Gelatin Hydrolysis Test: Introduction, Principle, Procedure and Result Interpretation

Gelatin Hydrolysis Test: Introduction, Principle, Procedure and Result Interpretation

Introduction of Gelatin Hydrolysis Test

Gelatin is a solidifying agent and is used in microbiology for culture media before the use of agar. It is nowadays replaced by agar due to having better-solidifying properties than gelatin but even in use for gelatin hydrolysis test to identify various organisms like Bacillus, Clostridium, Proteus, Pseudomonas, and Serratia. It is a protein derived from the animal protein collagen– a component of vertebrate connective tissue. Gelatin is a liquid at 28°C or higher.

Principle of Gelatin Hydrolysis Test

Gelatinase is a proteolytic enzyme that hydrolyzes gelatin into polypeptides and individual amino acids. This enzyme destroys the structure of the gelatin, and it becomes liquid.

Requirement for Gelatin Hydrolysis Test

  • Test organism ( pure or well-isolated colonies, 18-24 hours old culture)
  • Nutrient gelatin medium
  • Inoculating wire
  • Bunsen burner
  • Refrigerator
  • Incubator
  • Control strains

Positive Control: Pseudomonas aeruginosa ATCC 10145

Negative Control : Escherichia coli ATCC 25922

Procedure of Gelatin Hydrolysis Test

  1. Incubation temperatures vary according to the nature of organisms e.g for Enterobacteriaceae and fluorescent Pseudomonas 22°C and for non-fermenting, gram-negative rods 30°C
  2. . The medium should be solidified at room temperature. After touching several well-isolated colonies with a sterile needle, stab directly down the center of the tube to approximately 10 mm from the bottom. Repeat to inoculate heavily.
  3. Incubate the test and an uninoculated control tube for 48 hours.
  4. Gently remove the inoculated and uninoculated tubes from the incubator and refrigerate for at least 30 min or until the control tube solidifies.
  5. Note: a. Do not shake or invert the tubes prior to refrigeration. b. Gently invert to detect liquefaction by the test organism after 30 min of refrigeration.
  6. Reincubate a negative test for up to 2 weeks if indicated by the nature of the organism, and examined at regular intervals.

Result Interpretation for Gelatin Hydrolysis Test

Gelatin Hydrolysis Test Positive: At the end of the refrigeration period, the control tube will be resolidified and the test tube will remain liquid at least to the depth of the stab. Note: Some organisms only partially hydrolyze gelatin or liquefy just at the surface of the tube.

Gelatin Hydrolysis Test Negative: At the end of the refrigeration period, the control tube and the test tube will be resolidified, even at the top of the tube as shown above figure.

Control strains

  • Pseudomonas aeruginosa ATCC 10145—Liquefaction (Gelatin hydrlysis test positive)
  • Escherichia coli ATCC 25922—No  liquefaction ( Gelatin hydrolysis test negative)

Keynotes of Gelatin Hydrolysis Test

  1. P. fluorescens is gelatinase positive, but P. putida is gelatinase negative.
  2. Gelatin liquefaction test or gelatin hydrolysis test also  distinguishes the gelatinase-positive, pathogenic Staphylococcus aureus from the gelatinase-negative, non-pathogenic S. epidermidis
  3. Gelatin hydrolysis test positive bacteria are-  Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Clostridium perfringens, Clostridium tetani, Staphylococcus aureus, Pseudomonas, Serratia, and Proteus.
  4. This test is also useful to differentiate genera of gelatinase-producing bacteria like Serratia and Proteus from other members of  Enterobacteriaceae.

Limitation of Test

  1. Gelatinase usually acts at the surface of the tube medium. Shaking the tube while it is warm may result in a false-negative interpretation.
  2. Gelatin may vary in its gelling ability; therefore, incubate an uninoculated control with the test. The control must be refrigerated along with the test, prior to reading.

Further Readings

  1. Cowan and Steel’s, manual for the identification of medical bacteria
  2. Lynne S. Garcia, Clinical Microbiology Procedures Handbook
  3. https://www.researchgate.net/post/How_to_perform_a_Gelatin_liquefaction_test_for_bacterial_cultures
  4. https://www.himedialabs.com/TD/M060.pdf
  5. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/IFU60950.pdf
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