Calcium Stains

1. Silver nitrate method. Wash the formalin-fixed specimen under running water for 24 h and then in several changes of distilled water for 24 h. In a darkroom, immerse the specimen in a 1% solution of silver nitrate in distilled water and stain for 6-15 h. Rinse it in distilled water and then place it in 5% sodium hydrosulfite solution for 24 h. The specimen can now be exposed to light, washed, and mounted in 50% alcohol or Kaiserling solution,

2. Alizarin method. Immerse the specimen for 12 h in a 1:10,000 solution of alizarin red S with just enough potassium hydroxide to render the solution basic. For differentiation, transfer the specimen to a solution of equal parts of alcohol and glycerin and expose the jar to sunlight. Alizarin dyes stain calcium pink. After several days, mount the specimen in Kaiserling solution that is made alkaline by adding a small amount (1:1,000) of potassium hydroxide.

Specimens With Gouty Changes For the demonstration of deposits in gout or pseudogout, the murexide test is used. A sample of finely dispersed tissue fragments is heated with an equal volume of 25% nitric acid until the acid has evaporated. To the dry residue add 2-3 drops of 25% ammonium hydroxide solution and then the same amount of 20% sodium hydroxide solution. In the presence of urates, the dry residue will be bright red or orange, purple after addition of ammonium hydroxide, and blue-violet after addition of sodium hydroxide. For the preparation of museum specimens, the sample is dehydrated over 2 wk in several changes of absolute alcohol. Transfer into mounting fluid (see below). Deposits also can be displayed in their native state.

1. Procedure. Fix specimen, preferably in an anhydrous fixative such as alcohol. Although urate crystals are freely soluble in water, crystalline deposits are usually identifiable in the center of the specimens even after aqueous formalin fixation. The crystals often also resist the dehydration and staining procedure.

2. Mounting. Mount in plastic jar with undiluted glycerin. Seal without leaving air under the lid.

Gallbladder If the gallbladder can be obtained within a few hours after death, the oxidative greenish discoloration will not yet have occurred and can be prevented by the following procedure (23).

1. Procedure. Place specimen in 3% solution of sodium sulfite for 20 min. Rinse for a few minutes in running water. Place in 10% formalin solution for 12-24 h. Wash thoroughly and mount.

If the greenish color of biliverdin has already formed, a 5% sodium sulfite solution is used, to which 1% formalin is added. The specimen is left in this solution for 12 h. The subsequent steps remain the same.

2. Mounting fluid: 10 g potassium acetate, 5 g chloral hydrate, 10 mL glycerin, 90 mL tap water.

Instead of the sodium sulfite-formalin mixture, a saturated solution of calcium chloride can be used. The specimen should be soaked in this solution for 24-48 h (21).

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