• A lipid is a high-energy, low-molecular-weight molecule.
• After being taken in by the cell, they are processed via aerobic respiration to generate ATP (adenosine triphosphate), the energy currency of the cell (ATP).
• Other metabolic processes may also use the components to synthesise essential cellular protoplasm.
• However, degradation is necessary before bacteria can absorb them.
• Lipases (esterases) are extracellular hydrolyzing enzymes that cleave the ester bonds in this molecule by the addition of water, resulting in the breakdown of lipids like triglycerides into the constituents glycerol (an alcohol) and fatty acids.

Lipid Hydrolysis Test of Objectives

• To find out if the organism can break down lipids with water.
• To find out which bacteria can make the exoenzyme lipase.

Principle of Lipid Hydrolysis Test

• Most lipids are made of molecules that are not polar and don’t dissolve well in water.
• Lipids are made up of long chains of fatty acids and glycerol. Fats are one type of lipid. Fats are too big to fit through the cell membrane.
• In order to use fats, bacterial cells send lipases, which are exoenzymes, outside the cell. The lipases break down the fat into fatty acids and glycerol.
• Lipolytic bacteria are those that can make the enzyme exoenzyme lipase.
• When lipids are put into agar, they form an emulsion, which makes the mixture opaque. However, when lipids are broken down into their end products, glycerol and fatty acids, they don’t form an emulsion, so they don’t make the mixture opaque; instead, they make it clear.
• In the lipid hydrolysis test, the test bacteria are grown on agar plates with tributyrin as the lipid substrate. In the agar medium, the butyrin oil is mixed in a way that makes it hard to see.
• If the bacteria can hydrolyze lipids, the bacterial colonies break down the tributyrin in the medium around them into soluble glycerol and fatty acids (butyric acid), while the rest of the tributyrin is still intact in the other parts of the plates.
• The result of hydrolysis is clear, transparent zones around the colonies, while the rest of the plates are opaque, showing that the tributyrin has not been broken down.

Media Used in Lipid Hydrolysis Test

Tributyrin agar: Tributyrin agar is a differential medium that tests an organism’s ability to make lipase, an exoenzyme that breaks down tributyrin oil. Lipases break up fats and oils (fats). Tributyrin oil is a triglyceride, which is a type of lipid. In other lipase tests, fats like corn oil, olive oil, peanut oil, egg yolk, and soybean oil are used.

Lipids can be broken up into smaller pieces by the organisms that make lipase. Glycerol and three fatty acids are what make up triglycerides. These get broken up and can be turned into many different end products that the cell can use to make energy or do other things.

In the agar, the tributyrin oil makes a cloudy suspension. When an organism makes lipase and breaks down tributyrin, it leaves a clear halo around the area where it has grown.

Tributyrin agar composition

Ingredients	Gms / Litre
Peptone	5.000
Yeast extract	3.000
Agar	15.000
Final pH ( at 25°C)	7.5±0.2

Tributyrin agar Preparation

1. Mix 990 ml of purified or distilled water with 23 grammes of the powder.
2. Add 10 ml of Tributyrin (FD081). To dissolve the medium completely, mix it and bring it to a boil.
3. Use an autoclave for 15 minutes at 15 pounds of pressure (121°C) and 121°F.
4. Bring the temperature down to 45-50°C.
5. Mix well and pour into Petri plates that have been cleaned.

Note: If you want lipase to work right, you should use glass plates instead of paper plates. So, ONLY USE GLASS PLATES. DO NOT use plates made of plastic.

Lipid Hydrolysis Test Procedure

1. Inoculate the tributyrin agar medium with organisms streaked in a single line.
2. Immediately after streaking, incubate anaerobically in a gas pak jar and transfer to an incubator maintained at 35-37o C for 24-48 hours; for aerobes, incubate the plate at 35-37o C for 24-48 hours.
3. Observe the free space surrounding the bacterial development.

Lipid Hydrolysis Test Result and Interpretation

• Positive test: clear zone around the growth of bacteria.
• Negative test: There is no clear area around the growth of bacteria. 

Quality Control

• Clostridium perfringens ATCC 12924: negative test, no clear zone around colony
• Clostridium sporogenes ATCC 11437: Test positive, clear zone around colony
• Bacillus subtilis ATCC 6633: Test positive, clear zone around colony 

Limitations of Lipid Hydrolysis Test

• Colonies from pure culture should be tested using biochemical, immunological, molecular, or mass spectrometry for definitive identification.

Uses

• Useful for determining the identity of bacteria such as Enterobacteriaceae, Fusobacterium, Propionibacterium, Clostridium, Pseudomonas, Mycoplasma, Corynebacterium, and Staphylococcus that produce lipase.
• Useful for counting the number of lipolytic bacteria present in food and other samples.

References

• Cappuccino J.G. and Sherman N.  2008.  Microbiology: A Laboratory Manual, 8th ed. Pearson Benjamin Cummings, San Francisco, CA, USA.
• Brown A.E. 2009.  Benson’s Microbiological Applications: Laboratory Manual in General Microbiology, 11th ed. McGraw-Hill Companies, New York, NY, USA.
• delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/lipid.pdf
• https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Datasheet/1/91015dat.pdf