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Troubleshooting

Troubleshooting stains

ProblemSolution
Copper is masked (Rhodanine) Do not overstain with hematoxylin
Stained copper is difficult to distinguish from lipofuchsin (Rhodanine) Copper concentration is low, fading has occurred
Calcium salts have been removed (Von Kossa) Alcoholic iodine solution used for removal of mercury pigment will cause this
Formalin pigment will reduce silver (Von Kossa) Use of unbuffered formalin
Brown reaction product (Von Kossa) Use of artificial light
Formalin pigment will give positive reaction (Fontana Masson) Technique is not specific for melanin and argentaffin granules
Dirty gray background with loss of contrast (Fontana Masson) Overstaining
Nonspecific background staining (Schmorl) Use of glassware contaminated with iron-containing reagents
Background staining is undesirable (Schmorl) More specific staining with less background staining is obtained if solutions are fresh
Diffuse background staining (Prussian Blue) Be sure staining jar is chemically cleaned
Cloudy slides (Prussian Blue) Excess Nuclear Fast Red not adequately removed. Return sections to running water and wash well, redehydrate and clear.
Rapid fading of silver stains of microorganisms (Steiner and Steiner) Xylene substitutes will cause this
Sections have been overdeveloped (Warthin-Starry) Treat with iodine and sodium thiosulfate for color removal, then restain
Reticular fibers, red blood cells and other tissue structures may also be stained (Grocott Methenamine Silver) Slides are left in methenamine silver too long
Reduction of chromic acid solution, causes color of the solution to change from orange to brown. (Grocott Methenamine Silver) Failure to adequately remove alcohol during deparaffinization causes this. Discard solution.
Breakdown of solution and nonspecific staining (Grocott Methenamine Silver) Silver is overheated.
Free aldehyde groups can reduce the silver and give nonspecific staining (Grocott Methenamine Silver) Avoid Glutaraldehyde fixative
Increased background staining (Chromic Acid Schiffs) Insufficient oxidation with chromic acid
Reduced staining of fungal organisms (Chromic Acid Schiffs) Prolonged oxidation of chromic acid
Darkened chromic acid (Chromic Acid Schiffs) Reduction with alcohol remaining from rehydration step: wash thoroughly after the alcohol.
Poor staining (Hotchikiss McManus PAS Fungus) Oxidizing agent and Schiffs reagent must not be overused. Best if fresh oxidizing agent is used.
Excessive decolorization (Diff-Quik Giemsa) Prolonged time in last distilled water rinse or in the dehydrating alcohols causes this.
Formation of insoluble compounds (Gram) Sections allowed to dry at any stage causes this.
Acid-fast bacteria contamination (Kinyoun, Ziehl-Neelsen) No tap water should be used. Acid-Fast bacteria is found in tap water.
Organisms are masked (Kinyoun) Overstained with methylene blue. Take it back to acid-alcohol to remove the methylene blue, wash with water, then repeat counterstain step.
Diffuse violet background staining (Luxol Fast Blue-Cresyl Echt Violet Stain) Failure to add acetic acid to cresyl echt violet solution.
Decreased staining of Nissl substance (Luxol Fast Blue-Cresyl Echt Violet Stain) Failure to heat the cresyl echt violet
Protoplasmic astrocytes lose stainability (Cajal) Prolonged fixation
Crystal violet precipitate forms (Holzer Method) Remove with straight aniline oil.
Stain precipitate and a dirty background are obtained (Bodian method, Periodic Acid-Methenamine Silver, Gomori, and other silver stains) Use chemically clean glassware and nonmetallic forceps to avoid this.
Contrast is lost (Bodian Method) Do not overcounterstain with aniline blue.
Dissolved fat (Oil Red O, Sudan Black B) Organic solvents present in synthetic resinous mounting media causes this. Aqueous mounting media must be used.
Fat is displaced (Oil Red O, Sudan Black B) Pressure is placed on cover glass. If air bubbles are present, soak slides in warm water to remove coverslip.
Fat is melted and displaced (Oil Red O, Sudan Black B) Glycerin jelly used for mounting is overheated and causes this.
Uneven or interuppted staining (Periodic Acid-Methenamine Silver) Stopping the silver impregnation too soon results in this.
Masked silver stains and decrease of contrast (Periodic Acid-Methenamine Silver) Application of too much counterstain causes this.
Incomplete impregnation of reticular fibers (Gomori) Excess of ammonia decreased sensitivity. A hint of turbidity must remain in silver solution to avoid this.
Staining of reticulin is decreased (Gomori) Rinse between diamine silver solution and formaldehyde is prolonged.
Excessive background staining (Gomori) Insufficient rinse time between diamine silver solution and formaldehyde.
Cloudiness (Gomori) Unwashed slides. Wash slides well after nuclear fast red staining. Can only be removed by backing slides back up to water.
Silver staining of nuclei (Gomori) Helped by using acetified potassium permanganate.
Reticulin stains gray-black rather than sharp black (Gomori) Ammonium hydroxide has lost strength because of the loss of ammonia from solution. Fresh bottle should be opened.
Inhibited subsequent staining steps (Movat Pentachrome) Failure to completely remove alkaline alcohol with running water.
Overdifferentiated stain (Verhoeff) Restain at any step provided it has not been treated with alcohol.
Picric acid differentiates stain further (Verhoeff) Prolonged staining with van Gieson causes this.
Collagen does not stain red, and cytoplasm, muscle and collagen may all stain the same color (Verhoeff) Improper preparation of van Gieson solution. Picric acid is not saturated causing this.
Lack of sharp color differentiation between collagen and muscle (Van Gieson Picric Acid-Acid Fuchsin) Check saturated picric acid pH. Addition of .25 mL of hydrochloric acid to 100 mL van Gieson solution may sharpen color differentiation.
Decreased red staining (Masson Trichrome) Staining solution is aged or overused and should be discarded.
Blue staining of connective tissue appears faded (Masson Trichrome) Section has been overdifferentiated in acetic acid solution.
Poor staining (Masson Trichrome) Sections fixed in 10% NBF will do this if not mordanted in Bouin.
Red blood cells stain but neither the smooth muscle surrounding the blood vessels nor epithelium is stained red. (Masson Trichrome) Bad staining. Red blood cells should never be used to judge quality of stain. Repeat with fresh reagents.
Bleeding or diffusion into the surrounding mounting medium of basic aniline dyes. (Crystal Violet) Occurs with aqueous mounting media. Use modified apathy mounting medium. Or allow to air dry completely then dip in xylene and mount with synthetic resin.
Faint red birefringence (Congo Red) Sections are too thin.
Yellow birefringence (Congo Red) Sections are too thick.
Nonspecific staining (Alcian Blue) Rinsing with acid after the alcian blue solution prevents this.
Weak staining (Alcian Blue) Incomplete hydration causes this.
Nuclear staining (Alcian Blue) Prolonged staining in alcian blue solution causes this.
Cloudiness (Alcian Blue) Slides are improperly washed after nuclear fast red and then placed in alcohols causes this.
Carminophilic properties are obscured (Mayer Mucicarmine) Overstaining with hematoxylin or metanil yellow causes this.
Decreased staining (Mayer Mucicarmine) Aged and deteriorated mucicarmine solutions cause this. Discard and prepare fresh solutions.
Precipitation of dye (Best Carmine) Result of evaporation of ammonia in carmine solutions. Working solution should be filtered and used in a closed container.
Nonspecific staining (PAS) Highly chlorinated water is capable of oxidation, and if sections are transferred directly to this tap water, any loosely adsorbed Schiffs may be reoxidized to basic fuchsin.
Weak Schiffs reaction Chromate containing fixatives may overoxidize reactive groups, causing this.
False-negatives(PAS) Livers containing large amounts of glycogen should not be used, because the reaction can be weak but still very apparent, and poor or depleted reagent would not be detected.
Differential staining does not occur (Methyl Green-Pyronin Y) Acids and some fixatives may cause a depolymerization of DNA, resulting in a loss of ability to bind methyl green, when that occurs, the DNA will bind pyronin.
Masked Feulgen reaction (Feulgen) Counterstain should be applied very lightly to avoid this.
Created by: impossiblesum