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Gelatin hydrolysis test

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Saajida Sultaana

The gelatin hydrolysis test detects microorganisms that produce the enzyme gelatinase, which liquefies gelatin. Gelatin is derived from collagen and solidifies at temperatures below 25°C. The test uses a nutrient gelatin medium that is inoculated with the microbe. If the microbe produces gelatinase, the enzyme will hydrolyze the gelatin into polypeptides and amino acids, liquefying the medium. A positive result is indicated by liquefaction of the inoculated tube after refrigeration. A negative result shows no liquefaction. The test helps identify microbes like Bacillus subtilis that can produce extracellular proteolytic enzymes.

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Saajida Sultaana Mahusook *
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.
*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.
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
Procedure: 1. Prepare nutrient gelatin agar medium 2. Autoclave the test tubes at 121°C and 15 psi for 15 minutes. 3. Allow the tube to cool in an upright position. 4. Store the prepared medium at 2-8°C. 5. Inoculate the nutrient gelatin medium with a test inoculum by stabbing 4 to 5 times half inch into the medium. 6. Incubate the test and an un-inoculated tube (control) for 48 hour at 37°C. 7. 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.) 8. Re-incubate a negative test for up to 2 weeks and examine at regular intervals to check for liquefication.
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
*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

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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: 1. Prepare nutrient gelatin agar medium 2. Autoclave the test tubes at 121°C and 15 psi for 15 minutes. 3. Allow the tube to cool in an upright position. 4. Store the prepared medium at 2-8°C. 5. Inoculate the nutrient gelatin medium with a test inoculum by stabbing 4 to 5 times half inch into the medium. 6. Incubate the test and an un-inoculated tube (control) for 48 hour at 37°C. 7. 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.) 8. 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