GRAM REACTION (APNA)

K915 - 15 DISCS

K915B - 30 DISCS

APNA Discs are used in confirming the gram staining reaction of Gram-negative and Gram-positive aerobic and facultatively anaerobic bacteria. The presence of cell wall aminopeptidase is detected by the hydrolysis of L-alanine-p-nitroanilide indicating a gram-negative reaction.(1)

STORAGE:

Store tightly sealed at 2-8C with desiccant provided.

MATERIAL SAFETY DATA:

APNA discs contain L-alanine-p-nitroanilide in Tris buffer. This ingredient in it's pure state may be hazardous if inhaled, ingested or absorbed through the skin. The amount used in the disc does not present a hazard when used as directed.

QUALITY CONTROL:

The APNA disc is for invitro diagnostic use only. Observe aseptic techniques when working with clinical specimens and microbiological cultures. Discard used tests in a manner appropriate for biohazardous materials. Each lot of APNA discs should be tested prior to use with known Gram reaction organisms. Suggested organisms are:

Gram-positive -Staphylococcus aureus 25923

Gram-negative -Escherichia coli 25922

MATERIALS REQUIRED:

Key APNA discs are provided in packs of 15 or 30 discs. The tests require fresh growth on plated media appropriate for the specimen. A sterile loop or swab for harvesting, distilled water (neutral pH) and small test tubes are required but not provided.

PROCEDURE

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 2-3 drops of distilled water into a small test tube.

2. Add sufficient organisms being examined to produce a milky suspension.

3. Add 1 APNA disc to the suspension and incubate at 37C for 5 - 20 minutes.

INTERPRETATION OF RESULTS:

The development of a yellow color is a positive test, confirming the organism is gram-negative. No color change indicates a negative test and the organism is gram-positive. Optional: Add 1 drop of spot indole reagent (sold separately) to confirm reaction: blue/purple=positive, red or yellow=negative.

LIMITATIONS:

APNA discs will not give predictable reactions for some gram-positive cocci, microaerophilic, or anaerobic organisms. Tests done on yellow pigmented colonies may appear positive immediately, but this is the pigmentation and should not be recorded as positive. In this case, the test should be observed for a deepening of the yellow reaction to indicate positive and/or the results should be confirmed with reagent.

REFERENCES:

1. Carlone, G.M., M.J. Valdez, and M.J. Pickett. Method for Distinguishing Gram-positive from Gram- negative Bacteria. J. Clinical Microbiology. 16:1157-1159

2. Lennette, E.H., A. Balows, W.J. Haulser, and J.P. Truant (EDS) 1980. Manual of Clinical Microbiology, 3rd edition. American Society for Microbiology. 99kb