Gelatin Hydrolysis Test- Principle, Procedure, Types, Result, Uses

Gelatin hydrolysis test is a biochemical test that determines the ability of bacteria to produce gelatinase, an enzyme that breaks down gelatin. Gelatin is a protein derived from animal collagen, which forms a solid gel at low temperatures. Gelatinase hydrolyzes gelatin into smaller peptides and amino acids, which can be utilized by bacteria as a source of nutrients. Gelatin hydrolysis test is used as a presumptive test for the identification of various Gram-positive and Gram-negative bacteria, especially those that are involved in invasive infections. The test is based on the observation of the liquefaction of gelatin medium in the presence of gelatinase-producing bacteria. Alternatively, the test can also be performed by adding mercuric chloride to the gelatin medium, which precipitates gelatin and forms clear zones around the colonies of gelatinase-producing bacteria. Gelatin hydrolysis test is also known as gelatin liquefaction test or gelatinase test.

The main objectives of gelatin hydrolysis test are:

• To test an organism`s ability to liquefy gelatin by the production of gelatinase enzyme.
• To differentiate organisms into different groups based on their ability to hydrolyze gelatin.
• To identify some pathogenic bacteria that produce gelatinase as a virulence factor.

Gelatin hydrolysis test is based on the ability of some bacteria to produce an enzyme called gelatinase that can break down gelatin, a protein derived from animal collagen. Gelatin is a complex protein that forms a solid structure at lower temperatures, but liquefies when heated. Gelatinase is a proteolytic enzyme that hydrolyzes gelatin into smaller polypeptides and amino acids, which can be used by the bacteria as a source of nutrients. Gelatinase is also considered as a virulence factor in some pathogenic bacteria, as it helps them to invade the host tissues by dissolving the connective tissue matrix.

In the gelatin hydrolysis test, a medium containing gelatin is inoculated with the bacterial culture and incubated for a certain period of time. The presence or absence of gelatinase activity is then determined by observing the state of the medium after cooling it down. If the medium remains solid or gelled, it indicates that the bacteria do not produce gelatinase and thus cannot hydrolyze gelatin. This is a negative result for gelatin hydrolysis. On the other hand, if the medium becomes liquid or liquefied, it indicates that the bacteria produce gelatinase and thus can hydrolyze gelatin. This is a positive result for gelatin hydrolysis.

There are two methods to perform the gelatin hydrolysis test: the stab method and the plate method. In the stab method, a tube containing nutrient gelatin medium is inoculated with a needle and incubated at 37°C for 24-48 hours. The tube is then placed at 4°C for another 24 hours to allow the gelatin to solidify. The liquefaction of gelatin in the tube indicates a positive result, while the solidification of gelatin indicates a negative result. In the plate method, an agar plate containing nutrient gelatin medium is inoculated with a loop and incubated at 37°C for 24-48 hours. The plate is then flooded with mercuric chloride solution, which precipitates the unhydrolyzed gelatin. The formation of clear zones around the colonies indicates a positive result, while the absence of clear zones indicates a negative result.

Gelatin hydrolysis test can be used to test a variety of microorganisms, especially Gram-positive and Gram-negative bacteria that produce gelatinase enzyme. Some of the common microorganisms tested by this method are:

• Staphylococcus aureus: This is a Gram-positive coccus that causes various infections in humans and animals. It is positive for gelatin hydrolysis test, which helps to differentiate it from other nonpathogenic staphylococci that are negative for the test.
• Enterococcus faecalis: This is a Gram-positive coccus that is part of the normal flora of the gastrointestinal tract. It can cause opportunistic infections such as endocarditis, urinary tract infections, and wound infections. It is positive for gelatin hydrolysis test, which helps to identify it from other enterococci that are negative for the test.
• Bacillus subtilis: This is a Gram-positive rod that is widely distributed in soil and water. It is used as a model organism for studying bacterial genetics and physiology. It is positive for gelatin hydrolysis test, which indicates its ability to degrade collagen and other proteins.
• Escherichia coli: This is a Gram-negative rod that is part of the normal flora of the human intestine. It can cause various diseases such as diarrhea, urinary tract infections, and septicemia. It is negative for gelatin hydrolysis test, which helps to distinguish it from other enteric bacteria that are positive for the test.
• Pseudomonas aeruginosa: This is a Gram-negative rod that is ubiquitous in soil and water. It is an opportunistic pathogen that can cause infections in immunocompromised hosts such as cystic fibrosis patients. It is positive for gelatin hydrolysis test, which reflects its ability to produce extracellular enzymes and toxins.
• Serratia marcescens: This is a Gram-negative rod that is found in soil, water, and plants. It can cause nosocomial infections such as pneumonia, urinary tract infections, and septicemia. It is positive for gelatin hydrolysis test, which shows its ability to degrade connective tissue and invade host cells.

Media Used

Nutrient Gelatin Media is used for the demonstration of gelatin hydrolysis either by adding mercuric chloride or by the liquefaction of gelatin.

The composition of the Nutrient Gelatin Media is given below:

The pH of the medium is adjusted to 6.8 ± 0.2 at 25°C.

Reagent Used

Mercuric chloride (HgCl2) is used as a reagent to precipitate gelatin in the agar plate method.

The reagent is prepared by dissolving 5 grams of mercuric chloride in 100 ml of distilled water.

The reagent is stored in a dark bottle at room temperature.

Supplies Used

• Inoculating needle
• Incubator at 37°C
• Pipettes
• Petri dishes
• Test tubes

There are two methods to perform the gelatin hydrolysis test: the stab method and the plate method. Both methods use nutrient gelatin media as the medium for the test. The following steps describe the procedure for each method:

Stab method

1. Prepare the nutrient gelatin media by dissolving 128 grams of the dehydrated medium in 1000 milliliters of warm (50°C) distilled water. Heat the solution with agitation to bring it to boiling and dissolve the medium completely.
2. Dispense the medium into several test tubes and autoclave at 15 lbs pressure (121°C) for 15 minutes. Cool the tubed medium to 45-50°C in an upright position.
3. Inoculate the gelatin medium in the tube with 4-5 drops of a 24-hour broth culture of the test organism. Use a sterile inoculating needle to stab the medium to about half its depth.
4. Incubate the inoculated tubes at 37°C in air for 24-48 hours. If the organisms grow well at 25°C, incubate at 25°C instead.
5. After incubation, place the tubes at 4°C for another 24 hours to solidify the gelatin.
6. Observe the tubes for any liquefaction of gelatin.

Plate method

1. Prepare the nutrient gelatin media by dissolving 128 grams of the dehydrated medium in 1000 milliliters of warm (50°C) distilled water. Heat the solution with agitation to bring it to boiling and dissolve the medium completely.
2. Autoclave the medium in a beaker at 15 lbs pressure (121°C) for 15 minutes. Cool the medium to 45-50°C and pour into sterile Petri dishes. Allow the medium to solidify.
3. Inoculate the gelatin plates with a heavy inoculum of an 18-24 hour culture of the test organism. Use a sterile inoculating loop to streak the surface of the medium.
4. Incubate the plates at 37°C for 24-48 hours.
5. After incubation, flood the plates with mercuric chloride (HgCl2) solution and observe for any clear zones around the colonies.

The result of the gelatin hydrolysis test can be interpreted by observing the appearance of the gelatin medium after incubation and refrigeration. The result can be either positive or negative for gelatin hydrolysis, depending on the presence or absence of gelatinase enzyme in the tested organism.

A positive result for gelatin hydrolysis is indicated by the liquefaction or clearing of the gelatin medium. This means that the organism produces gelatinase enzyme that breaks down gelatin into polypeptides and amino acids. The liquefied medium can be easily poured out of the tube or shows clear zones around the colonies on plates.

A negative result for gelatin hydrolysis is indicated by the solidification or turbidity of the gelatin medium. This means that the organism does not produce gelatinase enzyme or produces it in insufficient amounts to hydrolyze gelatin. The solidified medium remains intact in the tube or shows no clear zones around the colonies on plates.

Some examples of control organisms for the gelatin hydrolysis test are:

• Positive result: Bacillus subtilis, Pseudomonas aeruginosa, Proteus vulgaris.
• Negative result: Escherichia coli, Staphylococcus aureus, Enterococcus faecalis.

Some factors that may affect the result interpretation of the gelatin hydrolysis test are:

• The temperature and duration of incubation. Some organisms may require longer incubation periods or lower temperatures to produce gelatinase enzyme.
• The quality and concentration of gelatin in the medium. Some media may have different gelling abilities or may contain inhibitors of gelatinase enzyme.
• The inoculum size and distribution. Too much or too little inoculum may affect the rate and extent of gelatin hydrolysis.
• The presence of other proteolytic enzymes that may interfere with gelatin hydrolysis.

Gelatin hydrolysis test is a simple and inexpensive method to test the ability of an organism to produce gelatinases, which are important enzymes for the degradation of collagen and other connective tissues in animals.

Gelatin hydrolysis test can help in the identification and differentiation of various bacteria based on their gelatinase production. Some examples are:

• The test can distinguish the gelatinase-positive, pathogenic Staphylococcus aureus from the gelatinase-negative, nonpathogenic Staphylococcus epidermidis and Staphylococcus saprophyticus.
• The test can differentiate genera of gelatinase-producing bacteria such as Serratia, Proteus, Pseudomonas, Flavobacterium, and Clostridium from other members of the family Enterobacteriaceae and Gram-positive bacilli.
• The test can also identify some species of Bacillus, Listeria, Aeromonas, and Vibrio that are capable of hydrolyzing gelatin.

Gelatin hydrolysis test can also provide some information about the virulence potential of some bacteria, as gelatinases are considered to be virulence factors that facilitate the invasion and dissemination of pathogens in the host tissues. For example:

• Gelatinase production by Staphylococcus aureus is associated with increased severity of infections such as endocarditis, osteomyelitis, and septicemia.
• Gelatinase production by Pseudomonas aeruginosa is correlated with increased tissue damage and mortality in burn wound infections.
• Gelatinase production by Clostridium perfringens is involved in the pathogenesis of gas gangrene and necrotizing enteritis.

Gelatin hydrolysis test has some limitations that should be considered while performing and interpreting the test results. Some of the limitations are:

• The test is not suitable for the detection of gelatinase activity in slow-growing or fastidious organisms, as they might not produce enough enzyme to liquefy the gelatin within the incubation period.
• The test is also not reliable for the identification of obligate anaerobes, as they might not grow well in the aerobic conditions of the test medium.
• The test is influenced by the temperature and pH of the medium, as gelatinase activity is optimal at neutral to alkaline pH and at temperatures above 20°C. Therefore, the tubes should be incubated at the appropriate temperature for the organism and chilled before reading the results.
• The test is also affected by the quality and concentration of gelatin in the medium, as different sources of gelatin might have different gelling and melting properties. Therefore, an uninoculated control tube should be used to check the gelling ability of the medium and to rule out false-positive results due to spontaneous liquefaction.
• The test is not specific for gelatinase production, as some organisms might produce other proteolytic enzymes that can also degrade gelatin. Therefore, the test should be confirmed by other biochemical tests or molecular methods for accurate identification of the organism.
• The test is not sensitive enough to detect low levels of gelatinase production, as some organisms might produce gelatinase only under certain conditions or in response to specific stimuli. Therefore, the test should be performed with fresh cultures and with adequate inoculum size to ensure sufficient enzyme production.

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