PYR Test: Principle, Procedure, Results

Pyrrolidonyl Arylamidase (PYR) test is a rapid test that is used for the presumptive identification of group A beta-hemolytic streptococci and enterococci. PYR test is also used for the identification of Escherichia coli (-ve), separating it from other indole-positive, lactose-positive, Gram-negative rods.

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


PYR test is available in different formats. 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.

PYR broth test Positive (1,4 and 5): brilliant red-fuchsia color. Negative (2&3)  yellow or weakly orange color
PYR broth test (image source: here)
Positive (1,4 and 5): brilliant red-fuchsia color. Negative (2&3) yellow or weakly orange color 

PYR Broth 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 35°C 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 the reagent.

PYR Disc Test

  1. Using forceps, place PYR disk in Petri dish.
  2. Moisten, but do not saturate, disk with sterile water.
  3. Using a sterile stick, remove one or two loopfuls of culture from a blood agar plate that is 24 to 48 hours old. Use several loopfuls for organisms that take 48 hours or more to grow.
  4. Smear the colonies onto PYR disk.
  5. Allow to react for 2 min (extend the time to 10 min for poorly growing organisms).
  6. After the incubation period, add 1 drop of cinnamaldehyde reagent and observe for red color.
PYR Disc Test
PYR Disc Test (Left-Positive; Right-Negative)

Results and interpretations

  1. Positive: the development of a deep cherry red color within a minute of addition of the reagent
  2. Negative: no color change or 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 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.


  • If the disc is too moist, a false-negative test can occur.
  • If selective media or tube biochemical agars are used to provide inocula, false-negative tests result may appear.

References and further readings

  1. Procop, G. W., & Koneman, E. W. (2016). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Seventh, International edition). Lippincott Williams and Wilkins.
  2. Andrea J. Linscott, 2016, Clinical Microbiology Procedures Handbook, 4th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.CMPH.ch2.1

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

3 thoughts on “PYR Test: Principle, Procedure, Results


  2. 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?

    1. Thank you Oleksandr for your comment. Sorry, I did not notice your comment earlier. The typo has now been corrected.

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