L-Pyrrolidonyl Arylamidase (PYR) test: Principle, Requirements, Procedure and Results




L-Pyrrolidonyl Arylamidase (PYR) test: Principle, Requirements, Procedure and Results
L-Pyrrolidonyl Arylamidase (PYR) test: Principle, Requirements, Procedure and Results source: microbiologyinfo.com

Principle of PYR test:

  • L-Pyrrolidonyl Arylamidase (PYR) test is one of the rapid tests employed for the identification of group a beta-hemolytic Streptococci and Enterococci on the basis of activity of the enzyme pyrolidonyl arylamidase.
  • It is also termed as pyrrolidonyl aminopeptidase.
  •  PYR is a bacterial enzyme
  • The test disk is impregnated with L-pyroglutamic acid-β-naphthylamide that is hydrolysed by PYR hence serves as a substrate for the detection of PYR
  • Hydrolysis of the L-pyroglutamic acid-β-naphthylamide yields beta-naphthylamine which in combination with the PYR Reagent (p-dimethylamino-cinnamaldehyde)  forms a bright pink to cherry red colour.
  • Either broth assay method or rapid disk method is used for the test.

Procedure:

  • Broth Method
    • Inoculate PYR broth with 2-5 colonies from overnight (18-24 hours) pure culture.
    • Incubate the tube aerobically at 35-37°C for 4 hours.
    • Add 2-3 drop of PYR reagent and observe for colour change.
    • Observe for the red colour development within 1-2 minutes.
  • Disk Method (Rapid)
    • With 10 µl sterile distilled water or deionized water, wet the PYR test disc on the strip.
    • Note: Do not flood the disk.
    • From 18-24 hrs culture, place 5-10 colonies of the tested strain on the surface of the disc with a loop and smear them lightly on it.
    • Incubate the disc for 1-2 minutes at room temperature.
    • Add 1 drop of N, N-dimethylaminocinnamaldehyde after incubation.
    • Observe for red colour development within 1-2 minutes.

Results interpretations:

  • Positive test:
    • The positive test is indicated by the appearance of bright pink or cherry red color within 1-2 mins. Examples: Citrobacter, Klebsiella etc.
  • Negative test:
    • The negative test is suggested by no any color change or a blue color because of indole reaction. Examples: Streptococcus bovis, S. equinus

Limitations:

  • PYR is only for the presumptive identification of group A Streptococci and group D enterococci from other streptococci thus other tests are recommended for complete identification.
  • If the disk or filter paper are too moist, a false-negative test can result.
  • Few isolates of lactococci and aerococci maybe PYRase positive.
  • If reactions are read after 20 seconds, non-specific color reactions may occur.

L-Pyrrolidonyl Arylamidase (PYR) test: Principle, Requirements, Procedure and Results