Leucine Aminopeptidase Test Microbiology

In this leucine aminopeptidase test microbiology post we have briefly explained Leucine Aminopeptidase test principle, objectives, requirements, procedure, uses and limitations of Leucine Aminopeptidase test.

Leucine Aminopeptidase Test Microbiology

Leucine Aminopeptidase test are used to characterise catalase-negative, gram-positive cocci in the laboratory. To distinguish between catalase-producing and gram-positive cocci, the Leucine Aminopeptidase test is frequently used in conjunction with PYR and other biochemical tests.

Streptococcus pneumoniae and Streptococcus pyogenes, as well as Pediococcus, Lactococcus, and Enterococcus species, are Leucine Aminopeptidase test positive, whereas other beta-hemolytic Streptococci, Aerococcus, and Leuconostoc species are Leucine Aminopeptidase test negative.

The discs are impregnated with leucine-ß-naphthylamide, which is degraded by the Leucine Amino Peptidase positive organism’s enzyme leucine aminopeptidase. When the p-dimethylaminocinnamaldehyde reagent is introduced, the ß-naphthylamine is released, which interacts with the p-dimethylaminocinnamaldehyde reagent to generate a highly visible red Schiff base.

Objective

To detect the presence of the enzyme leucine aminopeptidase for the preliminary characterization of catalase-negative, gram-positive cocci, especially non-beta-hemolytic cocci.

Principle

Leucine Aminopeptidase test is one of the quick tests used to identify the enzyme leucine amino peptidase.

Leucine Aminopeptidase test is used to characterise catalase-negative, gram-positive cocci, particularly non-beta hemolytic cocci, before further testing.

The substrate is a leucine-β-naphthalamide impregnated disc. Leucine Aminopeptidase test enzyme is in charge of hydrolyzing the substrate to produce -naphthalamine.

When the result is mixed with p-methyl (an aminocinnamaldehyde reagent), it generates a bright crimson Schiff’s base.

Requirements

Cinnamaldehyde reagent

Distilled water

Petri dish

Wooden applicator stick

Procedure

1. In a sterile petri dish, place an Leucine Amino Peptidase disc and allow it to warm to room temperature.

2. Use reagent grade water or a little amount of sterile distilled water to lightly dampen the Leucine Amino Peptidase disc.

3. Rub a small amount of several colonies of an 18 to 24 hour pure culture onto a small area of the Leucine Aminopeptidase test disc with a wooden applicator stick.

4. Allow for a 5-minute incubation period at room temperature. After the incubation period, add 1 drop of cinnamaldehyde reagent and read within one minute.

Results

Positive result: The Leucine Aminopeptidase test positive test is indicated by the development of a red/pink color.

Negative result: The Leucine Aminopeptidase test negative test is suggested by no change or slight yellow color.

Quality control

Expected Outcomes of the Organism: ATCC 29212 Enterococcus faecalis + ve – Red colour, ATCC 11563 Aerococcus viridans -ve – No colour change.

Limitations

1. Before performing the Leucine Aminopeptidase test, establish that the test organism is gram-positive coccus and catalase negative.

2. Streptococci should be tested prior to 48-hour incubation or subculture prior to testing to avoid false negatives.

Further Readings

Reference