Lecithinase Test (Nagler’s Reaction)- Principle, Procedure, Results

Lecithinase is a type of phospholipase enzyme that can hydrolyze lecithin, a major component of cell membranes. Some bacteria can produce lecithinase as a virulence factor that helps them to damage host cells and tissues. Lecithinase test (also known as Nagler’s reaction) is a biochemical test that can detect the presence of lecithinase in bacterial cultures. It is based on the observation that lecithinase-producing bacteria can cause a visible change in the appearance of egg yolk agar, a culture medium that contains lecithin from egg yolk.

Lecithinase test was first described by A. Nagler in 1901, who used it to differentiate between Clostridium perfringens (formerly known as C. welchii) and other clostridia. C. perfringens is a Gram-positive, anaerobic, spore-forming bacterium that causes gas gangrene and food poisoning in humans and animals. It produces a potent lecithinase called alpha-toxin (or phospholipase C) that can degrade phospholipids in cell membranes and cause tissue necrosis and hemolysis. Nagler observed that C. perfringens colonies on egg yolk agar produced a characteristic opaque halo around them, indicating the hydrolysis of lecithin in the medium.

Since then, lecithinase test has been widely used as a presumptive identification method for C. perfringens and other lecithinase-producing bacteria, such as Bacillus cereus, Bacillus anthracis, Pseudomonas aeruginosa, and Burkholderia spp. Lecithinase test can also help to differentiate between various species and strains of bacteria based on their lecithinase activity and the size and shape of the halo zone. Lecithinase test is simple, inexpensive, and easy to perform and interpret. However, it is not a confirmatory test and should be supplemented by other biochemical and molecular tests for accurate identification of bacteria.

In this article, we will discuss the principle, procedure, results, applications, and limitations of lecithinase test in detail. We will also provide some examples of bacteria that test positive and negative for lecithinase production. We hope that this article will help you to understand the importance and significance of lecithinase test in microbiology.

The main objectives of lecithinase test (Nagler’s reaction) are:

• To assess the ability of bacteria to produce lecithinase enzyme, which is a type of phospholipase that can hydrolyze lecithin and other phospholipids in the cell membranes of host cells and cause tissue damage and necrosis.
• To presumptively identify the bacteria that produce lecithinase enzyme, especially Clostridium perfringens, which is the causative agent of gas gangrene and food poisoning. Other bacteria that can produce lecithinase include some species of Bacillus, Pseudomonas, Burkholderia and Listeria.
• To differentiate between lecithinase-positive and lecithinase-negative bacteria based on their ability to cause a visible change in the egg yolk agar medium. Lecithinase-positive bacteria produce a milky white halo around their colonies, while lecithinase-negative bacteria do not. This test can help in narrowing down the possible identification of an unknown bacterial isolate.

The principle of Lecithinase Test (Nagler’s Reaction) is based on the ability of some bacteria to produce a toxic, phospholipase enzyme called lecithinase. This enzyme can hydrolyze lecithin, a major component of egg yolk, into phosphorylcholine and diglyceride. The diglyceride is insoluble in water and forms a white precipitate in the medium around the bacterial colonies. This results in an opaque halo that indicates a positive lecithinase test.

The egg yolk also contains another phospholipid called lecithovitellin, which is partly water-soluble and makes the medium translucent. The lecithinase enzyme can also hydrolyze lecithovitellin into phosphorylcholine and diglyceride, causing the same opaque halo effect. Therefore, the test is also known as Nagler’s reaction, after Arthur Nagler who first described it in 1948.

The lecithinase test is useful for presumptively identifying bacteria that produce this enzyme, such as Clostridium perfringens, Bacillus cereus, Pseudomonas aeruginosa and others. These bacteria are usually pathogenic and cause various infections in humans and animals. The lecithinase enzyme is one of the virulence factors that contribute to tissue damage and necrosis. Therefore, the detection of lecithinase production can help in the diagnosis and treatment of these infections.

The lecithinase test is performed on a solid medium containing egg yolk as the substrate for the enzyme. The medium is inoculated with the bacterial culture and incubated under appropriate conditions. After incubation, the presence or absence of an opaque halo around the bacterial growth is observed and recorded as a positive or negative result respectively. The size and shape of the halo may vary depending on the bacterial species and strain. Some bacteria may produce a small or faint halo that requires careful examination. A control plate with an uninoculated medium is also used to compare the results and confirm a negative test.

To perform the lecithinase test, you will need the following requirements:

• Culture medium: You will need an agar medium that contains lecithin, which is a phospholipid found in egg yolk. The most commonly used medium for this test is Egg Yolk Agar, which is prepared by adding emulsified egg yolk to a basal medium that contains proteose peptone, glucose, sodium chloride, magnesium sulfate, hemin and agar . Alternatively, you can use other modified media that contain egg yolk and other ingredients, such as mannitol or sodium taurocholate.
• Reagents: You will need a 50% v/v egg yolk emulsion in sterile distilled water to prepare the Egg Yolk Agar medium. You can either buy a ready-made egg yolk emulsion or prepare it yourself by sterilizing an egg, separating the yolk and mixing it with an equal volume of sterile water.
• Equipment: You will need standard laboratory equipment such as inoculating loops, spreaders, pipettes, Petri dishes, anaerobic jars or incubators and PPE (personal protective equipment).
• Test organism: You will need a pure culture of the bacterium that you want to test for lecithinase production. You can use a positive control organism such as Clostridium perfringens ATCC 12924 or a negative control organism such as Clostridium difficile ATCC 9689 to verify the validity of your test .

Egg Yolk Agar is a differential medium that contains lecithin, a phospholipid found in egg yolk. Lecithin is the substrate for the lecithinase enzyme produced by some bacteria. The medium also contains other nutrients and indicators to support bacterial growth and differentiation.

Egg Yolk Agar is prepared by adding emulsified egg yolk to a sterile Egg Yolk Agar Base. The base is a complex medium that contains proteose peptone, disodium hydrogen phosphate, potassium dihydrogen phosphate, glucose, sodium chloride, magnesium sulfate, hemin and agar. The base provides nitrogen, carbon, minerals and vitamins for bacterial metabolism. Hemin is added to enhance the growth of anaerobic bacteria.

The composition and preparation of Egg Yolk Agar Base and Egg Yolk Agar are as follows:

Composition of Egg Yolk Agar Base per 1000 mL

• Proteose peptone: 40.00 grams
• Disodium hydrogen phosphate: 5.00 grams
• Potassium dihydrogen phosphate: 1.00 grams
• Glucose (Dextrose): 2.00 grams
• Sodium chloride: 2.00 grams
• Magnesium sulfate: 0.10 grams
• Hemin: 0.005 grams
• Agar: 25.00 grams
• Final pH 7.6 ±0.2 at 25°C

Preparation of Egg Yolk Agar Base

• Measure the appropriate amount of Egg Yolk 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 (75.10 grams of the above composition in 900 mL distilled water).
• 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.

Preparation of Egg Yolk Agar

• In the above-prepared base, dispense 100 mL of sterile egg yolk emulsion (egg yolk and distilled water in a 1:1 ratio) when the mixture is around 40-45°C.
• Stir the mixture completely so that the egg emulsion is properly mixed with the basal medium.
• 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.

To ensure the validity and accuracy of the lecithinase test, it is important to use positive and negative control organisms along with the test organism. Control organisms are bacteria that are known to produce or not produce lecithinase enzyme under standard conditions. By comparing the results of the control organisms with the test organism, one can confirm or rule out any errors or discrepancies in the test procedure or the medium.

Positive control organisms are bacteria that produce lecithinase enzyme and show a positive result on the egg yolk agar medium. They produce a milky white halo around their colonies due to the hydrolysis of lecithin in the egg yolk. Some examples of positive control organisms are:

• Clostridium perfringens ATCC 13124
• Bacillus cereus ATCC 11778
• Pseudomonas aeruginosa ATCC 27853

Negative control organisms are bacteria that do not produce lecithinase enzyme and show a negative result on the egg yolk agar medium. They do not produce any halo around their colonies and the medium remains translucent. Some examples of negative control organisms are:

• Clostridium sporogenes ATCC 11437
• Bacteroides fragilis ATCC 25285
• Bacillus subtilis ATCC 6633

An un-inoculated egg yolk agar plate should also be used as a control plate to check for any contamination or degradation of the medium. The un-inoculated plate should show no growth or change in color or opacity.

The lecithinase test is performed on Egg Yolk Agar medium, which is prepared by adding 50% v/v egg yolk emulsion to the sterilized Egg Yolk Agar Base medium. The test can be done by either streaking or stabbing the inoculum on the agar surface. The following steps describe the procedure of the lecithinase test:

1. Using a sterile inoculating loop, pick up a heavy inoculum from a well-isolated colony of fresh culture (18 to 72 hours old culture).
2. 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 Egg Yolk Agar plate. Alternatively, you can stab the inoculum into the agar using a sterile needle.
3. 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. (Incubation temperature varies according to bacterial species. Incubate according to the following for better results: Bacillus spp., Gram-positive Rods and Anaerobes at 35°C; Non-glucose fermenting Gram-negative Rods (except P. aeruginosa) at 25°C; Glucose fermenting Gram-negative Rods including P. aeruginosa at 30°C)
4. Following incubation, observe the development of a milky white halo around the bacterial colony (line of growth).

The presence of a milky white halo indicates a positive lecithinase test, which means that the bacteria can produce lecithinase enzyme and hydrolyze lecithin in the egg yolk. The absence of a halo indicates a negative lecithinase test, which means that the bacteria cannot produce lecithinase enzyme or hydrolyze lecithin in the egg yolk.

The following image shows an example of a positive lecithinase test by Clostridium perfringens (left) and a negative lecithinase test by Clostridium difficile (right) on Egg Yolk Agar plates.

The result of the lecithinase test is based on the observation of a milky white halo around the bacterial colony or line of growth on the egg yolk agar medium. The halo indicates the hydrolysis of lecithin by the enzyme lecithinase produced by the bacteria. The size and intensity of the halo may vary depending on the bacterial species and the incubation conditions.

A positive lecithinase test is indicated by the presence of a milky white halo around the bacterial colony or line of growth. This means that the bacteria can produce lecithinase and degrade lecithin in the egg yolk. A positive result is usually seen within 24 to 48 hours of incubation, but some bacteria may require longer incubation periods to show a positive reaction.

A negative lecithinase test is indicated by the absence of a milky white halo around the bacterial colony or line of growth. This means that the bacteria cannot produce lecithinase or degrade lecithin in the egg yolk. A negative result can be confirmed by comparing the inoculated plate with an uninoculated control plate.

The following table summarizes the interpretation of the lecithinase test results:

The lecithinase test is a useful presumptive test for identifying and classifying some bacteria, especially Clostridium spp. and Bacillus spp. However, it is not a confirmatory test and should be supplemented with other biochemical tests for complete identification of an unknown organism. Some limitations of the lecithinase test are:

• Some glucose non-fermenting rods produce a small halo zone that is difficult to read.
• Lecithinase is diffusible and can spread all over the plate, making the result interpretation difficult.
• Some bacteria may require longer incubation periods or different temperatures to show lecithinase activity.
• Some strains of P. fluorescens are positive while some are negative for lecithinase production.

Therefore, it is important to follow the standard procedure and precautions while performing the lecithinase test and interpret the results with caution and reference to other tests.

Lecithinase is an enzyme that hydrolyzes lecithin, a phospholipid found in egg yolk and other biological membranes. Lecithinase production is a characteristic of some bacteria, especially those belonging to the genera Clostridium and Bacillus. Lecithinase test can help in the presumptive identification of these bacteria by observing the formation of a white precipitate around the colonies on egg yolk agar.

Some of the bacteria that test positive for lecithinase production are:

• Clostridium perfringens: This is a Gram-positive, anaerobic, spore-forming rod that causes gas gangrene, food poisoning and necrotic enteritis. It produces a large zone of lecithinase activity on egg yolk agar, which is also known as Nagler`s reaction.
• Pseudomonas aeruginosa: This is a Gram-negative, aerobic, motile rod that causes opportunistic infections in immunocompromised patients. It produces a small zone of lecithinase activity on egg yolk agar, which can be enhanced by adding iron salts to the medium.
• Bacillus cereus: This is a Gram-positive, aerobic, spore-forming rod that causes food poisoning and eye infections. It produces a moderate zone of lecithinase activity on egg yolk agar, which can be distinguished from B. anthracis by its motility and hemolytic activity.
• Bacillus anthracis: This is a Gram-positive, aerobic, spore-forming rod that causes anthrax, a serious zoonotic disease. It produces a moderate zone of lecithinase activity on egg yolk agar, which can be distinguished from B. cereus by its non-motility and non-hemolytic activity.
• Bacillus thuringiensis: This is a Gram-positive, aerobic, spore-forming rod that produces insecticidal toxins and is used as a biological pesticide. It produces a moderate zone of lecithinase activity on egg yolk agar, which can be distinguished from B. cereus and B. anthracis by its parasporal crystals.
• Burkholderia spp.: These are Gram-negative, aerobic, motile rods that cause various infections in humans and animals. Most of them produce a small zone of lecithinase activity on egg yolk agar.

Some of the bacteria that test negative for lecithinase production are:

• Clostridium sporogenes: This is a Gram-positive, anaerobic, spore-forming rod that is used as an indicator organism for sterilization processes. It does not produce any zone of lecithinase activity on egg yolk agar.
• Clostridium botulinum: This is a Gram-positive, anaerobic, spore-forming rod that causes botulism, a life-threatening neuroparalytic disease. It does not produce any zone of lecithinase activity on egg yolk agar.
• Clostridium difficile: This is a Gram-positive, anaerobic, spore-forming rod that causes pseudomembranous colitis and antibiotic-associated diarrhea. It does not produce any zone of lecithinase activity on egg yolk agar.
• Pseudomonas putida: This is a Gram-negative, aerobic, motile rod that is widely distributed in soil and water. It does not produce any zone of lecithinase activity on egg yolk agar.
• Bacteroides fragilis: This is a Gram-negative, anaerobic, non-spore-forming rod that is part of the normal flora of the human colon. It does not produce any zone of lecithinase activity on egg yolk agar.
• Bacillus subtilis: This is a Gram-positive, aerobic, spore-forming rod that is widely used as a model organism in microbiology and biotechnology. It does not produce any zone of lecithinase activity on egg yolk agar.

This list is not exhaustive and there may be other bacteria that show positive or negative results for lecithinase test. Therefore, it is important to perform other biochemical tests to confirm the identity of the bacteria.

Quality control is an essential aspect of any laboratory test to ensure the accuracy and reliability of the results. For the lecithinase test, the following quality control measures should be followed:

• Use only fresh and pure cultures of bacteria for inoculation. Avoid using old or contaminated cultures as they may give false results.
• Use appropriate culture media and reagents for the test. Check the expiry date and storage conditions of the media and reagents before use. Prepare the Egg Yolk Agar medium as per the instructions and sterilize it properly. Do not add the egg yolk emulsion to the medium before autoclaving or when it is too hot as it may affect the quality of the medium.
• Use positive and negative control organisms for the test. Clostridium perfringens ATCC 12924 is a recommended positive control organism as it produces a clear and distinct milky white halo around its colonies. Clostridium difficile ATCC 9689 is a recommended negative control organism as it does not produce any halo around its colonies. The control organisms should be inoculated and incubated under the same conditions as the test organisms. The results of the control organisms should match the expected results; otherwise, the test should be repeated or investigated for possible errors.
• Use an un-inoculated Egg Yolk Agar medium plate as a negative control plate to confirm a negative result. The un-inoculated plate should show no change in color or opacity after incubation. If the un-inoculated plate shows any sign of lecithin hydrolysis, such as a milky white appearance or precipitation, it indicates that the medium is contaminated or degraded and should not be used for testing.
• Observe and record the results carefully and objectively. Use a magnifying glass or a microscope if necessary to see the halo around the colonies. Compare the results with the control plates and interpret them accordingly. Do not rely on subjective judgments or assumptions while reading the results. Report any unusual or unexpected results and seek expert advice if needed.
• Document and maintain records of all the steps and results of the test. Include information such as date, time, name of the tester, name of the organism, source of the culture, type of medium, incubation conditions, results, interpretation, and any comments or observations. Follow the standard operating procedures and guidelines of the laboratory while performing and reporting the test.
• Dispose of the used materials and waste properly after completing the test. Follow the biosafety and infection control measures of the laboratory while handling and disposing of potentially infectious materials. Sterilize or disinfect all the equipment and surfaces that came in contact with the cultures or media. Wash your hands thoroughly after performing the test.

By following these quality control measures, you can ensure that your lecithinase test results are accurate and reliable. You can also avoid errors or discrepancies that may affect your identification and classification of bacteria. Remember that quality control is not only a responsibility but also a benefit for you as a laboratory professional.

• The egg yolk emulsion should be sterile and free of contamination. It is advisable to use a commercially available product or prepare it under aseptic conditions.
• The egg yolk agar medium should be prepared and dispensed carefully to avoid bubbles or cracks on the surface. The medium should not be overheated or overcooked as it may affect the lecithin content and clarity of the medium.
• The inoculation of the test organism should be done with a heavy inoculum to ensure confluent growth and clear halo formation. A single colony or a loopful of culture should be used for inoculation. The inoculation should be done gently to avoid scratching or damaging the surface of the medium.
• The incubation temperature and atmosphere should be appropriate for the test organism. Different bacteria may require different temperatures and oxygen levels for optimal growth and lecithinase production. The incubation period should also be sufficient to allow the development of visible halos around the colonies.

• The interpretation of the results should be done by comparing the inoculated plates with an uninoculated control plate. The presence or absence of halos should be observed carefully and recorded. A positive result is indicated by a milky white halo around the colonies, while a negative result is indicated by no halo formation. The size and shape of the halos may vary depending on the bacterial species and strain. Some bacteria may produce small or irregular halos that are difficult to read. Some bacteria may also produce other reactions on the egg yolk agar medium, such as lipase activity (indicated by iridescence or opalescence), protease activity (indicated by clearing or liquefaction), or pigment production (indicated by color change). These reactions should not be confused with lecithinase activity and should be confirmed by other tests if necessary.

  Applications of Lecithinase Test (Nagler`s Reaction)

Lecithinase test (Nagler`s reaction) is a useful biochemical test for the identification and classification of bacteria based on their ability to produce lecithinase enzyme. Some of the applications of this test are:

• Lecithinase test can help to differentiate and identify species of food-borne pathogenic bacterial genera like Clostridium and Bacillus . For example, Clostridium perfringens, the causative agent of gas gangrene and food poisoning, is positive for lecithinase production, whereas Clostridium difficile, the causative agent of antibiotic-associated diarrhea, is negative.
• Lecithinase test can also help to identify other lecithinase-producing bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes, etc . These bacteria are associated with various infections such as skin infections, wound infections, urinary tract infections, respiratory infections, meningitis, etc.
• Lecithinase test can be used to detect coagulase-positive strains of S. aureus, because there is a high correlation between lecithinase activity and coagulase activity. Coagulase-positive S. aureus are more virulent and cause more severe infections than coagulase-negative S. aureus.
• Lecithinase test can be used to differentiate between certain species within the genus Bacillus. For example, Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are positive for lecithinase production, whereas Bacillus subtilis is negative.

Limitations of Lecithinase Test (Nagler’s Reaction)

Lecithinase test is a useful biochemical test for assessing the ability of bacteria to produce lecithinase enzyme and presumptively identify some bacterial species. However, it has some limitations that should be considered while interpreting the results. Some of the limitations are:

• Lecithinase test is not a confirmatory test; hence, it needs to be supplemented with other biochemical tests for the complete identification of an unknown organism. For example, C. perfringens can be differentiated from other lecithinase-positive clostridia by its double zone of hemolysis on blood agar and its negative indole and gelatinase tests.
• Some glucose non-fermenting rods, such as Pseudomonas spp. and Burkholderia spp., produce a small halo zone around their colonies that may be difficult to read. The size and clarity of the halo zone may depend on the inoculum size, incubation time and temperature, and the composition of the medium. Therefore, careful observation and comparison with positive and negative controls are necessary for accurate results.
• Lecithinase is a diffusible enzyme; hence, it can diffuse all over the plate and make the result interpretation difficult. A negative plate must be compared to an un-inoculated medium plate to confirm a negative result. Alternatively, a filter paper impregnated with lecithin can be placed on the surface of the medium after inoculation to localize the lecithinase activity around the colonies.
• Some bacteria may require a longer period of incubation (up to one week) for showing lecithinase activity. For example, Bacillus anthracis may take 4 to 7 days to produce a clear halo zone on egg yolk agar. Therefore, premature discarding of negative plates may lead to false-negative results.
• Some strains of P. fluorescens are positive while some are negative for lecithinase production. Therefore, other tests, such as oxidase and fluorescence tests, are needed to identify this species.
• Some bacteria may produce other enzymes that can hydrolyze lecithin or other components of egg yolk agar, such as lipases or proteases. This may result in false-positive results or interference with lecithinase activity. For example, Staphylococcus aureus produces both lecithinase and lipase that can cause an opaque halo around its colonies on egg yolk agar. However, S. aureus can be distinguished from other lecithinase-positive bacteria by its coagulase and catalase tests.

These limitations indicate that lecithinase test is not a definitive test for identifying bacteria and should be used in conjunction with other tests for accurate diagnosis and classification. Lecithinase test is a simple and inexpensive test that can provide valuable information about the enzymatic activity and pathogenic potential of bacteria, but it should be performed and interpreted with caution and care.

Lecithinase test is a useful biochemical test for assessing the ability of bacteria to produce lecithinase enzyme and presumptively identify some bacterial species. However, it has some limitations that should be considered while interpreting the results. Some of the limitations are:

• Lecithinase test is not a confirmatory test; hence, it needs to be supplemented with other biochemical tests for the complete identification of an unknown organism. For example, C. perfringens can be differentiated from other lecithinase-positive clostridia by its double zone of hemolysis on blood agar and its negative indole and gelatinase tests.
• Some glucose non-fermenting rods, such as Pseudomonas spp. and Burkholderia spp., produce a small halo zone around their colonies that may be difficult to read. The size and clarity of the halo zone may depend on the inoculum size, incubation time and temperature, and the composition of the medium. Therefore, careful observation and comparison with positive and negative controls are necessary for accurate results.
• Lecithinase is a diffusible enzyme; hence, it can diffuse all over the plate and make the result interpretation difficult. A negative plate must be compared to an un-inoculated medium plate to confirm a negative result. Alternatively, a filter paper impregnated with lecithin can be placed on the surface of the medium after inoculation to localize the lecithinase activity around the colonies.
• Some bacteria may require a longer period of incubation (up to one week) for showing lecithinase activity. For example, Bacillus anthracis may take 4 to 7 days to produce a clear halo zone on egg yolk agar. Therefore, premature discarding of negative plates may lead to false-negative results.
• Some strains of P. fluorescens are positive while some are negative for lecithinase production. Therefore, other tests, such as oxidase and fluorescence tests, are needed to identify this species.
• Some bacteria may produce other enzymes that can hydrolyze lecithin or other components of egg yolk agar, such as lipases or proteases. This may result in false-positive results or interference with lecithinase activity. For example, Staphylococcus aureus produces both lecithinase and lipase that can cause an opaque halo around its colonies on egg yolk agar. However, S. aureus can be distinguished from other lecithinase-positive bacteria by its coagulase and catalase tests.

These limitations indicate that lecithinase test is not a definitive test for identifying bacteria and should be used in conjunction with other tests for accurate diagnosis and classification. Lecithinase test is a simple and inexpensive test that can provide valuable information about the enzymatic activity and pathogenic potential of bacteria, but it should be performed and interpreted with caution and care.

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