Objective of Lecithinase Test
The objective is to determine the ability of microorganisms to produce the enzyme lecithinase and to identify the bacteria which are capable of producing lecithinase enzyme.
Principle of Lecithinase Test
Lecithinases or phospholipases are enzymes released by bacteria that have the ability to destroy animal tissues and play a role in pathogenecity. Lecithinase, which is also called phospholipase C, is such an enzyme that splits the phospholipid lecithin. Phospholipid complexes are usually emulsifying agents occurring in tissues, serum and egg yolk. Lecithin is a normal component of the egg yolk. Lecithinase activity is used to characterize several gram positive and gram negative bacteria. Bacterial lecithinases break down this lecithin to an insoluble diglycerides resulting in an opaque halo, surrounding the colony when grown on the egg yolk agar medium.
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. In egg yolk agar, the lipoprotein component Lecithovitellin can also be split by lecithinase into phosphorylcholine and an insoluble diglyceride, which results in the formation of a precipitate in the medium. This precipitate occurs as a white halo, surrounding the colony that produces lecithinase enzyme. The opalescence created is due to the release of free fat.
Bacillus cereus, aerobic spore producer is a strong lecithinase positive organism, can be identified using Mannitol-egg yolk-polymyxin (MYP) agar plates. B. cereus is typically mannitol-negative, produce pink-red colonies in MYP agar with a zone of precipitate around the colonies, indicating lecithinase-positive activity. For the identification of Clostridium perfringens and differentiating it from other Clostridia species, Egg Yolk Agar is used. It is an enriched, non-selective, and differential medium, and is also used in the Nagler Test for the presumptive identification of Clostridium perfringens. It is packed in oxygen – free, reduced state in order to prevent the formation of toxic oxidized by-products that will damage the obligate anaerobes and inhibit the growth of more fastidious species. A positive lecithinase test is characterized by the appearance of a white, opaque, diffuse zone that extends into the medium surrounding the colonies.
Media Used in Lecithinase Test
Egg yolk agar medium
|Pancreatic Digest of Casein||15.0gm|
|Vitamin K 1||10.0gm|
|Papaic Digest of Soybean Meal||5.0gm|
|Egg Yolk Emulsion||100.0ml|
Final pH 7.0 +/- 0.3 at 25ºC.
Procedure of Lecithinase Test
- Take a loopful of test organism and streak it as a straight line on the plate.
- Incubate anaerobically in a gas pak jar immediately after streaking and transfer into the incubator maintained at 35-37o C for 24-48 hours for anaerobes and for aerobes incubate the plate at 35-37o C for 24-48 hours.
- Examine the plate for the opalescent halo surrounding the inoculums.
Result Interpretation of Lecithinase Test
Positive lecithinase test: appearance of a white, opaque, diffuse zone that extends into the medium surrounding the colonies.
Negative lecithinase test: absence of a white, opaque zone extending from the edge of the colony.
Limitations of Lecithinase Test
- Maintenance of anaerobic condition 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.
- perfringenstype 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 are used.
Quality Control of Lecithinase Test
Clostridium perfringens, ATCC13124: lecithinase positive
Clostridium sporogenes, ATCC11437: lecithinase negative