GELATIN HYDROLYSIS TEST

Material required: 

1. Gelatin Agar(Peptic digest of animal tissue 5 g/L, Beef extract 3 g/L, Gelatin 120 g/L, Final pH ( at 25°C) 6.8±0.2),
2. Boiling test tubes
3. Cotton Plug
4. Inoculation needle
5. Culture(Bacillus subtilis and Escherichia coli).

Theory:

Gelatin is a protein derived from the connective tissues of vertebrates, that is, collagen. It is produced when collagen is boiled in water. Gelatin hydrolysis detects the presence of gelatinases. Gelatinases are proteases secreted extracellularly by some bacteria which hydrolyze or digest gelatin. This process takes place in two sequential reactions.
In the first reaction, gelatinases degrade gelatin to polypeptides.

Then, the polypeptides are further converted into amino acids.

 
 FIG Reactions involved in gelatin hydrolysis. 

The bacterial cells can then take up these amino acids and use them in their metabolic processes.
Gelatin hydrolysis is detected using a nutrient gelatin medium. This medium contains peptic digest of animal tissue (peptone), beef extract, and gelatin. Gelatin serves as both solidifying agent and substrate for gelatinase activity. When nutrient gelatin tubes are stab-inoculated with a gelatinase-positive bacterium, the secreted gelatinases will hydrolyze the gelatin resulting in the liquefaction of the medium. Since gelatin is digested and is no longer able to gel, the medium will remain liquid when placed inside a refrigerator or in an ice bath. A nutrient gelatin medium inoculated with a gelatinase-negative bacterium will remain solid after the cold treatment. The medium can be inoculated with both aerobic and anaerobic bacteria and incubated as appropriate.

TABLE  List of common bacteria and their reactions to the gelatin hydrolysis test performed on nutrient gelatin

Species	Growth	Liquefaction
Bacillus subtilis	+	+
Clostridium perfringens	+	+
Escherichia coli	+	-
Proteus vulgaris	+	+
Serratia liquefaciens	+	+
Staphylococcus aureus	+	+

Procedure:

1. Prepared gelatine agar and distributed in three boiling tubes (two for two bacteria and one for control).
2. Autoclaved at 121 ˚C for 15 minutes.
3. Sterilize the needle in the blue flame of the Bunsen burner till red hot and then allowed to cool. 
4. Inoculate Bacillus subtilis and Escherichia coli in two nutrient gelatin tubes(Test) and keep one tube uninoculated as(control). 
5. Incubate at 37 ^˚C .
6. After incubation gently place the two tubes in the refrigerator for 15-30 minutes.   
7. After 15-30 minutes, if the gelatin remains LIQUID, the result is POSITIVE
8. If the gelatin is SOLID, the result is negative.   

 

EXPECTED RESULT: