Leucine Aminopeptidase (LAP) Test: Principle, Procedure, Results

Leucine Aminopeptidase (LAP) test is used to identify catalase-negative gram-positive cocci. Specifically, it differentiates Aerococcus and Leuconostoc from Streptococcus, Enterococcus, Lactococcus, and Pediococcus.

This test is mostly used for the preliminary characterization of Streptococcus-like organisms. This test is only one of a battery of useful tests for identifying catalase-negative, Gram-positive cocci. For example, the streptococcal identification panel (API Rapid Strep) also contains a LAP test.

Principle

The LAP disk is a rapid test for detecting the enzyme leucine aminopeptidase. Leucine-beta-naphthylamide impregnated disk serves as a substrate for the detection of leucine aminopeptidase. Following hydrolysis of the substrate by enzyme, the resulting beta- naphthylamine produces a red color upon adding cinnamaldehyde reagent.

Quality control

Before using, perform QC on each new lot or shipment of LAP disks and color reagent. Since the test is performed infrequently and most Gram-positive cocci are LAP positive, verify potency by testing the positive control each time a negative test is obtained using test organisms.

1. Enterococcus faecalis ATCC 29212—LAP positive
2. Aerococcus viridans ATCC 11563—LAP negative

Method

1. Before incubation, slightly dampen the LAP disk with reagent-grade water. Do not supersaturate the disk.
2. Using a wooden applicator stick, rub a small amount of several colonies of an 18 to 24-hour pure culture onto a small area of the LAP disk.
3. Incubate at room temperature for 5 minutes.
4. After the incubation period, add 1 drop of cinnamaldehyde reagent.

Expected results

1. Positive: Development of a deep red to reddish-purple color within 3 minutes after adding cinnamaldehyde reagent.
2. Weak reactions are pink.
3. Negative: No color change or development of a slight yellow color.

LAP Test Results

The LAP test result of some catalase-negative, Gram-positive cocci is as follows:

• LAP negative: Leuconostoc
• LAP positive: Streptococcus, Enterococcus, Lactococcus, and Pediococcus
• LAP variable: Aerococcus

Limitations

1. LAP test is one of the useful tests for identifying catalase-negative, Gram-positive cocci and is most helpful when kit tests yield uncommon identifications or Gram stain results do not show cocci in chains.
2. If the inoculum is insufficient, false-negative results may be seen.

References and further readings

1. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814
2. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition