Lipid Hydrolysis Test (Lipase Test)- Principle, Procedure, Results

Lipid Hydrolysis Test (Lipase Test) is the biochemical test used to detect the ability of bacteria to produce exoenzyme lipase and hydrolyze lipids.

Lipids are important components of the biological system. A lipid is a vast group containing fats, oils, fat-soluble vitamins, sterols, phospholipids, sterols, and many other hydrophobic and amphipathic chemical compounds. Fats are the major members of lipids that are found as stored food or energy source in animals and plants. Among all the lipids, triglycerides are the main constituents of animal and plant fats. These molecules are susceptible to bacterial hydrolysis. Bacterial hydrolysis of fats results in free fatty acids mostly characterized by unpleasant odor and test. 

Hydrolysis of lipids by bacteria is an enzymatic hydrolysis process caused by an extracellular enzyme system called lipases. Lipase production is, however, not seen in all bacteria; only lipolytic bacteria can synthesize lipase enzymes. This can be easily tested in a lab by using a biochemical testing method called the lipid hydrolysis test or lipase test. In this test, we use ‘tributyrin’, a triglyceride component mainly found in animal fats, as a substrate to assess the ability of bacteria to produce lipases. It is mainly done for the characterization of bacteria isolated from food sources.     

Objectives of Lipid Hydrolysis Test

• To detect the ability of bacteria to produce exoenzyme lipase and hydrolyze lipid 

Principle of Lipid Hydrolysis Test

Triglyceride tributyrin is one of the simplest lipids found in natural animal and plant fats and oils that can be hydrolyzed by every lipolytic bacterium. It is incorporated in the tributyrin agar medium as the substrate to detect lipase production. Tributyrin is water insoluble and forms the culture medium turbid or opaque. However, bacterial lipase enzyme will hydrolyze the tributyrin converting it to water-soluble butyric acid. This hydrolysis will make the medium transparent. Hence, if the test bacteria are lipolytic i.e. able to synthesize lipase enzyme, a clear transparent zone of tributyrin hydrolysis will be formed around the bacterial colonies. 

Requirements for Lipid Hydrolysis Test

Culture Media

Tributyrin agar medium is the medium of choice for the detection of lipolytic bacteria (lipase production). Although Egg Yolk Agar medium can be used (and is in use) to detect lipid hydrolysis together with lecithinase production, tributyrin medium is easy to read for a positive result.

Composition of Tributyrin Agar Base per 1000 mL

Peptone- 5.00 grams

Yeast Extract- 3.00 grams

Agar- 15.00 grams

Final pH 7.5 ±2  at 25°C

(Reference: Tributyrin Agar Base w/o Tributyrin (himedialabs.com))

Preparation of Tributyrin Agar Medium

• Measure the appropriate amount of Tributyrin Agar Base powder (or the media components) and mix in the water of the required volume in a conical flask (or glass bottle) according to the instruction of the manufacturing company (23.0 grams of the above composition in 990 mL distilled water). 
• Dispense 10 mL of “Tributyrin” in the mixture.  
• Stir well using a magnetic stirrer or manually and heat to boiling so that all the components and agar dissolve completely in water. 
• Autoclave the flask or bottle at 121°C and 15 lbs pressure for 15 minutes and let it cool to around 40 – 45°C.
• Pour about 25 mL of the medium mixture into a 10 cm diameter sterile petri plate and let the medium solidify properly at room temperature. 

b. Reagents

Tributyrin is required for the lipase test. Tributyrin is a triglyceride (ester) formed by the acylation of glycerol by butyric acid at the three hydroxy groups of the glycerol. It is also called “Glycerol Tributyrate” or “Propane – 1, 2, 3 –  triyl tributyrate” and has the molecular formula of C₁₅H₂₆O₆.   

c. Equipment

PPE and other general laboratory materials

d. Test Organism (Sample Bacteria)  

Positive Control: Staphylococcus aureus ATCC 12600

Negative Control: Clostridium difficile ATCC 9689/ Clostridium perfringens ATCC 12924

Procedure of Lipid Hydrolysis Test

• Using a sterile inoculating loop, pick up a heavy inoculum from a well-isolated colony of fresh culture (18 to 72 hours old culture). 
• Inoculate the sample organism plate by drawing either a straight line or forming a circular inoculation of a size of a dime over the surface of the Tributyrin Agar plate.
• Incubate the plates at 35±2°C for about 24 to 48 hours aerobically for aerobes or facultative, and for about 72 hours anaerobically for anaerobes. 
• Following incubation, observe the formation of a clear zone of tributyrin hydrolysis around the bacterial colony.

Result and Interpretation of Lipid Hydrolysis Test

• A positive test is indicated by the formation of a clear transparent zone of hydrolysis (tributyrin hydrolysis) around the bacterial colony (in the line). 
• A negative test is indicated by no clear zone of hydrolysis around the bacterial growth line (colonies).

Lipase Test Positive Bacteria

Moraxella catarrhalis, S. aureus, S. saprophyticus, C. botulinum, C. sporogenes, Bacillus subtilis, Proteus mirabilis, Pseudomonas aeruginosa, etc. 

Lipase Test Negative Bacteria

C. perfringens, C. difficile, E. coli, K. pneumoniae, K. oxytoca, etc.   

Quality Control

A clear transparent zone of hydrolysis is produced around the line of growth (colonies) of Staphylococcus aureus ATCC 12600.

No zone of hydrolysis is produced around the line of growth (colonies) of Clostridium perfringens ATCC 1292.

Precautions

• It is better to use heavy inoculum for bigger zone size and quick results.
• Be sure to incubate for at least 3 days at 35±2⁰C. Anaerobic bacteria may show results after 3 days. 

Applications of Lipid Hydrolysis Test

• Identification of possible lipolytic bacteria in foods and dairy products. 
• Differentiation and identification of members of mainly Corynebacterium, Clostridium, Bacillus, and Moraxella genera. 

Limitations of Lipid Hydrolysis Test

• It is not a confirmatory test; hence, it requires other biochemical test results for the complete identification of the unknown bacteria.
• Require a longer incubation period. 
• Fastidious organisms don’t grow in the medium and are difficult to grow.

References

1. Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
2. Aneja K.R. 2003. Experiments in Microbiology, Plant Pathology and Biotechnology, fourth revised edition, New Age International (P) limited, Ansari road, Daryaganj, New Delhi-110002.
3. Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
4. Philipp R. Melendez, Royce H. Johnson, Bacteremia and Septic Arthritis Caused by Moraxella catarrhalis, Reviews of Infectious Diseases, Volume 13, Issue 3, May-June 1991, Pages 428–429, https://doi.org/10.1093/clinids/13.3.428
5. Lipid Hydrolysis Test – Principle, Procedure, Uses and Interpretation (microbiologyinfo.com)
6. Lipid Hydrolysis Test: Result, Principle, and Procedure (researchtweet.com)
7. Lipid Hydrolysis Test on Bacteria to find-out their Ability to Hydrolyse Lipids (With Figure) (yourarticlelibrary.com)
8. Lipid Hydrolysis Test: Objective, Principle, Procedure, Uses And Results Interpretation – BIOCHEMINSIDER
9. Determination of lipase activity | Microbiology (upatras.gr)
10. National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 6050, Tributyrin. Retrieved February 19, 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Tributyrin.