PRINCIPLE:

ALN detects the enzyme L-alanyl-L-alanyl aminopeptidase in bacterial cells. It is useful to rapidly separate Fusobacterium species (negative) from most Bacteroides species (positive). (See LIMITATIONS for B. ureolyticus.)

Hydrolysis of L-alanyl-L-alanyl ßeta Naphthylamide releases pure naphthylamide which is red after adding PEP (cinnamaldehyde) reagent.

MSDS:

ALN Discs should be used only by trained individuals. Unbound naphthylamide in considered hazardous: the disc contains the bound form and unbound napthylamide 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-dimethylamino- cinnamaladehyde 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 ALN discs are in packs of 25 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 ALN disc is 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 ALN and positive indole.

QUALITY CONTROL:

Each lot of discs should be tested with organisms of known reactivity prior to use. Suggested organisms are: B. fragilis ATCC 25285 and Fusobacterium nucleatum ATCC 10953.

LIMITATIONS:

B. ureolyticus, which is ALN negative, can be differentiated from Fusobacterium by its negative reaction to spot indole and PYR.

Capnocytophaga species are ALN positive and can cause confusion with anaerobic gram negative bacilli. The colonial morphology, ability to grow at 35C in 5% carbon dioxide, and PRO reaction are helpful in eliminating these organisms.

PYR, spot indole, and PRO are all available from KEY Scientific. Published tables are available which list tests useful for definitive identification of this group of organisms.

It should be emphasized that this test is only one of a battery of tests for identifying catalase negative gram positive cocci. Further biochemical characterization and serological grouping may be necessary for specific identification.

REFERENCES:

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

2. Baranowski, J, et al. Separation of Anaerobic Gram Negative and Gram Positive Organisms using L-Alanyl-L-alanyl-ß- naphthylamide. Abstr. Ann Meet. ASM, 1984 C:140:260.

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

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