NAPHTHYLAMIDE WEE-TABS

PRINCIPLE/DISCUSSION:

Some bacteria produce enzymes which hydrolyze various chromogenic substrates. Enzymatic hydrolysis of the arylamide in the tablet releases free á-naphthylamine which is detected and shown by the color change after adding reagent. 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.

ACTIVE INGREDIENTS

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

L-pyroglutamic acid a-naphthylamide (PYR)

L-arginine a-naphthylamide (ARG)

L-phenylalanine a-naphthylamide (PAL)

L-proline a-naphthylamide (PRO)

L-serine a-naphthylamide (SER)

Glycine a-naphthylamide (GLY)

Leucyl-Glycine a-naphthylamide(LGY)

N-glutaryl GLY GLY phe ß naphthylamide (CHY)

Na-benzoyl D L arginine ß naphthylamide (TRY)

L-cysteine-naphthylamide(CYS)

PEP reagent contains p-dimethyl-amino-cinnamaldehyde in hydrochloric acid.

MATERIAL SAFETY DATA:

Naphthylamide compounds have been identified as possible carcinogens and should be handled accordingly. Avoid excessive handling. When used only as directed there is no hazard involved. PEP reagent is poisonous and corrosive and stains clothing and hands. Handle with care. Consult poison control center if ingested.

MATERIAL REQUIRED:

All tests require fresh 24-48 hour growth on media not available from KEY. 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

Pipettes

Each product is sold ready-to-use, 1 tablet per tube, 28 tubes per bottle.

K1083 ARG

K1086 CHY

K1595 CYS

K1305 GLY

K1376 LGY

K1525 PAL

K1535 PRO

K1536 PYR

K1575 SER

K1084 TRY

K2375 PEP-10 ml bottles

K982375 PEP DropIt vials-3 ml each.

ALSO AVAILABLE:dual and triple test tablets containing the above substrates.

TEST SETUP

(1) For direct inoculation, add 3-5 drops of distilled water to the test tube. (Either aerobically or in a gas chamber).

(2) 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.

(3) Incubate all tests aerobically, uncovered, at 37C for 2 hours. For dual test tablets, or less inoculum, incubating an additional hour will produce brighter reactions.

(4) After incubation, add 1 drop of PEP reagent and observe for color development to dark pink or red.

TIPS:

1) Be sure not to pick up any of the culture media with the organism as this can affect the test.

2) *A loopful means to fill the eye of a 1 ul loop. Less organism may be used but will take longer to develop.

3) If doing multiple tests of the same organism, make a suspension equal to a #3-#5 McFarland in 1-2 ml. of distilled water. Add 3-5 drops of this suspension to the test tube containing the tablet. The suspension will remain reactive for up to 3 days.

4) If results are ambiguous after completion, vortexing or shaking the test vigorously will enhance the color development. A denser suspension will also produce brighter reactions.

(5) Note that PEP is also the reagent used for spot indole, Because of this cross reaction, indole-positive / naphthylamide-positive organisms may show a deep purple instead of pink or red and indole-positive, naphthyamide-negative organisms may show green.

(6) Naphthylamide tablets may be added to any glycosidase tablet for simultaneous testing or used as a stand-alone test or spot test:

SPOT TEST:

To use as a spot test, smear the organism onto a piece of filter paper, then add 1 drop of the rehydrated solution (SETUP step 1) onto the smear. Wait 5 minutes, then add PEP reagent. A positive test will turn pink to red within 1 minute.

STORAGE:

Store tightly covered in the original jar with desiccant. Refrigerate at 2-8C.

QUALITY CONTROL:

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

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.