Gelatin liquefaction test

GELATIN LIQUIFACTION TEST

AIM:

            To determine the ability of an organism that produce gelatinases.

PRINCIPLE:

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:

1.      A heavy inoculum of test bacteria is inoculated (18- to 24-hour-old) by stabbing 4-5 times on the tube containing nutrient gelatin medium.

2.      The inoculated tube is incubated along with an uninoculated medium at 35°C, for up to 2 weeks.

3.      the tubes daily from the incubator shall be removed and placed in ice bath or refrigerator (4°C) for 15-30 minutes (until control is gelled) every day checked 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.       The tubes are tilted 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)