ONPG-PDA

Cat. no. R93 ONPG-PDA, 13x100mm Tube, 0.5ml 20 tubes/box

INTENDED USE

Hardy Diagnostics ONPG-PDA test is a rapid combination test recommended for use in differentiating members of the Enterobacteriaceae and other microorganisms based on beta-D-galactosidase and phenylalanine-deaminase activity.

SUMMARY

The ability of a bacteria to ferment lactose depends on two enzymes, permease and beta-galactosidase. Permease allows lactose to enter the bacterial cell wall where it is then broken down into glucose and galactose by beta-galactosidase. (1) The glucose and galactose can then be metabolized by the bacteria. O-nitrophenyl-beta-D-galactopyranoside (ONPG) is incorporated into this test and acts as the substrate for the beta-galactosidase. If the galactosidase enzyme is present, the ONPG is hydrolyzed releasing o-nitrophenol and this yields a yellow color in alkaline solution.

Phenylalanine-deaminase (PDA) activity has also been demonstrated to be useful in the taxonomy of the Enterobacteriaceae. (2,3) This test detects the formation of phenylpyruvic acid from phenylalanine-deamination by members of the Proteus , Morganella , and Providencia groups as well as a few other species. (4) The amino acid phenylalanine undergoes oxidative-deamination, catalyzed by an amino acid oxidase enzyme to yield phenylpyruvic acid. To test for the production of this acid, 10% ferric chloride is then added to the medium. The ferric chloride chelates with the phenylpyruvic acid to form a green color. (5,6)

FORMULA

Ingredients per liter of deionized water:*

Disodium Phosphate 9.46gm
Phenylalanine 4.0gm
o-nitrophenyl-beta-D-galactopyranoside (ONPG) 2.0gm
Monopotassium Phosphate 0.907gm

Final pH 8.0 +/- 0.3 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at < -10 o C. away from direct light. Product will arrive in the users laboratory non-frozen; the determination of shelf life has taken this transit period into consideration. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed.

Product is light sensitive . Store frozen immediately and protect from light.

PRECAUTIONS

PROCEDURE

1. Using a heavy inoculum from a pure 18-24 hour culture, inoculate the test medium.

2. Incubate aerobically at 35ºC.

3. Examine for a yellow color development at 1 hour and up to 2 hours. The ONPG result must be interpreted before the addition of the 10% Ferric Chloride.

4. After 2 hours incubation, add 4 to 5 drops of 10% Ferric Chloride (Cat. no. Z63) and examine immediately for a dark green color development. Do not shake tubes.

INTERPRETATION OF RESULTS

ONPG:
Positive: Yellow color change before the addition of Ferric Chloride.
Negative: No color change before the addition of Ferric Chloride.

PDA:
Positive: Dark green color development after the addition of Ferric Chloride.
Negative: Yellow or no color change after the addition of Ferric Chloride.

LIMITATIONS

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents (Ferric Chloride, 10%), are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Shigella sonnei
ATCC ® 9290**
E 1-2hrs 35°C Aerobic ONPG (+) / PDA (-)
Proteus mirabilis
ATCC ® 12453**
E 1-2hrs 35°C Aerobic ONPG (-) / PDA (+)
Salmonella enterica
ATCC ® 14028**
E 1-2hrs 35°C Aerobic ONPG (-) / PDA (-)

**Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

User Quality Control

PHYSICAL APPEARANCE

ONPG-PDA should appear clear and colorless, with no precipitate.

Positive ONPG Test

Shigella sonnei (ATCC ® 9290) growing in ONPG-PDA (Cat. no. R93). Incubated aerobically for two hours at 35ºC. The yellow color development was indicative of a positive ONPG reaction.

Negative ONPG Test

Proteus mirabilis (ATCC ® 12453) growing in ONPG-PDA (Cat. no. R93). Incubated aerobically for two hours at 35ºC. No yellow color development was indicative of a negative ONPG reaction.

Negative PDA Test

Shigella sonnei (ATCC ® 9290) growing in ONPG-PDA (Cat. no. R93). Incubated aerobically for two hours at 35ºC. Five drops of 10% Ferric Chloride (Cat. no. Z63) was added to the tube. No green color development was indicative of a negative PDA reaction.

Positive PDA Test

Proteus mirabilis (ATCC ® 12453) growing in ONPG-PDA (Cat. no. R93). Incubated aerobically for two hours at 35ºC. Five drops of 10% Ferric Chloride (Cat. no. Z63) was added to the tube. The green color development was indicative of a positive PDA reaction.

REFERENCES

1. Gottschalk, G. 1986; p. 97-98, 178-179. Bacterial Metabolism, 2nd ed. Springer-Verlag, New York.

2. Singer, J. and B.E. Volcani.1955. J. Bacteriol.; 69:303.

3. Ben Hamida, F. and L. LeMinor. 1956. Ann. Inst. Pasteur; 90:671-673.

4. Buttiaux, R., R. Osteux, R. Fresnoy and J. Moriamez. 1954. Ann. Inst. Pasteur; 87:375.

5. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

6. Balows, A., K.L. Hermann, H. Isenberg, H.J. Shadomy and W.J. Hausler, Jr. 1991. Manual of Clinical Microbiology , 5th ed. ASM, Washington, D.C.

7. 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.

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

9. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

10. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

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


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040416gr