GLYCOSIDASE WEE-TABS (NITROPHENOL TESTS)

28 TESTS -

DISCUSSION:

Some bacteria produce enzymes which hydrolyze various chromogenic substrates. Because the organism produces these enzymes during the growth process and the presence of the enzymes can be detected rapidly, more substrates can be used in a wide variety of applications which would normally require special growth conditions. KEY glycosidase tablets provide an easy inexpensive way to detect these enzymes.

PRINCIPLE:

When bound to nitrophenol, the hydrolysis of the colorless aryl-substituted glycoside or phospoester releases the nitrophenol base with its yellow color. The most familiar test using this principle is o-Nitrophenol-ß-d-galactopyranoside (ONPG) used for enterics and Neisseria species.

ACTIVE INGREDIENTS

The tablets contain aproximately 0.05 mg. of one or more of the following substrates in a Sodium Chloride and Dicalcium phosphate base:

p-Nitrophenol-N-acetyl a-d-glucosaminide (NAG)

o-Nitrophenol-a-d-arabinopyranoside (aARA)

p-Nitrophenol-a-l-fucopyranoside (aFUC)

p-Nitrophenol-ß-d-fucopyranoside (ßFUC)

o-Nitrophenol-a-d-galactopyranoside (aGAL)

o-Nitrophenol-ß-d-galactopyranoside (ONPG)

p-Nitrophenol-a-d-glucopyranoside (aGLU)

p-Nitrophenol-ß-d-glucopyranoside (ßGLU)

o-Nitrophenol-a-d-mannopyranoside (aMAN)

p-Nitrophenol phosphate (alkaline phosphatase) (PNP)

o-Nitrophenol-ß-d-xylopyranoside (aXYL)

MATERIAL SAFETY DATA:

None of the nitrophenol bases are known at this time to be hazardous in any manner. Used tests should be discarded with biohazard waste. Unused discs may be discarded with normal waste.

MATERIAL REQUIRED:

All tests require fresh 24 hour growth on solid media not available from KEY. Broth media are not acceptable. Consult a suitable manual for recommended media for the specimen. The following items are required but not provided:

Inoculating loop

Distilled water, pH 7.0-7.2

Each product is sold ready-to-use, 28 tubes per pack. The catalog numbers of the tablets are listed in the QUALITY CONTROL section. These substrates are also available in dual and triple combination tests for economy of setup.

SINGLE TEST SETUP

(1) If performing a single test, add 3-5 drops of distilled water to the test tube. For multiple tests of the same organism, harvest sufficient colonies into 1-2 ml of distilled water to make a suspension equal to #5 McFarland. This suspension may be stored aerobically for later use. (up to 5 days). (2) If doing a single test, inoculate heavily with a loopful of organism from a fresh pure 24 hour culture plate or slant. Mix with the loop until the organism is in suspension. For multiple tests, add 3-5 drops of the prepared suspension to the tube containing the tablet, shaking to disintegrate the tablet.

3) Incubate all tests aerobically, uncovered, at 37C for 2 hours.

TIPS:

Vortexing or shaking the test vigorously will enhance color development. A denser suspension will also produce brighter and faster reactions. Pale reactions, which might be considered questionable, may be enhanced to a brighter yellow by adding 1 drop of 3% KOH to the finished test. In this case, negative reactions will show no color change.

INTERPRETATION OF RESULTS

No change at 2 hours is negative. The appearance of a yellow color at any time during the 2 hours is a positive glycosidase test. Tests may be held up to 24 hours. After 24 hours only very bright yellow is positive and pale yellow is negative. PO4 tablets should only be considered positive at any time if very deep yellow.

STORAGE:

Glycosdiase tablets may be stored @0-80F. Store tightly covered with desiccant.t room temperature. PO4 and PEP reagent should be refrigerated.

QUALITY CONTROL:

Each lot of tablets should be tested with known positive and negative organisms. Some suggested strains are listed. Dispose of all used material in a manner appropriate for biohazardous material.

GLYCOSIDASE: POSITIVE NEGATIVE

K1046 aARA Prevotella oris Prevotella denticola

K1235 aFUC Bacteroides fragilis Bacteroides ureolyticus

K1230 ßFUC Streptococcus intermedius Streptococcus constellatus

K1242 aGLU Bacteroides fragilis Bacteroides ureolyticus

K1275 ßGLU Bacteroides fragilis Bacteroides ureolyticus

K1442 aMAN Arcanobacterium spp. Corynebacterium spp

K1463 NAG Bacteroides fragilis . Bacteroides ureolyticus

K1490 ONPG Bacteroides fragilis Bacteroides ureolyticus

K1045 PNP Bacteroides fragilis Bacteroides ureolyticus

K1692 ßXYL Escherichia coli Klebsiella pneumoniae

REFERENCES

(1) Manual of Clinical Microbiology, Fifth Edition, Chapter 36, Enterobacteriaceae

(2) Kilian, M and Bulow, P. 1976. Rapid Diagnosis of Enterobacteriacae, Acta path. microbio. Scan, Sect B, 84:245-251

(3) Wadsworth Anaerobic Bacteriology Manual, 5th Edition, 1993,Glucosidase tests, page 152. 951010K