BIOL 2325L: Fundamentals of Microbiology Laboratory

Name
Sec./Gro
up

Bailie Guidry
4/A

Lab 8 Report: Differential Tests, Nutrient Utilization,
Combination Differential Media
Introduction:

As catalysts, enzymes speed the rate of the biochemical reactions, but are
not altered or consumed by the reactions they catalyze. Enzymes contain an
active site, which is a three-dimensional cleft in the molecule. The substrate fits
into the active site on the enzyme and is converted into the product of the reaction.
Some bacteria secrete enzymes which degrade or modify a variety of substrates.
Enzymes used in catabolic reactions (reactions by which complex substances are
broken down into simple substances) or anabolic reactions (reactions by which
simple substances are converted into complex substances) may either be
constitutive or induced. Constitutive enzymes are produced at all times, whether
their required substrates are present or not. Inducible enzymes, on the other hand,
are only produced in the presence of the appropriate substrate.
The biochemical activities of bacteria can be divided into intracellular
reactions (occurring inside the cell) and extracellular reactions (occurring outside the
cell). Enzymes that act inside of a cell are known as endoenzymes, while those that
act outside of the cell are called exoenzymes (those excreted by organisms). The
starch test is used to demonstrate the hydrolytic activities of exoenzymes. Starch
is a high molecular weight polymer of glucose linked together by glycosidic bonds,
called α-(1,4) linkages and α-(1,6) linkages. Starch has two forms: amylose, which
has no α-(1,6) linkages and therefore, no branches in its structure, and
amylopectin, which has branching chains of α-(1,4) linked glucose attached to α(1,6) linkages at branch points. Both forms, amylose and amylopectin, can be
degraded by bacteria. Bacteria use enzymes called amylase and maltase to break
starch into smaller units (ultimately glucose) that can be transported into the cell.
The gelatin test is used to test for the presence of a protease called
gelatinase. Proteases are enzymes capable of breaking down proteins to smaller
chains of amino acids, in some cases to the individual amino acids. If bacteria
produce gelatinase, the enzyme will break down gelatin in the extracellular
medium. Once broken down, the gelatin will no longer solidify, even at low
temperatures.
The urease test is used to identify certain intestinal flora, including many
Gram negative, non-lactose forming, organisms. Urea is a nitrogenous compound
found in urine which functions in the detoxification and removal of ammonia,
generated during amino acid metabolism in animals. Urease hydrolyzes urea by
the following chemical reaction:

NH2-C=O-NH2 + 2H2O
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CO2 + H2O + 2NH3
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BIOL 2325L: Fundamentals of Microbiology Laboratory

The ammonia, produced by the breakdown of urea, generates hydroxyl ions upon
reaction with water:

NH3 + H2O

NH4 + OH-

Hemolysins are enzymes produced which break down cell membranes.
Hemolysin production can be detected by growing bacteria on blood agar plates,
nutrient agar containing 5% blood (usually from sheep). After growth on blood agar,
bacteria are divided into three categories based on appearance:
Non-hemolytic No change occurs in the appearance of the blood agar surrounding
the bacteria because no hemolysins are produced. This is also
known as gamma-hemolytic.
Alpha (α)-hemolytic The production of alpha hemolysins results in the partial
destruction of the red blood cell membranes. An opaque, greenishbrown, zone is present around the growth due to oxidation of
hemoglobin to methemoglobin.
Beta (β)-hemolytic The production of beta hemolysins results in the total
destruction of the red blood cell membrane. A clear zone will
surround bacteria producing beta hemolysins. Beta hemolysis is
useful in the diagnostic identification of Streptococcus pyogenes,
the organism responsible for strep throat, scarlet fever, rheumatic
fever, glomerulonephritis and necrotizing fasciitis.
If whole blood is collected and allowed to sit for several hours, it will clot.
Clotting is the result of the formation of a fibrin mesh by a complex biochemical
pathway involving numerous proteins, including calcium and, Vitamin K. This
pathway can be blocked by the addition of an anticoagulant to the blood. The
enzyme coagulase converts fibrinogen to fibrin, even in the presence of an
anticoagulant. It is produced by Staphylococcus aureus, but not by other members
of the genus Staphylococcus (or other common bacteria). Coagulase can be
detected by incubation of bacteria in plasma for 1-24 hours. If the plasma remains
liquid, the test is negative. If the plasma becomes semi-solid, it is positive.
The citrate utilization test is a differential cultural test which identifies
genera within the bacterial family Enterobacteriaceae that are able to utilize sodium
citrate as a sole source of carbon. Citrate is the sole source of carbon in the
Simmons citrate medium, while inorganic ammonium salt (NH4H2PO4) is the sole
fixed nitrogen source. When an organic acid, such as citrate, is used as a carbon and
energy source, alkaline carbonates and bicarbonates are produced. The breakdown
of the ammonium salt produces ammonia and ammonia hydroxide. The
production of these bases raises the pH, and an indicator, bromothymol blue,
changes from green to blue in color. Even if the medium does not change color,
growth on citrate agar is a positive result, since only bacteria which use citrate as a
carbon source grow.
An enzyme that catalyzes the depolymerization of DNA into small fragments is
called a deoxyribonuclease, or DNase. The ability to produce this enzyme can be
determined by culturing and observing an organism on DNase Test Agar. DNase
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BIOL 2325L: Fundamentals of Microbiology Laboratory

agar contains an emulsion of DNA, peptides as a nutrient source and methyl green
dye. The dye and polymerized DNA form a complex that gives the agar a blue-green
color. Bacterial colonies that secrete DNase will hydrolyze DNA in the medium into
smaller fragments, unbound from the methyl green dye. This results in clearing
around the growth.
SIM medium is used for the determination of three bacterial activities: sulfur
reduction, indole production and motility. The semisolid medium includes casein and
animal tissue (amino acid source), iron and sulfur in the form of sodium thiosulfate.
The enzyme thoisulfate reductase catalyzes the reduction of the sulfur (in the
form of sulfate at the end of the anaerobic respiratory ETC). The enzyme
cysteine desulferase catalyzes the purtrefaction of the amino acid cysteine to
pyruvate. Both systems produce hydrogen sulfide gas. When either reaction
occurs in SIM medium, the H2S that is produced combines with iron, in the form of
ferrous ammonium sulfate, to form ferric sulfide, a black precipitate. Any
blackening of the medium is an indication of sulfur reduction and a positive test.
Indole production in the medium is made possible by the presence of the
amino acid tryptophan (contained in the casein and animal protein). Bacteria
possessing the enzyme tryptophanase can hydrolyze tryptophan to pyruvate,
ammonia, and indole. The hydrolysis of tryptophan in SIM medium can be detected
by the addition of Kovac’s reagent after a period of incubation. When a few drops
of the Kovac’s reagent are added to the tube, the components react with any indole
present and produce a compound that turns the reagent layer red. The formation of
red color in the reagent layer indicates the presence of tryptophanase and is a
positive reaction.
Determination of motility in SIM medium is made possible by the reduced
agar concentration and the method of inoculation. The medium is inoculated with a
single stab from an inoculating loop or needle. Motile organisms are able to move
about in the semisolid medium and can be detected by the radiating growth
pattern extending outward in all directions from the central stab line

Research Questions (cite sources):
1. Why is the DNase test readable after 24 hours, while other tests (i.e., gelatinase)
take up to a week?
Deoxyribonuclease was the enzyme that catalyzed the DNAse reaction. This allowed
the bacteria to replicate quicker than the other tests because the other tests did not
contain the enzyme (Unknown Author, 2012).

2. What is an allosteric site (of an enzyme)? What is its purpose?
Allosteric site means “other site” or “other structure.” An allosteric site is the site
that other molecules, other than a substrate, will bind to an enzyme and not to their
active site. When molecules bind to the allosteric site, it inhibits the enzyme and
changes the active site so that a substrate cannot attach (Enzyme Regulators, n.d.)
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BIOL 2325L: Fundamentals of Microbiology Laboratory

Objectives:
The didactic objectives included becoming familiar with the concepts of catabolic
and anabolic reactions, endoenzymes, exoenzymes, ureases, reductases, hydrolytic
enzymes, and hemolysins. The practical objectives included observing and
describing the processes, and by-products of utilization of various nutrients by
bacterial species, as well as understanding safety and disposal procedures relating
to this experiment.

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BIOL 2325L: Fundamentals of Microbiology Laboratory

Methods:
1. What color change indicates a positive citrate test?
A color change from green to royal blue indicated a positive citrate test.

2. What color change indicates a positive indole test?.
A color change to a red/pink color in the reagent layer of the SIM deep indicated a
positive indole test.

Results:
Fill in the following tables with your observations.
Starch Hydrolysis:
Organism
Bacillus cereus

Result
+ or -

Appearance/Observatio
ns
Black

Bacillus subtilis

+

Yellow

Gelatin Hydrolysis (gelatinase production):
Organism
Bacillus cereus

Result
+ or -

Appearance/Observatio
ns
solid

Bacillus subtilis

-

solid

Bacillus cereus

Result
(alpha, beta, gamma)
Beta

Appearance/Observatio
ns
Complete transparency

Bacillus subtilis

alpha

Partial break down

Hemolysins:

Organism

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BIOL 2325L: Fundamentals of Microbiology Laboratory

Urease Acitvity:
Organism
Proteus vulgaris
Enterobacter
aerogenes

Result
+ or -

Appearance/Observatio
ns
Yellow

-

yellow

Result
+ or +

Appearance/Observatio
ns
Semi-solid

-

Liquid

Result
+ or -

Appearance/Observatio
ns
Green color

+

Blue color

Result
+ or -

Appearance/Observatio
ns
Snowflake pattern
appearance
Snowflake pattern
appearance

Coagualse:
Organism
Staphylococcus aureus
Enterobacter
aerogenes

Citrate Utilization:
Organism
Staphylococcus aureus
Enterobacter
aerogenes

DNA Hydrolysis:
Organism
Staphylococcus aureus
Enterobacter
aerogenes

-

SIM Medium (reduction of sulfur):
Organism
Enterobacter
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Result
+ or -

Appearance/Observatio
ns
Yellow
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BIOL 2325L: Fundamentals of Microbiology Laboratory
aerogenes
Citrobacter freundii

+

Black growth

Result
+ or -

Appearance/Observatio
ns
No color change

-

No color change

Result
+ or +

Appearance/Observatio
ns
Growth to side

+

Growth to side

SIM Medium (production of indole):
Organism
Enterobacter
aerogenes
Citrobacter freundii

SIM Medium (motility):
Organism
Enterobacter
aerogenes
Citrobacter freundii

Conclusions:
Most tests produced expected results, but there were a few that were false results.
For the Urease Activity test, Proteus vulgaris should have produced a positive result
with a pink color, but instead presented as a false negative yellow. During the DNA
hydrolysis test, Staphylococcus aureus presented a false negative result. There was
no clear zone growth around the bacteria, which lead to a false negative result.
Citrobacter freundii presented with a false negative result as no color change was
observed.

References (use APA Format):
Unknown
Author
(2012).
Biochemical
Tests.
http://www.vetbact.org/vetbact/?biochemtest=1#id27
Enzyme
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Regulators.

Elmhurst

Vetbact.

College.
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(n.d.)

BIOL 2325L: Fundamentals of Microbiology Laboratory

http://www.elmhurst.edu/~chm/vchembook/573regulate.html
Leboffe, Michael and Pierce, Burton (2013). Brief Microbiology Theory and
Application: Customized for Our Lady of the Lake College. (Second edition).
Englewood, CO: Morton Publishing.

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