The PYR test is a biochemical test that detects the presence of pyrrolidonyl aminopeptidase (PYRase) enzyme in bacteria. PYRase is an enzyme that hydrolyzes the synthetic substrate L-pyrrolidonyl-β-naphthylamide (PYR) and releases β-naphthylamide, which reacts with a color reagent to produce a bright pink or cherry red color. The PYR test is used to differentiate and presumptively identify certain groups of bacteria based on their ability to produce PYRase.

The PYR test is especially useful for the identification of Streptococcus pyogenes (Group A Streptococcus), which is the causative agent of streptococcal pharyngitis (strep throat), scarlet fever, rheumatic fever, and other infections. S. pyogenes is PYR positive, while other β-hemolytic streptococci such as Streptococcus agalactiae (Group B Streptococcus) and Streptococcus bovis (Group D Streptococcus) are PYR negative. The PYR test can also help to distinguish Enterococcus spp. (PYR positive) from other non-enterococcal streptococci (PYR negative).

In addition, the PYR test can be used to identify some Gram-negative bacteria such as Citrobacter spp., Klebsiella spp., and Yersinia spp., which are PYR positive, from other Gram-negative bacteria such as Escherichia coli, Salmonella spp., and Shigella spp., which are PYR negative. The PYR test can also differentiate E. coli (PYR negative) from other indole-positive, lactose-positive, Gram-negative bacilli such as Enterobacter spp. and Serratia spp. (PYR positive).

The PYR test can be performed by two methods: the tube method and the rapid method. The tube method uses a liquid or solid culture medium that contains the PYR substrate and requires incubation for 4 to 24 hours before adding the color reagent. The rapid method uses a filter paper disk that is impregnated with the PYR substrate and requires only 2 to 10 minutes of incubation before adding the color reagent. Both methods are simple, inexpensive, and reliable for the detection of PYRase activity in bacteria.

The purpose of this article is to explain the principle, media, procedure, results, uses, quality control, precautions, applications, and limitations of the PYR test in detail. This article will help microbiologists, students, researchers, and health professionals to understand and perform the PYR test effectively and accurately for the identification of bacteria.

The PYR test is based on the ability of some bacteria to produce an enzyme called pyrrolidonyl aminopeptidase (also known as pyroglutamyl aminopeptidase or L-pyrrolidonyl arylamidase). This enzyme can hydrolyze (break down) a synthetic substrate called L-pyrrolidonyl-β-naphthylamide (PYR), which is present in the culture media or impregnated on a disk. The hydrolysis of PYR releases a compound called β-naphthylamide, which has a yellow color.

The β-naphthylamide can then react with another reagent called N, N-dimethylaminocinnamaldehyde (DMACA), which is added after incubation. The DMACA reagent forms a Schiff base with β-naphthylamide, resulting in a bright pink or cherry red color. This color change indicates a positive PYR test, meaning that the bacteria have the pyrrolidonyl aminopeptidase enzyme and can hydrolyze PYR.

The PYR test is useful for differentiating and identifying some groups of bacteria based on their enzymatic activity. For example, it can help to distinguish Streptococcus pyogenes (group A streptococcus) from other β-hemolytic streptococci, as S. pyogenes is PYR positive while most other streptococci are PYR negative. It can also help to identify Enterococcus species, which are also PYR positive, from other non-enterococcal streptococci. Additionally, it can help to differentiate some Gram-negative bacilli, such as Citrobacter and Klebsiella (PYR positive) from Salmonella and E. coli (PYR negative).

The PYR test is simple, rapid and inexpensive to perform, and it has high sensitivity and specificity for some bacterial groups. However, it is not sufficient for the complete identification of bacteria, and it may give false results under certain conditions. Therefore, it should be used in conjunction with other biochemical tests and morphological characteristics to confirm the identity of the sample bacteria.

To perform a PYR test, you will need the following materials and equipment:

• A sample of bacteria that you want to test for the presence or absence of pyrrolidonyl aminopeptidase enzyme. The sample should be a well-isolated colony from a fresh culture (18 to 24 hours old) or a 0.5 McFarland standard suspension of bacteria in sterile saline.
• A culture medium that contains the substrate L-Pyrrolidonyl-β-naphthylamide (PYR broth or PYR agar). You can either prepare the medium yourself by following the instructions given in point 5 or purchase ready-made PYR broth or agar from a reputable supplier.
• A PYR reagent that consists of 0.01% or 1% p-N, N-dimethylaminocinnamaldehyde in hydrochloric acid. You can either prepare the reagent yourself by following the instructions given in point 7 or purchase ready-made PYR reagent from a reputable supplier.
• A PYR impregnated disk (for the rapid method or disk test) that contains L-Pyrrolidonyl-β-naphthylamide and an indicator dye. You can purchase ready-made PYR disks from a reputable supplier.
• Sterile distilled water to moisten the PYR disk and to prepare the bacterial suspension if needed.
• Sterile inoculating loops or needles to transfer the bacterial sample to the medium or disk.
• Test tubes with caps or cotton plugs to hold the PYR broth (for the tube method).
• Petri plates to hold the PYR agar (for the tube method) or the PYR disk (for the rapid method).
• Forceps to handle the PYR disk.
• An incubator to maintain the optimal temperature for bacterial growth (35±2°C).
• A timer to measure the incubation time and the reaction time.
• Positive and negative control organisms to ensure the validity and accuracy of the test results. The recommended positive control is Streptococcus pyogenes ATCC 19615 and the recommended negative control is E. coli ATCC 25922.
• Personal protective equipment (PPE) such as gloves, goggles, lab coat, etc. to prevent contamination and exposure to potentially pathogenic bacteria and chemicals.

The culture media used in the PYR test are either liquid or solid media that contain a chromogenic substrate called L-Pyrrolidonyl-β-naphthylamide (PYR). This substrate is hydrolyzed by the enzyme pyrrolidonyl aminopeptidase (PYRase) which is produced by some bacteria. The hydrolysis releases β-naphthylamide, which reacts with a reagent to form a pink or red color. The presence or absence of this color indicates whether the bacteria are PYR positive or negative.

There are two main types of culture media used in the PYR test: PYR broth and PYR agar. Both media have similar compositions and functions, but differ in their physical state and method of inoculation. PYR broth is a liquid medium that is inoculated with a loopful of bacterial suspension and incubated for 4 hours (or 18 to 24 hours for PYR agar) before adding the reagent. PYR agar is a solid medium that is inoculated with a loopful of bacterial colony and incubated for 10 minutes before adding the reagent.

Another type of culture medium used in the PYR test is the PYR impregnated disk. This is a filter paper disk that contains the PYR substrate and can be moistened with sterile water and inoculated with a loopful of bacterial colony. The disk is incubated for 2 minutes (or 10 minutes for slow-growing bacteria) before adding the reagent.

The advantage of using PYR broth or agar over PYR disk is that they allow for more bacterial growth and enzyme production, which may increase the sensitivity and specificity of the test. The advantage of using PYR disk over PYR broth or agar is that it is faster and simpler to perform, which may save time and resources. However, all three types of culture media can be used to perform the PYR test depending on the availability and preference of the laboratory.

PYR broth is used for the broth (tube) method of the PYR test. It contains the following components per 1000 mL of distilled water:

• 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

The chromogenic mixture consists of L-Pyrrolidonyl-β-naphthylamide and a pH indicator (bromocresol purple). The final pH of the medium is 7.8±0.2 at 25°C.

To prepare PYR broth, follow these steps:

1. Measure the appropriate amount of PYR broth powder (or the media components) and mix in the water of the required volume in a conical flask (or glass bottle) according to the instruction of the manufacturing company.
2. Stir well using a magnetic stirrer or manually and heat to boiling so that all the components dissolve completely in water.
3. Dispense about 5 mL (or the required volume) of the medium in test tubes, tighten the cap of the test tubes, or cotton-plug them.
4. Autoclave the tubes at 121°C and 15 lbs pressure for 15 minutes and let them cool to around room temperature (below 40°C).

Alternatively, PYR agar medium can be used instead of PYR broth. PYR agar will have all the components of PYR broth with 15.0 grams of agar powder to make it solidified. The preparation steps are similar to those of PYR broth, except that the medium is poured into sterile petri plates after autoclaving and allowed to solidify.

The main reagent used in the PYR test is PYR reagent, which is a solution of 0.01% p-N, N-dimethylaminocinnamaldehyde (or 1% p-N, N-dimethylaminocinnamaldehyde) in distilled water and concentrated hydrochloric acid. This reagent is also known as PYR indicator or PYR chromogen.

The PYR reagent reacts with the β-naphthylamide that is released from the hydrolysis of the L-Pyrrolidonyl-β-naphthylamide substrate by the pyrrolidonyl aminopeptidase enzyme. The reaction produces a bright pink or cherry red color that indicates a positive PYR test.

The composition of PYR reagent per 100 mL is as follows:

• N, N-dimethylaminocinnamaldehyde: 0.01 g (or 1.0 g)
• Concentrated hydrochloric acid: 1.0 mL
• Distilled water: 99.0 mL

The preparation of PYR reagent involves the following steps:

• Add 0.01 g (or 1.0 g) of N, N-dimethylaminocinnamaldehyde in 99.0 mL of distilled water and stir well until dissolved.
• Slowly add 1.0 mL of concentrated hydrochloric acid with constant stirring.
• Store the reagent at 2 to 8°C in a dark place.

The PYR reagent should be used within 6 months of preparation and should be discarded if it becomes cloudy, discolored, or precipitated.

Another reagent used in the PYR test is the PYR impregnated disk, which is a filter paper disk that contains the L-Pyrrolidonyl-β-naphthylamide substrate. The disk is used for the rapid method or disk test of the PYR test.

The disk can be prepared by soaking filter paper disks in a solution of L-Pyrrolidonyl-β-naphthylamide and drying them at room temperature. Alternatively, commercially available PYR disks can be used.

The disk should be stored at 2 to 8°C in a sealed container and should be used within 3 months of preparation or expiry date. The disk should not be used if it becomes moist, discolored, or contaminated.

The PYR reagent is a solution of N, N-dimethylaminocinnamaldehyde in hydrochloric acid and water. It is used to detect the presence of β-naphthylamide, which is released by the hydrolysis of L-Pyrrolidonyl-β-naphthylamide substrate by pyrrolidonyl aminopeptidase enzyme. The reagent reacts with β-naphthylamide to form a bright pink or cherry red color Schiff base, indicating a positive PYR test.

To prepare the PYR reagent, follow these steps:

• Add 1.0 grams of N, N-dimethylaminocinnamaldehyde in 99.0 mL of distilled water and stir well until it dissolves completely.
• Slowly add 1.0 mL of concentrated hydrochloric acid to the solution with constant stirring. The solution should turn yellowish.
• Transfer the solution to a dark glass bottle and label it as PYR reagent. Store it at 2 to 8°C in a dark place.
• Before using the reagent, check its color and clarity. It should be clear and yellowish. If it is cloudy or has a different color, discard it and prepare a fresh one.

The PYR reagent is stable for up to 6 months if stored properly. However, it is recommended to prepare it in small batches and use it within a month for optimal results.

Some precautions to take while preparing and using the PYR reagent are:

• Wear appropriate personal protective equipment (PPE) such as gloves, goggles, and lab coat while handling the chemicals and the reagent.
• Avoid contact with skin, eyes, and mucous membranes as the reagent is corrosive and may cause irritation or burns.
• Do not inhale the vapors of the reagent as they may cause respiratory problems or damage the mucous membranes.
• Use a calibrated pipette or dropper to measure and dispense the reagent accurately. Do not use metal or plastic utensils as they may react with the reagent or contaminate it.
• Do not mix the reagent with other chemicals or solutions as they may interfere with the reaction or cause false results.
• Dispose of the used reagent and materials according to the local regulations and safety guidelines.

The equipment required for the PYR test are:

• PPE and other general laboratory materials such as gloves, goggles, lab coat, etc.
• Sterile inoculating loop or needle
• Sterile petri plate and forceps (for the rapid method)
• Incubator
• Test tubes and rack (for the tube method)
• Autoclave
• Magnetic stirrer or manual stirrer (for preparing the broth or agar medium)

The test organisms used in the PYR test are:

• Sample bacteria that need to be identified or differentiated based on their ability to produce pyrrolidonyl aminopeptidase enzyme
• Positive control: Streptococcus pyogenes ATCC 19615
• Negative control: E. coli ATCC 25922

The sample bacteria can be obtained from various sources such as clinical specimens, environmental samples, food samples, etc. The sample bacteria should be well-isolated and grown on a suitable culture medium such as blood agar plate (BAP) for 18 to 24 hours at 35±2°C before performing the PYR test. The sample bacteria should also be Gram-stained and checked for their morphology and arrangement under a microscope.

The positive control is a bacterium that is known to produce pyrrolidonyl aminopeptidase enzyme and gives a positive PYR test result. The positive control is used to verify that the test procedure and reagents are working properly and that the test results are reliable.

The negative control is a bacterium that is known to not produce pyrrolidonyl aminopeptidase enzyme and gives a negative PYR test result. The negative control is used to check for any false positive reactions due to contamination or improper handling of the test materials.

The positive and negative controls should be inoculated and incubated in the same conditions as the sample bacteria and tested with the same reagent. The controls should also be obtained from a reputable source and stored properly according to the manufacturer`s instructions.

Procedure for conducting a PYR test using tube and rapid methods

There are two methods for performing a PYR test: the tube method and the rapid method. The tube method uses PYR broth or agar as the culture medium, while the rapid method uses PYR impregnated disks. The procedure for each method is as follows:

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 culture) and inoculate the PYR broth.
• (Alternatively, prepare 0.5 McFarland standards suspension of sample bacteria and transfer a loop full of the suspension to the PYR broth.)
• Incubate aerobically at 35±2°C for about 4 hours (18 to 24 hours for PYR Agar).
• Add 1-2 drops of PYR reagent.
• Observe for color change after 1 to 2 minutes of addition of the reagent.

Rapid Method (PYR Disk Method)

• Place a PYR disk in a sterile petri plate with 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: -hemolytic colonies from BAP) and rub them onto the PYR disk.
• (Transfer several loops full of bacteria if the sample bacteria is slow-growing bacteria that needs 48 hours or more hours of incubation for growth.)
• Incubate the plate aerobically for 2 minutes at room temperature. (Allow incubation for about 10 minutes for slow-growing bacteria.)
• Add 1 to 2 drops of PYR reagent over the PYR disk.
• Observe for color change after 1 to 2 minutes of addition of the reagent.

The PYR test is based on the detection of pyrrolidonyl aminopeptidase enzyme in bacteria. This enzyme hydrolyzes the substrate L-pyrrolidonyl-β-naphthylamide (PYR) and releases β-naphthylamide, which reacts with N, N-dimethylaminocinnamaldehyde (PYR reagent) to form a bright pink or cherry red color. The presence or absence of this color indicates whether the bacteria are PYR positive or negative.

A positive PYR test is indicated by the development of a bright pink or cherry red color over the disk or broth (agar) within 1 to 2 minutes of adding the PYR reagent. This means that the bacteria have pyrrolidonyl aminopeptidase enzyme and can hydrolyze the PYR substrate. A positive PYR test is characteristic of some bacteria such as Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Citrobacter spp., Klebsiella spp., Yersinia spp., Staphylococcus haemolyticus, etc.

A negative PYR test is indicated by no color change (or the formation of blue, yellow, or orange color) after adding the PYR reagent. This means that the bacteria do not have pyrrolidonyl aminopeptidase enzyme and cannot hydrolyze the PYR substrate. A negative PYR test is characteristic of some bacteria such as E. coli, Streptococcus agalactiae, Streptococcus bovis, Salmonella spp., Staphylococcus aureus, etc.

A slight (faint or weak) pink color after adding the PYR reagent is also considered as a negative reaction. This may be due to the presence of other enzymes that can partially hydrolyze the PYR substrate but not enough to produce a strong color reaction. A slight pink color may also be due to the use of too little inoculum or too moist disk.

The interpretation of the PYR test results should be done in conjunction with other biochemical tests and phenotypic characteristics to confirm the identification of the sample bacteria. The PYR test is useful for differentiating some groups of bacteria such as Group A and Group D Streptococci (PYR positive) from other Streptococci; E. coli (PYR negative) from other indole-positive, lactose-positive, Gram-negative bacilli; Citrobacter spp. (PYR positive) from other H2S-positive Gram-negative rods; Salmonella spp. (PYR negative), etc.

The PYR test is a simple and rapid method for presumptive identification of some bacteria. However, it has some limitations such as false negative results due to inadequate inoculum, moist disk, or selective media; false positive results due to contamination or cross-reaction; unspecific results due to early reading or indole-positive bacteria; etc. Therefore, quality control measures and precautions should be taken while conducting and interpreting the PYR test.

The PYR test can be used to differentiate between various groups of bacteria based on their ability to produce pyrrolidonyl aminopeptidase enzyme. The following table summarizes some of the common bacteria that test positive or negative for PYR.

Some of the bacteria that test positive for PYR are clinically important pathogens that cause infections such as pharyngitis, endocarditis, urinary tract infections, septicemia, and plague. Therefore, the PYR test can be a useful tool for the presumptive identification and diagnosis of these infections.

However, the PYR test is not sufficient for the definitive identification of bacteria, as some bacteria may show variable or ambiguous results. For example, some strains of Enterobacter spp., Proteus spp., and Serratia spp. may give weak positive or negative results depending on the culture conditions and reagent quality. Moreover, some bacteria may produce other enzymes that can interfere with the PYR reaction and cause false positive or negative results. For example, some indole-positive bacteria such as E. coli may produce a blue color instead of a pink color due to the reaction of indole with the PYR reagent. Therefore, the PYR test should be interpreted in conjunction with other biochemical tests and phenotypic characteristics to confirm the identification of bacteria.

Quality control is an essential part of any laboratory test to ensure the accuracy and reliability of the results. Quality control measures for the PYR test include the following:

• Use of positive and negative control organisms to verify the performance of the media, reagents, and test procedure. Streptococcus pyogenes ATCC 19615 is used as a positive control and E. coli ATCC 25922 is used as a negative control for the PYR test. The positive control should produce a bright pink or cherry red color within 1 to 2 minutes of adding the PYR reagent, while the negative control should show no color change or produce a blue color.
• Use of fresh and pure cultures of sample bacteria to avoid false results due to contamination or degradation of the enzyme. The sample bacteria should be taken from an 18 to 24 hours old culture grown on a non-selective medium such as blood agar or nutrient agar.
• Use of appropriate inoculum size and incubation time to ensure sufficient enzyme production and reaction. A heavy inoculum (1 or 2 loops full) of sample bacteria should be transferred to the PYR broth or disk and incubated for about 4 hours (or 10 minutes for slow-growing bacteria) before adding the PYR reagent. A lower inoculum or shorter incubation time may result in false negative results due to weak or delayed color development.
• Use of proper storage and handling conditions for the media, reagents, and disks to prevent deterioration or contamination. The PYR broth or agar should be stored at 2 to 8°C in a dark place and used within the expiry date. The PYR reagent should be stored at 2 to 8°C in a dark place and protected from light exposure. The PYR disks should be stored at room temperature in a dry place and used within the expiry date.
• Use of standardized test procedure and interpretation criteria to avoid errors or discrepancies. The test procedure should follow the manufacturer`s instructions or standard operating procedures. The color change should be observed within 1 to 2 minutes of adding the PYR reagent and compared with the control organisms. A bright pink or cherry red color indicates a positive reaction, while no color change or a blue color indicates a negative reaction. A faint pink color should also be considered as negative.

By following these quality control measures, the PYR test can provide reliable results for the identification and differentiation of bacteria based on their ability to produce pyrrolidonyl aminopeptidase enzyme.

• The PYR reagent should be stored in a cold and dark place to prevent degradation or oxidation. The reagent should also be checked for its expiry date before use.
• The PYR disk should not be saturated with water, as this may dilute the substrate and affect the reaction. The disk should be moistened slightly with sterile distilled water before inoculation.
• The inoculum should be taken from a well-isolated colony of fresh culture (preferably from a blood agar plate) and transferred to the PYR disk or broth in a heavy amount. Using too little inoculum may result in a false negative reaction due to the formation of a faint pink color, which is considered negative.
• The inoculated disk or broth should be incubated for the recommended time and temperature according to the method used. For the rapid method, the incubation time is usually 2 minutes at room temperature, while for the tube method, it is 4 hours at 35°C. For slow-growing bacteria, longer incubation times may be required.
• The PYR reagent should be added after the incubation period and observed for color change within 1 to 2 minutes. Reading the results before or after this time interval may give unspecific results. The development of a bright pink or cherry red color indicates a positive reaction, while no color change or any other color (blue, yellow, orange) indicates a negative reaction. A slight or faint pink color is also regarded as negative.
• The test results should be interpreted in conjunction with other biochemical tests and phenotypic characteristics of the sample bacteria. The PYR test alone is not sufficient for the complete identification of bacteria and may give false positive or negative results in some cases. For example, some strains of Staphylococcus haemolyticus may test positive for PYR, while some strains of Enterococcus faecalis may test negative. Therefore, other tests such as catalase, coagulase, bile esculin hydrolysis, etc. should be performed to confirm the identification of bacteria.

The PYR test is a useful biochemical test for the identification and differentiation of various bacteria based on their ability to produce pyrrolidonyl aminopeptidase enzyme. Some of the applications of the PYR test are:

• For identification of Streptococcus pyogenes among other β-hemolytic Streptococci. S. pyogenes is the causative agent of streptococcal pharyngitis (strep throat), scarlet fever, rheumatic fever, and other infections. It is important to identify S. pyogenes quickly and accurately to initiate appropriate antibiotic therapy and prevent complications.
• For differentiation of Group A and Group D Streptococci (PYR positive) from other Streptococci. Group A Streptococci (GAS) are also known as S. pyogenes and are responsible for various human diseases. Group D Streptococci (GDS) include Enterococcus spp. and Streptococcus bovis group, which are associated with urinary tract infections, endocarditis, bacteremia, and other infections. Other Streptococci such as Group B (S. agalactiae), Group C (S. dysgalactiae), and Group G (S. anginosus) are PYR negative and can be differentiated from GAS and GDS by the PYR test.
• For identification of E. coli among other indole-positive, lactose-positive, Gram-negative bacilli. E. coli is a common inhabitant of the human intestinal tract and can cause various infections such as urinary tract infections, diarrhea, septicemia, meningitis, etc. E. coli is indole-positive and lactose-positive, which means it can produce indole from tryptophan and ferment lactose to produce acid and gas. However, there are other bacteria that share these characteristics such as Klebsiella spp., Citrobacter spp., Enterobacter spp., etc. The PYR test can help to identify E. coli among these bacteria as E. coli is PYR negative while the others are PYR positive.
• For differentiation of Citrobacter spp. (PYR positive) from other H2S-positive Gram-negative rods; Salmonella spp. (PYR negative). Citrobacter spp. and Salmonella spp. are both H2S-positive, which means they can produce hydrogen sulfide gas from sulfur-containing compounds such as thiosulfate or cysteine. This can be detected by the blackening of the media or the disk due to the formation of ferrous sulfide precipitate. However, Citrobacter spp. and Salmonella spp. differ in their PYR test results as Citrobacter spp. are PYR positive while Salmonella spp. are PYR negative.

Despite its usefulness, the PYR test also has some limitations that should be considered while performing and interpreting the test results. Some of the limitations are:

• It is not enough for the complete identification of bacteria; hence, requires other biochemical tests to confirm the identification of the sample bacteria. The PYR test only indicates the presence or absence of pyrrolidonyl aminopeptidase enzyme in bacteria, which is not a definitive marker for bacterial identification. There may be other bacteria that are not included in the list of PYR positive or negative bacteria that can give similar results due to their enzyme production or lack thereof.
• It gives a false negative result if the disk is too moist. The moisture on the disk can dilute the reagent and reduce its sensitivity to detect β-naphthylamide released from the substrate by the enzyme action. Therefore, it is important to moisten the disk slightly with sterile distilled water and not saturate it.
• It gives false negative results if the inocula (sample bacteria) are taken from selective or tube-biochemical test media. The selective or tube-biochemical test media may contain inhibitors or substrates that can interfere with the enzyme activity or reagent reaction in the PYR test. Therefore, it is recommended to use fresh cultures from non-selective media such as blood agar plates for inoculation.
• The use of lower (inadequate) inoculum may give a false negative result. The inoculum size should be sufficient to ensure adequate enzyme production and substrate hydrolysis in the PYR test. Using too little inoculum may result in weak or no color change after adding the reagent due to insufficient β-naphthylamide formation.
• Reading results before 1 minute may give unspecific results. The color change in the PYR test should be observed after 1 to 2 minutes of adding the reagent as the reaction may take some time to develop. Reading results too early may lead to false negative or false positive results due to incomplete or non-specific reactions.
• E. coli and other indole-positive bacteria form blue color over the media and disk, which can be confusing (is regarded as PYR negative). The blue color is due to the reaction of indole (produced by indole-positive bacteria from tryptophan) with the reagent, which forms a blue Schiff base. This color is different from the pink or red color that indicates a positive PYR test. However, some inexperienced or careless observers may mistake the blue color for a positive result or disregard it as a negative result. Therefore, it is important to distinguish the blue color from the pink or red color and confirm the indole production by other methods such as Kovac`s reagent or Ehrlich`s reagent.

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