PYR Test: Principle, Media, Procedure, Results, Uses

Table of Contents

• Introduction
• Objectives
• Principle of PYR Test
• Requirements for PYR Test
• Culture Media
• Reagents
• PYR Impregnated Disk (for the rapid method or disk test)
• Equipment
• Test Organisms (Sample Bacteria)
• Procedure of PYR Test
• Tube Method (PYR Broth or Agar Method)
• Rapid Method (PYR Disk Method)
• Result and Interpretation of PYR Test
• PYR Positive Bacteria
• PYR Negative Bacteria
• Quality Control
• Precautions
• Applications of PYR Test
• Limitations of PYR Test

Introduction: 

• The PYR test (L-Pyrrolidonyl-β-naphthylamide test) is a biochemical test used to detect the ability of bacteria to produce the enzyme pyrrolidonyl aminopeptidase using L-Pyrrolidonyl-β-naphthylamide (PYR) as a substrate.
• It was first reported in 1981 by Godsey J, Schulman R, and Erique LA, who used PYR as a substrate to identify Streptococcus pyogenes and Enterococci.
• This test is now widely used in clinical and research laboratories for diagnostic purposes.
• It is primarily used for the detection of Streptococcus pyogenes, Enterococcus spp., E. coli, and for the identification of Staphylococcus spp.
• There are two methods currently in use for performing the PYR test:
• The tube method, which uses PYR broth.
• The disk method, which uses a PYR-impregnated disk.

Objectives

• Assess the ability of bacteria to produce the enzyme pyrrolidonyl aminopeptidase.
• Biochemically differentiate and presumptively identify sample bacteria using this assessment.

Principle of PYR Test

• If bacteria can synthesize the pyrrolidonyl aminopeptidase enzyme, the L-Pyrrolidonyl-β-naphthylamide substrate in the broth or disk is hydrolyzed.
• This hydrolysis releases β-naphthylamide.
• The released β-naphthylamide reacts with N, N-dimethylaminocinnamaldehyde.
• This reaction forms a bright pink or cherry red Schiff base.
• The appearance of this color indicates a positive PYR test.

Requirements for PYR Test

a. Culture Media

PYR broth is used for the broth (tube) method of the PYR test.

Composition of PYR Broth per 1000 mL

• Beef Heart Infusion: 500.00 grams
• Peptic Digest of Animal Tissue: 20.00 grams
• Dextrose: 2.00 grams
• Disodium Phosphate: 0.40 grams
• Sodium Chloride: 2.00 grams
• Sodium Carbonate: 2.50 grams
• Chromogenic Mixture: 0.10 grams
• Final pH: 7.8 ± 0.2 at 25°C

(Alternatively, PYR agar medium can be used. PYR agar will have all the components of PYR broth with the addition of 15.0 grams of agar powder to make it solidified.)

Preparation of PYR Broth

• Measure the appropriate amount of PYR broth (agar) powder (or the media components).
• Mix in the required volume of water in a conical flask (or glass bottle) according to the instructions of the manufacturing company.
• Stir well using a magnetic stirrer or manually and heat to boiling to ensure all components dissolve completely in water.
• Dispense about 5 mL (or the required volume) of the medium into test tubes.
• Tighten the caps of the test tubes or cotton-plug them.
• Autoclave the tubes at 121°C and 15 lbs pressure for 15 minutes.
• Let the tubes cool to around room temperature (below 40°C).

b. Reagents

PYR Reagent

• Use 0.01% p-N, N-dimethylaminocinnamaldehyde (or 1% p-N, N-dimethylaminocinnamaldehyde) as the PYR reagent.

Composition of PYR Reagent:

• N, N-dimethylaminocinnamaldehyde: 1.0 grams
• Concentrated Hydrochloric Acid (HCl): 1.0 mL
• Distilled Water: 99.0 mL

Preparation of PYR Reagent:

• Add 1.0 gram of N, N-dimethylaminocinnamaldehyde to 99.0 mL of distilled water.
• Slowly add 1.0 mL of concentrated HCl with constant stirring.
• Mix thoroughly until the N, N-dimethylaminocinnamaldehyde is completely dissolved.
• Store the solution at 2 to 8°C in a dark place.

PYR Impregnated Disk (for the rapid method or disk test)

c. Equipment

• Test tubes
• Forceps
• Weighing Machine
• Autoclave
• Bunsen burner
• Incubator
• Inoculating loop
• Petri Plate
• PPE and other general laboratory materials

d. Test Organisms (Sample Bacteria)

• Positive Control: Streptococcus pyogenes ATCC 19615
• Negative Control: E. coli ATCC 25922

Procedure of PYR Test

Tube Method (PYR Broth or Agar Method)

• Using a sterile inoculating loop, pick sample bacteria from a well-isolated colony of fresh culture (18 to 24 hours old).
• Inoculate the PYR broth with the bacteria.
• (Alternatively, prepare a 0.5 McFarland standard suspension of the sample bacteria and transfer a loopful of the suspension to the PYR broth.)
• Incubate aerobically at 35±2°C for about 4 hours (or 18 to 24 hours for PYR Agar).
• Add 1-2 drops of PYR reagent to each tube.
• Observe for a color change within 1 to 2 minutes after adding the reagent.

Rapid Method (PYR Disk Method)

• Place a PYR disk in a sterile petri plate using forceps.
• Moisten the disk with sterile distilled water.
• Using a sterile inoculating loop, pick up 1 or 2 loops full of well-isolated sample bacteria, preferably from Blood Agar Plate (B-hemolytic colonies from BAP), and rub them onto the PYR disk.
• (Transfer several loops full of bacteria if the sample bacteria is slow-growing and requires 48 hours or more of incubation for growth.)
• Incubate the plate aerobically at room temperature for 2 minutes.
• (Extend the incubation to about 10 minutes for slow-growing bacteria.)
• Add 1 to 2 drops of PYR reagent directly onto the PYR disk.
• Observe for a color change within 1 to 2 minutes after adding the reagent.

Result and Interpretation of PYR Test

• A bright pink or cherry red color developing over the disk or in the broth (agar) within 1 to 2 minutes after adding the PYR reagent indicates a positive reaction (PYR positive).
• No color change or the formation of blue, yellow, or orange colors after adding the PYR reagent indicates a negative reaction (PYR negative).
• The development of a slight or faint pink color also indicates a negative reaction.

PYR Positive Bacteria

• Streptococcus pyogenes
• Enterococcus faecalis
• Enterococcus faecium
• Citrobacter spp.
• Klebsiella spp.
• Yersinia spp.
• Staphylococcus haemolyticus

PYR Negative Bacteria

• E. coli
• Streptococcus agalactiae
• Streptococcus bovis
• Salmonella spp.
• Staphylococcus aureus

Quality Control

• Streptococcus pyogenes ATCC 19615: Results in the formation of a bright pink or red color on the broth (or over the disk/agar) within 1-2 minutes of adding the PYR reagent.
• E. coli ATCC 25922: Does not change color (remains unchanged) after the addition of the PYR reagent.

Precautions

• Store the PYR reagent in cool, dark conditions.
• Moisten the PYR disk slightly; do not saturate it with water.
• Ensure to transfer a sufficient inoculum and rub it thoroughly over the disk. Using too little inoculum might lead to false negative results, characterized by the formation of a faint pink color.
• Do not interpret faint pink color as indicating a positive reaction.

Applications of PYR Test

• To distinguish Streptococcus pyogenes from other β-hemolytic Streptococci.
• To differentiate Group A and Group D Streptococci (PYR positive) from other Streptococci.
• For identifying E. coli among other Gram-negative bacilli that are indole-positive and lactose-positive.
• To distinguish Citrobacter spp. (PYR positive) from other H2S-positive Gram-negative rods, such as Salmonella spp. (PYR negative).

Limitations of PYR Test

• Additional biochemical tests are necessary to confirm the complete identification of bacteria.
• Excess moisture on the disk can lead to false negative results.
• Taking inocula from selective or tube-biochemical test media can result in false negative results.
• Using inadequate inoculum may lead to false negative results.
• Results should not be interpreted before 1 minute to avoid nonspecific outcomes.
• E. coli and other indole-positive bacteria may exhibit a blue color on the media and disk, which is considered PYR negative and can be confusing.