Objectives of Lipid Hydrolysis Test
- To determine the ability of the organism to hydrolyse lipid.
- To identify bacteria capable of producing the exoenzyme lipase.
Principle of Lipid Hydrolysis Test
Lipids generally are nonpolar molecules that do not dissolve well in water. Fats are one type of lipids that are large polymers of fatty acids and glycerol that are too large to enter the cell membrane. In order to utilize fats, bacterial cells secrete exoenzymes known as lipases outside of the cell that hydrolyze the lipid to fatty acids and glycerol. These bacteria capable of producing exoenzyme lipase are called lipolytic bacteria. The lipids form an emulsion, when dispensed in agar, producing opacity while their hydrolysed end products, glycerol and fatty acids do not form such emulsion with agar, for which they do not produce such opacity; rather produce transparency.
In the lipid hydrolysis test, the test bacteria are grown on agar plates containing tributyrin as the lipid substrate. Tributyrin oil forms an opaque suspension in the agar medium. If the bacteria has the ability to hydrolyse lipids, the bacterial colonies hydrolyse the tributyrin in the medium in the areas surrounding them to soluble glycerol and fatty acids (butyric acid), while the rest of the areas of the plates contain unhydrolysed tributyrin. The result of hydrolysis is demonstrated as a transparent clear zones around the colonies and the rest of the areas of the plates remain opaque indicative of unhydrolysed tributyrin region.
Media Used in Lipid Hydrolysis Test
Tributyrin agar: Peptic digest of animal tissue 5.0 gm/L, Yeast extract 3.0 gm/L, Agar 15.0 gm/L, Final pH ( at 25°C) 7.5±0.2
Procedure of Lipid Hydrolysis Test
- Inoculate the tributyrin agar medium with single line streaking of organism
- Incubate anaerobically in a gas pak jar immediately after streaking and transfer into the incubator maintained at 35-37o C for 24-48 hours for anaerobes and for aerobes incubate the plate at 35-37o C for 24-48 hours.
- Observe for the clear zone around the bacterial growth.
Result Interpretation of Lipid Hydrolysis Test
- Positive test: clear zone around the bacterial growth.
- Negative test: absence of clear zone around the bacterial growth.
Quality Control of Lipid Hydrolysis Test
Clostridium perfringens ATCC 12924 – negative test, absence of clear zone around colony
Clostridium sporogenes ATCC 11437 – positive test, clear zone around colony
Bacillus subtilis ATCC 6633 – positive test, clear zone around colony