HardyDisk™ UREA/PDA DISKS

Cat. no. Z7331 Urea/PDA Disks 50 disks/cartridge

INTENDED USE

HardyDisk™ Urea/PDA Disks are used to determine an organism's ability to hydrolyze urea to ammonia, and to deaminate phenylalanine to phenylpyruvic acid. The test is useful in differentiating Proteus, Providencia, and Morganella species from most other members of the Enterobacteriaceae.(5,6)

SUMMARY

Urea is a waste product of protein metabolism that is excreted in the urine. The enzyme urease, hydrolyzes urea to form ammonia. This causes an increase in pH, which is detected by the phenol red indicator turning a dark pink to red color. Strong urease activity is characteristic, and mostly limited to all Proteus species.(6) However, Morganella spp., and some Klebsiella, Yersinia, Citrobacter, Enterobacter, Providencia, and Pantoea spp. may also be positive.

Phenylalanine is an amino acid that, upon deamination by oxidase enzymes, results in the formation of phenylpyruvic acid. The deamination of phenylalanine to phenylpyruvic acid is detected by the addition of a Ferric Chloride Reagent (Cat. no. Z63) that reacts with the by-product to produce a light to deep green color. In the family Enterobacteriaceae, only members of the Proteus, Providencia and Morganella group are capable of deaminating phenylalanine. (5)

HardyDisk™ Urea/PDA Disks are a rapid and reliable method to detect urease activity and phenylalanine deamination in Enterobacteriaceae, and has the advantage of two tests in one tube. This makes the product an excellent way to inexpensively rule out lactose negative organisms when screening stool for Salmonella and Shigella, which will be negative for both urease and phenylalanine deaminase.

FORMULA

Each HardyDisk™ Urea/PDA Disk is prepared by impregnating appropriate concentrations of urea, phenylalanine and phenol red onto a 0.25 inch diameter filter paper disk.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Product should not be used if there are any signs of deterioration, discoloration, or if the expiration date has passed. Protect the product from light, excessive heat, and moisture.

PRECAUTIONS

PROCEDURE

Specimen Collection: This product is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organisms. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.

Method of Use:

1. Using an 18-24 hour culture, prepare a heavy suspension (equivalent to a McFarland 3.0 turbidity standard) of the test organism in 0.3ml of sterile saline in a 12x75mm plastic test tube. See limitations.

2. Add a single Urea/PDA disk to the bacterial suspension.

3. Incubate aerobically at 35-37ºC. for two hours.

4. After incubation, if a red color develops, indicating a positive urease test, add two drops of 1N hydrochloric acid (1N HCl) to clear the urea reaction. If there is no color development, no HCl is needed to clear the tube.

5. After interpretation of the urease test, add two drops of 10% Ferric Chloride Reagent (Cat. no. Z63) to test for phenylalanine deaminase.

6. A green color development indicates a positive test for phenylalanine deaminase activity.

INTERPRETATION OF RESULTS

Urea - pink to red color development after incubation indicates a positive reaction, no color change after incubation indicates a negative reaction.

Phenylalanine - dark green color development after the addition of 10% Ferric Chloride Reagent (Cat. no. Z63) indicates a positive reaction, no color change after the addition of 10% Ferric Chloride Reagent indicates a negative reaction.

LIMITATIONS

The use of plastic test tubes are recommended for this test, as false-positive reactions for the urease test were observed when glass test tubes were used, due to the alkaline properties of glass.

A significant number of Yersinia, Enterobacter, and Citrobacter spp. strains may be urease positive when the urease test is incubated at room temperature.(3)

Urea reaction will be unreadable if it is not read before the addition of the ferric chloride reagent. Some very rare strains of Salmonella enterica will be positive for urease.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, plastic test tubes, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms
Inoculation Method*
Incubation
Results
Time
Temperature
Atmosphere
Proteus mirabilis

ATCC ® 12453
D
2hr
35°C
Aerobic
Urea (+) / PDA (+)
Klebsiella pneumoniae

ATCC ® 13883
D
2hr
35°C
Aerobic
Urea (+) / PDA (-)
Escherichia coli

ATCC ® 25922
D
2hr
35°C
Aerobic
Urea (-) / PDA (-)

User Quality Control

PHYSICAL APPEARANCE

HardyDisk™ Urea/PDA Disks are 0.25 inch (in diameter) filter paper disks, and should appear orange-yellow in color.

Positive Urease Test

Proteus mirabilis (ATCC® 12453) suspended in 0.3mL of saline with a HardyDisk™ Urea/PDA Disk (Cat. no. Z7331) and incubated aerobically for 2 hours at 35ºC. The pink to red color development represents a positive test for urea hydrolysis.

Positive PDA Test

Proteus mirabilis (ATCC® 12453) suspension from the tube pictured on the left with one drop of 1N HCl added to clear the urea reaction color. Subsequently, two drops of Ferric Chloride Reagent (Cat. no. Z63) was added. The dark green color development represents a positive test for phenylalanine deamination.



Negative Urease Test

Escherichia coli (ATCC® 25922) suspended in 0.3mL of saline with a HardyDisk™ Urea/PDA Disk (Cat. no. Z7331) and incubated aerobically for 2 hours at 35ºC. The absence of pink color development represents a negative test for urea hydrolysis.

Negative PDA Test

Escherichia coli (ATCC® 25922) suspension from the tube pictured on the left with two drops of Ferric Chloride Reagent (Cat. no. Z63) added. Absence of green color develolpment represents a negative test for phenylalanine deamination.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Baron, E.J., et al. 1994. Bailey and Scott's Diagnostic Microbiology, 9th ed. C.V. Mosby Company, St. Louis, MO.

4. Howard, B.J., et al. 1987. Clinical and Pathogenic Microbiology, 2nd ed. C.V. Mosby Company, St. Louis, MO.

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. MacFaddin, J.F. 2000. Urease Test / Phenylalanine Deaminase Test, p. 424-425 / p. 388-391. Biochemical Tests for Identification of Medical Bacteria, 3rd ed. Lippincott Williams & Wilkins, Philadelphia, PA.

7. Ederer, G.M., J.H. Chu, and D.J. Blazevic. 1971. Rapid test for urease and phenylalanine deaminase production. Appl. Microbiol.; 21:545.

8. Golden, M. and A. Glenn. 1962. J. Bacteriol.; 84:870-871.


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080816gr