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Gelatin Hydrolysis Test Procedure

In this gelatin hydrolysis test procedure post we have briefly explained gelatin hydrolysis test principle, objectives, requirements, gelatin hydrolysis test procedure, uses and limitations of gelatin hydrolysis test.

Gelatin Hydrolysis Test

Nutrient gelatin is a differential media used to assess an organism’s ability to create gelatinase, an exoenzyme that hydrolyses gelatin. Although gelatin is typically associated with gelled salads and sweets, it is essentially a connective tissue protein. Gelatin is a semisolid material when the temperature is below 32°C (or a few degrees below that). It is a viscous liquid at temperatures over 32°C.

Gelatinase breaks down gelatin into smaller polypeptides, peptides, and amino acids that can pass the cell membrane and be used by the organism. When gelatin is broken down, it no longer has the ability to solidify. Even if the gelatin is refrigerated, if an organism can break down gelatin, the portions where the organism has grown will remain liquid.

Principle

The main goal of this gelatin hydrolysis test is to determine whether or not an organism has the ability to manufacture the enzyme gelatinase. Gelatin is a globular protein made from collagen after it has been hydrolyzed. Collagen is the most abundant protein in human and animal connective tissues and tendons.

At 50°C, gelatin dissolves in water, exists as a liquid above 25°C, and solidifies as a gel below 25°C. Even a temperature of 4°C will not restore the gel-like characteristics of gelatin once it has been hydrolyzed. Gelatin is a peptide-linked polymer of amino acids.

Adding acidic HgCl2 to the mixture detects the presence of gelatin protein (mercuric chloride). With gelatin protein, HgCl2 creates a white precipitate. An extracellular gelatinase-producing bacterium hydrolyses gelatin, releasing constituent amino acids and short peptides.

Microorganisms then make use of these amino acids. With amino acids, HgCl2 does not produce white precipitate; hence the bacterium that hydrolyses gelatin does not produce white precipitate with HgCl2.

Media Composition

Ingredients gms/Litre: Peptone-5.000, HM peptone-3.000, Gelatin-120.000, Final pH (at 25°C) 6.8±0.2.

Media Preparation

128 grams purified / distilled water, warmed to 50°C, suspended in 1000 ml Bring to a boil to completely dissolve the medium.

Fill test tubes with the mixture. Autoclave at 15 lbs pressure (121°C) for 15 minutes to sterilise. In an upright position, allow the tubed medium to cool to 45-50°C.

Requirements

  1. 1% gelatin agar
  2. Mercuric chloride (HgCl2)
  3. 24 hours culture of Bacillus spp and E.coli

Gelatin Hydrolysis Test Procedure

Agar plate

1. Prepare 1 percent gelatin agar media. Using an inoculating loop, inoculate the gelatin agar plate with the provided organism (coli and Bacillus in separate plates). Make a single centre streak in the plate.

2. Incubate the plates for 24-48 hours at 37°C. HgCl2 solution is poured into the plates. After a few minutes, have a look at the plates. Gelatin hydrolysis is indicated by a distinct halo-zone around the infected area; positive test.

Test tube

1. In a test tube, prepare gelatin medium. Stabbing the media many times with inoculating wire to inoculate the gelatin tube with the supplied organism.

2. Incubate the tube for 24-48 hours at room temperature (25°C). Place the tube in the refrigerator for 30 minutes at 4° after incubation for 24-48 hours.

3. Keep an eye on the tube for signs of gelatin hydrolysis. Even after being frozen at 4°C, the hydrolyzed tube remains liquid.

Results

Gelatin Hydrolysis Test

Positive Result

Within 14 days, partial or entire liquefaction of the infected tube at 4°C (the control tube must be totally solidified). Clear zones around gelatinase-positive colonies on plates suggest gelatin hydrolysis (Clostridium perfringens, Bacillus subtilis, Proteus vulgaris, and Staphylococcus aureus).

Negative Result

At 4°C, the tube solidifies completely. There are no defined zones around colonies on plates. (Escherichia coli).

Applications

1. The main goal of this gelatin hydrolysis test is to determine whether or not an organism has the ability to manufacture the enzyme gelatinase.

2. Staphylococcus aureus and Staphylococcus epidermidis can be distinguished using the gelatin hydrolysis test.

3. Serratia marcescens, Proteus vulgaris, and Proteus mirabilis can all be distinguished from other enterics using this gelatin hydrolysis test.

Limitations

1. Gelatin hydrolysis test medium, some organisms may grow slowly or not at all. Because gelatin is liquid at temperatures over 20°C, findings must be determined after refrigeration.

2. Gelatinase normally works at the medium’s surface. If you shake the tube while it’s warm, you can get a false-negative result.

Further Readings

Reference