Done by : Huda Elsharatha Number : Dr Abdelrauf El manama

Principle: Gelatin is a protein that is derived from collagen. The gelatin molecule is to large to enter the cell as a whole so the exoenzyme gelatinase

Purpose: To differentiate between organisms that produce the exoenzyme gelatinase and those that do not 1. vibrio (vibrio cholera) 1. vibrio (vibrio cholera) 2. Pseudomonas (pseudomonas aeruginosa) 2. Pseudomonas (pseudomonas aeruginosa) 3. Serratia 3. Serratia

vibrio

pseudomonas

serratia

Gelatinase Gelatinase Gelatin causes liquids to solidify at temperatures below 28 degrees Celsius. At temperatures above 28 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 3-4 days Negative :no liquefaction after 30 days

The Gelatin Strip Method The gelatin strip method employs strips of clear blue plastic covered with a gray-green 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). 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. 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