Grocott-Gomori’s Methenamine Silver Staining

The main objectives of Grocott-Gomori’s Methenamine Silver Staining are:

• To demonstrate the presence of fungi in a given sample, such as tissue sections, smears, or aspirates.
• To demonstrate the presence of specific fungal species that cause opportunistic infections in immunocompromised or immunosuppressed patients, such as Pneumocystis jiroveci and Histoplasma spp.

Fungi are eukaryotic microorganisms that have a cell wall composed of polysaccharides, such as chitin and glucan. These polysaccharides can be detected by using silver and methenamine, which are the main components of Grocott-Gomori’s Methenamine Silver Staining. This staining technique is based on the principle of argentaffin reaction, which is the ability of some cells to reduce silver ions to metallic silver, forming a black precipitate. By using this staining technique, fungal cells can be visualized as black structures against a pale green background.

Grocott-Gomori’s Methenamine Silver Staining is a popular and sensitive method for identifying fungal organisms in histology and microbiology. It can differentiate various fungal morphologies, such as hyphae, spores, yeast-like cells, and arthroconidia. It can also distinguish fungal cells from bacterial cells, which do not stain with this technique. Moreover, it can identify some fungi that are difficult to detect by other methods, such as Pneumocystis jiroveci and Histoplasma spp. These fungi are known to cause pneumonia and systemic infections in patients with impaired immunity, such as those with HIV/AIDS, cancer, organ transplantation, or corticosteroid therapy.

Therefore, Grocott-Gomori’s Methenamine Silver Staining is a useful and reliable technique for demonstrating the presence and diversity of fungi in various samples and for diagnosing fungal infections in clinical settings.

The principle of this staining technique is based on the oxidation of polysaccharides in the fungal cell wall by chromic acid, which produces aldehydes. These aldehydes then reduce the alkaline-hexamine-silver complex to form black metallic silver deposits on the fungal structures. This reaction is similar to the periodic acid-Schiff (PAS) reaction, but it uses silver instead of fuchsin as the dye. The silver staining is more sensitive and specific for fungi than the PAS staining, as it can differentiate various fungal morphologies and species.

The reduction of silver by the fungal cell wall is also known as the argentaffin reaction, which means that the cells have the ability to bind and reduce silver ions to metallic silver. Argentaffin cells are normally found in the epithelial lining of the lungs, intestines, and skin, where they produce melanin. However, some fungi also have this property, which makes them visible by the Grocott-Gomori’s methenamine silver stain.

The staining procedure can be performed by two methods: the conventional method at room temperature or the microwave method. The conventional method takes longer but requires less equipment, while the microwave method is faster but requires a microwave oven and special containers. Both methods involve the following steps:

• Hydration of tissue sections with distilled water
• Oxidation of polysaccharides with chromic acid
• Washing with water and sodium bisulfite
• Rinsing with distilled water
• Staining with pre-heated working silver solution
• Rinsing with distilled water
• Toning with gold chloride
• Rinsing with distilled water
• Fixing with sodium thiosulfate
• Washing with tap water
• Counterstaining with light green
• Dehydration, clearing, and mounting

The result of the staining is that fungal organisms appear black against a pale green background. The inner parts of mycelia and hyphae may stain pink-red or rose, while mucin may stain dark gray. The stain can be used to identify various fungal species, such as Pneumocystis jiroveci, Histoplasma spp., Candida spp., Aspergillus spp., Cryptococcus spp., and others.

Some cells in the body have the ability to take up silver from a solution and then reduce it to a visible metallic state, without the aid of a reducing agent. This is called an argentaffin reaction, and it is used to identify certain types of cells, such as neuroendocrine cells, melanin-producing cells, and some bacteria and fungi. The argentaffin reaction is based on the presence of organic compounds in the cell cytoplasm that can act as electron donors and reduce silver ions to metallic silver. The resulting silver granules appear black under the microscope and contrast with the surrounding tissue.

The argentaffin reaction is different from the argyrophil reaction, which requires an external reducing agent to produce the same effect. The argyrophil reaction is more sensitive and can detect more types of cells than the argentaffin reaction. However, the argentaffin reaction is more specific and can distinguish between different types of argentaffin cells based on their staining intensity and distribution.

The argentaffin reaction is used in histology to demonstrate the presence of fungi in tissue sections, as well as some bacterial and parasitic infections. The fungal cell wall contains polysaccharides that can undergo oxidation by chromic acid to form aldehydes, which then reduce the alkaline-hexamine-silver complex. The fungi appear black against a green background stained by light green. The argentaffin reaction can also be used to identify Pneumocystis jiroveci and Histoplasma spp., which are yeast-like fungi that cause opportunistic infections in immunocompromised patients.

The argentaffin reaction can also be used to diagnose carcinoid tumors, which are neuroendocrine tumors that secrete serotonin and other hormones. Carcinoid tumors are derived from argentaffin cells that are normally found in the gastrointestinal tract, lungs, appendix, and other organs. The argentaffin cells in carcinoid tumors retain their ability to reduce silver and appear black with various silver staining methods. The argentaffin reaction can help differentiate carcinoid tumors from other types of tumors that may have a similar appearance.

The argentaffin reaction can also be used to detect melanin, which is a brown-black pigment produced by melanocytes in the skin, hair, eyes, and some parts of the central nervous system. Melanin can protect the cells from ultraviolet radiation and oxidative stress, but it can also be associated with some diseases such as melanoma, albinism, and vitiligo. The argentaffin reaction can demonstrate the presence and distribution of melanin in tissue sections by showing black granules in the cytoplasm of melanocytes or melanoma cells.

The argentaffin reaction is a simple and useful technique for identifying certain types of cells and organisms in histological specimens. However, it has some limitations and precautions that should be considered. For example, some substances such as formaldehyde or mercury can interfere with the reaction and cause false-positive or false-negative results. Also, some of the reagents used for the argentaffin reaction are corrosive, toxic, or carcinogenic and should be handled with care. Finally, the argentaffin reaction should be interpreted in conjunction with other histological features and clinical information to avoid misdiagnosis or confusion.

The main reagents used in Grocott-Gomori’s Methenamine Silver Staining are:

• Chromium trioxide solution: This is an oxidizing agent that converts polysaccharides in the fungal cell wall into aldehydes. It is also known as chromic acid or chromate anhydride .
• Sodium bisulfite solution: This is a reducing agent that removes excess chromium trioxide from the tissue sections. It is also known as sodium hydrogen sulfite .
• Silver nitrate solution: This is a source of silver ions that react with the aldehydes in the fungal cell wall to form metallic silver. It is also known as lunar caustic .
• Methenamine solution: This is a source of hexamine or hexamethylenetetramine, which acts as a base and a catalyst for the silver nitrate reduction. It is also known as urotropin or hexamine .
• Borax solution: This is a source of sodium tetraborate, which acts as a buffer and a stabilizer for the silver nitrate solution. It is also known as sodium borate or simply borax .
• Gold chloride solution: This is a toning agent that enhances the contrast and specificity of the silver stain. It is also known as chloroauric acid or gold(III) chloride .
• Sodium thiosulfate solution: This is a fixing agent that removes unreacted silver ions from the tissue sections. It is also known as sodium hyposulfite or hypo .
• Light green stock solution: This is a counterstain that colors the background and non-fungal elements in pale green. It is also known as light green SF yellowish or acid green 5 .

The reagents for Grocott-Gomori’s Methenamine Silver Staining can be obtained from commercial kits or prepared manually according to the following steps:

• Chromic Acid (1.4%): Dissolve 4 g of chromium trioxide in 100 ml of distilled water. Store in a dark glass bottle at room temperature. This solution is stable for several months.
• Silver Solution: Mix 23 ml of 3% methenamine/hexamine, 1.25 ml of 5% silver nitrate, and 3 ml of 5% borax in a glass beaker. Add 25 ml of distilled water and stir well. Transfer to a dark glass bottle and store at room temperature. This solution is stable for one week.
• Sodium Chloroaurate (0.2%): Dissolve 1 g of gold chloride in 500 ml of distilled water. Store in a dark glass bottle at room temperature. This solution is stable for several months.
• Sodium Thiosulfate (4.2%): Dissolve 2 g of sodium thiosulfate in 100 ml of distilled water. Store in a glass bottle at room temperature. This solution is stable for several months.
• Working Light Green Stock Solution: Mix 10 ml of 1% light green in 1% acetic acid with 40 ml of distilled water. Store in a glass bottle at room temperature. This solution is stable for one week.

Note: Prepare the solutions depending on the concentrations you need for staining. Use fresh distilled water and clean glassware to avoid contamination and precipitation.

There are two types of procedures for Grocott-Gomori’s Methenamine Silver Staining: the conventional procedure and the microwave procedure. Both procedures aim to oxidize the fungal cell wall polysaccharides with chromic acid, reduce the silver solution with hexamine, and tone the sections with gold chloride and sodium thiosulfate. The main difference is the duration and temperature of each step.

Conventional Procedure

• Hydrate sections with distilled water.
• Oxidize the sections with 4% aqueous chromic acid at room temperature for 1 hour.
• Wash in water for a few seconds.
• Treat the sections with 1% sodium metabisulfite for 1 minute.
• Wash in smoothly running tap water for 3 minutes.
• Rinse thoroughly in distilled water.
• Put the slides in pre-heated working silver solution in a water bath at 60°C for 15 to 20 minutes until the sections turn yellowish-brown. Check microscopically to see if fungi turn dark brown.
• Rinse well in distilled water.
• Tone the sections with 0.2% gold chloride for 2 minutes.
• Rinse well in distilled water.
• Treat the sections with 2% sodium thiosulfate for 2 minutes.
• Wash with smoothly running tap water for 5 minutes.
• Counterstain the sections with light green for 15 seconds.
• Rinse off the excess light green solution with alcohol.
• Dehydrate, clear and mount.

Microwave Procedure

• Hydrate the sections to distilled water.
• Place slides in 40 ml of 1.4% aqueous chromic acid in a loosely covered plastic Coplin jar.
• Microwave at 150 Watt for 2 minutes and 30 seconds.
• Dip the slides up and down in the Coplin jar and let them stand for an extra 2 minutes.
• Wash in smoothly running tap water for 30 seconds.
• Add 3.2% sodium metabisulfite to the sections and leave for 10 seconds and wash with tap water for 30 seconds.
• Preheat the working silver solution at 450 watts for 60 seconds and rinse the slides thoroughly with distilled water. Then place the slides sections in the hot silver solution.
• Microwave the sections at 150 watt for 30 seconds.
• Dip slides up and down in the Coplin jar and allow to stand for another 1 minute. Rinse with distilled water and check under the microscope. Note: Dip back the slides in the silver solution if they are not stained sufficiently and check every 1 minute until the fungi stain dark brown.
• Tone the sections with 3.2% gold chloride for 30 seconds and rinse with distilled water.
• Add 4.2% of sodium thiosulfate to the section for 1 minute and wash with tap water for 15 seconds.
• Counterstain the section with light green solution for 15 seconds.
• Rinse off the light green solution with alcohol.
• Dry and cover.

The Grocott-Gomori’s Methenamine Silver Staining highlights the fungal organisms by staining the polysaccharides in their cell walls. The fungi appear black or dark brown against a pale green background. The inner parts of the mycelia and hyphae may stain pink-red or rose. The mucin may stain dark grey. The staining intensity and specificity may vary depending on the type and quality of the tissue sample, the duration and temperature of the silver solution, and the counterstain used .

The following table summarizes the expected results for some common fungal species and nonfungal organisms that may be detected by GMS stain :

The interpretation of Grocott-Gomori`s methenamine silver staining depends on the identification of the black or dark brown coloration of the fungal cell walls due to the reduction of silver ions by the aldehyde groups. The shape, size and arrangement of the fungal elements can also provide clues to the type of fungus present in the sample. However, fungal morphology on GMS is usually not specific enough to allow definitive species identification, and other methods such as culture or molecular testing are preferred for confirmation.

Some examples of fungal organisms that can be detected by GMS are:

• Pneumocystis jiroveci: a yeast-like fungus that causes pneumocystis pneumonia (PCP) in immunocompromised patients. It appears as clusters of small (2-5 µm) round or oval cysts with thin walls and intracystic bodies .
• Histoplasma capsulatum: a dimorphic fungus that causes histoplasmosis, a systemic infection that affects the lungs and other organs. It appears as small (2-4 µm) round or oval yeast cells with narrow-based buds within macrophages or extracellularly .
• Aspergillus spp.: a group of filamentous fungi that cause various infections such as aspergillosis, allergic bronchopulmonary aspergillosis (ABPA) and invasive pulmonary aspergillosis (IPA). They appear as septate hyphae with acute-angle branching (45°) and occasional conidial heads .
• Candida spp.: a group of yeast-like fungi that cause various infections such as candidiasis, thrush and invasive candidiasis. They appear as round or oval yeast cells with pseudohyphae and blastoconidia .

GMS can also stain some nonfungal organisms, such as bacteria, mycobacteria, Strongyloides larvae and viral inclusions. Therefore, it is important to correlate the GMS findings with other stains (such as Gram stain, Ziehl-Neelsen stain or immunohistochemistry) and clinical information to avoid misinterpretation.

Additionally, GMS can be affected by artifacts, mimickers and other pigments that can cause false-positive or false-negative results. Some common sources of confusion are:

• Melanin: a pigment produced by melanocytes or melanoma cells that can stain black with GMS. It can be differentiated from fungi by its irregular shape, lack of cell wall and negative Fontana-Masson stain .
• Hemosiderin: a pigment derived from hemoglobin that can stain brown with GMS. It can be differentiated from fungi by its granular appearance, presence in macrophages or blood vessels and positive Prussian blue stain .
• Formalin pigment: an artifact caused by the interaction of formalin and hemoglobin that can stain brown with GMS. It can be differentiated from fungi by its amorphous shape, random distribution and negative Schmorl`s stain .
• Silver precipitate: an artifact caused by incomplete washing of silver solution that can stain black with GMS. It can be differentiated from fungi by its globular shape, lack of cellular detail and negative Schiff reagent .

To avoid these pitfalls, it is recommended to use proper fixation, staining and washing techniques, as well as appropriate controls and quality assurance measures.

Grocott-Gomori’s Methenamine Silver Staining involves the use of several chemicals and dyes that can be hazardous to your health and safety. Therefore, you should follow these precautions when performing the staining procedure:

• Wear gloves, goggles and laboratory coats to protect your skin, eyes and clothing from exposure to corrosive, toxic or carcinogenic substances .
• Keep hot uncapped solutions under the fume hood to avoid inhalation of vapors or fumes .
• Avoid contact and inhalation of dyes and chemicals, especially chromic acid, sodium metabisulfite, silver nitrate, gold chloride and light green . These substances can cause irritation, inflammation, allergic reactions, gastrointestinal distress, kidney damage or cancer .
• Dispose of the waste solutions and stained slides according to the local regulations and guidelines for hazardous materials.
• In case of accidental contact or ingestion of any of the reagents, seek medical attention immediately and follow the instructions on the safety data sheets.

Grocott-Gomori’s Methenamine Silver Staining is a widely used staining method in histology and microbiology to demonstrate the presence and morphology of fungal organisms in tissue sections and smears. The stain is particularly useful for identifying fungi that have polysaccharides in their cell walls, such as Pneumocystis jiroveci, Histoplasma spp., Candida spp., Aspergillus spp., Cryptococcus neoformans, and Blastomyces dermatitidis . The stain also helps to differentiate fungal cells from bacterial cells, which do not stain with silver.

The stain can also be used to detect other organisms that have argentaffin properties, such as Leishmania spp., Toxoplasma gondii, Helicobacter pylori, and Legionella pneumophila. However, these organisms are usually negative or weakly positive with the stain and require additional confirmation with other methods.

The stain can also reveal some non-biological structures that may be present in the tissue samples, such as elastic fibers, collagen fibers, reticular fibers, basement membranes, and amyloid deposits. These structures may show aberrant staining with silver due to their chemical composition or oxidation state. Therefore, it is important to interpret the stain results carefully and correlate them with other histological and clinical findings.

Some of the applications of Grocott-Gomori’s Methenamine Silver Staining are:

• Diagnosis of fungal infections in immunocompromised patients, such as those with HIV/AIDS, organ transplant recipients, cancer patients, or patients on immunosuppressive drugs .
• Diagnosis of opportunistic infections caused by Pneumocystis jiroveci, which causes a form of pneumonia called Pneumocystis pneumonia (PCP) or pneumocystosis .
• Diagnosis of systemic mycoses caused by Histoplasma spp., which can affect various organs such as the lungs, liver, spleen, bone marrow, and lymph nodes .
• Diagnosis of cutaneous and subcutaneous mycoses caused by various fungi that invade the skin and underlying tissues, such as dermatophytes, Sporothrix schenckii, and Madurella mycetomatis .
• Diagnosis of mucosal and oral mycoses caused by Candida spp., which can cause thrush, esophagitis, vaginitis, and denture stomatitis .
• Diagnosis of invasive and allergic bronchopulmonary aspergillosis caused by Aspergillus spp., which can cause asthma-like symptoms, lung nodules, cavities, and hemoptysis .
• Diagnosis of cryptococcosis caused by Cryptococcus neoformans, which can cause meningitis, encephalitis, lung lesions, and skin ulcers .
• Diagnosis of blastomycosis caused by Blastomyces dermatitidis, which can cause skin lesions, lung infections, bone infections, and disseminated disease

• It is an aggressive stain that fixes the stained sections firmly and can be used for future reference and observation.
• It can differentiate various fungal morphologies of fungi.
• It has a higher sensitivity and specificity than other stains for detecting fungi and other polysaccharide-rich microorganisms.
• It can identify yeast-like fungi such as Pneumocystis jiroveci, which are difficult to detect by other methods.

Grocott-Gomori’s Methenamine Silver Staining is a useful technique for identifying and differentiating fungal cells in tissue sections, but it also has some limitations that should be considered. Some of the limitations are:

• The chemical reagents used in the staining process can cause irritations of the skin, eyes, mucous membranes and respiratory tract if they come in contact with them or if they are inhaled. Some of the reagents, such as chromic acid and light green SF yellowish, are also carcinogenic and can increase the risk of cancer with prolonged exposure. Therefore, proper safety precautions should be taken when handling these reagents, such as wearing gloves, goggles and laboratory coats, and working under a fume hood or a well-ventilated area.
• The staining procedure can be time-consuming and labor-intensive, especially the conventional method that requires heating the silver solution in a water bath for 15 to 20 minutes and checking the staining progress microscopically. The microwave method can reduce the staining time to about 5 minutes, but it still requires careful monitoring and adjustment of the microwave power and time. Moreover, the staining procedure can be affected by various factors, such as the quality and concentration of the reagents, the thickness and fixation of the tissue sections, the pH and temperature of the solutions, and the duration and intensity of the staining steps. Therefore, optimal staining conditions should be established and followed consistently to ensure reliable and reproducible results.
• The staining specificity can be compromised by some factors that can interfere with the argentaffin reaction or produce false-positive or false-negative results. For example, some non-fungal structures or substances, such as melanin, argentaffin cells, mucin and lipofuscin, can also reduce the silver solution and stain black or dark grey, which can be confused with fungal cells. To avoid this confusion, careful examination of the morphology and location of the stained structures is necessary. On the other hand, some fungal cells may not stain well or at all with this technique, such as those with thick cell walls or low polysaccharide content. To overcome this limitation, additional stains or methods may be needed to confirm or complement the diagnosis of fungal infections.

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