Lecithinase test or Nagler’s reaction is a biochemical test used to identify organisms that liberate phospholipases (lecithinases) e.g. Clostridium perfringens. The alpha (α) toxin of C. perfringens has phospholipase activity and hence, helps in the differentiation of C. perfringens from other Clostridium spp that also produce lecithinase (C.baratti, C.absonum, C.bifermantans, C.sordelli, and C.novyi) by neutralization of lecithin c activity by an antitoxin.
Bacillius cereus also exhibits strong lecithinase activity but can be differentiated by its strong hemolytic property on sheep blood agar and motility. Among Bacillus species, B. thuringiensis and B.anthracis are lecithinase positive or weakly positive. B. anthracis, however, is a nonmotile organism and produces nonhemolytic colonies. Bacillus sphaericus is a negative producer of Lecithinase.
Principle: Bacterial lecithinase break down this lecithin (a normal component of egg yolk) to insoluble diglycerides resulting in an opaque halo, surrounding the colony when grown on the egg yolk agar medium.
Modified egg yolk agar is a differential and enriched medium used in the isolation and presumptive differentiation of different species based on their lecithinase and lipase production and proteolytic activity. The degradation of lecithin present in the egg yolk results in the formation of opaque precipitate around the colonies. The lipase enzyme hydrolyzes the fats within the egg yolk, which results in an iridescent sheen on the colony surface.
- Media: Basal egg yolk media is prepared by dissolving the standard amount in distilled water in which 10% of egg yolk is added after autoclaving and cooling the media before dispensing into sterile Petri dishes. Various modifications of the original like modified egg yolk agar media, mannitol egg yolk media, etc are also available and can be used for specified purposes.
- Others: Inoculating loop, spreader, pipettes
- Equipment: Anaerobic gas pak or candle jar or anaerobic incubator for anaerobic incubation
- Label and dry an egg yolk media plate and mark the plate into two halves.
- Inoculate 60 µl of Clostridium perfringens type A antitoxin in half of the plate, spread over the surface of agar using a spreader, and allow to absorb and dry.
- Mark the side of the plate in which the antitoxin is inoculated.
- Streak the test organism in a straight line from the toxin-free agar half of the plate to toxin containing side. Repeat the same procedure with control strains on the same plate.
- Incubate anaerobically at 35-37°C for 24-48 hrs.
- Examine the plate for an opalescent halo around the inoculum and inhibition by antitoxin.
Result and interpretation:
|A zone of opacity in the antitoxin-free half only but not on another half due to neutralization of the alpha-toxin.||Lecithinase positive|
|A zone of opacity on both sides of the plate or no reaction on the agar.||Lecithinase negative|
Following strains can be used for quality control testing in naglers reaction
- Clostridium perfringens ATCC 13124 – Lecithinase positive
- Clostridium sporogenes ATCC 11437 – Lecithinase negative
- Bacteroides fragilis ATCC 25285 – No activity on agar
Limitations of the test:
- Maintenance of anaerobic conditions is compulsory.
- A negative lecithinase test should be compared to an un-inoculated control plate, as lecithinase can diffuse throughout the entire agar plate and make interpretation difficult.
- C. perfringens type A antitoxin is not specific for C. perfringens ; a positive Nagler reaction can also be produced by C. bifermentans , C. sordelli , and C. baratti if heavy inoculum is used.