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

Leucine Aminopeptidase (LAP) Test: Introduction, Principle, Procedure, Result Interpretation and Limitations

Introduction of Leucine Aminopeptidase (LAP) Test

LAP stands for leucine aminopeptidase which is an enzyme useful for deﬁnitive identiﬁcation of catalase-negative, gram-positive cocci of Aerococcus and Leuconostoc (LAP test negative) from other organisms in the group like Streptococcus, Enterococcus, Lactococcus, and Pediococcus (almost always positive).

Principle of Leucine Aminopeptidase Test

Hydrolysis of the leucine-pnaphthylamide substrate by the enzyme LAP on a paper disk inoculated with the bacteria like Streptococcus, Enterococcus, Lactococcus, and Pediococcus releases leucine and free b-naphthylamide. The bnaphthylamide combines with cinnamaldehyde reagent to form a pink to cherry-red color.

Requirements for Leucine Aminopeptidase Test

Test organisms -Fresh growth less than 24 hours old of catalase-negative, gram-positive cocci. If growth is poor, a culture that has been incubated for up to 72 hours may be used.
LAP Disk
0.01% p-Dimethylaminocinnamaldehyde (the same reagent as used in PYR test)
Sterile loop or stick
Petri dish or slide
Control strains

Positive Control (PC):Enterococcus faecalis ATCC 29212

Negative Control (NC): Aerococcus viridans ATCC 11563

Procedure of Leucine Aminopeptidase Test

Place disk onto a petri dish or a slide.
Moisten (do not saturate the disk) with a loopful of distilled water.
Using a sterile stick or loop, smear with the suspected isolate.
Incubate at room temperature for 5 minutes.
Add a drop of cinnamaldehyde reagent and wait 2 minutes to observe the color.

Result and Interpretation of Leucine Aminopeptidase Test

LAP test positive: Development of a deep red to reddish-purple after addition of cinnamaldehyde reagent

LAP test Negative: No color change or development of a yellow color

Enterococcus faecalis ATCC 29212: Positive

Aerococcus viridans ATCC 11563: Negative

Limitations of Leucine Aminopeptidase Test

False negatives may result from insufﬁcient inoculum.
This test is only one of a battery of 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 chaining cocci.

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