Objectives of Lipase Test
- The objective is to identify bacteria capable of producing the exoenzyme lipase.
- The Lipase Test is used to detect and enumerate lipolytic bacteria, especially in high-fat dairy products.
- A variety of other lipid substrates, including corn oil, olive oil, and soybean oil, are used to detect differential characteristics among members of Enterobacteriaceae, Clostridium, Staphylococcus, and Neisseria.
- Several fungal species also demonstrate lipolytic ability.
Principle of Lipase Test
Fats are formed by ester linkage between three molecules of fatty acids and one molecule of glycerol. Simple fats are known as triglycerides or triacylglycerols. Triglycerides are composed of glycerol and three long chain fatty acids. Enzymes that break simple fat into its component fatty acid and glycerol are knows as lipase. Many bacteria can be classified based on their ability to produce and secrete lipases. A variety of simple fats can be used for this determination, depending on the type of organism being tested; however, tributyrin is the simplest naturally occurring triglyceride and thus, a common constituent of lipase testing media.
Tributyrin is too large to enter the cell, so a lipase is released to break it down prior to cellular uptake. After hydrolysis the glycerol can be converted to dihydroxyacetone phosphate, an intermediate of glycolysis. The fatty acids are catabolized by a process called β-oxidation and may be converted into a variety of end-products that can be used by the cell in energy production or other processes. Tributyrin oil is a type of lipid called a triglyceride. Other lipase tests use different fat sources such as corn oil, olive oil, peanut oil, egg yolk, and soybean oil.
Tributyrin agar is a differential medium that tests the ability of an organism to produce an exo-enzyme, called lipase that hydrolyzes tributyrin oil. Tributyrin agar is prepared as an emulsion so that the agar will appear opaque. When the plate is inoculated with a lipase-positive organism, clear zones will appear around the growth as evidence of lipase activity. If no clear zones appear, the organism is lipase-negative.
Media Used in Lipase 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 Lipase 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 Lipase Test
Positive Lipase test: clear zone around the bacterial growth
Negative Lipase test: absence of clear zone around the bacterial growth
Limitations of Lipase Test
- Zones of opacity are small with non-glucose-fermenting rods.
- Some microorganisms may require up to 1 week to produce a positive lipase reaction.
Quality Control of Lipase Test
Staphylococcus aureus ATCC 25923, positive, clear zone around colony
Clostridium perfringens ATCC 12924, negative, absence of clear zone around colony
Clostridium sporogenes ATCC 11437, positive, clear zone around colony.