list (using either MLA or APA format).
10
10
5
10
5
10
Title: ​Identification of Unknown Bacterial Species #8 Using Traditional Biochemical Techniques
Abstract
The identification of bacteria is an essential part of the microbiology field in
modern health practices from clinical to sanitation. The rapid identification of
bacteria and their traits has been used to help prevent foodborne illness outbreaks
and contamination as well as treating and eliminating these illnesses. In this
experiment the traditional biochemical techniques were explored through practical
application in an attempt to identify an unknown bacteria labeled Unknown #8. The
tests used to identify the unknown included: FTM, SIM, Gram Staining, Kligler’s
Iron Agar (KIA), Citrate Test, and Urea Hydrolysis. The main results obtained from
these tests were that the unknown was a motile gram-positive bacteria with the
characteristics of a facultative aerobe. The bacteria also was incapable of breaking
down urea, due to lack of the enzyme urease, and the inability to utilize citrate as the
sole carbon source. After reviewing these results the species was suspected of being
​ Escherichia Coli ​which was then confirmed by the instructor.
Introduction
Identifying bacterial species is the first step towards treatment of bacterial illnesses, providing the
needed info to develop treatments and respond to outbreaks. Beginning in the 1880’s when simple
staining techniques were developed for the viewing and identification of bacterial species, more detailed
and more efficient methods have arose for identification. (Pommerville, 2016). Since then these
techniques have been used to help not only individual cases and patients in a clinical setting but to
respond to outbreaks of illness in areas such as the food industry. Lab techniques have been taken and
used to create rapid detection procedures when food contamination cause outbreaks of diseases such as
salmonella. By using what is already known about the bacteria, and gathering selected identification
techniques, tools have been provided to food producers in order to have a constant and effective
monitoring and response system for these dangerous bacteria (Lee et al. 2015).
When identifying bacteria two types of methods may be used, biochemical or molecular.
Traditional biochemical methods rely on phenotypic identifications, using gram staining and culture and
biochemical methods. Through multiple tests attributes can be compared to manuals such as ​Bergey’s
Manual​ of Determinative Bacteriology ​to identify the species of the unknown bacteria. Molecular
identification, known as 16S rDNA sequencing, uses analysis of the 16S rDNA genes in bacteria (Woo et
al. 2008). Both methods are still utilized in the field as each has its own benefits and drawbacks.
Traditional biochemical techniques are accurate and relatively inexpensive, but limit the identification to
bacteria that have been identified previously and only bacteria that can be cultured. Molecular tests help
to Identify novel species and can be much faster. However the use of molecular identification is also often
unused due to a lack of automation in the process and the higher cost associated with it (Awong-Taylor et
al. 2008).
The purpose of this laboratory experiment was to identify unknown bacterial species #8 using
traditional biochemical techniques. The experiment also provided experience in the setup, execution, and
possible results of these traditional techniques through hands on practice.
Methods:
Traditional biochemical tests were conducted in order to identify the unknown bacterial species
#8. Proper aseptic technique was followed for all tests under supervision of the instructor. After all tests
the results were recorded in a lab notebook and on a characteristic list provided by the instructor which
listed the possible outcomes for all tests. The tests conducted included: Gram Staining, FTM, Nutrient
Gelatin, MR-VP, Simmons Citrate, Oxidase Test, Catalase Test, Nitrate Reduction, Urea Hydrolysis,
Starch Hydrolysis, Kligler’s Iron Agar, and SIM.
Gram Staining: ​To prepare for the Gram staining a smear slide was prepared using an inoculating loop.
The slide was then air dried and heat set using a gas lighter passed under the slide. The stain was
performed over a sink with the Crystal violet stain, Gram’s Iodine, Decolorizer, and safranin bottles
ordered and set to the side with water for rinsing. Crystal violet was placed on the smear for 20 seconds,
then tapped off into the waste cup in the sink, and rinsed with water. Next Gram’s Iodine was placed on
the smear for one minute, then tapped into the waste cup and rinsed with water. Next the Decolorizer was
placed on the smear for ten seconds then tapped off into waste cup and rinsed with water. Last the
Safranin was placed on the smear for one minute then tapped into the waste cup and rinsed off with water.
The smear was then viewed under a compound light microscope using a 40X, 100X, and 400X
magnification to observe a purple color for Gram positive results, or a pink color for Gram negative
results. The results were recorded for color and significance.
FTM:​ A Fluid Thioglycollate Medium tube provided by the instructor was used to determine the
aerotolerance of the unknown bacterial species. The FTM tube was inoculated with an inoculating needle
using the stab technique. The inoculated sample was incubated for 48 hours at 37°C. The tube was then
observed for growth in specific regions for aerobic, or anaerobic oxygen requirements, or general growth
for facultative requirements.
Nutrient Gelatin: ​ To perform this test an inoculation needle was used to transfer a sample of the unknown
from a culture plate to a Nutrient Gelatin tube using stab technique. The tube was then incubated for 7
days at 37°C. The tube was then observed for liquefaction of the gelatin medium indicating a presence of
the enzyme gelatinase. Positive results were cooled to ensure accuracy of the result. The results were then
recorded for indication of the presence of the enzyme in the unknown organism.
MR-VP:​ This test was conducted to observe the type of fermentation, either mixed-acid or 2,3-butanediol,
used by the unknown organism. A Methyl Red- Vogues Proskauer tube was inoculated using an
inoculating loop to transfer the sample from a culture to the tube. The tube was then incubated for 48
hours at 37°C. 1 ml of the inoculated medium was then transferred to a separate tube. 18 drops of both
Barritt’s Reagent A and B were added to the new tube and incubated for 30 minutes to test for
2,3-butanediol designated by a change in color by a pH indicator. While incubating the original tube was
tested for mixed acid fermentation by adding 4 drops of the Methyl Red which would be indicated by a
change in color due to a pH indicator. The results were then recorded for any color change to indicate
fermentation or a lack of to indicate no fermentation.
Simmons Citrate: ​This test was performed using a citrate slant medium. The sample was transferred from
a culture using an inoculating needle and a stab and streak technique. The test was conducted to test for
the ability of the unknown to utilize citrate, indicated by a prussian blue color. The tube was incubated for
48 hours at 37°C for 48 hours. The tube was then observed and results were recorded for color indicating
the ability of the unknown to utilize citrate.
Oxidase Test: ​This test was run using an oxidase reagent and a cotton swab which was used to extract a
sample of the unknown from culture. 2 to 3 drops of the oxidase reagent were then dropped on the sample
and after 30 seconds the sample was observed for the presence of a purple color indicating presence of the
oxidase reagent or lack of.
Catalase Test: ​This test was performed using a smear on a glass slide and hydrogen peroxide. The slide
was prepared using a wooden pick to transfer a sample of the unknown from culture to the slide. 3 to 4
drops of hydrogen peroxide was then dropped on the sample. In the presence of catalase enzyme the
hydrogen peroxide would bubble. Results were observed and recorded for any bubbling reaction taking
place.
Nitrate Reduction: ​This test was performed using a nitrate broth medium with a durham tube placed
inside. The tube was inoculated using an inoculating loop, and incubated for 48 hours at 37°C. Results
were then observed for the presence of a gas bubble in the durham tube indicating the formation of any
gas from nitrate reduction. For negative results 2 to 3 drops of both reagent A and B were added to test for
any partial reduction of nitrate indicated by a red color change. Any results or lack of was recorded for
whole or partial nitrate reduction.
Urea Hydrolysis:​ A urea medium test tube was used for this test which indicates a breakdown in urea with
a hot pink color. The tube was inoculated using an inoculating needle to transfer the sample from culture
to tube using the stab technique. THe tube was then incubated for 48 hours at 37°C. Results were
observed and recorded for color change indicating the breakdown of urea and presence of urease.
Starch Hydrolysis:​ This test was performed using a starch agar plate inoculated using an inoculating
needle to transfer the sample from a culture to the plate with a straight scratch. The plate was then
incubated for 48 hours at 37°C. Gram’s iodine was then used to coat the plate, with a red orange halo
around the bacteria indicating breakdown of starch. Results were then recorded for red-orange color
indicating a positive result.
Kligler’s Iron Agar:​ This test was performed with a KIA slant tube. The medium is a multiple test
medium used to test for the fermentation of glucose, lactose, as well as the production of hydrogen
sulfide, each indicated by a unique change in color. The tube was inoculated using an inoculating needle
and a stab and streak technique. The tube was then incubated for 48 hours at 37°C then observed for any
color change. Any results were recorded for possible fermentation or hydrogen sulfide production.
SIM:​ This test was performed using a Sulfide Indole Motility medium, a multiple test medium that tests
for the motility of the unknown as well as indole production and sulfur reduction. The tube was
inoculated using an inoculating needle and incubated for 48 hours at 37°C. The tube was then observed
for motility and results recorded for any movement in organism growth. 2-3 drops of Kovac’s reagent
were added and observed for any color change due to the production of indole. Results were recorded for
possible indole production.
Results
Figure 1: Example of Gram-Staining on unknown Bacteria
The image seen in Figure 1 above is an example of a gram negative bacteria stain of an unknown
bacteria. The image was obtained using gram-staining technique and a brightfield microscope at 1000x
magnification using an oil immersion lense. The cells appeared dark pink and were clustered closely
together in large scattered groups.
Table 1: Purpose, Expected, and Observed properties of tests used to IDentify Unknown #8
Test
Purpose
Expected
Observed
FTM
This test is used to determine
oxygen requirements of the
unknown organisms by
observing the location of
growth in the tube.
Aerobic organisms
were expected to grow
towards the top of the
FTM tubes, anaerobes
were expected to grow
towards the bottom,
and facultative
organisms were
expected to grow
throughout the tube.
No growth was
observed in the FTM
tube. However the
instructor noted that
normally the organism
grows to create a
cloudy appearance
throughout the tube
which would suggest a
facultative aerobe.
Nutrient
Gelatin
This test is used to determine
the presence of gelatinase in
the unknown organism
through liquefaction of the
gelatin agar medium.
[Extra]
MR-VP
This test is used to determine
if the unknown performs
fermentation of either glucose
or 2,3-butanediol via two
separate reagent additions.
[Extra]
Simmons
Citrate
To determine if the unknown
is capable of utilizing citrate
indicated by a prussian blue
developement.
A negative result would
appear as a deep green
color in the tube. A
positive result would
appear a deep prussian
blue.
Oxidase Test
To determine if the oxidase
enzyme is present in the
unknown indicated by a
change in color to a
purple/blue.
[Extra]
Catalase Test
To determine if the catalase
enzyme is present in the
unknown through the
observation of hydrogen
sulfide reacting with the
unknown to form bubbles.
[Extra]
The tube remained a
deep green color
suggesting a negative
result for the ability of
the organism to reduce
citrate
Nitrate
Reduction
To determine if the organism
is capable of reducing nitrate.
Complete reduction is visible
after incubating partial
reduction can also be tested
for using reagents.
[Extra]
Urea Hydrolysis
To test for the presence of the
urease enzyme in the
unknown indicated by a hot
pink color.
Starch
Hydrolysis
To test for the ability of the
unknown to breakdown starch
indicated by a bright
orange-red halo around the
bacterial growth.
[Extra]
Kligler Iron
Agar
To test for the ability of the
unknown to ferment lactose
or glucose indicated by a pink
or yellow color. Also tests for
the production of hydrogen
sulfide indicated by a black
color.
A positive result for
lactose fermentation is
indicated by an entirely
yellow tube, while
glucose only is
indicated by a yellow
slant and a red butt. A
negative result for both
would present as an
entirely red-orange
slant and tube.
The tube presented
entirely yellow with no
black suggesting the
ability of the unknown
to ferment both glucose
and lactose with no
hydrogen sulfide
production.
SIM
This test determines both the
motility of the unknown
visible by cloudiness
surrounding the inoculation
stab as well as indole
production indicated by a
change in color with the
addition of a reagent.
Positive motility results
are shown by the
spread of the unknown
from the inoculation
stab line. Positive
indole production
presents as a change to
The SIM test resulted
with a positive result
for motility with the
organism spreading
from the line of
inoculation.
A negative result would
present as remaining an
uncolored medium. A
positive result would
present as a hot pink
color throughout the
medium.
The tube remained
uncolored and slightly
cloudy. This result
suggested that urease is
not present in the
unknown organism.
Figure 2: Flowchart of Identifying Unknown #8 Using Traditional Biochemical Tests.
The process of using test results to identify Unknown #8 as Escherichia Coli is pictured in a
flowchart in Figure 2 above. After failed attempts at gram-staining, the instructor noted that the unknown
is gram negative and rod shaped. The instructor then also noted that the unknown presents as a facultative
aerobe. After running a SIM test it was determined that the Unknown was motile and a citrate test was
used to obtain a negative result for utilization of citrate. A KIA test resulted in positive results for
fermentation of lactose and a urease test returned a negative result for the breakdown of urea. Using these
test results and Bergey’s Manual of Determinative Bacteriology it was determined that the Unknown #8
was ​Escherichia Coli.
Discussion
As an essential part of the field of microbiology identification of unknown bacteria has come a
long way in both traditional biological techniques and molecular methods. Both are used in many
situations in order to quickly identify pathogens and address any associated issues (Lee et al. 2015)
To begin the identification process the first step used was a gram-staining procedure in order to
categorize bacteria into either a gram-positive or gram-negative classification. This test utilizes a stain
and/or counter stain in order to create a visible color in the cell wall of the unknown bacteria. The color
varies depending on the amount of peptidoglycan present in the cell wall. Thicker walls that contain
higher levels of peptidoglycan present as a purple color due to the thick walls retaining the initial crystal
violet stain. A negative result occurs when the cell has a thinner wall that contains low amounts of
peptidoglycan and only retains the counterstain safranin which presents as a light …
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