- 02 Apr 2017
Objective: To determine the ability of an organism to produce proteolytic-like enzymes (gelatinases) which break down gelatin. Gelatinase destroys (hydrolyzes) the gel and causes its collapse and liquefaction.
1. Obtain two solidified gelatin butts but keep them in the refrigerator until just prior to inoculation. Pick up a heavy inoculum from your pure culture and stab one of the butts to a depth of 2 inches. The other tube should not be inoculated and used as a control.
2. Incubate both the test and control tubes simultaneously at the optimal growth temperature for the organism for 48 hours to 14 days.
3. At the end of each 48-hour period, place both tubes (test bacterium and control) in a refrigerator for about 1 hour to determine whether digestion of gelatin (liquefaction) has occurred. Make the transfer from incubator to refrigerator without shaking the tubes. Check tubes daily up to 2 weeks unless liquefaction occurs sooner.
Gently tilt the tubes. The test is positive if the medium of the test organism is liquefied (gelatin breakdown) while that of the control has remained solid (lack of gelatin hydrolysis). The result of the test is negative if the medium of the test organism is as solid as that of the control.
-- Always run a control tube in parallel with organism being tested.
-- Gelatin is solid when incubated at 20°C or less and liquid at 30°C or greater. Gelatin changes from a gel (solid state) to a liquid at about 28°C. Therefore, if gelatin tubes are incubated at 30°C or greater, they must first be placed in a refrigerator for an hour and cooled before an interpretation of liquefaction is made.
-- Do not shake gelatin tubes while warm since growth and liquefaction of gelatin frequently occur only on the surface layer. If the gelatin is shaken and allowed to be mixed with the warm fluid of the medium, there is a possibility that a positive result may be overlooked, and thereby a false negative result may be obtained.