LAP Test- Principle, Procedure, Results, Uses

The LAP test is a biochemical test that is used to assess the ability of bacteria to synthesize leucine aminopeptidases enzymes. These enzymes are involved in the hydrolysis of amino acids from the N-terminal end of peptides and proteins. The LAP test can also be used to differentiate and presumptively identify catalase-negative Gram-positive cocci bacteria, such as Streptococcus spp., Enterococcus spp., Lactococcus spp., and Pediococcus spp.

The LAP test is based on the ability of some bacteria to produce leucine aminopeptidase (LAP) enzymes that can break down the amino acid leucine. The LAP test uses a special disk that contains leucine-p-naphthylamide, a synthetic substrate for LAP enzymes. When the bacteria are inoculated onto the disk, they release LAP enzymes that hydrolyze leucine-p-naphthylamide into leucine and p-naphthylamide. The p-naphthylamide is then detected by adding a chemical reagent called N,N-dimethylaminocinnamaldehyde (also known as PYR reagent), which reacts with p-naphthylamide to form a bright pink or cherry red color.

Reagents

• LAP Disks: These are disks impregnated with leucine-p-naphthylamide, which is the substrate for the LAP enzymes.
• LAP Reagent: This is a solution of 0.01% N,N-dimethylaminocinnamaldehyde in hydrochloric acid.
• Distilled water: This is used to moisten the LAP disk and to dilute the LAP reagent if needed.

Equipment

• PPE and other general laboratory materials
• Test Organisms: Well-isolated colonies of the sample bacteria, positive and negative control organisms

1. Place a LAP disk in a sterile petri plate with forceps.
2. Moisten the disk with sterile distilled water.
3. Using a sterile inoculating loop, pick up 1 or 2 loops full of well-isolated sample bacteria and rub them onto the LAP disk.
4. Incubate the plate aerobically for 5 minutes at room temperature.
5. Add 1 to 2 drops of LAP reagent over the seeded LAP disk.
6. Observe for color change after 2 minutes of addition of the reagent.

The result of the LAP test is based on the color change of the LAP disk after the addition of the LAP reagent. The color change indicates the presence or absence of leucine aminopeptidase enzymes in the sample bacteria.

To ensure the accuracy and reliability of the LAP test, it is important to use quality control strains along with the sample bacteria. The recommended quality control strains for the LAP test are:

• Positive control: Enterococcus faecalis ATCC 29212
• Negative control: Aerococcus viridans ATCC 11563

• Store the LAP disk and reagent in cold and dark conditions.
• Do not saturate the LAP disk with distilled water, just moisten it slightly.
• Transfer a heavy inoculum and rub it over the disk.
• Use fresh cultures of bacteria for the test.
• Read the results within 2 minutes of adding the LAP reagent.
• Do not use the LAP test for bacteria that are catalase-positive, Gram-negative, or non-cocci.

The LAP test is mainly used for the definitive differentiation and identification of catalase-negative, Gram-positive cocci bacteria. The LAP test can help to distinguish these bacteria from other catalase-negative, Gram-positive cocci such as Leuconostoc, Aerococcus and Tetragenococcus, which are usually LAP negative.

• The LAP test is only applicable to catalase-negative, Gram-positive cocci bacteria.
• The LAP test may give false negative results if the inoculum is insufficient or if the bacteria are inhibited by the disk or the reagent.
• The LAP test is not enough for the complete identification of bacteria.

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