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Name
Sec./Gro
up
Bailie Guidry
4/A
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 -(1,4) linkages and -(1,6) linkages. Starch has two forms: amylose, which
has no -(1,6) linkages and therefore, no branches in its structure, and
amylopectin, which has branching chains of -(1,4) linked glucose attached to (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
Rev. 10/14 DS
The ammonia, produced by the breakdown of urea, generates hydroxyl ions upon
reaction with water:
NH3 + H2O
NH4 + OH-
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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 Kovacs reagent after a period of incubation. When a few drops
of the Kovacs 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
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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.
Rev. 10/14 DS
Page 4 of 8
Methods:
1. What color change indicates a positive citrate test?
A color change from green to royal blue indicated a positive citrate test.
Results:
Fill in the following tables with your observations.
Starch Hydrolysis:
Organism
Bacillus cereus
Result
+ or -
Appearance/Observatio
ns
Black
Bacillus subtilis
Yellow
Result
+ or -
Appearance/Observatio
ns
solid
Bacillus subtilis
solid
Bacillus cereus
Result
(alpha, beta, gamma)
Beta
Appearance/Observatio
ns
Complete transparency
Bacillus subtilis
alpha
Hemolysins:
Organism
Rev. 10/14 DS
Page 5 of 8
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
Result
+ or -
Appearance/Observatio
ns
Yellow
Page 6 of 8
Black growth
Result
+ or -
Appearance/Observatio
ns
No color change
No color change
Result
+ or +
Appearance/Observatio
ns
Growth to side
Growth to side
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.
Regulators.
Elmhurst
Vetbact.
College.
Page 7 of 8
(n.d.)
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.
Rev. 10/14 DS
Page 8 of 8