Objective
To detect the presence of the enzyme leucine aminopeptidase for the preliminary characterization of catalase-negative, gram-positive cocci, especially non-beta-hemolytic cocci.
Principle of Leucine Amino Peptidase (LAP) Test
Leucine aminopeptidase (LAP) test is a rapid test for the detection of enzyme leucine aminopeptidase. 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. Leucine- β- napthalamide impregnated disk serves as a substrate for the detection of leucine aminopeptidase. The enzyme LAP hydrolyzes the substrate resulting in the production of β- naphthylamine which upon addition of p-dimethyl, aminocinnamaldehyde reagent form a highly visible red-colored Schiff base.
Procedure of Leucine Amino Peptidase (LAP) Test
- Aseptically place a LAP disk in a sterile Petri dish and allow the disk to warm to room temperature.
- Moisten the LAP Disk with a small amount of sterile distilled water.
- Inoculate with several colonies from an overnight culture plate.
- Incubate at room temperature for five minutes
- Add one drop of cinnamaldehyde reagent
- Observe for the color change in the disk
Result of Leucine Amino Peptidase (LAP) Test
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Positive test: Development of a red color within 1 minute after adding cinnamaldehyde reagent.
Negative test: No color change or development of a slight yellow color.
Limitations
- The test result depends on the integrity of the substrate-impregnated disk.
- LAP test is often used in conjunction with PYR and other biochemical tests to help differentiate between catalase-negative, gram-positive cocci.
Quality Control
Positive: Enterococcus faecalis (ATCC29212)
Negative: Aerococcus viridans (ATCC11563)
