GELATIN HYDROLYSIS TEST

GELATIN HYDROLYSIS TEST

AIM

      To determine the ability of an organism to produce proteolytic enzyme Gelatinase that liquefy Gelatin.

PRINCIPLE       

         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. Robert Koch used nutrient gelatin as an early type of solid growth medium. Gelatin dissolves in water at 50 °C and exists as liquid above 25 °C and solidifies or gels when cooled below 25 °C. 

        In 1926, Frazier described the very first method to detect the ability of microorganisms to liquefy gelatin. Clarke (1953) described a simplified plate method using 10 % leaf gelatin and HgCl ₂–HCl solution for the detection of gelatin-liquefying bacteria and compared it with the gelatin stab method and the Frazier’s plate method. This comparison showed that hydrolysis of gelatin was generally more accurate and rapid in the simplified plate method (3 days) than in the stab method (up to 14 days) and Frazier’s plate method (up to 4 days). The plate test, however, did not eliminate the use of acidic mercuric chloride to visualize the digestion of gelatin. Green and Larks (1955) also reported a quick method for the detection of gelatin-liquefying bacteria using formalin-denatured gelatin-charcoal. In this method, gelatin hydrolysis was observed when charcoal particles were liberated and settled to the bottom of the culture tube. This method was found to be quicker than the gelatin stab method.

        Gelatin hydrolysis test is a great way to highlight proteolysis by bacteria. Gelatin hydrolysis test is used to detect the ability of an organism to produce Gelatinase that liquefy Gelatin.  Hydrolysis of Gelatin indicates the presence of Gelatinase (proteolytic enzyme). This process takes place in two sequential reactions. In the first reaction, Gelatinase degrade Gelatin to Polypeptides. Then, the Polypeptides are further converted into Amino acids.  The bacterial cells can then take up these amino acids that can cross the cell membrane and be utilized by the organism.

Figure – 1: Conversion of Gelatin into Polypeptides and Amino acids by Gelatinase

      Gelatin hydrolysis test distinguishes the Gelatinase positive, pathogenic Staphylococcus aureus from the Gelatinase negative, non-pathogenic Staphylococcus epidermidis. Gram positive, spore - forming, rod shaped, 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 Aeromonas hydrophila, Flavobacterium sp., Pseudomonas sp., Serratia sp. and Proteus sp. from other members of the family Enterobacteriaceae.

MATERIALS REQUIRED

1. Test bacteria
2. Nutrient Gelatin medium
3. Inoculation loop
4. Incubator
5. Refrigerator

PROCEDURE

a)     Inoculate an inoculum of test bacteria (18 to 24 hours old) by stabbing 4 - 5 times (half inch) on the tube containing Nutrient gelatin medium.

b)     Incubate the inoculated tube along with an uninoculated medium at 37 °C for upto 2 weeks.

c)     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 25 °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).

d)     Tilt the tubes to observe if gelatin has been hydrolyzed.

OBSERVATION AND RESULTS

·    Positive Result - Partial or complete liquefaction of the inoculated tube at 4 °C. The control tube remains solidified even after exposure to cold temperature.

·   Negative Result - At the end of the refrigeration, the control tube and the test tube both remain completely solidified.

Figure – 2: Gelatin Liquefaction Test (Source: studyblue.com)