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PYR Test Principle and Procedure

In this PYR test principle and procedure post we have briefly explained PYR test principle, objectives, requirements, procedure, uses and limitations of Pyrrolidonyl Arylamidase test.

Pyrrolidonyl Arylamidase Test

Pyrrolidonyl Arylamidase test is a rapid colorimetric method based on the activity of the enzyme pyrolidonyl arylamidase for presumptive identification of enterococci and Group A Streptococcus. The substrate for pyrolidonyl arylamidase detection is L-pyroglutamic acid β-napthylamide, which is impregnated into the test disc.

The substrate is hydrolyzed to produce beta-naphthylamide, which reacts with the Pyrrolidonyl Arylamidase test Reagent (p-dimethylamino-cinnamaldehyde) to produce a brilliant pink to cherry red colour. The presumptive identification of group A streptococci and Enterococcus spp. is possible with a positive Pyrrolidonyl Arylamidase test.


Casein peptone is a source of growth factors and trace minerals. The osmotic balance is maintained by sodium chloride. S. Enterococcus spp. and pyogenes L-pyrrolidonyl aminopeptidase hydrolyzes the substrate L-pyroglutamic acid β—naphthylamide (PYR) to generate a free β-naphthylamine. After that, the amine reacts with the cinnamaldehyde reagent to produce a vivid red end product.

Media Composition

Casein Peptone – 20.0  g, Sodium Chloride – 2.0   g, Beef Heart Infusion – 3.1  g,    Disodium Phosphate – 0.4   g, Sodium Carbonate- 2.5  g, L-pyroglutamic acid-β-naphthylamide – 0.1 g, Dextrose-2.0 g, Demineralized Water – 1000.0 ml. pH 7.8 ± 0.2 @ 25°C

Broth Preparation

In 1000 mL pure / distilled water, dissolve 37 grams. If required, heat the medium to completely dissolve it. Mix thoroughly and pour into tubes or flasks as needed. Autoclave at 15 lbs pressure (121°C) for 15 minutes to sterilise.


Broth Method

1. Use the right techniques to make sure the test isolate is streptococci or enterococci. Use 3-5 colonies from a pure, 18-24 hour culture to inoculate the broth.

2. Incubate for 4 hours at 33-37°C in an aerobic environment. Add 1-2 drops of PYR Reagent (REF R21258) to the tube after incubation. Within 1-2 minutes, look for a red colour to appear in Pyrrolidonyl Arylamidase test.

Disk Method

1. Wet the Pyrrolidonyl Arylamidase test disc on the strip with 10 l sterile distilled water or deionized water. Note: Do not flood the disk.

2. Place 5-10 colonies of the tested strain on the surface of the disc with a loop after 18-24 hours of culture and lightly smear them on it.

3. At room temperature, incubate the disc for 1-2 minutes. After incubation, add 1 drop of N, N-dimethylaminocinnamaldehyde. Within 1-2 minutes, look for red colour development in Pyrrolidonyl Arylamidase test.



Positive & negative Pyrrolidonyl Arylamidase test (streptococcus pyogenes is PYR positive). Image Source:

Positive test

The positive Pyrrolidonyl Arylamidase test is indicated by the appearance of bright pink or cherry red color within 1-2 mins. Examples: Citrobacter, Klebsiella etc. Enterococcus faecalis, Streptococcus pyogenes.

Negative test

The negative Pyrrolidonyl Arylamidase test is suggested by no any color change or a blue color because of indole reaction. Examples: Streptococcus bovis, S.equinus,Streptococcus agalactiae, Streptococcus gallolyticus.


1. A positive Pyrrolidonyl Arylamidase test indicates a high likelihood of enterococci and S. pyogenes detection. Other species, such as gram positive cocci, may, nevertheless, hydrolyze PYR.  For a conclusive identification, additional biochemical tests may be required.

2. Pyrrolidonyl Arylamidase test can be used to distinguish group A streptococci and Enterococcus spp. from other streptococci in a presumptive manner.  For a full identification, further testing with a pure culture is recommended.

3. If the disc is too wet, a false negative test can occur.  When selective media is utilised to provide inoculate, false negative tests might occur.

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