Gelatin liquefaction Test: Introduction, Principle, Procedure and result

Gelatin Liquefaction Test: Introduction, Principle, Procedure and Result Interpretation

Introduction of Gelatin Liquefaction Test

Gelatin is a solidifying agent and is used in microbiology for culture media before the use of agar. It is nowadays replaced by agar due to having better-solidifying properties than gelatin but even in use for gelatin liquefaction test to identify various organisms like Bacillus, Clostridium, Proteus, Pseudomonas, and Serratia. It is a protein derived from the animal protein collagen– a component of vertebrate connective tissue. Gelatin is a liquid at 28°C or higher.

Principle of Gelatin Liquefaction Test

Gelatinase is a proteolytic enzyme that hydrolyzes gelatin into polypeptides and individual amino acids. This enzyme destroys the structure of the gelatin, and it becomes liquid.

Requirement for Gelatin Liquefaction Test

Test organism ( pure or well-isolated colonies, 18-24 hours old culture)
Nutrient gelatin medium
Inoculating wire
Bunsen burner
Refrigerator
Incubator
Control strains

Positive Control: Pseudomonas aeruginosa ATCC 10145

Negative Control : Escherichia coli ATCC 25922

Procedure of Gelatin Liquefaction Test

Incubation temperatures vary according to the nature of organisms e.g for Enterobacteriaceae and ﬂuorescent Pseudomonas 22°C and for non-fermenting, gram-negative rods 30°C
. The medium should be solidiﬁed at room temperature. After touching several well-isolated colonies with a sterile needle, stab directly down the center of the tube to approximately 10 mm from the bottom. Repeat to inoculate heavily.
Incubate the test and an uninoculated control tube for 48 hours.
Gently remove the inoculated and uninoculated tubes from the incubator and refrigerate for at least 30 min or until the control tube solidiﬁes.
Note: a. Do not shake or invert the tubes prior to refrigeration. b. Gently invert to detect liquefaction by the test organism after 30 min of refrigeration.
Reincubate a negative test for up to 2 weeks if indicated by the nature of the organism, and examined at regular intervals.

Result Interpretation for Gelatin Liquefaction Test

Gelatin Liquefaction test Positive: at the end of the refrigeration period, the control tube will be resolidiﬁed and the test tube will remain liquid at least to the depth of the stab. Note: Some organisms only partially liquefy gelatin or liquefy just at the surface of the tube.

Gelatin Liquefaction test Negative: at the end of the refrigeration period, the control tube and the test tube will be resolidiﬁed, even at the top of the tube as shown above figure.

Control strains

Pseudomonas aeruginosa ATCC 10145—liquefaction (Gelatin Liquefaction test positive)
Escherichia coli ATCC 25922—no liquefaction ( Gelatin Liquefaction test negative)

Keynotes of Gelatin Liquefaction Test

P. ﬂuorescens is gelatinase positive, but P. putida is gelatinase negative.
Gelatin liquefaction test or gelatin hydrolysis test also distinguishes the gelatinase-positive, pathogenic Staphylococcus aureus from the gelatinase-negative, non-pathogenic S. epidermidis
Gelatin liquefaction test positive bacteria are- Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Clostridium perfringens, Clostridium tetani, Staphylococcus aureus, Pseudomonas, Serratia, and Proteus.
This test is also useful to differentiate genera of gelatinase-producing bacteria like Serratia and Proteus from other members of Enterobacteriaceae.

Limitation of Test

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.

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