Lipid Hydrolysis Test: Principle, Procedure, and Result

Lipid hydrolysis or lipase test helps determine the ability of bacteria to produce lipase and hydrolyze lipids. Lipids are fatty, waxy, and oily bio-compounds. These include fats and triglycerides.  

Some bacteria catabolizes lipid, especially in high-fat dairy product, to generate energy (ATP). These bacteria may also use lipids for synthesizing various biomolecules by entering different pathways.

Since lipids are complex fatty acids, they require further degradation before bacteria metabolize them. The extracellular enzyme lipase produced by bacteria helps in the hydrolysis of lipids.  

The bacteria that can produce lipase belong to Enterobacteriaceae, Clostridium, Staphylococcus, and Neisseria. Many fungal species also demonstrate lipolytic ability. 

Principle of Lipid Hydrolysis Test

Lipid is complex biological compounds that include fats and oils. The hydrolysis of fats occurs in the presence of the enzyme lipases. Some bacteria can produce this enzyme, whereas others cannot. 

Although many oils can be used for lipid hydrolysis tests, tributyrin oil is the most commonly used lipid for determining the lipolytic effects of the bacteria. The oil is simple fat or triglycerides or triacylglycerols.   

Triglycerides comprise glycerol and three long-chain fatty acids. Although tributyrin is simple fat, it is large and cannot enter the cell. Some cells can secrete the enzyme lipase to break down tributyrin before cellular uptake.

The breaking down of tributyrin releases glycerol converted into dihydroxyacetone phosphate, an intermediate product of glycolysis. Beta-oxidation catabolizes the fatty acids; two carbons combine with Coenzyme A to form acetyl Coenzyme A.

The acetyl CoA enters into the Krebs cycle to produce energy. Glycerol and fatty acids are useful in both anabolic and catabolic pathways alternatively. Tributyrin agar is opaque, and when the plate is inoculated with lipase-positive organisms, clear zones appear around the growth. The absence of the clear indicates the organism does not produce lipase. 

The testing lipid hydrolysis can also occur in spirit blue agar. The composition of the agar is an emulsion with tributyrin oil and spirit blue dye as a color indicator. The media is opaque and light blue. Lipase-positive bacteria produce clear halos around the growth. However, the lightening of the medium is not considered positive growth.          

Materials Required for Lipid Hydrolysis Test

A lipid hydrolysis test requires some laboratory equipment and materials. The equipment and materials needed for the test are as follows: 

  1. Tributyrin agar base: It has peptone, yeast extract, and agar. The pH of the base is 7.5 ± 2 at 25℃. 
  2. Tributyrin: It is also known as glycerol tributyrate or propane-1,2,3-triyl tributyrate. Its molecular formula is C15H26O6.   
  3. Petri plates: The use of Petri plates is for preparing culture media or tributyrin agar.  
  4. Test tubes: The test tubes are used for preparing bacterial inoculum .    
  5. Autoclave: This equipment is used for sterilizing tributyrin agar during preparation.  
  6. Incubator: Tributyrin plate inoculated with bacterial isolates is incubated during the test.  
  7. Beaker: The glassware is used to transfer liquid during the tributyrin agar preparation. 
  8. Inoculating loop: The inoculating loop of standard size is used for streaking bacterial inoculum in the tributyrin agar.  
  9. Bunsen burner: It is used to sterilize inoculating loops and preparing tributyrin agar
  10. Analytical balance: It is used for weighing powder for preparing tributyrin agar. 
  11. Conical flask: It is used for preparing tributyrin agar. 

Composition of Tributyrin Agar Base

Peptone5.00 g
Yeast Extract3.00 g
Agar15.00 g
Final pH7.5±2 at 25°C

Procedure of Lipid Hydrolysis Test

The lipid hydrolysis test involves streaking bacterial inoculum in tributyrin agar. The clear zone around the growth indicates a positive for lipase production.  

Preparation of Tributyrin Agar

Before conducting a lipid hydrolysis test, preparing tributyrin agar is an important step. The steps for preparing tributyrin agar depend on the manufacturing company. The steps for the preparation of tributyrin agar manufactured by Himedia are as follows:

  1. In 990 ml of distilled water, mix 23 grams of tributyrin agar base. 
  2. After that, add 10 ml of tributyrin to the mixture above.
  3. Then, boil the solution to mix the base and tributyrin thoroughly.
  4. Sterilize the solution by autoclaving at 121℃ or 15 lbs pressure for 15 minutes. 
  5. Cool the solution to 40-45℃.
  6. After that, pour the solution into sterile Petri plates.  

Steps of Lipid Hydrolysis Test

Lipase test
Above: Positive for lipase test (presence of clear zone around the growth).
Below: Negative lipase test (absence of clear zone around the growth).

Following are the steps for performing a lipid hydrolysis test:

  1. In a tributyrin agar, inoculate a loopful of freshly isolated bacterial samples in a straight streak line or circle the size of a dime using a sterile inoculation loop.
  2. Incubate the streaked plate at 37℃ for 24-48 hours for aerobic bacteria and 72 hours for anaerobic bacteria.
  3. After incubation, observe the plate for a clear zone and interpret the result.


There are some precautionary steps to follow during performing lipid hydrolysis tests; they are as follows:

  1. While sterilizing the inoculation loop, heating should be done until the loop turns red hot. 
  2. Cooling down to the right temperature is necessary before picking up the bacterial colony.
  3. Maintaining proper incubation time is required. At least incubate for four days for anaerobic bacteria. 

Result Interpretation

The lipid hydrolysis test’s result interpretation is based on the presence or absence of the clear zone around the bacterial growth.

  1. Positive result: There is a clear zone around the inoculation area. Staphylococcus aureus, S saprophyticus, Clostridium botulinum, Bacillus subtilis, Pseudomonas aeruginosa, etc., are bacteria that show positive results. 
  2. Negative result: There is no clear zone around the inoculation area. Clostridium perfringens, C difficile, Escherichia coli, Klebsiella pneumoniae, K oxytoca, etc, are bacteria that shows negative result.   

Quality Control

The quality control bacteria used for this test are as follows:

  1. Positive control: Staphylococcus aureus ATCC 12600 is used as the positive control.
  2. Negative control: Clostridium perfringens ATCC 1292 is used as the negative control.

Uses of Lipid Hydrolysis Test

The lipid hydrolysis test is a biochemical test to determine the lipolytic properties of some bacteria. It helps identify the bacteria. Its use is as follows:

  1. Differentiating lipolytic bacteria from non-lipolytic bacteria, especially in dairy industries. 
  2. Identification of bacteria of the genus Clostridium, Corynebacterium, Bacillus, and Moraxella. 

Limitation of Lipid Hydrolysis Test

The lipid hydrolysis test is a practical test for identifying lipolytic bacteria. However, it also has some limitations, which are as follows:

  1. The time for incubation is longer compared to another biochemical test.
  2. The lipid hydrolysis test is not confirmatory. Further biochemical testing is required to confirm the bacteria .
  3. The growth of fastidious bacteria is not possible. 


  1. Leboffe, M.J. and Pierce, B.E. (2011) “Differential media,” in A Photographic Atlas for the 4th edition microbiology laboratory. 4th edn. Englewood, CO: Morton Pub. Co., pp. 77–78. 
  2. Madigan, M.T., Brock, T.D. and Stahl, D.A. (2011) Brock Biology of Microorganisms. 13th edn. Boston: Pearson/Benjamin Cummings. 
  3. Tributyrin Agar Base W/O Tributyrin (no date) HiMedia Leading BioSciences Company. HiMedia. Available at:

Ashma Shrestha

Hello, I am Ashma Shrestha. I had recently completed my Masters degree in Medical Microbiology. Passionate about writing and blogging. Key interest in virology and molecular biology.

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