Pyrrolidonyl Arylamidase (PYR) Test: Principle, procedure and results

Pyrrolidonyl Arylamidase (PYR) test is a rapid test which is used for the presumptive identification of group A beta-hemolytic Streptococci and Enterococci. PYR test is available in different format.

Original PYR test used to take 16-20 hours but now 4-hour broth assay is available, which is described in this post. Other formats include rapid (10-15 minutes) tests, in which PYR reagent impregnated filter paper disks or strips are inoculated with the organisms to be tested.

The substrate for the PYR test is L-naphthylamide-β-naphthylamide which is hydrolyzed by a specific bacterial aminopeptidase enzyme.

Hydrolysis of the substrate by this enzyme releases free β-naphthylamide, which is detected by the addition of N, N-dimethylaminocinnamaldehyde. This detection reagent couples with the naphthylamide to form a red Schiff base.

Procedure of PYR test

  1. With a sterile bacteriologic loop, pick up the growth of two to three morphologically similar colonies and emulsify them in the small volume of PYR broth
  2. Incubate the tube at 35oC for 4 hours
  3. Add one drop of PYR reagent and observe for color change.
  4. The reaction should be read and recorded 1 minute after the addition of reagent.

Results and interpretations

  1. Positive: the development of a deep cherry red color within a minute of addition of the reagent
  2. Negative: A yellow or orange color

Quality control

  1. Positive control: Enterococcus faecalis, Streptococcus pyogenes
  2. Negative control: Streptococcus agalactiae

Note: It is essential that testing be performed before the PYR test to determine that the organism is a streptococcus (i.e. gram positive cocci, catalase-negative).
Other organisms (e.g., some aerococci, staphylococci, nutritionally variant streptococci, Arcanobacterium haemolyticum) may also be PYR positive.


About Acharya Tankeshwar 460 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.


  1. Thanks a lot for very useful information! One doubt do exists – it seems that “L-naphthylamide-β-naphthylamide” is uncorrect name of reagent (substrate). It should be “pyrrolidonyl-beta-naphthylamide”. Or both reagents can be used in this test?

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