RAPID ORNITHINE

Cat. no. K279 Rapid Ornithine, 15x103mm Tube, 1ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Rapid Ornithine can be used to detect ornithine decarboxylase activity in as little as two to four hours. This test will assist in the identification of Enterobacteriaceae and Staphylococcus lugdunensis.

SUMMARY

Conventional methods for detecting ornithine decarboxylase activity require an extended period of incubation. The tests generally involve the fermentation of glucose, which lowers the pH of the medium to the optimum hydrogen ion concentration for decarboxylase activity. Decarboxylation results in the formation of amines that will raise the pH. Positive reactions are usually obtained after 18 to 24 hours of incubation, but some strains require up to four days incubation. An overlay of mineral oil acts as a barrier to oxygen and prevents alkalinization of the surface of the medium.

Fay and Barry modified the conventional decarboxylase medium by removing the glucose and decreasing the pH to 5.5.(7) A small volume of the broth is heavily inoculated and then overlayed with sterile mineral oil. With the Hardy Diagnostics Rapid Ornithine, results can be obtained after only two to five hours of incubation at 35 to 37ºC.

The test can be used to determine ornithine decarboxylase activity in the family Enterobacteriaceae and can be used to differentiate Staphylococcus lugdunensis from other species of Staphylococcus. It is especially useful to differentiate Proteus mirabilis from other Proteus spp., which are ornithine decarboxylase negative. Some strains of Proteus require five hours of incubation to detect a positive result.

Decarboxylase basal media consists of peptones and yeast extract which supply nitrogenous and other nutrients necessary for bacterial growth. Bromcresol purple is a pH indicator. The amino acid ornithine is added to detect the production of the enzyme ornithine decarboxylase.

FORMULA

Ingredients per liter of deionized water:*

L-Ornithine 10.0gm
Meat Peptone 5.0gm
Yeast Extract 3.0gm
Bromcresol Purple 1.0gm

Final pH 5.6 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2 to 8ºC. away from direct light. Media should not be used if there are any signs of deterioration, discoloration or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

1. Inoculate a Rapid Ornithine tube with two to three colonies (18-24 hours old) and mix well.

2. Overlay with at least 0.5ml of sterile mineral oil (Cat. no. Z80).

3. Incubate aerobically with loose caps at 35 to 37ºC., for two to four hours. (See Limitations)

INTERPRETATION OF RESULTS

For reference, compare an uninoculated tube with inoculated tubes.

Positive reaction - development of a dark purple to violet color. Must be darker than the uninoculated reference tube.

Negative reaction - no development of color (remains pale yellow to light grayish-purple) or a development of yellow color.

Organisms expected to give a positive result:

Organisms expected to give a negative result:

LIMITATIONS

Incubating tubes with tight caps may lead to false negative reactions with some Proteus spp.

Some strains of Proteus require up to five hours of incubation to detect positive results.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, sterile mineral oil (Cat. no. Z80), other culture media, 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
Staphylococcus lugdunensis
ATCC® 49576**
E 2-4hr 35°C Aerobic Positive
Proteus mirabilis
ATCC® 12453
E 2-5hr 35°C Aerobic Positive
Staphylococcus epidermidis
ATCC® 12228**
E 2-4hr 35°C Aerobic Negative
Proteus vulgaris
ATCC® 8427
E 2-5hr 35°C Aerobic Negative

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

USER QUALITY CONTROL

Physical Appearance

Rapid Ornithine should appear clear and pale yellow to light grayish-purple in color.

LEFT: Positive; RIGHT: Negative

LEFT: Staphylococcus lugdunensis (ATCC® 49576)
Positive reaction at four hours.
RIGHT: Staphylococcus epidermidis (ATCC® 12228)
Negative reaction at four hours.

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

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

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

6. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

7. Fay, G.D and Barry, A.L. 1972. Rapid Ornithine Decarboxylase Test for the Identification of Enterobacteriaceae. Applied Microbiology; Vol. 23, p. 710-713.


ATCC is a registered trademark of the American Type Culture Collection.

041116gr