Gelatin Hydrolysis Test

Generally, gelatin is used to define an animal protein or a collagen which is a component of a vertebrate in a connective tissue. Gelatin is used as a solidifying agent in a food for a long day ago, Organisms that produces proteolytic enzyme, specifically gelatinases help in hydrolysing the gelatin into a polypeptide and individual amino acids.

During this process, gelatin losses its structure and converts in to liquid form. Robert Koch used gelatin in his culture in the form of a nutrient gelatin which is one of the oldest solid culture media.

Gelatin usually dissolves in a water at 50 degree Celsius and it exists as the liquid at a temperature of above 25 degree Celsius and it further solidifies and forms a gel like substance when it is cooled below 25ºC.

What is Gelatin Hydrolysis Test?

Gelatin hydrolysis test is also called as Gelatin liquefication test, as this test involves the process of liquefication of gelatin in the presence of an enzyme gelatinase.

Gelatinase is considered as one of the most important enzymes in various pathogenic organisms as it is produced extracellularly, and hydrolysis gelatin which is derived from the connective tissues of the vertebrates in the form of collagen.

This enzyme also works as a virulent factor which dissolves the connective tissues of the host and aids in producing invasive infections.

Gelatin is a protein that hydrolyses in the presence of an enzyme gelatinase by breaking down the complex structure into monomeric amino acids.

This test is being followed from early days in the form of presumptive test to identify the pathogenic organisms like Serratia, Pseudomonas, Flavobacterium and Clostridium.

Gelatin Hydrolysis Test Principle

Gelatin is a type of protein derived from the animal tissues, collagen and it forms a solid structure at low temperature. This protein is metabolised or degraded by a group of enzymes known as gelatinase.

Gelatinases are the proteolytic enzymes which liquifies the gelatin into polypeptides and individual amino acids.

The degradation of the gelatin into polypeptides, is followed by covering the polypeptides into amino acids.

Gelatinase is very important in bacteria as gelatin is comparatively a large polymer and thus it cannot be transported into the cell membranes, as gelatinase breaks this compound into the smaller peptides it can easily be transported into the cell and it is utilised by the bacteria.

In hydrolysis test, media containing gelatin is used and its hydrolysis is detected either by liquification of the media or by flooding the media with mercuric chloride, as mercuric chloride helps in precipitating the gelatin and make the hydrolysed area to look clear.

This test is commonly used to determine the ability of an organism to produce the extracellular proteolytic enzymes, gelatinases, which hydrolyses the gelatin, a component of the vertebrate connective tissue.

This reaction usually occurs in a series of two steps, in first reaction process, Gelatinases hydrolyses gelatin into polypeptides and further the polypeptides are converted into amino acids.

The amino acids are taken up by the cells and they are used for their own metabolic purposes. Generally, the presence of gelatinases can be detected using a nutrient gelatin medium.

When an organism produces gelatinase, the enzymes liquifies the growth medium by liquifying the gelatin that is present in the medium.

Gelatin Hydrolysis Test and Microorganism

Gelatin hydrolysis test is generally used to detect the micro-organisms Gram negative rods require gelatine for the identification of specific fluorescent pseudomonas. Such as Pseudomonas putida (negative) from pseudomonas fluorescens (positive). Whereas the gram-positive rods are needed for identifying the pathogens in species level.

Gelatin Hydrolysis Test Materials

Nutrient Gelatin media is generally used for the purpose of demonstrating the hydrolysis of gelatin by adding mercuric chloride or by using liquification of gelatin.

Ingredients	Gram/litre
Tryptose	20.0
Gelatin	10.0
Magnesium sulphate	0.01
Beef extract	3.0
Agar	15.0

Reagents:

• Mercuric chloride

Supplies:

• Inoculating needle

• Incubator

• Pipettes

Gelatin Hydrolysis Test Procedure

1. Preparation of the media

First, the media is prepared. 128 grams of dehydrated medium mixed with 1000 millilitres of warm distilled water in a beaker.

The prepared solution is heated with agitation to bring about the boiling point and the media is let to dissolve completely.

Then the prepared medium is dispensed into series of test tubes and it is autoclaved at 15lbs pressure for about 15 minutes. If agar plate method is used, the medium is autoclaved in the beaker.

The tubes are cooled after autoclaving to about 49 to 50 degree Celsius by keeping it in an upright position.

2. Gelatin Hydrolysis

Gelatin hydrolysis can be identified by nutrient gelatin slab method or by agar plate flooding method by using mercuric chloride.

a. Stab Method of Gelatin Hydrolysis Test

Gelatin medium in a tube is inoculated with 4 to 5 drops of a 24-hour broth medium.The inoculated tubes are then incubated at a temperature of about 37 ºC in the air for 24 to 48 hours, If the organisms have the capability to grow at 25ºC then the incubation should be done at 25ºC. After the first incubation, the tubes are placed at 4ºC for one 24 hours.

b. Plate Method of Gelatin Hydrolysis Test

In plate method, heavy inoculum of a culture is taken using an inoculating loop and it is inoculating using nutrient gelatin medium. And the plates are kept in an incubator, by setting a temperature of 37ºC for about 24 to 48 hours.

Gelatin Hydrolysis Test Result

1. Tube Method: In positive result, either partial or total liquefication of the gelatin can be observed in the tubes. In negative result, complete solidification of gelatin is seen at a temperature of 4ºC.

2. Plate Method: In positive result, clear zone around the colonies is noted after adding mercuric chloride. If there is no clear zone after adding mercuric chloride, it indicates the negative result.

Gelatin Hydrolysis Test Uses

 Gelatin hydrolysis test is usually used to test the capability of an organism to produce gelatinase.

 This test also helps in identification of Serratia, Pseudomonas, Flavobacterium and clostridium.

 Gelatin hydrolysis test also helps in distinguishing the gelatinase positive staphylococcus aureus from the gelatinase negative non-pathogenic S. epidermidis.

 Using this test; genera of bacteria such as Serratia and proteus are differentiated from other members of Enterobacteriaceae family