LAP (Leucine Aminopeptidase) Test

Leucine aminopeptidase (LAP) is an enzyme that is employed in a test for the presumptive identification of Streptococci. LAP is a peptidase that hydrolyzes peptide bonds near free amino groups. LAP is known as leucine aminopeptidase because it responds most efficiently with leucine. Leucine- β-naphthylamide is hydrolyzed to yield β-naphthylamine. A red color forms with the addition of the paradimethylaminocinnamaldehyde reagent.

There are commercially available rapid tests that utilize filter paper disks soaked with the substrate. The LAP test, which is frequently used in conjunction with the PYR test, is particularly useful in distinguishing Aerococcus and Leuconostoc spp. from other gram-positive cocci. LAP-positive bacteria include Streptococcus, Enterococcus, and Pediococcus spp., while LAP-negative bacteria include Aerococcus and Leuconostoc spp.

Principle

The LAP disk is a simplistic test for detecting the enzyme leucine aminopeptidase. Leucine-beta-naphthylamide moistened disks serve as a substrate for the detection of leucine aminopeptidase. The enzyme hydrolyzes the substrate, and the resulting beta-naphthylamine interacts with the cinnamaldehyde reagent to produce a red color.

Requirements

• LAP Disks
• 0.01% p-dimethylaminocinnamaldehyde
• Sterile loop or stick to obtain colony
• Reagent grade distilled water (neutral pH)
• Petri dish

Procedure

1. Place the disk on the slide or in the petri dish. Using a loopful of distilled water, moisten (but not completely wet) the disk.
2. Smear the presumptive isolate on a sterile stick or loop.
3. Incubate at room temperature for 5 min.
4. Wait 2 minutes to observe the color after adding 1 drop of cinnamaldehyde developer reagent.

Interpretation

Limitation

This test is one of the most important for detecting catalase-negative, Gram-positive cocci, and it is especially beneficial when kit tests give unusual results or Gram stain findings do not indicate chain based cocci. False negatives may occur as a result of inadequate inoculum. The integrity of the substrate impregnated disk influences the test result.

Lap Test Results of Some Gram-Positive bacteria

References

1. Bailey Scotts Diagnostic Microbiology 14^th Edition
2. Clinical Microbiology Procedures Handbook; Amy L Leber
3. Textbook of Diagnostic Microbiology 6^th Edition