Urease
test:
Urea
is a nitrogen containing compound that is produced during the decarboxylation
process of the amino acid arginine in the urea cycle. Urea is highly soluble in
water and is thus it is an efficient way for the human body to excess nitrogen.
This excess urea is then taken away from the body with the help of the kidneys
as a part of urine.
Certain bacteria produce the enzyme urease during its
metabolism process and that will break down the urea in the medium to ammonia
and carbon dioxide:
Some
enteric bacteria produce the enzyme urease, which splits the urea molecule into
carbon dioxide and ammonia. The urease test is useful in identifying the genera
Proteus, Providentia, and Morganella, which liberate this enzyme.
Urease,
which is produced by some micro organisms, is an enzyme that is especially
helpful in the identification of Proteus vulgaris, although other
organisms may produce urease, their action on the substrate urea tends to be
slower than that seen with Proteus species. Therefore this test serves
to rapidly distinguish members of this genus from other lactose non fermenting
enteric micro organisms.
Urease
is a hydrolytic enzyme that attacks the nitrogen and carbon bond in amide
compounds such as urea and forms the alkaline end product ammonia. The presence
of urease is detectable when the organisms are grown in a urea broth medium
containing the pH indicator phenol red. As the substrate urea is split into its
products, the presence of ammonia creates an alkaline environment that causes
the phenol red to turn to deep pink. This is a positive reaction for the
presence of urease. Failure of deep pink colour to develop is evidence of a
negative reaction.
The
ability of an organism to move by itself is called motility. Motility is
closely linked with chemotaxis, the ability to orientate along certain chemical
gradients. Eucaryotic cells can move by means of different locomotor
organelles such as cilia, flagella, or pseudopods. Prokaryotes move by means of
propeller-like flagella unique to bacteria or by special fibrils that produce a
gliding form of motility. Almost all spiral bacteria and about half of the
bacilli are motile, whereas essentially none of the cocci are motile.
The
medium mainly used for this purpose is SIM medium ( Sulphide Indole
Motility medium) which is a combination differential medium that tests three
different parameters, sulphur reduction, indole production and motility. This
media has a very soft consistency that allows motile bacteria to migrate
readily through them causing cloudiness. In soft agar tubes non-motile bacteria
will only grow on the inoculated region. Motile bacteria will grow along the
stab line and will tend to swim out away from the stabbed area. Therefore, a
negative result is represented by growth in a distinct zone directly along the
stab. A positive result is indicated by diffuse or cloudy growth mainly at the top
and bottom of the stabbed region.
SIM
agar may also be used to detect the presence of H2S
production. The SIM medium contains peptones and sodium thiosulfate
as substrates, and ferrous ammonium sulfate,
Fe(NH4)SO4, as the H2S
indicator. Cysteine is a component of the peptones used in SIM
medium. Sufficient agar is present to make the medium
semisolid. Once H2S is produced, it combines
with the ferrous ammonium sulfate, forming an insoluble, black
ferrous sulfide precipitate that can be seen along the line of the stab
inoculation. If the organism is also motile, the entire tube may
turn black. This black line or tube indicates a positive H2S
reaction; absence of a black precipitate indicates a negative reaction.
Gelatin
Hydrolysis Test:
Gelatin,
a protein derived from the animal protein collagen. It has been used as a
solidifying agent in food for a long time. Robert Koch used nutrient gelatin as
an early type of solid growth medium. One problem is that many bacteria have
the ability to hydrolyze (liquefy) gelatin. This gelatin liquefaction ability
(or inability) forms the basis for this test. Some microorganisms possess an
enzyme called gelatinase, which breaks down gelatin into amino acids. Gelatin
deeps contain the substrate gelatin, which is a protein produced by the
hydrolysis of collagen. Organisms which hydrolyze gelatin will cause the
gelatin to liquefy.
The
gelatin hydrolysis tests for an organism's ability to break down the protein
gelatin which is derived from collagen. Gelatin causes the media to
thicken, especially at cooler (below 28oC) temperatures.
If the organism can release gelatinase enzymes the gelatin is broken down or
liquefied. The media is checked over a period of about a week after
inoculation and incubation at room temperature, for gelatinase activity.
The tube is placed on ice for a few minutes and if the media fails to solidify
it is considered a positive test. The gelatinase reaction may be
slow or incomplete.
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