Gelatin Hydrolysis Test: Principle, Procedure and expected results

Gelatin is a protein derived from the animal protein collagen– component of vertebrate connective tissue. It has been used as a solidifying agent in food for a long time. Gelatin hydrolysis test is a great way to highlight proteolysis by bacteria

Principle of Gelatin hydrolysis test

Gelatin hydrolysis test is used to detect the ability of an organism to produce gelatinase (proteolytic enzyme) that liquefy gelatin.  Hydrolysis of gelatin indicates the presence of gelatinases.

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.

Uses of Gelatin Hydrolysis test 

Gelatin hydrolysis test is helpful in identifying and differentiating species of Bacillus, Clostridium, Proteus, Pseudomonas, and Serratia.

It distinguishes the gelatinase-positive, pathogenic Staphylococcus aureus from the gelatinase-negative, non-pathogenic S. epidermidis . Gram-positive, spore-forming, rodshaped, aerobic or anaerobic bacteria such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Clostridium perfringens and Clostridium tetani, are also positive for gelatin hydrolysis.

The test can also be used to differentiate genera of gelatinase-producing bacteria such Serratia and Proteus from other members of the family Enterobacteriaceae.

Procedure /Method 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 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 up to 2 weeks.
  3. Remove the tubes daily from the incubator and place in ice bath  or refrigerator (4°C) for 15-30 minutes (until control is gelled) every day to check for gelatin liquefaction.(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 refrigerator at 4°C).
  4. Tilt the tubes to observe if gelatin has been hydrolyzed.

Expected results

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

Gelatin Hydrolysis Test: Above tube: Positive
Below Tube: Negative

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

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




Bacillus subtilis


Clostridium perfringens



Escherichia coli



Proteus vulgaris



Serratia liquefaciens



Staphylococcus aureus



Control organisms