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
- Result and Interpretation of PYR Test
- 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
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.
