Lipid hydrolysis test – Its Principle, procedure and Interpretations

Introduction

The lipid hydrolysis test is a biochemical assay used in microbiology to determine the ability of microorganisms to hydrolyze lipids. Lipids, such as triglycerides, are complex molecules consisting of glycerol and fatty acid chains. Microorganisms produce enzymes called lipases that can break down these lipids into glycerol and free fatty acids through a process known as hydrolysis.

The lipid hydrolysis test specifically focuses on the activity of microbial lipases. It is  a class of hydrolases that specifically target ester bonds present in triglycerides, phospholipids, and other lipid molecules. Through hydrolysis, lipases cleave the ester bonds, releasing the fatty acids and glycerol. 

It provides valuable information about the metabolic capabilities of microorganisms and their ability to utilize lipid substrates for growth and energy production. By assessing the presence or absence of lipid hydrolysis, this test aids in the identification and differentiation of various microorganisms.

Enzymes Involved in Lipid Hydrolysis

Several microbial enzymes are involved in lipid hydrolysis, with lipases being the primary catalysts. Lipases are produced by various microorganisms, including bacteria, fungi, and some yeasts. These enzymes are secreted by microorganisms into their environment or associated with their cell surface. Lipases have evolved to function optimally at the interface between lipid substrates and water, owing to their unique structure and hydrophobic binding sites.

In addition to lipases, esterases also contribute to lipid hydrolysis. Esterases catalyze the hydrolysis of ester bonds, including those present in lipids. While lipases primarily target triglycerides, esterases have a broader substrate specificity and can act on various ester linkages in lipids, including phospholipids and sterol esters.

 Principle of the Lipid Hydrolysis Test:

The principle of the lipid hydrolysis test is based on the detection of lipase activity, which indicates the ability of microorganisms to hydrolyze lipids. The test typically involves the inoculation of microorganisms onto a specific agar medium containing lipids, such as tributyrin or spirit blue agar. The lipids present in the medium serve as the substrate for lipase enzymes produced by the microorganisms.

When lipase enzymes are secreted by the microorganisms, they catalyze the hydrolysis of the ester bonds present in the lipids. This hydrolysis results in the release of free fatty acids from the lipid substrate. The free fatty acids can then diffuse into the surrounding agar medium.

To visualize the hydrolysis of lipids, a pH indicator or a lipid-specific dye, such as spirit blue dye, is incorporated into the agar medium. The pH indicator changes color or the dye precipitates in the presence of free fatty acids, indicating a positive result for lipid hydrolysis.

The detection of clear zones or halos around bacterial colonies on the agar medium indicates the hydrolysis of lipids and thus the presence of active lipase enzymes. The size of the clear zones can vary depending on the efficiency of the lipase activity. Larger clear zones indicate more pronounced lipid hydrolysis.

Materials and Reagents Required

To perform the lipid hydrolysis test, the following materials and reagents are required:

1. Agar plates: Typically, tributyrin agar or spirit blue agar is used for lipid hydrolysis testing. These agars contain lipids as the substrate for hydrolysis.
2. Nutrient Agar: Used for subculturing and maintaining bacterial cultures.
3. Inoculation loop or needle: Used to transfer microorganisms onto the agar plates.
4. Incubator: Required for providing appropriate temperature and conditions for microbial growth.
5. Staining reagents (optional): Some variations of the lipid hydrolysis test involve staining techniques to enhance the visibility of hydrolysis zones.

Quality control

Positive Control: Staphylococcus aureus ATCC 12600

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

Preparation of Media for Lipid Hydrolysis Test:

Tributyrin Agar: To prepare tributyrin agar, follow these steps:

1.  Prepare nutrient agar according to the manufacturer’s instructions.
2. Melt the nutrient agar in a water bath or microwave.
3. Allow the agar to cool down to around 45-50°C.
4. Add tributyrin to the agar at a concentration of 1-2% (w/v).
5. Mix the agar and tributyrin thoroughly.
6. Pour the agar into sterile Petri dishes and allow it to solidify.

Spirit Blue Agar: To prepare spirit blue agar, follow these steps:

1. Prepare nutrient agar according to the manufacturer’s instructions.
2. Melt the nutrient agar in a water bath or microwave.
3. Allow the agar to cool down to around 45-50°C.
4. Add spirit blue dye to the agar at a concentration of 0.01% (w/v).
5. Mix the agar and spirit blue dye thoroughly.
6. Pour the agar into sterile Petri dishes and allow it to solidify.

Procedure of lipid hydrolysis test 

1. Sterilize the inoculation loop or needle by passing it through a flame until it turns red hot. Allow it to cool briefly.
2. Select the microbial culture to be tested for lipid hydrolysis. This can be a pure culture of a known microorganism or a clinical sample.
3. Transfer a small amount of the microbial culture onto the inoculation loop or needle.
4. Streak the inoculation loop or needle onto the surface of the prepared lipid hydrolysis agar plate. Make sure to spread the culture evenly over the agar surface.
5. Repeat the streaking process for multiple cultures, ensuring proper labeling and identification for each plate.
6. Incubate the inoculated lipid hydrolysis agar plates at an appropriate temperature for lipid hydrolysis, typically around 37°C.
7. Incubate the plates for 24-48 hours to allow sufficient time for microbial growth and hydrolysis of lipids to occur.
8. Observe the plates.

Interpretation of Results

Positive Results 

A positive result in the lipid hydrolysis test is indicated by the presence of clear zones or halos around bacterial colonies on the agar plate. These clear zones suggest that the tested microorganism has the ability to produce lipases and hydrolyze lipids.

Negative Results 

Negative results in the lipid hydrolysis test indicate the absence of lipid hydrolysis by the tested microorganism. 

Example of bacteria that give positive and negative results 

Positive Bacteria	Negative Bacteria
Pseudomonas aeruginosa	Lactobacillus acidophilus
Staphylococcus aureus	Streptococcus mutans
Burkholderia cepacia	Listeria monocytogenes
Enterococcus faecalis	Salmonella enterica
Candida albicans	Shigella flexneri
Streptococcus pneumoniae	Clostridium difficile
Bacillus subtilis	Neisseria meningitidis
Escherichia coli	Helicobacter pylori
Proteus mirabilis	Bifidobacterium longum
Mycobacterium tuberculosis	Campylobacter jejuni

Applications of Lipid Hydrolysis

Lipid hydrolysis has a wide range of applications in various fields. Here are some of the key areas where the lipid hydrolysis test finds practical utility:

1. Microbial Identification and Differentiation: The lipid hydrolysis test is widely used in microbiology laboratories for the identification and differentiation of microorganisms. Different species and strains of microorganisms exhibit varying abilities to hydrolyze lipids. By assessing the presence or absence of lipid hydrolysis, microbiologists can narrow down potential microbial species and distinguish them from closely related organisms. This information is invaluable in clinical, environmental, and food microbiology.
2. Clinical Microbiology: In clinical settings, the lipid hydrolysis test serves as a valuable diagnostic tool. Lipase-producing bacteria, such as Pseudomonas aeruginosa, can cause various infections, including urinary tract infections, wound infections, and pneumonia. The detection of lipase production through the lipid hydrolysis test aids in the accurate identification of such pathogenic bacteria, enabling appropriate treatment strategies.
3. Food and Beverage Industry: Lipid hydrolysis testing plays a crucial role in quality control within the food and beverage industry. Lipolytic bacteria can cause spoilage in various food products, leading to changes in taste, texture, and odor. By testing for lipid hydrolysis, manufacturers can detect the presence of lipolytic microorganisms and take timely measures to prevent product deterioration and maintain quality.
4. Oil and Fat Processing: The lipid hydrolysis test is widely utilized in the oil and fat processing industry. Lipases present in microorganisms can contribute to the degradation and rancidity of oils and fats, affecting their quality and shelf life. By assessing the hydrolytic activity of microorganisms on lipids, producers can evaluate the freshness and stability of their products, implement appropriate storage and processing conditions, and ensure optimal quality of oils and fats.
5. Environmental Microbiology: The lipid hydrolysis test finds applications in environmental microbiology, particularly in wastewater treatment plants. Microorganisms involved in wastewater treatment utilize lipids as a carbon source for their metabolic activities. By assessing the efficiency of lipid degradation through the lipid hydrolysis test, researchers and engineers can gain insights into the performance of microbial communities, optimize treatment processes, and enhance overall wastewater treatment efficiency.
6. Bioremediation: Lipid hydrolysis is a crucial step in bioremediation processes. Some microorganisms possess the ability to degrade and metabolize lipids found in contaminated environments, including hydrocarbon pollutants. By assessing the lipid hydrolysis activity of microorganisms, researchers can identify potential candidates for bioremediation strategies and enhance the removal of lipid-based contaminants from the environment.
7. Research and Development: Lipid hydrolysis testing is employed in research and development activities across various disciplines. It serves as a tool to understand the metabolic capabilities of microorganisms, study enzyme kinetics, evaluate enzyme inhibitors, and explore novel lipase-producing microorganisms. Researchers utilize the lipid hydrolysis test to investigate lipid metabolism, enzymatic pathways, and the role of lipases in microbial physiology.

Limitations

1. Need more biochemical tests for further confirmations 
2. Fastidious bacteria cannot grow in media 

References 

• Cappuccino J.G. and Sherman N. 2008. Microbiology: A Laboratory Manual, 8th ed. Pearson Benjamin Cummings, San Francisco, CA, USA.
• Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814
• Procop, G. W., Church, D. L., & Koneman, E. W. (2020). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. Jones & Bartlett Learning.