Lipid Hydrolysis Test – Principle, Procedure, Result

It is a biochemical test that is used to determine the ability of microorganism to produce and secrete the enzyme lipase. It is also referred to as lipase test. The complex lipids such as triglycerides are large molecules and these cannot pass through the bacterial cell membrane. Due to this reason some bacteria releases the enzyme lipase into the surrounding medium. This enzyme hydrolyses the lipids into smaller units like glycerol and fatty acids which can be absorbed by the cell.

In this test the organism is inoculated on a special medium such as Tributyrin agar which contains lipid substrate in the form of emulsion making the medium opaque. If the organism is lipase positive the lipid present in the medium is hydrolysed and a clear zone or halo is formed around the bacterial growth. If the organism is lipase negative the medium remains opaque with no clear zone. This test is used for identification and differentiation of different bacterial genera such as Staphylococcus Bacillus Clostridium and Pseudomonas.

Objectives of Lipid Hydrolysis Test

• To determine the ability of organism to hydrolyze lipids such as triglycerides into simpler compounds.
• To detect the production of extracellular enzyme lipase by microorganism.
• To differentiate lipase producing bacteria from non-lipolytic organisms.
• To help in identification and characterization of bacterial genera such as Staphylococcus Bacillus Clostridium Corynebacterium Pseudomonas and Moraxella.
• To detect lipolytic microorganisms involved in spoilage of food products especially dairy products like milk butter and cheese.

Principle of Lipid Hydrolysis Test

It is based on the ability of microorganism to produce and secrete the enzyme lipase. Lipase is an extracellular enzyme (exoenzyme) which helps in the breakdown of complex lipid molecules. The medium used in this test such as Tributyrin agar or Spirit Blue agar contains lipid substrate in the form of emulsion. Due to the presence of this lipid emulsion the medium appears opaque or turbid.

When a lipase producing organism grows on the medium the enzyme is released outside the cell. This enzyme hydrolyses the insoluble triglycerides present in the medium into simpler and water soluble products such as glycerol and free fatty acids. As a result of this breakdown the opaque lipid emulsion is dissolved and a clear transparent zone is formed around the growth of the organism. This clear halo indicates a positive lipid hydrolysis test.

In some media like egg yolk agar the lipase activity is not seen as a clear zone. Instead it is observed as an iridescent or pearly sheen on the surface of the colony. This appearance is due to accumulation of insoluble fatty acids produced during lipid hydrolysis.

Requirements for Lipid Hydrolysis Test

• Lipid agar medium such as Tributyrin agar or Spirit Blue agar.
• Lipid substrate like tributyrin corn oil olive oil cottonseed oil or egg yolk emulsion mixed in the medium.
• Fresh bacterial culture of test organism (18–72 hours old).
• Positive control organism such as lipase producing bacteria.
• Negative control organism which does not produce lipase.
• Sterile inoculating loop or inoculating needle.
• Bunsen burner or spirit lamp for maintaining aseptic condition.
• Incubator maintained at suitable temperature (generally 35–37°C).
• Anaerobic jar or GasPak system when anaerobic organisms are tested.
• Sterile petri plates for pouring and culturing the medium.
• Marker or wax pencil for labelling the plates.
• Disinfectant solution such as alcohol or bleach for cleaning purpose.
• Personal protective equipment like lab coat gloves and safety glasses.

Tributyrin Agar

Composition (per 1 litre)

• Peptone – 5.0 g
• Yeast extract – 3.0 g
• Agar – 12.0–15.0 g
• Tributyrin – 10.0 mL
• Distilled water – 990 mL
• Final pH – 7.5 ± 0.2

Preparation

1. Weigh the required amount of peptone yeast extract and agar and dissolve in 990 mL of distilled water.
2. Add 10 mL of tributyrin to the medium and mix properly.
3. Heat the medium with continuous stirring until complete dissolution of agar occurs.
4. Sterilize the medium by autoclaving at 121°C for 15 minutes at 15 lbs pressure.
5. Allow the medium to cool to about 45–50°C and shake gently at intervals to maintain uniform emulsion.
6. Pour the medium into sterile petri plates under aseptic condition.
7. Allow the plates to solidify properly. The prepared medium appears turbid or opaque due to lipid emulsion.

Procedure of Lipid Hydrolysis Test

1. Clean the working area properly and maintain aseptic condition using spirit or disinfectant.
2. Take a fresh pure culture of the test organism (18–72 hours old).
3. Sterilize the inoculating loop and allow it to cool.
4. Pick a heavy inoculum of the organism using sterile inoculating loop.
5. Inoculate the lipid agar plate (Tributyrin agar or Spirit Blue agar) by streaking a straight line or a small circular spot on the surface of the medium.
6. Do not disturb or break the surface of the agar while inoculation.
7. Incubate the plates in inverted position at suitable temperature (generally 35–37°C).
8. For anaerobic organisms place the plates in anaerobic jar immediately after inoculation.
9. Incubate aerobic cultures for 24–48 hours. Incubation may be extended up to 7 days if required.
10. After incubation observe the medium for clear zone around the growth indicating lipid hydrolysis.
11. In egg yolk agar observe the colony surface for pearly or iridescent sheen which indicates lipase activity.

Result of Lipid Hydrolysis Test

Positive result (Lipase present)

• Clear transparent zone or halo is formed around the bacterial growth on Tributyrin agar or Spirit Blue agar.
• The opacity of the medium is lost near the growth due to hydrolysis of lipid substrate.
• In egg yolk agar an iridescent or pearly sheen is seen on the surface of the colony.
• In Spirit Blue agar sometimes deep blue colour or blue ring may appear around the growth.

Negative result (Lipase absent)

• No clear zone is observed around the growth.
• The medium remains opaque or turbid up to the margin of the colony.
• In egg yolk agar no pearly or iridescent sheen is formed.

Related reactions (Egg yolk agar)

• Lecithinase positive reaction shows opaque white precipitate around the colony.
• Proteolysis produces a clear zone due to protein degradation which should not be confused with lipid hydrolysis.

Organisms showing Lipid Hydrolysis Test

Positive organisms

• Staphylococcus aureus
• Staphylococcus epidermidis
• Staphylococcus saprophyticus
• Bacillus subtilis
• Pseudomonas aeruginosa
• Clostridium sporogenes
• Clostridium botulinum
• Fusobacterium necrophorum
• Moraxella catarrhalis
• Penicillium commune

Negative organisms

• Escherichia coli
• Clostridium perfringens
• Clostridium difficile
• Klebsiella pneumoniae
• Klebsiella oxytoca
• Salmonella Typhimurium
• Proteus mirabilis
• Bacteroides fragilis

Quality Control of Lipid Hydrolysis Test

Positive controls (lipase positive)

– Staphylococcus aureus (ATCC 25923 or 12600): Shows luxuriant growth with clear transparent zone around colony on tributyrin agar and spirit blue agar.

– Staphylococcus epidermidis (ATCC 12228): Exhibits luxuriant growth with clear zone around colony.

– Clostridium sporogenes (ATCC 11437 or 3584): On egg yolk agar produces iridescent pearly sheen on colony surface and medium. On tributyrin agar clear zone is formed.

– Bacillus subtilis (ATCC 6633): Shows good growth with distinct clear zone.

– Pseudomonas aeruginosa (ATCC 27853): Shows good growth with clear zone around colony.

– Fusobacterium necrophorum (ATCC 25286): Produces positive lipase reaction with iridescent sheen on egg yolk agar.

Negative controls (lipase negative)

– Escherichia coli (ATCC 25922): Shows luxuriant growth but no clear zone and medium remains opaque.

– Proteus mirabilis (ATCC 25933): Shows luxuriant growth but no zone around colony.

– Clostridium perfringens (ATCC 12924 or 13124): On egg yolk agar opaque zone is formed due to lecithinase activity but no iridescent sheen is seen. On tributyrin agar no clear zone is observed.

– Salmonella Typhimurium (ATCC 14028): Shows good growth without any clear zone.

– Bacteroides fragilis (ATCC 25285): Shows no lipase reaction or growth inhibition depending on selective agents used.

Precautions of Lipid Hydrolysis Test

• Lipid agar medium should be mixed properly during preparation to maintain uniform emulsion.
• Do not use media showing separation of lipid layer cracking or contamination.
• While cooling the medium shake gently to prevent settling of lipid substrate.
• Use glass petri plates whenever recommended for better lipase activity.
• For anaerobic organisms pre-reduce the medium before inoculation.
• Use a heavy inoculum to obtain clear and reliable result.
• Sterilize the inoculating loop properly and allow it to cool before use.
• Avoid touching the sides of the plate during inoculation to prevent contamination.
• Incubate the plates for sufficient time and do not report negative result early.
• Always include positive and negative control organisms.
• In egg yolk agar carefully differentiate lipase activity from proteolysis or lecithinase reaction.
• Maintain proper aseptic technique throughout the procedure.
• Wear lab coat gloves and follow laboratory safety measures.

Uses of Lipid Hydrolysis Test

• To determine the ability of microorganism to produce and secrete the enzyme lipase.
• To help in identification of lipase producing bacteria.
• To differentiate between lipolytic and non-lipolytic organisms.
• To differentiate closely related bacterial species such as Clostridium perfringens and Clostridium sporogenes.
• To identify bacteria belonging to genera like Staphylococcus Bacillus Clostridium Corynebacterium Pseudomonas and Moraxella.
• To detect lipolytic microorganisms present in food and dairy products such as milk butter and cheese.
• To study spoilage of fat rich food materials due to lipid breakdown.
• To assist in presumptive identification of anaerobic organisms using egg yolk agar and Nagler reaction.

Advantages of Lipid Hydrolysis Test

• It is useful for differentiation and identification of bacterial species based on lipase production.
• It helps in classification of bacteria belonging to genera like Staphylococcus Bacillus Clostridium Pseudomonas Moraxella and Corynebacterium.
• It assists in detection of pathogenic organisms where lipase acts as a virulence factor.
• It is useful in food microbiology for detection of lipolytic organisms causing spoilage and rancidity.
• It helps in quality control of dairy and fat rich food products.
• Egg yolk agar allows detection of lipase lecithinase and proteolytic activity on single medium.
• The test is simple easy to perform and easy to interpret.
• Clear zone formation provides direct visual evidence of lipid hydrolysis.
• It is useful for screening of lipase producing microorganisms for industrial applications.

Limitations of Lipid Hydrolysis Test

• It is not a confirmatory test and cannot identify organism on its own.
• Additional biochemical or molecular tests are required for final identification.
• Lipase activity may be slow and delayed especially in anaerobic bacteria.
• Negative result cannot be confirmed until prolonged incubation up to 7 days.
• Fastidious organisms may not grow well on lipid agar leading to false negative result.
• In egg yolk agar clear zone may be due to proteolysis and not due to lipase activity.
• High concentration of glucose in medium may suppress lipase production.
• Improper preparation or storage of medium may disturb lipid emulsion and affect results.
• Interpretation of results may be difficult due to diffusion of enzymes in the medium.