The Lecithinase Test (Nagler’s Reaction) is a biochemical test used to assess the ability of bacteria to produce a toxic phospholipase enzyme called lecithinase.
Lecithinase (Phospholipase C) is a lipolytic enzyme produced by some bacteria that hydrolyzes lecithin.
Lecithin is a group of fatty acids (glycerophospholipids) and lipoproteins found in animal and plant tissues, considered toxic, and plays a significant role in bacterial pathogenesis.
Not all bacterial species produce lecithinase, even within the same genus.
This property is utilized to classify bacteria into two groups: lecithinase producers and non-producers.
The test is generally used to differentiate and identify species of food-borne pathogenic bacterial genera such asClostridium speciesandBacillus species.
Objectives of Lecithinase Test (Nagler’s Reaction)
Assess the ability of bacteria to produce the lecithinase enzyme.
Presumptively identify the bacteria.
Principle of Lecithinase Test (Nagler’s Reaction)
The egg yolk is a good source of lecithin and contains a significant amount of lecithovitellin.
Lecithovitellin is partly water-soluble and initially makes the medium translucent.
The lecithinase enzyme produced by bacteria splits lecithovitellin into phosphorylcholine and water-insoluble diglyceride.
The diglyceride settles as a precipitate in the medium around colonies of bacteria-producing lecithinase, forming an opaque white halo.
This development of an opaque halo indicates the hydrolysis of lecithovitellin and signifies a positive result.
Requirements for Lecithinase Test (Nagler’s Reaction)
a. Culture Medium
Agar medium containing lecithin is necessary for detecting lecithinase enzyme production.
Egg Yolk Agar medium is preferred, prepared by adding emulsified (50%) egg yolk to the sterilized Egg Yolk Agar Base medium.
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 gram
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 (75.10 grams for 900 mL distilled water) and mix it in a conical flask or glass bottle.
Stir well using a magnetic stirrer or manually, and heat to boiling to dissolve all components and agar completely.
Autoclave at 121°C and 15 lbs pressure for 15 minutes, then cool to around 40 – 45°C.
Preparation of Egg Yolk Agar
Dispense 100 mL of sterile egg yolk emulsion (1:1 ratio of egg yolk and distilled water) into the cooled base (40-45°C).
Stir the mixture thoroughly to mix the egg emulsion with the basal medium.
Pour about 25 mL of the medium mixture into a 10 cm diameter sterile petri plate and allow it to solidify at room temperature.
b. Reagents
50% v/v egg yolk in sterile distilled water is required for making the Egg Yolk Agar medium.
It is advisable to buy the readymade egg yolk emulsion to avoid contamination, but it can be prepared in the lab as follows:
Sterilize a hen egg by rubbing it with spirit.
In a sterile condition, scrub the egg and soak it in 95% ethanol for 1 hour.
Break the egg within a sterile zone and aseptically separate the yolk.
Dispense the yolk in a sterile beaker and add an equal volume of sterile distilled water.
Using a sterile inoculating loop, pick up a heavy inoculum from a well-isolated colony of fresh culture (18 to 72 hours old).
Inoculate the sample organism plate by drawing either a straight line or forming a circular inoculation (the size of a dime) over the surface of the Egg Yolk Agar plate.
Incubate the plates at 35±2°C for about 24 to 48 hours aerobically for aerobes or facultative bacteria, and for about 72 hours anaerobically for anaerobes.
Incubation temperature varies according to bacterial species. Follow these guidelines 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.
After incubation, observe the development of a milky white halo around the bacterial colony.
Result and Interpretation of Lecithinase Test (Nagler’s Reaction)
Positive Lecithinase Test:
Indicated by the development of a milky white halo in the medium around the bacterial colony (line of growth).
Negative Lecithinase Test:
Indicated by the absence of a halo around the bacterial colony (line of growth).
Lecithinase Test Positive Bacteria:
Clostridium perfringens
Pseudomonas aeruginosa
Most Burkholderia spp.
Bacillus cereus
Bacillus thuringiensis
Bacillus anthracis
Lecithinase Test Negative Bacteria:
Clostridium sporogenes
Clostridium butyricum
Clostridium botulinum
Clostridium difficile
Pseudomonas putida
Bacteroides fragilis
Bacillus subtilis
Quality Control
Positive Control:
Clostridium perfringens ATCC 12924 produces a milky white halo around its colonies.
Negative Control:
Clostridium difficile ATCC 9689 grows but does not produce any halo around its colonies.
Control Plate:
An un-inoculated Egg Yolk Agar medium plate is necessary to confirm a negative result.
Precautions
Whenever possible, use readymade egg yolk suspension. Lab-made suspensions pose a high contamination risk.
Do not add egg yolk emulsion to the medium base prior to autoclaving or when the medium is hot.
Maintain the incubation temperature and oxygen/carbon dioxide level according to the sample organism's requirements.
Prefer using a heavy inoculum to promote confluent growth and the development of a clear halo.
Incubate an un-inoculated control plate to ensure proper assessment and verification of results.
Applications of Lecithinase Test (Nagler’s Reaction)
Useful for identifying and classifying Clostridium spp. and Bacillus spp. based on their lecithinase production.
Helps in the identification of pathogens such as Clostridium perfringens, Staphylococcus aureus, Listeria monocytogenes, and Pseudomonas aeruginosa based on their lecithinase activity.
Limitations of Lecithinase Test (Nagler’s Reaction)
It is not a confirmatory test; therefore, it necessitates additional biochemical test results for the comprehensive identification of an unknown organism.
Glucose non-fermenting rods may produce a small halo zone, leading to difficulties in interpretation.
Lecithinase is diffusible, potentially spreading throughout the plate and complicating result interpretation. Comparison with an un-inoculated medium plate is essential to confirm a negative result.
Some strains may require a longer incubation period, up to 1 week, to exhibit lecithinase activity.
Variability exists within strains of Pseudomonas fluorescens; some may test positive for lecithinase activity while others may test negative.
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