Download Lab 9 – gelatin_test

Principle:
Gelatin is a protein that is derived from the 
hydrolysis of collagen.
Its incomplete protien because it lake tryptophan.
The gelatin molecule is to large to enter the cell as a
whole so the exoenzyme gelatinase can break
down it. However it produce agel in water below
25C , but when it hydrolyzed it lose the ability to
form agel.
Purpose:
To differentiate between organisms that produce
the exoenzyme gelatinase and those that do
not
1. vibrio (vibrio cholera)
2. Pseudomonas (pseudomonas aeruginosa)
3. Serratia
vibrio
pseudomonas
serratia
Gelatinase
Gelatin causes liquids to solidify at temperatures
below 25 degrees Celsius. At temperatures above
25 degrees C gelatin is a liquid. Some bacteria
produce gelatinase,
Gelatinase :enzyme that hydrolyzes gelatin
Two methods used to determine gelatinase
production
The gelatin stab method
The gelatin strip method
The Gelatin Stab Method
The gelatin stab method employs nutrient gelatin
deep tubes that contain 12% gelatin. A heavy
inoculum from a pure culture of the test organism
is stabbed into the media. The gelatin media is
incubated for at least 48 hours, and then placed
into the refrigerator for approximately 30
minutes. If the gelatin is still intact (the bacteria
did not produce gelatinase), the media will solidify
in the refrigerator and a negative test result is
recorded. If the organism has produced sufficient
gelatinase, the tube will remain liquid (at least
partially) and not solidify in the refrigerator. A
positive test result is recorded.
Some organisms may produce gelatinase in rather
small quantities. Thus, a tube with a negative
gelatinase result should be reincubated for 30dys.
Whenever desired, the tube may be refrigerated
and results observed. If the tube is still negative
after 30days of incubation (completely solidifies
when refrigerated), it can be reasonably concluded
that this organism does not produce gelatinase.
1. Gelatin Stab Method
The tubes to the right depict a gelatinase negative (A) and gelatinase positive (B and C) reactions.
The 'Serratia marcescens' on the left is positive for gelatinase production, as
evidenced by the liquidation of the media. The 'Salmonella typhimurium' on the
right is negative, as evidenced by the solidity of the media.
RESULTS
Positive strong :liquefaction occurs with 3days
Positive weak :liquefaction occurs in 3-30 days
Negative :no liquefaction after 30 days
The Gelatin Strip Method
The gelatin strip method employs X ray strips which
consist of of clear blue plastic covered with a graygreen coating of gelatin
Upon incubation with a gelatinase producing
bacteria, the gelatin coating is slowly hydrolyzed
and the blue plastic strip becomes visible (a
positive result). There will not be any blue plastic
visible on the gelatin strip in an organism that is
unable to produce gelatinase (a negative result).
Some organisms may produce gelatinase in rather
small quantities. Thus, a negative gelatin strip
tube should be reincubated for up to two weeks.
2. Gelatin Strip Method