Gelatin Hydrolysis Test- Principle, Procedure, Types, Result, Uses

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Gelatin Hydrolysis Test Definition

Gelatin hydrolysis test is a biochemical test performed as a presumptive test for the identification of Staphylococcus, Enterococcus, and other Gram-positive bacilli.

  • Gelatin hydrolysis test is also termed as the Gelatin Liquefaction test as it involves the liquefaction of gelatin in the presence of the gelatinase enzyme.
  • Gelatinase is an important enzyme in various pathogenic organisms as it is produced extracellularly, which hydrolyses gelatin which is derived from the collagen found in the connective tissues of vertebrates.
  • These enzymes might work as a virulence factor that dissolves the connective tissues of the host’s cells which aids in the invasive infections.
  • Gelatin is a protein that liquefies in the presence of gelatinase enzyme as the enzyme breaks down the complex structure of gelatin into monomeric amino acids.
  • The test has been used for years as a presumptive test for the identification of a wide variety of organisms like SerratiaPseudomonasFlavobacterium, and Clostridium.

Objectives of Gelatin Hydrolysis Test

  • To test an organism’s ability to liquefy gelatin by the production of gelatinase enzyme.
  • To differentiate organisms into different groups based on their ability to hydrolyze gelatin.

Principle of Gelatin Hydrolysis Test

  • Gelatin is a protein derived from animal connective tissue, collagen, which forms a solid structure at lower temperatures.
  • The protein is metabolized or degraded by a group of enzymes called gelatinase.
  • Gelatinases are proteolytic enzymes that hydrolyze gelatin into polypeptides and individual amino acids.
  • The degradation of gelatin, like most proteins, occurs in two steps; the first step involves the degradation of gelatin into polypeptides, followed by the conversion of polypeptides into amino acids.
  • Gelatinase is important in most bacteria as the gelatin is a large polymer and thus cannot be transported into the cell.
  • The enzyme, thus, breaks down gelatin into smaller units, which can be transported into the cell and utilized by the bacteria.
  • In the hydrolysis test, media with gelatin is used, and its hydrolysis is observed either by the liquefaction of the media or by flooding with mercuric chloride.
  • The mercuric chloride added to the medium precipitates gelatin while the areas where the gelatin is hydrolyzed appear clear.

Microorganisms Tested

  • Gram-negative rods that require gelatin for identification, especially for the separation of the fluorescent Pseudomonas: Pseudomonas putida (negative) from Pseudomonas fluorescens (positive).
  • Gram-positive rods as needed for identification to the species level.

Media, Reagent, and Supplies Used

Media Used

  • Nutrient Gelatin Media is used for the demonstration of gelatin hydrolysis either by adding mercuric chloride or by the liquefaction of gelatin.
  • The composition of the Nutrient Gelatin Media is given below:
S.N Ingredients Gram/liter
1. Tryptose 20.0
2. Beef extract 3.0
3. Gelatin 10.0
4. Manganese sulfate 0.01
5. Agar 15.0
Final pH at 25°C: 6.8 ±0.2

Reagent Used

  • Mercuric chloride (HgCl2)

Supplies Used

  • Inoculating needle
  • Incubator at 37°C
  • Pipettes

Procedure of Gelatin Hydrolysis Test

A. Preparation of the media

  • About 128 grams of the dehydrated medium is taken in a beaker with 1000 milliliters of warm (50°C) distilled water.
  • The solution is then heated with agitation to bring it to boiling in order to dissolve the medium completely.
  • The medium is dispensed into several test tubes and autoclaved at 15 lbs pressure (121°C) for 15 minutes. In the case of the agar plate method, the medium is autoclaved in the beaker.
  • The tubed medium is cooled to 45-50°C in an upright position.

B. Gelatin Hydrolysis

Gelatin hydrolysis can be observed whether via nutrient gelatin stab method or by flooding the agar plates with mercuric chloride.

1. Stab method

  • The gelatin medium in the tube is inoculated with 4-5 drops of a 24-hour broth medium.
  • The inoculated tubes are incubated at 37°C in air for 24-48 hours. If the organisms grow well at 25°C, the incubation has to be done at 25°C.
  • After the first incubation, the tubes are to be placed at 4°C for another 24 hours.

2. Plate method

  • Heavy inoculum of an 18-24 hour culture is taken with an inoculating loop and inoculated on nutrient gelatin medium.
  • The plates are then incubated at 37°C for 24-48 hours.

Result Interpretation of Gelatin Hydrolysis Test

Tube test

  • Partial or total liquefaction of gelatin in the test tubes indicates a positive result.
  • Complete solidification of gelatin at 4°C represents a negative result.

Gelatin Hydrolysis Test- Principle, Procedure and Result Interpretation

Figure: Gelatin hydrolysis. Positive; liquefaction at top of the tube. Image Source: Bailey and Scott’s Diagnostic Microbiology. Elsevier.

Plate Method

  • A clear zone around the colonies after the addition of mercuric chloride (HgCl2) indicates a positive result on agar plates.
  • No clear zones around the colonies after the addition of mercuric chloride are representative of a negative result.

Gelatin Hydrolysis Test Plate Method

Figure: Gelatin hydrolysis positive reaction. Image Source: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen.

Control organisms

  • Positive result: Bacillus subtilis.
  • Negative result: Escherichia coli.

Uses of Gelatin Hydrolysis Test

  • Gelatin hydrolysis test is used to test the ability of an organism to produce gelatinases.
  • Gelatin hydrolysis test helps in the identification of SerratiaPseudomonasFlavobacterium, and Clostridium.
  • The test distinguishes the gelatinase-positive, pathogenic Staphylococcus aureus from the gelatinase-negative, nonpathogenic Staphylococcus epidermidis.
  • The test can be used to differentiate genera of gelatinase-producing bacteria such as Serratia and Proteus from other members of the family Enterobacteriaceae.

Limitations of Gelatin Hydrolysis Test

  • Gelatinase mostly acts at the surface of the tube medium. Shaking the tube while it is warm might result in a false-negative result.
  • Gelatin may vary in its gelling ability; therefore, an uninoculated control should be inoculated with the test. The control must be refrigerated along with the test, prior to reading.
  • False-positive results may occur with the tube method in some media or saline on prolonged incubation, i.e. greater than 4 hours. This can be detected in the uninoculated control tube.
  • The plate method is not recommended for the determination of gelatin liquefaction by fastidious species and obligate anaerobes.
  • If the tubes are incubated at temperatures greater than 20°C the tubes must be chilled below 20°C before reactions can be carried out.

References and Sources

  • Nutrient Gelatin. M060. HiMedia Laboratories.
  • Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook,– 
  • Thomas Edison E. dela Cruz, Jeremy Martin O. Torres. 2012. Gelatin hydrolysis test protocol.
  • Joseph J. McDade, R. H. Weaver. RAPID METHODS FOR THE DETECTION OF GELATIN HYDROLYSIS. Journal of Bacteriology. Jan 1959, 77 (1) 60-64.
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About Author

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Anupama Sapkota

Anupama Sapkota has a bachelor’s degree (B.Sc.) in Microbiology from St. Xavier's College, Kathmandu, Nepal. She is particularly interested in studies regarding antibiotic resistance with a focus on drug discovery.

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