RICHARD-ALLAN SCIENTIFIC COMPANY 88039 Epredia

Modified Grocott’s Methenamine Silver Stain Kit (Chromic Acid) is intended for use as an aid to identify fungal organisms and basement membranes in tissue sections for the diagnosis of general pathology specimens. Fungal cell walls are rich in polysaccharides known as glucans. Treatment with either periodic acid or chromic acid oxidizes these polysaccharides to form aldehyde groups; however, chromic acid is the stronger oxidizer. Chromic acid will achieve a greater degree of oxidation, rendering some chemical groups non-reactive with silver so that they are not demonstrated within the final stain. This greater level of oxidation also results in reduced background staining of basement membranes and collagen fibers, as well as more effective demonstration of Pneumocystis jiroveci (formerly classified as Pneumocystis carinii) and Histoplasma capsulatum (the causative organism of Histoplasmosis). After oxidation, Sodium Metabisulfite Solution removes residual Chromic Acid Solution from the tissue. The aldehyde groups react with the silver ions present in the Working Methenamine Silver Solution and reduce them to form visible metallic silver. Gold Chloride Solution tones the sections and intensifies the reduced silver by forming a silver-gold complex. Sodium Thiosulfate Solution removes any unreduced silver from the tissue sections, followed by Fast Green Stain Solution which produces a light green background to further enhance contrast.

Potassium Permanganate Solution is the oxidizing agent in this technique. It is followed by an Oxalic Acid Solution to remove the excess permanganate. This step serves to enhance the subsequent staining of the reticular fibers. A sensitizing step using Ferric Ammonium Sulfate Solution impregnates the fibers, creating a metal-organic bond that is replaced by silver during subsequent treatment with Working Ammoniacal Silver Solution. To develop the visible silver, a Reducing Solution of dilute formaldehyde is used. The tissue structures that have been impregnated with the silver solution will now appear brown to black. Gold Chloride Solution is used to “tone” the sections, producing better contrast and clarity as the gold reacts and combines with the reduced silver. Unreduced silver is removed via treatment with Sodium Thiosulfate Solution. An optional nuclear fast red counterstain provides background staining as well as nuclear detail.

The Amyloid Stain kit is a special stain technique to identify amyloid in tissue sections. Amyloid, a fiber-like protein, can be stained with Congo Red solution. Sodium Hydroxide is added to the solution to break down hydrogen bonds between adjacent amyloid fibers allowing more bonding sites for the Congo Red. A light counterstain with Mayer’s hematoxylin allows contrast. Amyloid can be best detected by the use of green birefringence under a polarized lens of a light microscope. Care should be taken to section tissue sections at a thickness of 7-10 microns for optimal staining.

Clarifier™ 1 is a reagent designed to eliminate background staining sometimes caused by excessive adhesives in the water bath such as gelatin. Clarifier™ 1 selectively removes hematoxylin staining from excess adhesive without affecting nuclear staining. This product is only effective with progressive staining and should not be used with regressive stains. Clarifier™ 1 is designed for use after staining with progressive hematoxylins. After tissues have been sufficiently stained with a progressive hematoxylin, slides are removed from the stain and excess hematoxylin is removed by rinsing the sections in water. The sections are then placed into Clarifier™ 1 for 30 seconds to 1 minute. A 1-minute water rinse should follow as this stops the reaction and rinses excess acid from the tissue section. If Clarifier™ 1 is not successful in removing the background staining, use Clarifier™ 2 in its place with a possible time reduction.

Clarifier™ 2 is an aqueous glacial acetic acid rinse designed to eliminate background staining sometimes caused by excessive adhesives in the water bath such as gelatin. Clarifier™ 2 selectively removes hematoxylin staining from excess adhesive without affecting nuclear staining. This product is only effective with progressive staining an should not be used with regressive stains. Clarifier 2 was designed to be used in histology only. Clarifier™ 2 is designed for use after staining with progressive hematoxylins. After tissues have been sufficiently stained with a progressive hematoxylin, slides are removed from the stain and excess hematoxylin is removed by rinsing the sections in water. The sections are then placed into Clarifier™ 2 for 20 seconds to 40 seconds. A 1-minute water rinse should follow as this stops the reaction and rinses excess acid from the tissue section.