Skip to content
Home » Lipase Assay Protocol

Lipase Assay Protocol

In this lipase assay protocol post we have briefly explained lipase activity assay principle, objectives, requirements, lipase activity assay, uses and limitations.

Lipase Activity Assay

Lipase activity assay is used to discover organisms that can produce the lipase enzyme. Triglycerides are hydrolysed by this enzyme, which produces glycerol and three long-chain fatty acids.

These substances are tiny enough to pass through the cell wall of bacteria. Glycerol is a glycolysis intermediate that can be transformed. Fatty acids can be catabolized, and their fragments can enter the Kreb’s cycle.

Principle

Clostridium perfringens is isolated and detected using Egg Yolk Agar Base, which is a modest modification of McClung Toabe Agar Base.

Hemin is added to Egg Yolk Agar Base, which makes it different from the original composition. The required nutrients, as well as carbonaceous and nitrogenous compounds, are provided by proteose peptone.

Phosphates act as a buffer for the medium, whereas sodium chloride keeps the osmotic balance in check. Along with sulphates, magnesium sulphate is a source of divalent cations. Glucose is a carbohydrate that provides energy. Hemin promotes the growth of anaerobic bacteria.

Lecithinase producing organisms break down the lecithin in the egg yolk emulsion, resulting in an insoluble opaque precipitate that forms surrounding the colonies. Lipase-producing organisms degrade free fatty acids, resulting in an iridescent shine on the colonies’ surface.

Medium

Ingredients Gms/Litre: Proteose peptone – 40.000, Disodium hydrogen phosphate – 5.000, Potassium dihydrogen phosphate – 1.000, Sodium chloride – 2.000, Magnesium sulphate – 0.100, Dextrose (Glucose) – 2.000, Hemin – 0.005, Agar – 25.000. Final pH (at 25°C) 7.6±0.2.

Procedure

Take a loopful of the test organism and smear it across the plate in a straight line. For anaerobes, immediately after streaking, incubate anaerobically in a gas pak jar and transfer to an incubator maintained at 35-37C for 24-48 hours; for aerobes, incubate the plate at 35-37C for 24-48 hours. Check for the creation of an iridescent sheen on the plate.

Results

Positive test: When the plate is held at an angle to a light source, an iridescent sheen (oil on water) appears immediately surrounding colonies, indicating a positive lipase test.

Negative test: The absence of an iridescent sheen indicates a negative lipase test.

Application

1. Lipolytic bacteria are detected and counted using the lipase activity assay, which is especially useful in high-fat dairy products.

2. Differential characteristics among members of the Enterobacteriaceae, Clostridium, Staphylococcus, and Neisseria are detected using a range of lipid substrates, including maize oil, olive oil, and soybean oil. Lipolytic capacity is also demonstrated by a number of fungus species.

Limitations

1. It is advised that biochemical, immunological, molecular, or mass spectrometry tests be done on colonies from pure culture for complete identification lipase activity assay. Lipase activity assay can take up to a week for some microbes to produce a positive lipase reaction.

Further Readings

Reference