PrinciplePrinciple::

GelatinGelatin is a protein that is derived from the is a protein that is derived from the hydrolysis of collagenhydrolysis of collagen..

Its incomplete protien because it lake tryptophanIts incomplete protien because it lake tryptophan..

The gelatin molecule is to large to enter the cell as a The gelatin molecule is to large to enter the cell as a whole so the exoenzyme gelatinase can break whole so the exoenzyme gelatinase can break down it. However it produce agel in water below down it. However it produce agel in water below 25C , but when it hydrolyzed it lose the ability to 25C , but when it hydrolyzed it lose the ability to

form agelform agel . .

PurposePurpose::

To differentiate between organisms that produce To differentiate between organisms that produce the exoenzyme gelatinase and those that do the exoenzyme gelatinase and those that do notnot

11 . .vibrio (vibrio cholera)vibrio (vibrio cholera)

22 . .Pseudomonas (pseudomonas aeruginosa)Pseudomonas (pseudomonas aeruginosa)

33 . .SerratiaSerratia

vibriovibrio

pseudomonaspseudomonas

serratiaserratia

GelatinaseGelatinase  

Gelatin causes liquids to solidify at temperatures Gelatin causes liquids to solidify at temperatures below 25 degrees Celsius. At temperatures above below 25 degrees Celsius. At temperatures above 25 degrees C gelatin is a liquid. Some bacteria 25 degrees C gelatin is a liquid. Some bacteria produce gelatinaseproduce gelatinase,,

Gelatinase :Gelatinase :enzyme that hydrolyzes gelatinenzyme that hydrolyzes gelatinTwo methods used to determine gelatinase Two methods used to determine gelatinase productionproduction The gelatin stab methodThe gelatin stab method

The gelatin strip methodThe gelatin strip method

The Gelatin Stab MethodThe Gelatin Stab Method The gelatin stab method employs nutrient gelatin The gelatin stab method employs nutrient gelatin deep tubes that contain 12% gelatin. A heavy deep tubes that contain 12% gelatin. A heavy inoculum from a pure culture of the test organism inoculum from a pure culture of the test organism is stabbed into the media. The gelatin media is is stabbed into the media. The gelatin media is incubated for at least 48 hours, and then placed incubated for at least 48 hours, and then placed into the refrigerator for approximately 30 into the refrigerator for approximately 30 minutes. If the gelatin is still intact (the bacteria minutes. If the gelatin is still intact (the bacteria did not produce gelatinase), the media will solidify did not produce gelatinase), the media will solidify in the refrigerator and a negative test result is in the refrigerator and a negative test result is recorded. If the organism has produced sufficient recorded. If the organism has produced sufficient gelatinase, the tube will remain liquid (at least gelatinase, the tube will remain liquid (at least partially) and not solidify in the refrigerator. A partially) and not solidify in the refrigerator. A

positive test result is recordedpositive test result is recorded . .

Some organisms may produce gelatinase in rather Some organisms may produce gelatinase in rather small quantities. Thus, a tube with a negative small quantities. Thus, a tube with a negative gelatinase result should be reincubated for 30dys. gelatinase result should be reincubated for 30dys. Whenever desired, the tube may be refrigerated Whenever desired, the tube may be refrigerated and results observed. If the tube is still negative and results observed. If the tube is still negative after 30days of incubation (completely solidifies after 30days of incubation (completely solidifies when refrigerated), it can be reasonably concluded when refrigerated), it can be reasonably concluded that this organism does not produce gelatinasethat this organism does not produce gelatinase..

11 . .Gelatin Stab MethodGelatin 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.

 

RESULTSRESULTS

Positive strong :liquefaction occurs with 3daysPositive strong :liquefaction occurs with 3days

Positive weak :liquefaction occurs in 3-30 daysPositive weak :liquefaction occurs in 3-30 days

Negative :no liquefaction after 30 daysNegative :no liquefaction after 30 days

The Gelatin Strip MethodThe Gelatin Strip Method

The gelatin strip method employs X ray strips which The gelatin strip method employs X ray strips which consist of of clear blue plastic covered with a gray-consist of of clear blue plastic covered with a gray-green coating of gelatingreen coating of gelatin

Upon incubation with a gelatinase producing Upon incubation with a gelatinase producing bacteria, the gelatin coating is slowly hydrolyzed bacteria, the gelatin coating is slowly hydrolyzed and the blue plastic strip becomes visible (a and the blue plastic strip becomes visible (a positive result). There will not be any blue plastic positive result). There will not be any blue plastic visible on the gelatin strip in an organism that is visible on the gelatin strip in an organism that is

unable to produce gelatinase (a negative result)unable to produce gelatinase (a negative result) . .Some organisms may produce gelatinase in rather Some organisms may produce gelatinase in rather small quantities. Thus, a negative gelatin strip small quantities. Thus, a negative gelatin strip

tube should be reincubated for up to two weekstube should be reincubated for up to two weeks..  

22 . .Gelatin Strip MethodGelatin Strip Method