Objective:
To determine the ability of bacteria
that produces extracellular hydrolytic enzyme Gelatianse that degrade gelatin.
Principle:
Gelatin is a protein obtained
from animal protein collagen, a major component of connective tissues and
tendons of humans and animals. It is an incomplete protein with lacking of
essential amino acid tryptophan. Gelatin maintains its gel properties below 25 ͦ
C and exists as a solid in nature. At temperature above 25 ͦ C, gelatin is a liquid.
Some microorganisms capable of
producing proteolytic extracellular enzyme gelatinase which hydrolysis this
gelatin into amino acids. After this degradation, it will not return to gel
characteristics even at very low temperature 4 ͦ C.
Materials required:
24
Hours old bacterial cultures (Bacillus sp. and E.coli )
Nutrient
gelatin
Procedure:
-Prepare sterilized nutrient gelatin in test tubes
and allow to solidify.
-Inoculate the organisms into gelatin tubes by
stabbing
-Incubate the tubes at 37 ͦ C for 24 hours (May
require upto 14 days to liquefy the gelatin.
-Following incubation, place the tubes in
refrigerator at 4 ͦ C for 30 min.
-Observe the liquefaction (Liquid formation) in
tubes
(If bottom is
resolidifies, slant the tubes and notice the surface of the medium is liquid or
not)
Result:
Bacillus
sp.,
is Gelatin hydrolysis positive (Gelatin liquefied)
E.coli
is Gelatin hydrolysis positive (non liquefied)
Interpretation:
Bacillus
sp. shows positive result as the medium remained liquefied after refrigeration.
This result indicates that the organism produce gelatinase enzyme. E.coli
give negative result as the medium remains solid after refrigeration.
Gelatin
Hydrolysis Positive Organisms:
Bacillus sp.
Clostridium perfringens
Proteus vulgaris
Staphylococcus aureus
Gelatin Hydrolysis Negative Organisms:
E.coli
Staphylococcus epidermidis
Enterobacter aerogenes
Nutrient Gelatin Composition:
Peptone - 5
g
Beef extract - 3
g
Gelatin - 120 g
Distilled water - 1000ml
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