Gelatin Hydrolysis Test: Principle, Procedure, Results

Gelatin is a protein derived from the animal protein collagen-a component of vertebrate connective tissue. It has been used as a solidifying agent in food for a long time. Organism producing proteolytic enzymes, and gelatinases, hydrolyze gelatin into polypeptides and individual amino acids. In the process, gelatin loses its structure and becomes liquid.

Principle of Gelatin hydrolysis test

A gelatin hydrolysis test is used to detect the ability of an organism to produce gelatinases that liquefy gelatin. This process takes place in two sequential reactions. In the first reaction, gelatinases degrade gelatin to polypeptides. Then, the polypeptides are further converted into amino acids. The bacterial cells can then take up these amino acids and use them in their metabolic processes.

Quality Control

1. Inspect gelatin agar tubes for contamination, dehydration, and lack of liquefaction at refrigeration temperatures before storage and before use.
2. Perform QC on each new lot of media prior to using them.
3. Use an uninoculated control with each use.
4. Organisms
   1. Pseudomonas aeruginosa ATCC 10145—liquefaction (positive)
   2. Escherichia coli ATCC 25922—no liquefaction (negative)

Procedure of Gelatin hydrolysis test

There are several methods for determining gelatinase production, all of which make use of gelatin as the substrate. The standard and most commonly employed method is the nutrient gelatin stab method.

1. Inoculate a heavy inoculum of test bacteria (18- to 24-hour-old) by stabbing 4-5 times (half an inch) on the tube containing nutrient gelatin medium.
2. Incubate the inoculated tube along with an uninoculated medium at 35°C, or at the test bacterium’s optimal growth temperature, for 48 hours.
3. Gently remove the tubes daily from the incubator and place them in an ice bath or refrigerator (4°C) for 30 minutes or until the control tube solidifies.
   (Gelatin normally liquefies at 28°C and above, so to confirm that liquefaction was due to gelatinase activity, the tubes are immersed in an ice bath or kept in a refrigerator at 4°C).
4. After 30 minutes of refrigeration, tilt the tubes gently to observe liquefaction by the test organism.
5. Reincubate a negative test for up to 2 weeks if indicated by the nature of the organism, and examine at regular intervals.

Expected results

Positive: Partial or total liquefaction of the inoculated tube (uninoculated control medium must be completely solidified) even after exposure to the cold temperature of ice bath or refrigerator (4°C).

Negative: Complete solidification of the inoculated tube even after exposure to the cold temperature of an ice bath or refrigerator (4°C)

Common bacteria and their reactions to the gelatin hydrolysis test performed on nutrient gelatin.

Uses of Gelatin Hydrolysis Test 

Gelatinase production is useful as a taxonomic aid in the classification and identification of both Gram-negative rods and Gram-positive rods.

• Gelatin liquefaction test can also be used to differentiate genera of gelatinase-producing bacteria such Serratia and Proteus from other members of the family Enterobacteriaceae.
  • Proteus spp. and Serratia spp. are usually gelatinase positive.
• Gelatin hydrolysis test is helpful in identifying and differentiating species of Bacillus, Clostridium, and, Pseudomonas.
  • Gram-positive, spore-forming, rod-shaped, aerobic, or anaerobic bacteria such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Clostridium perfringens, and Clostridium tetani, are also positive for gelatin hydrolysis.
  • P. fluorescens is gelatinase positive, but P. putida is gelatinase negative.
• It distinguishes the gelatinase-positive, pathogenic Staphylococcus aureus from the gelatinase-negative, non-pathogenic S. epidermidis.

Limitations

• Gelatinase usually acts at the surface of the tube medium. Shaking the tube while it is warm may result in a false-negative interpretation.
• 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.

References and further readings

• Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
• Koneman’s Color Atlas and Textbook of Diagnostic Microbiology
• Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814