Principle of  Leucine amino peptidase (LAP) test:

Leucine amino peptidase (LAP) test is used for the preliminary characterization of catalase negative, gram-positive  cocci.  This is a rapid test for detection of enzyme leucine aminopeptidase. Leucine- β- napthalamide impregnated disk serves as a substrate for the detection of leucine aminopeptidas.  This enzyme hydrolyse the substrate resulting in production of β- naphthylamine which upon addition of p-dimethyl, aminocinnamaldehyde reagent form a highly visible red colored Schiff base.
LAP test is often used in conjunction with PYR and other biochemical tests to help differentiate between catalase negative, gram-positive cocci. In general, Streptococcus pneumoniae and Streptococcus pyogenes, Pediococcus, Lactococcus, and Enterococcus sps are all LAP positive, while other beta-hemolytic Streptococci, Aerococcus  sps and Leuconostoc sps are LAP negative.

Procedure/ Method of LAP  test:

1. Aseptically place a LAP disk in a sterile petri dish, and allow disk to warm to room temperature.
2. Moisten the LAP Disk with a small amount of sterile distilled water.
3. Inoculate with several colonies from an overnight culture plate.
4. Incubate at room temperature for five minutes
5. Add one drop of cinnamaldehyde reagent.

Interpretation of Results:

Positive: Development of a red/pink color with 1 minute after addition of cinnamaldehyde reagent

Negative: No change or a slight yellow color

Notes:

• Confirm that the test organism is a gram positive coccus and is catalase negative before performing the LAP test.
• Ensure that adequate inoculum is used or false negatives may occur.
• Streptococci should be tested prior to 48-hour incubation or sub-cultured prior to testing.

Control organisms

Positive control: Enterococcus faecalis ATCC 29212

Negative Control: Aerococcus viridans ATCC 11563