K9466 TRYPSIN - 50 DISCS

NOTE: These substrates are also available in dual and triple test WEE-TABS for greater economy. See "Multiple test tablets "

PRINCIPLE:

K9466 discs detect the trypsin enzyme in bacterial cells. Hydrolysis of Na-Benzoyl- DL-arginine-ß-naphtylamide releases pure naphthylamide which is red after adding PEP (cinnamaldehyde) reagent.

K9467 discs detect the chymotrypsin enzyme. In this test the substrate hydrolyzed is N-Glutaryl-Gly-Gly-Phe-ß- naphthylamide.

These tests are useful in the identification of anaerobes, specifically of value for Porphyromonas species.(1)

TRY is also useful in the speciation of non-fermentative aerobic gram-negative bacilli and when used with other chromogenic tests produced by KEY can expedite identification.

MSDS

Discs should be used only by trained individuals. Unbound naphthylamide in considered hazardous: the disc contains the bound form and unbound naphthylamide is present only in a positive test. Do not handle the used test- discard in a manner appropriate for biohazardous materials.

PEP reagent is a 0.1% solution of p-dimethylaminocinna-maldehyde in hydrochloric acid and methanol. Hydrochloric acid can cause irritation or burns. In case of contact flush with water. PEP reagent will stain skin and clothing.

STORAGE:

Store discs at 0-8 degrees C. Do not use beyond the expiration date.

MATERIALS REQUIRED:

Key TRY and CHY discs are in packs of 50 discs with PEP reagent provided. The tests require fresh growth on media appropriate for the specimen. A sterile loop or stick for harvesting, a slide, and distilled water are required but not provided.

PROCEDURE

The discs are for invitro diagnostic use only. Observe aseptic techniques when working with clinical specimens and microbiological cultures. The discs should be white to cream colored. If discs have changed colors do not use them. For best results use fresh cultures less than 48 hours old.

1. Place a disc onto a clean slide and moisten slightly.

2. Using a sterile stick or loop, smear the disc with a visible paste of the suspected isolate. False negatives may result from insufficient inoculum.

3. Incubate at room temperature for 5 minutes.

4. Add 1 drop of PEP reagent and wait 2 minutes to observe color.

INTERPRETATION:

After adding reagent, a positive test will be a deep red to deep purple while a negative test will be colorless, yellow, or bluegreen: the bluegreen color indicates a negative enzyme test and positive indole.

QUALITY CONTROL:

Each lot of discs should be tested with organisms of known reactivity prior to use. We recommend:

Porphyromonas gingivalis, ATCC 33277 (TRY +, CHY -) and Porphyromonas levii, ATCC 29147, (TRY -, CHY +)

REFERENCES:

1. Hudsbeth, M.K., Gerardo, S. Hunt, Citron, D.M. and Goldstein, E.J.C., October 1997, Growth Characteristics and a Novel Method for Identification of Porphyromonas Species Isolated from Infected Dog and Cat Bite Wounds in Humans. Journal of Clinical Micro,Vol 35, No. 10, Pg. 2450-24531.

2. Lennette, E.H., et al, 1985 Manual of Clinical Microbiology, 4th Ed., ASM, Washington,D.C.

3. Finegold, S.M. et al, 1986. Diagnostic Microbiology 7th ed. C.V. Mosby Co. St. Louis, MO

3. Sutter, V.I. and W. T. Carter. 1972 Evaluation of media and reagents for indol-spot tests in anaerobic bacteriology. Am. J. Clin. Pathol. 58:335-338