Wednesday, 4 July 2018
Tuesday, 3 July 2018
Gelatin liquefaction test
GELATIN LIQUIFACTION TEST
AIM:
To determine the
ability of an organism that produce gelatinases.
PRINCIPLE:
Gelatin hydrolysis test is used
to detect the ability of an organism to produce gelatinase (proteolytic enzyme)
that liquefy gelatin. Gelatin is a protein derived from the connective tissues
of vertebrates, that is, collagen. It is produced when collagen is boiled in
water. Gelatin hydrolysis indicates the presence of gelatinases. This
process takes place in two sequential reactions.In the first reaction,
gelatinases degrade gelatin to polypeptides
Then, the polypeptides are further converted
into amino acids.
.bmp)
The bacterial cells can then take up these
amino acids and use them in their metabolic processes.
Procedure /Method of Gelatin hydrolysis test:
1. A heavy inoculum of
test bacteria is inoculated (18- to 24-hour-old) by stabbing 4-5 times on the
tube containing nutrient gelatin medium.
2.
The inoculated tube is incubated along with an uninoculated medium at
35°C, for up to 2 weeks.
3. the tubes daily from
the incubator shall be removed and placed in ice bath or refrigerator
(4°C) for 15-30 minutes (until control is gelled) every day checked for
gelatin liquefaction.(Gelatin normally liquefies at 28°C and above, so to
confirm that liquefaction was due to gelatinase activity, the tubes are
immersed in an ice bath or kept in refrigerator at 4°C).
4.
The tubes are tilted to
observe if gelatin has been hydrolyzed.
Expected
results
Positive: Partial or total
liquefaction of the inoculated tube (uninoculated control medium must
be completely solidified) even after exposure to cold temperature of ice
bath or refrigerator (4°C)
Negative: Complete
solidification of the inoculated tube even after exposure to cold temperature
of ice bath or refrigerator (4°C)
urease test
UREASE TEST
AIM:
To determine the ability
of the organism to hydrolyse urea by the action of urease enzyme.
PRINCIPLE:
Urea is a nitrogen
containing compound that is produced during decarboxylation of the amino acid
arginine in the urea cycle. Urea is a major organic waste product of
protein digestion in most vertebrate and is excreted in urine. Some bacteria
have the ability to produce an enzyme urease as part of its metabolism to break
down urea. The urease is a hydrolytic enzyme which attacks the carbon and
nitrogen bond with the liberation of ammonia and carbondioxide. It is useful
diagnostic test for identifying bacteria, especially to distinguish members of
the genus Proteus from Gram negative pathogens. Proteus
vulgaris is an important and fast producer of urease.
Urease test is performed
by growing test organism on urea agar slant or urea broth with phenol red as
indicator with pH6.8. During the incubation period, the organism capable of
producing urease enzyme hydrolyses urea and produce ammonia that raises the pH
level. As the pH increases, the phenol red changes from yellowish orange as
initial color of medium to deep pink. Failure of development of a pink
coloration due to no ammonia production is evidence of a inability of organism
to produce urease enzyme.
MATERIALS REQUIRED:
Urea broth medium, Bacterial Culture; Proteus and E.coli,
Inoculation loop, Test tubes
Media Components
Enzymatic digest of
gelatin (1 g), dextrose (1 g), NaCl (5 g), KH2PO4 (2 g), urea (20 g), phenol
red (0.012 g), per 1000 mL, pH 6.8.
PROCEDURE:
1.
The surface of a urea agar slant is streaked with a portion
of a well-isolated colony or inoculate test organism on urea broth
containing phenol red as indicator.
2.
The urea agar slant or urea broth is incubated at 37°C for 24-48
hours.
3.
The development of pink color is examined.
4.
In case of unknown result incubation shall be lenthened for 7 days
to check for slow urease production.
RESULTS:
Positive: Deep pink coloration, Light Orange, Magneta
Negative: No Color change, Yellowish orange coloration
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