Unknown

Assignment
15SP Microbiology AC65
BIO-175 AC65

Eric Williams

Purpose
There are three purposes for this assignment. The first is to show a true
understanding of the information that has been presented. This lab pulls on
information from the entire course and a complete understanding of that
information is needed to complete it. The second purpose is to demonstrate the
hands-on skill and procedures covered throughout the course. One of the main
advantages of a laboratory section is to take the information out of merely the
theoretical and present it as a practical skill. Lastly, this assignment mimics how
practicing doctors and biologists would go about identifying an infection of an
unknown agent on a patient or a given environment.

Materials
Staining
Microscope slides
Wire loop
Slide holder (clothespin)
Wash bottle
Gram staining kit
Spore-staining kit consisting of a
bottle
each of 5% malachite green and
safranin
Microscope
Bunsen burner
Marking pen
Methylene blue (Loefflers)
Bibulous paper
Electric hot plate and small
beaker (25
ml)
Depression slide
Cultural Characteristics
Nutrient Broth tube

Physiological characteristics
2 MRVP tubes
Barritt's reagent
Blood plate
Citrate tube
Dextrose tube
EmB plate

H202

Lactose tube
Litmus milk
Mannitol salt plate
methyl red indicator
Nitrate broth tube
Pipets

Spirit blue plate

Starch plate
Sucrose tube

7
2 tubes of semisolid or SIM
medium
Agar slant tube
Gelatin Stab tube
1 petri plate of soft nutrient agar
(2025 ml of soft agar per plate)

Principles of Biochemical Tests

Below is a description of all tests that were performed in order to correctly
identify the unknown organism. Because many microorganisms certain
morphological cultural and physiological characteristics, no one test could decisively
determine the identity of the unknown bacteria. However, when completed in
conjunction with one another, the following tests can be used to demonstrate the
unique characteristics of a given organism in such a way that a confident
identification can be made.

Fermentation
(Lactose, Dextrose and Sucrose)
Fermentation is a metabolic process that converts sugar to acids, gases, or
alcohol. Micro-organisms can be identified by the different forms of sugars they are
able to ferment and what the byproducts are. A Durham tube with a pH indicator
can be used to assess if a microbe is able to ferment a sugar to produce acid and
the inverted tube would capture any gasses produced as a result. Lactose, sucrose,
and dextrose were used to test the unknown organisms ability to perform
fermentation. A positive result would cause a color change in the pH indicator for
each carbohydrate and the presence of an air bubble would show the production of
gas.

Hydrolysis
(Starch, Blood and Spirit blue)
Hydrolysis is the excretion of exoenzymes to break down large molecules into
smaller units that can be absorbed into a microorganism. The presence of various
hydrolytic enzymes can be used as a basis for identifying an unknown organism.
Starch, blood, and Spirit Blue were used to determine which forms of nutrients could
be hydrolyzed by the unknown organism. A positive result, or the presence of
hydrolysis, is made evident by the detection of the hydrolytic enzyme necessary to
break down the specific type of nutrient. A positive for starch is the presence of
amylases. The presence of beta-hemolysis is a positive for a blood agar. The
breakdown of Spirit Blue can be identified as positive if lipase is detectable.

Selective and Differential mediums

(EmB and Mannitol salt agars)
EmB and Mannitol salt are both selective agar plates, meaning they allow the
grown of specific microorganism while inhibiting the growth of others. The use of
differential agar plates is vital in the identification of unknown organisms over a
short period of time. EmB is a blend of two stains, eosin and methylene blue, in a
6:1 ratio. This results in the slightly inhibited growth of gram-positive bacteria as
well as organisms that ferment lactose and those that do not, which can be
distinguished by the two indicator dyes, eosin and methylene blue. Containing a
high concentration (~7.5%-10%) of salt (NaCl), Mannitol Salt agar is selective for
gram-positive bacterium Staphylococci and Micrococcaceae. This is because the
NaCl inhibits the growth of most other bacteria. Mannitol salt is also a differential
medium for mannitol-fermenting staphylococci. This is due to the fact that it
contains carbohydrate mannitol and the indicators phenol red and a pH indicator
that can detect acid produced by mannitol-fermenting Staphylococci. When

inoculated with Staphylococcus aureus, the agar will produce yellow colonies with
yellow zones, whereas other Staphylococci produce small pink or red colonies with
no color change to the medium.

IMViC
(Indole, Methyl Red, Voges-Proskauer, and Citrate Utilization)
The IMViC tests are a group of individual tests used in microbiology lab
testing to identify an organism in the coliform group. Each of the letters in "IMViC"
stands for one of these tests except for the lowercase "i", which is added for ease of
pronunciation: Indole, Methyl Red, Voges-Proskauer, and Citrate Utilization. The
indole test performed to determine the ability of an organism to convert tryptophan
into the indole. The test is done by adding 5 drops of Kovac's reagent (isoamyl
alcohol, para-Dimethylaminobenzaldehyde, concentrated hydrochloric acid) to the
culture broth. A positive result is shown by the presence of a red or red-violet color
in the surface alcohol layer of the broth. The Methyl Red Test is used to identify the
production of stable acids by mechanisms of mixed acid fermentation of glucose.
Mixed Acids Fermentation results in a buildup of an acid and a significant drop in the
pH of the medium. Voges-Proskauer test is used to detect acetoin in a bacterial
broth culture. The test is performed by adding alpha-naphthol and potassium
hydroxide to the Voges-Proskauer broth, which has been inoculated with bacteria. A
positive result is indicated by a cherry-red color of the broth. Lastly, the citrate test
detects the ability of an organism to use citrate as the sole source of carbon and
energy. A positive is shown by growth on the medium even without a change in
color.

Additional tests
(Nitrate reduction, Catalase detection, Litmus Milk, and H2S production)
Although not formally organized into a group of tests, nitrate reduction, the
presence of motility, litmus milk, and catalase production were also used to further
identify the unknown organism. The nitrate reductase test is used to differentiate
between bacteria based on their ability to reduce nitrate (NO3) to nitrite (NO2)
through anaerobic respiration. To test for nitrate reduction cultures are grown in a
Durham tube of beef extract medium containing potassium nitrate. Gas captured in
the inverted tube is indicative of nitrate reduction. Moreover, partial reduction of
nitrate to nitrite can be assessed by adding sulfanilic acid (reagent A) followed by
dimethyl-alpha-naphthylamine (reagent B). The addition of sulfanilic acid and
dimethyl-alpha-naphthylamine while nitrite is present will result in a dark red color
change. Litmus milk is a milk-based medium used to distinguish between different
species of bacteria. The lactose (milk sugar), litmus (pH indicator), and casein (milk
protein) contained within the medium can all be metabolized by different types of
bacteria. Physical changes to the milk after inoculation and incubation can
determine if the bacterium can ferment lactose, reduce litmus, form clots, form gas,
or start peptonization. Lastly, the catalase test is done to detect the ability produce
the enzyme catalase. Hydrogen peroxide is used to detect the presence of catalase
enzyme. If the bacteria are capable of producing catalase, a small amount of
hydrogen peroxide added to a bacterial isolate will result in oxygen bubble
formation.
The H2S test is used to determine whether the microbe reduces sulfurcontaining compounds to sulfides during the process of metabolism. Sulfide-IndoleMotility (SIM) medium is often used to detect hydrogen sulfide production because
of it contains sulfates to serve as the substrate for detecting sulfide production. The

medium also has abundant tryptophan as a substrate for indole production, and its
content of 0.5% agar is sufficient for bacterial motility, thereby allowing detection of
motility. Motility is a microbes ability to propel itself across a given space, which is
evidence of specific physical characteristics that can aid in identification. If regions
of the agar have turned black after a 24-hour incubation period the organism is
producing hydrogen sulfide. Several other characteristics of the inoculated
organism can be identified using the Sulfide-Indole-Motility medium. Cracks in the
agar indicate gas production from the fermentation of sugars. Also, if the red agar
has turned yellow, acids are being produced from at least one of the sugars
contained within.

Results
Morphological characteristics
Cell shape
Arrangement
Spores
Grams stain
Motility

Coccus
Clusters
Negative
Positive
negative

Cultural Characteristics
Nutrient agar
Agar Slant
Nutrient broth
Gelatin stab
Oxygen requirements

Rounded with raised margins

Filiform
Membranous
Blended and without liquefaction
Facultative anaerobic

Physiological Characteristics

IMViC

Hydrolysis Fermentation

Tests

Results

Dextrose
Lactose
Sucrose
EmB

Acid no gas
Acid no gas
Acid no gas
Little to no growth

Mannitol Salt

Yellow

Starch
Blood
Spirit blue

Negative for amylose

Beta hemolytic
Negative

Indole
Methyl Red
Voges-Proskauer
Citrate Utilization
Nitrate
H2S Production
Catalase

Negative
Negative
Positive
Negative
Positive
Negative
Positive

Litmus Milk

Reaction
Acid
Alkaline
Coagulation
Reduction
Peptonizatoin
No Change

Time
Positive after 7 days
Negative
Positive after 7 days
Negative
Negative
Negative

Identification of Unknown Organism

The unknown organism was tested using all experimental methods presented

in the chart above. Based on the results of the performed test, Staphylocous
aureus is the best description of the unknown organism.

Conclusion
The pathogenicity of an organism in terms of exposure route, number of
bacteria involved in the infection, the hosts defense mechanisms, etc. are
established by the particular virulence factors it possesses. Among the many
factors that contribute to an organisms ability to cause disease, phagocytosis by
neutrophils is crucial to the hosts immune response to invading bacteria since it
leads to intracellular destruction of bacteria through production of oxygen radicals
and proteolytic enzymes. Although Staphylococcus aureus has several defining
factors that make it distinguishable from other forms of bacteria, it can be difficult
to differentiate from other coagulase-positive staphylococci from a single test.
However, a combination of tests involving colonies indicative of hemolytic and
coagulase activity as well as a catalase-positive result set Staphylococcus aureus
apart from other strains.
In order to provide an initial direction that would guide the rest of the
identification process, a Gram stain was performed to display the properties of the
unknowns cell wall. The experiment revealed clusters of gram-positive bacterial
species, which in this case were determined to be cocci given the shape. To further
narrow down the possible unknowns, the isolate was cultured in a selective medium
characteristic of mannitol salt agar, which both inhibits and encourages the growth
of specific bacteria. If an organism is able to ferment mannitol, the subsequent
drop in pH creates an acidic byproduct that manifests in the yellow colonies
characteristic of Staphylococcus aureus. Further differentiation past the class

involved a catalase test to detect whether or not the enzyme catalase was present.
All Staphylococcus species produce a positive reaction, which exhibit the
decomposition of hydrogen peroxide, an otherwise potent antimicrobial agent, into
less reactive oxygen and water so that the bacteria can survive unharmed in its
host. Staphylococcus aureus further interferes with host defense mechanisms by
producing several protein toxins with cytotoxic and immunogenic properties, and
potentially surface-associated factors that stimulate adherence and evade the
hosts attempt at defense. This reaction and degradation of hydrogen peroxide in
turn facilitate the production of supertoxins, which can present as a multitude of
complications ranging from impetigo to multiple system organ failure. One of the
final indications of the specific type of Staphylococcus isolate was demonstrated by
the positive coagulation results, which signifies a pathogen highly resistant to
phagocytosis due to a protective barrier of fibrin. The hemolytic properties
indicated formation of plasma clots and lysis of red blood cells, beta hemolysis, and
served to confirm the identification of Staphylococcus aureus as the unknown.

References and Citations

Brown, Alfred E., and Heidi Smith. Benson's Microbiological Applications: Laboratory
Manual in General Microbiology: Short Version. N.p.: n.p., n.d. Print.
Wikipedia contributors. "Catalase." Wikipedia, The Free Encyclopedia. Wikipedia,
The Free Encyclopedia, 24 Mar. 2015. Web. 6 May. 2015.
Wikipedia contributors. "Citrate test." Wikipedia, The Free Encyclopedia. Wikipedia,
The Free Encyclopedia, 7 Jun. 2013. Web. 6 May. 2015.
Wikipedia contributors. "Fermentation." Wikipedia, The Free Encyclopedia.
Wikipedia, The Free Encyclopedia, 25 Apr. 2015. Web. 6 May. 2015.
Wikipedia contributors. "Hydrogen sulfide." Wikipedia, The Free Encyclopedia.
Wikipedia, The Free Encyclopedia, 26 Apr. 2015. Web. 6 May. 2015.
Wikipedia contributors. "Hydrolysis." Wikipedia, The Free Encyclopedia. Wikipedia,
The Free Encyclopedia, 29 Apr. 2015. Web. 6 May. 2015.
Wikipedia contributors. "Indole test." Wikipedia, The Free Encyclopedia. Wikipedia,
The Free Encyclopedia, 1 Apr. 2015. Web. 6 May. 2015.
Wikipedia contributors. "Litmus milk." Wikipedia, The Free Encyclopedia. Wikipedia,
The Free Encyclopedia, 7 Aug. 2013. Web. 6 May. 2015.
Wikipedia contributors. "Methyl red." Wikipedia, The Free Encyclopedia. Wikipedia,
The Free Encyclopedia, 9 Feb. 2015. Web. 6 May. 2015.
Wikipedia contributors. "Nitrate reductase test." Wikipedia, The Free Encyclopedia.
Wikipedia, The Free Encyclopedia, 5 Jan. 2014. Web. 6 May. 2015.
Wikipedia contributors. "VogesProskauer test." Wikipedia, The Free Encyclopedia.
Wikipedia, The Free Encyclopedia, 6 May. 2015. Web. 6 May. 2015.

Rubric
Unknown Assignment
Name: Eric Williams

Unknown Organism: Staphylocous

aureus

Score:

Criteria:

Cover sheet

Name
Course number and name
Title of report
Date

Purpose

(5 points)
The purpose of the lab is stated with a
high degree of clarity.
(5 points)

Materials

All materials used are accurately listed.

(10 points)

Principles of biochemical tests

Principles of all biochemical tests used

are clearly explained.
(10 points)

Test Performance

All necessary biochemical tests are

performed.
(10 points)

Results

Identification of Unknown organism

The results are reported in a table form

to include the following:
Test name
Test result
Reason for any inconclusive/ incorrect
result
(10 points)
Both genus and species of unknown
organism are correctly identified.
(5 points)

7
Conclusion

Mechanics

Clear description of importance of

biochemical testing in unknown
identification. Two examples of
infections caused by the unknown
organism are discussed.
(5 points)
There are no errors in spelling,
punctuation and grammar. The report
is written in third person.
(5 points)

Adherence to guidelines

Punctuality

Total Points:

Report is typed
Double spaced
Organized using heading
Separate reference page
(5 points)
Report is turned in on or before the due
date.
(5 points)