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

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 Serratia, Pseudomonas, Flavobacterium, 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:

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.

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.

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

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 Serratia, Pseudomonas, Flavobacterium, 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, 3.17.1.1–3.17.48.3. 
• 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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