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
- 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
- Incubate the tube at 35°C for 4 hours.
- Add one drop of PYR reagent and observe for color change.
- The reaction should be read and recorded 1 minute after the addition of the reagent.
PYR Disc Test
- Using forceps, place PYR disk in Petri dish.
- Moisten, but do not saturate, disk with sterile water.
- 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.
- Smear the colonies onto PYR disk.
- Allow to react for 2 min (extend the time to 10 min for poorly growing organisms).
- After the incubation period, add 1 drop of cinnamaldehyde reagent and observe for red color.
Results and interpretations
- Positive: the development of a deep cherry red color within a minute of addition of the reagent
- Negative: no color change or yellow or orange color
- Positive control: Enterococcus faecalis, Streptococcus pyogenes
- 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
- Procop, G. W., & Koneman, E. W. (2016). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Seventh, International edition). Lippincott Williams and Wilkins.
- Andrea J. Linscott, 2016, Clinical Microbiology Procedures Handbook, 4th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.CMPH.ch2.1