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

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Introduction of LAP (Leucine Aminopeptidase) 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).

Enterococcus faecium lactose fermenting colony on CLED agar
Fig. Enterococcus faecium lactose fermenting colony on CLED agar
Gram positive cocci in singles, pairs and short chains of Enterococcus faecium in Gram staining of culture
Fig. Gram-positive cocci in singles, pairs, and short chains of Enterococcus faecium in Gram staining of culture (Mag. 3000X)

Principle of LAP 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 LAP (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 the 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 LAP Test

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

Result and  Interpretation of LAP (Leucine Aminopeptidase) Test

LAP test positive: Development of a deep red to reddish-purple after the 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

Enterococcus faecalis in Gram Staining of Culture at a Magnification of 4000X
Fig. Enterococcus faecalis in Gram Staining of Culture at a Magnification of 4000X

 LAP (Leucine Aminopeptidase) Test-Positive and Negative Bacteria

 LAP (Leucine Aminopeptidase) Test-Positive and Negative Bacteria
Fig.  LAP (Leucine Aminopeptidase) Test-Positive and Negative Bacteria

Limitations of the LAP 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.
Enterococcus faecium in Saline Wet Mount Microscopy of Culture at a magnification of 1600X
Fig. Enterococcus faecium in Saline Wet Mount Microscopy of Culture at a magnification of 1600X

Bibliography

  1. Cowan and Steel’s, manual for the identification of medical bacteria
  2. Lynne S. Garcia, Clinical Microbiology Procedures Handbook
  3. www.dalynn.com/dyn/ck_assets/files/tech/DL10.pdf
  4. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/IFU21129.pdf
  5. https://eportal.mountsinai.ca/Microbiology/manual/tech/tech23.pdf
  6. https://www.sigmaaldrich.com/catalog/product/sial/75554?lang=en&region=NP

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