Gelatin hydrolysis is helpful in identifying and differentiating species of Bacillus, Clostridium, Proteus, Pseudomonas, and Serratia.  It also 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.
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. Gelatin is a protein derived from the connective tissues of vertebrates, that is, collagen. It is produced when collagen is boiled in water. Gelatin hydrolysis 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.
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)
Negative: Complete solidification of the inoculated tube even after exposure to cold temperature of ice bath or refrigerator (4°C)

Control organisms

Positive Control: Proteus vulgaris
Negative Control: Enterobacter aerogenes
Composition of Nutrient gelatin medium Peptone                                      5.0 g/liter
Beef extract                                3.0 g/liter
Gelatin                                    120.0 g/liter
Final pH: 6.8 ± 0.2 at 25°C.

Preparation of Nutrient gelatin medium

  • Prepare medium by mixing all ingredients in 1,000 ml of distilled or deionized water and heating gently to dissolve.
  • Dispense 2 to 3 ml of medium into 13- by 100-mm culture tubes.
  • Autoclave medium at 121°C.(15 psi) for 15 minutes.
  • Allow the tubed medium to cool in an upright position before use.
  • Store the prepared medium at 2 to 8°C. Note:Tubed medium stored at 2 to 8°C. may be used until its expiration date. Do not use tubed medium if it shows signs of microbial contamination, discoloration, drying, or other signs of deterioration.

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