PYR Broth Test and Disc method For Steptococcus pyogenes

PYR broth  is generally used for the isolation and identification of Streptococcus pyogenes .It is highly sensitive test and often replaces bacitracin and salt tolerance tests.

It is a presumptive test for both group A and D enterococcal streptococci which determine the hydrolysis activity of enzyme L-pyrrolidonyl arylamidase (PYR) produced by Streptococcus pyogenes but not by other beta haemolytic streptococci were free b-naphthylamide is then detected by addition of the diazo dye complex, N ,Dimethylaminocinnamaldehyde.A Development of a red colour is indicative of PYR hydrolysis .

Principle of PYR Broth

  • Beef heart infusion and peptic digest of animal tissue provide nitrogenous nutrients.
  • The sugar dextrose is the carbohydrate serving as an energy source.
  • Disodium phosphate serves as buffering agent
  • As for sodium chloride,it maintains osmotic balance.
  • Chromogenic mixture provides substrate for PYR enzyme.

Composition of PYR Broth

IngredientsGrams/Litres
Beef heart infusion from500.000
Peptic digest of animal tissue 20.000
Dextrose2.000
Sodium chloride2.000
Disodium phosphate0.400
Sodium carbonate2.500
Chromogenic mixture0.100
Final pH at temperature at 25°C7.8±0.2

Preparation of PYR Broth

  • Start by Suspending 37 grams in 1000 ml distilled water
  • Heat if necessary to dissolve the medium completely.
  • Now mix well and dispense as desired.
  • Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Appearance of PYR Broth

  • It appears as a Light yellow coloured clear solution

PYR Test  Procedure using PYR Broth

  • Pick  3-5 colonies of the suspected organisms  from a 18-24 hours pure culture plate and Inoculate on PYR broth
  • Now incubate the tube aerobically at 35-37°C for 4 hours.
  • Add 1-2  drops of PYR reagent directly into the inoculated PYR broth
  • Observe for the red color development within 1-2 minutes.
Also read  Stool examination (Wet mount vs Lugol iodine staining) Method

PYR Test  Procedure using Disk Method

  • Start by wetting the PYR test disc on the strip with 10 µl sterile distilled water or deionized water.

Take Note not to flood the disk

  • Using a wire loop,pick up 5-10 colonies of the suspected organism from a 18-24 hours pure culture and make a light smear on the surface of the disc
  • Incubate the disc for 1-2 minutes at room temperature.
  • After incubation, add 1 drop of PYR reagent (N, N-dimethylaminocinnamaldehyde)
  • Observe for red color development within 1-2 minutes.

Interpretation of PYR Test

PositiveBright pink or cherry-red color within 1-2 minutes.
NegativeNo color change or a blue color due to a positive indole reaction
Broth-PYR-Test result
Broth-PYR-Test result
PYR Disc Method result
PYR Disc Method result

Cultural Test Response

Streptococcus pyogenespositive, red colouration
Enterococcus faecalispositive, red colouration
Escherichia coli negative
Streptococcus agalactiaenegative

Reference

  • Facklam R. R., Thacker L. G, Fox B., Eriquez L., 1982, J. Clin. Microbiol., 15 (6), a, 987-990.
  • MacFaddin J. F., 2000, Biochemical Tests for Identification of Medical Bacteria, 3rd Edition, Lippinocott Williams and Wilkins, N.Y. 407-410.
  • Koneman E. W., Allen S. D., Janda W. M., Schreckenberger P. C., Winn W. C. Jr., 1992, Colour Atlas and Textbook of Diagnostic Microbiology, 4th Ed., J. B. Lippinccott Company.
  • Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Eds.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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