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Troubleshooting
Troubleshooting stains
| Problem | Solution |
|---|---|
| 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. |
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