<b>DUAL-TEST NITROPHENOL AND AMINOPEPTIDASE</b></b>

DUAL-TEST NITROPHENOL AND AMINOPEPTIDASE

COMBINED TABLETS

PRINCIPLES/DISCUSSION:

Some bacteria produce enzymes which hydrolyze various chromogenic substrates. When these substrates are bound to another base, the hydrolysis will produce a color reaction, some apparent immediately and others by the use of special equipment or the addition of color developers. The dual test tablets make use of these principles and, by combining non-interferring substrates, produce two chromogenic reactions in one product and reduce the cost per test.

1. The primary tests are nitro-phenol bound substrates. Organisms containing the necessary enzymes hydrolyze these substrates, releasing the yellow nitro-phenol which is visible without addition of any reagent. The nitro-phenol test will be the one listed first in the product name.

2. The last tests are aminopeptidase tests. Organisms containing the enzymes necessary for hydrolysis of the applicable arylamide release free beta-naphthylamide which is detected by the addition of aminopeptidase reagent. These will be listed last on the labels.

MATERIAL SAFETY DATA:

Each tablet is consists of approximately 0.5 mgs. each of the applicable substrates with inert fillers and tableting compounds. None of the ingredients is hazardous in this form.

Tablets contain the following chemicals in different combinations:

Nitrophenol substrates: (1st test - yellow)

ONPG (o-nitro-phenol-ß-D galactopyranoside)

NAG (p-nitro-phenol-N-acetyl-ß-D-

Glucosaminide)

AFU (P-nitrophenol-alpha-D-fucopyranoside)

AGLU (0-nitrophenyl-alpha-D-glucopyranoside)

Naphthylamide substrates: (2nd test - red or purple positive)

PRO (proline-ß-naphthylamide)

PYR (pyrrolidonyl-ß-naphthylamide)

TRY(Na-Benzoyl-DL-arginine-ß-naphtylamide-"trypsin")

CHY(N-Glutaryl-Gly-Gly-Phe-ß naphthylamide-

"chymotrypsin")

LGY (Leucyl glycine ß naphthylamide)

SER (Serine ß naphthylamide)

ARG (Arginine ß naphthylamide)

PAL (Phenylalanine ß naphthylamide)

MATERIALS REQUIRED:

Each substrate is sold separately, 1 tablet per tube, 28 tubes per bottle. Usage requires 24 hour growth on media not available from KEY. Consult a current reference manual for the correct media to use. The following items are also required but not provided:

Microbiology loop, needle, or sterile swab for harvesting

Distilled water, neutral pH

K2375 PEP reagent

PROCEDURE:

1) Add at least .25 ml but not more than 0.5 ml (4-6 drops) of distilled water to the tube containing the tablet.

2) Inoculate heavily from fresh 24 hour growth on a plate or slant: the heavier the inoculum the brighter and faster the reaction. Emulsify the organism well. It is not necessary for the tablet to dissolve.

3) Incubate for at least 2 hours. Do not read after more than 28 hours as false positives may occur.

INTERPRETATION:

(Read and interpret in the exact sequence listed)

1) Nitro-phenol tests: Observe for a yellow color indicating a positive test. Colorless is negative.

2) Aminopeptidase: Perform the aminopeptidase test by adding 2 drops of PEP reagent directly to the tube and reincubating for 15 minutes. Ambiquous results of the aminopeptidase may be intensified by vortexing a few seconds after addition of the reagent. Red, blue, or purple is positive. Green, turquoise, or yellow is negative. Brightest and easiest to read reactions are done by dipping a swab into the tube then dropping 1 drop of reagent onto the swab. Swab reaction develop within 1 minute.

References / Footnotes:

(1) Manual of Clinical Microbiology, Fifth Edition

(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. 9804K

.25 ml but not more than 0.5 ml (about 5 drops) of distilled water to the tube containing the tablet.