Preparation of the Elastase Diffusion Plate

PROCEDURE FOR PREPARATION OF THE ELASTASE DIFFUSION PLATE

The procedure is sufficient to prepare 10 elastase diffusion plates.

Materials Required

1.     Elastin-Fluorescein or Elastin-Rhodamine, particle size Pass 400 mesh (smaller than 37 micron)

2.     Agar, high purity

3.     Acetone, reagent grade

4.     Buffer, 0.2 M Tris pH 8.8 at 37º C containing 0.02% NaN3

5.     10 cm² plastic disposable Petri plates. Ten required

6.     Hot-plate equipped with a magnetic stirrer.

Procedure

1.     To a dry 250 ml flask, add 600 mg of substrate. Add 5 ml of acetone and swirl the contents gently so that all particlesare wetted. Add 100 ml buffer and swirl until all particles are dispersed. Heat with stirring to 90º-95º C to expel the acetone. Cool to about 80º C.|

2.     Add 2.5 gm agar and heat with stirring until the suspension boils. Reduce the heat and maintain a temperature of 85º-95º C.

3.     DO NOT PIPET BY MOUTH. Place 10 ml of the hot suspension into the bottoms of level Petri plates. Cover, allow to cool to room temperature.

4.     Cut 4 mm wells in the solidified gel. Cover the surface of the gel with a square of polyethylene film. Place a circle of water-saturated filter paper on top of the film. Cover and seal the plate within a polyethylene bag. Store 2º-5º C. DO NOT FREEZE. Stable about 4 months.