Microbiology 2325L

Identification of Unknown Bacteria

Unknown Code: C14
Abstract:
The unknown organism given was in a petri dish labeled C14. Experiments performed on
the organism in the Microbiology laboratory along with research regarding the results, led to the
identification of the organism. Following identification, research began, which illustrated
diseases caused by the organism and treatments to fight those diseases.
Background:
Table 1: Testing results for Serratia marcescens
Test

Gram Stain

Urease Test

Purpose of test

Reagents/Media

Observations

Results

The purpose of
the Gram stain
test was to
determine the
Gram stain
reaction of the
bacterium.

Crystal violet,
Grams Iodine, 95%
ethanol, and
Safranin were used
in the Gram stain
test.

The organism
appeared
bacillusshaped, stained
purple in color
and could
either be
arranged in
chains or
clusters.

Gram negative

The purpose of
the urease test is
to determine if
the organism is
positive for the
urease enzyme

Nutrient broth
deeps, pH indicator,
phenol red.

The broth was

yellow in color.

Negative for
the urease
enzyme

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and therefore
belongs to the
Genus Proteus.
After receiving the unknown organism in plate C14, careful observations were made
regarding the appearance of the bacterium on the plate. The bacterium initially appeared to be
raised and light pink in color. After observing the organism on the same plate a week later, it still
appeared raised, but the color changed from light pink to a darker brick color.
The first experiment performed to determine the identity of the organism was a Gram
stain. The Gram stain is used to differentiate between cells by differences in the cell wall
structure. Gram positive organisms have a thicker peptidoglycan layer in their cell wall than
Gram negative organisms, which drastically helps in differentiating between the two.
During this procedure a sterile inoculating loop was used to prepare a bacterial smear on
a slide, which was then heat fixed. After heat fixing, the primary stain, Crystal Violet was used
to flood the smear, coloring all cells on the slide purple. A mordant, Grams Iodine, was then
used to bind to the Crystal Violet and the cell wall. Next, a decolorizer, 95% Ethanol was used to
selectively remove dye from cells based on certain characteristics of the cell wall. Gram positive
organisms remain the color of the primary stain due to their thick peptidoglycan layer, while
Gram negative organisms are colorless after the 95% ethanol is used. The final reagent in the
Gram stain experiment was the counterstain, Safranin, which binds to colorless Gram negative
organisms causing them to appear pink in color.
Unknown organism, C 14 appeared pink in color following the Gram stain experiment,
proving it is a Gram negative bacterium. This finding helped to eliminate the Gram positive
organisms on the list of possible unknown organisms.

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While looking at the organism more closely, under a microscope, it was noticed that the
appearance of the unknown organism was bacillus and the arrangement of the bacterium was
either in chains or clusters, but it was hard to tell. There were no visible spores.
A second experiment was performed to assist in the identification of the unknown
organism. This test was the urease test, which was used to test the organism for the urease
enzyme, which hydrolyzes urea, a nitrogenous compound found in urine. Organisms from the
Genus, Proteus, are positive for the urease enzyme. Ammonia and hydroxyl ions resulting from
the breakdown of urea cause an increase in the pH of organisms positive for the urease enzyme.
A pH indicator, phenol red, changes from yellow to red, due to the increase in pH in organisms
positive for the urease enzyme.
After performing the urease test on unknown organism C 14, the media remained yellow
in color, disproving the production of the urease enzyme. The results of this test were helpful in
eliminating Proteus mirabilis from the list of possible unknown organisms.
Unknown Organism:
The unknown organism labeled C 14 is Serratia marcescens. Serratia marcescens is a
Gram negative, bacillus organism with short rods, which presents itself as pink or red at room
temperature. S. marcescens belongs in the Enterobacteriaceae family and occurs naturally in
soil, water, and in the intestines. It is a human pathogen and is involved in nosocomial
infections, including wound infections and urinary tract infections. Researching this bacterium
was a little difficult, because most of the articles were very similar or not from reputable sources.

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S. marcescens

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