University of Nebraska-Medical Center

Clinical Laboratory Science Program

CLS 418 & 419

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For student use only

PYR Hydrolysis Procedure

Principle
The PYR test is used in the identification of streptococci. It is useful in the identification of Group A
streptococci and the differentiation of Enterococcus from Group D streptococcus. Enterococcus species
are salt-tolerant, hydrolyze esculin in the presence of bile, and also hydrolyze L-pyrrolidonyl-betanapthylamide (PYR).
The substrate used for the PYR test is L-pyrrolindonyl-B-naphthylamide. This compound is hydrolyzed by
the L-pyrroglutamyl-aminopeptidase enzyme. The hydrolysis of the substrate by this enzyme produces a
beta-naphthylamine. When the beta-naphthylamine combines with a cinnamaldehyde reagent, a bright
red color is produced.
Specimen Collection and Preparation
Testing should be performed on colonies taken from a blood agar plate and growth must be less than 24
hours old, 15-18 hours being optimal.
Reagents
1. Sterile Sticks or inoculation loop
2. Sterile normal saline
3. Slides
4. PYR kit
Storage
1. PYR kit - PYR discs and reagents should be stored at 2-8o C in the original container.
Quality Control
Quality control should be performed per lot/shipment date.
Positive control - Enterococcus faecalis ATCC 29212
Negative control - Streptococcus agalactiae ATCC 12386
Expected results
1. Enterococcus faecalis ATCC 29212 a development of a pink or cherry red color with 1 minute after
addition of the color developer
2. Streptococcus agalactiae ATCC 12386 no color change within 1 minute after addition of the color
developer
Procedure
1. Bring disks to room temperature.
2. Place PYR disc on a disposable glass microscopic slide.
3. Moisten the disc by adding a small volume of sterile normal saline (5-10 uL) directly to the disc.
NOTE: Do not flood the disc.
4. With a sterile loop or stick, pick up several well isolated colonies from an 18-24 hour sheep blood
plate. Do not gouge the surface of the agar. Gently rub a heavy visible inoculum onto small area of
the disc. False negative will occur if the inoculum is too small.
5. Incubate the inoculated disc at room temperature for 2 minutes.
6. Dispense 1 drop of color developer onto the disc.
Interpretation of Test
1. A positive result is indicated by the development of a pink or cherry-red color within 1 minute after
addition of the color developer.
2. A negative result is indicated by no color change within 1 minute after addition of the color developer
References
1. Bellows A, Hausler W, Kerrmann K. Manual of Clinical Microbiology, 5th ed., ASM, 1991.
2. Remel package insert.