Gelatin hydrolysis test

GELATIN HYDROLYSIS TEST

1. Saajida Sultaana Mahusook GELATIN HYDROLYSIS TEST

2. Aim Gelatin hydrolysis test  is used to detect the ability of a microorganism to produce  gelatinase   that liquefy  gelatin Principle Gelatin is a protein derived from the animal protein collagen, a component of connective tissue and tendons in human and other animals. It has been used as a solidifying agent in food for a long time. Robert Koch used nutrient gelatin as an early type of solid growth medium. Gelatin dissolves in water at 50 degree Celsius and exists as liquid above 25°C and solidifies or gels when cooled below 25°C . This test is used to determine the ability of an organism to produce extracellular proteolytic enzymes, gelatinases that hydrolyze gelatin. The reaction occurs in two sequential steps: in first reaction gelatinases hydrolyze gelatin into polypeptides and then polypeptides are further converted into amino acids as shown in figure.

3. The amino acid is taken up by the cell and used for metabolic purposes. The presence of gelatinases is detected using a nutrient gelatin medium. This medium is a simple medium composed of gelatin, peptone and beef extract. When nutrient gelatin tube is stab-inoculated with a gelatinase positive organisms, the secreted gelatinases will liquefy the gelatin, resulting in the liquefaction of the medium. While the gelatinase negative organisms do not secrete enzymes and do not liquefy the medium.

4. Materials required Gelatin nutrient agar medium Gelatin                        120g/l peptone                       5g/l Beef extract                3g/l Final pH:                     6.8±0.2 at 25°C . Test tubes Inoculating loop Bunsen Burner

5. Procedure: Prepare nutrient gelatin agar medium Autoclave the test tubes at 121°C and 15 psi for 15 minutes. Allow the tube to cool in an upright position. Store the prepared medium at 2-8°C . Inoculate the nutrient gelatin medium with a test inoculum by stabbing 4 to 5 times half inch into the medium. Incubate the test and an un-inoculated tube (control) for 48 hour at 37°C. Gently remove the inoculated and uninoculated tubes from the incubator and place in ice bath or refrigerate for at least 30 min. ( NOTE: i. Do not shake or invert the tubes prior to refrigeration. ii. Gently invert to detect liquefaction by the test organism after 30 min of refrigeration.) Re-incubate a negative test for up to 2 weeks and examine at regular intervals to check for liquefication .

6. Result Positive:   After incubation, partial or complete liquefaction of the inoculated tube at 4°C. The control tube remain solidified. Examples:   Aeromonas hydrophila , Bacillus subtilis Negative:   After incubation, both the control tube and the test tube remain completely solidified. Examples:   Micrococcus roseus , Escherichia coli

7. Reference https ://microbiologynotes.com/gelatin-hydrolysis-test-principle-uses-media-procedure-and-result / MICROBIOLOGY A LABORATORY MANUAL, James G. Cappuccino and Natalie Sherman , tenth edition