Grocott-Gomori’s Methenamine Silver Staining

• A histology stain called Grocott-Gomori's Methenamine Silver (GMS) stain is primarily used to identify carbohydrates in fungal bacteria.
• György Gömöri, a doctor from Hungary who invented the staining technique, is honoured by having his staining process named after him.
• When used to identify Pneumocystis jiroveci, a fungus known to cause an opportunistic infection called pneumocytosis in immunosuppressed and compromised patients, it was initially applied to assess missing tissues and diseases in the liver and the rectum (Nadworny, Wang, Tredget, & Robert, 2010).
• Goromi has a better sensitivity for identifying fungus and other polysaccharide-rich microorganisms in paraffin prepared sections than other stains like Periodic Acid-Schiffs stain and Gridley Stain.
• Chromic acid and Gomori's methenamine-silver nitrate make up the bulk of the chemicals used in traditional Grocott staining.
• Additionally, it has been used to the detection of fungus in tissue samples.
• Its use in histology makes it perfect for finding and displaying fungus in tissues, aspirates, and smears.

Table of Contents

• Objectives
• Principle of Grocott-Gomori’s Methenamine Silver Staining
• Reagents
• Preparation of solution
• Procedure of Grocott-Gomori’s Methenamine Silver Staining
• Conventional Procedure
• Microwave Procedure
• Results
• Interpretation
• Applications of Grocott-Gomori’s Methenamine Silver Staining
• Advantages
• Limitations

Objectives

• To demonstrate that fungi are present in a particular sample.
• To demonstrate the existence of Histoplasma spp. and Pneumocystis jiroveci.

Principle of Grocott-Gomori’s Methenamine Silver Staining

The polysaccharides that make up the fungal cell wall interact with chromatin acid to undergo oxidation and produce aldehydes. The decrease of the alkaline-hexamine-silver combination serves as evidence for this.

The reduction process in Grocott's alkaline hexamine-silver solution results in precipitates of silver ions, which give the fungi's cell walls their dark colour. The capacity of cells to convert silver solution into metallic silver and create a black tissue component is known as the argentaffin response. The epithelial lining of the lungs, intestines, and melanin include argentaffin cells. The fate of the stain is determined by this response.

There are two types of staining techniques:

1. The conventional method at room temperature
2. Microwave method

Reagents

Chromium trioxide, sodium bisulfite, silver nitrate, methenamine, borax, gold chloride, sodium thiosulfate, and light green stock solutions are some examples of solutions.

Preparation of solution

NOTE: For manual preparation, these reagents often come in kits, and some solutions are already made.

• Chromic Acid(1.4%): Chromium trioxide 4g + 100ml Distilled water
• Silver solution: 3% Methanamine/hexamine 23ml + 5% Silver Nitrate 1.25ml + 5% Borax(Sodium tetraborate) 3ml  +25ml distilled water
• 3.2% Sodium Chloroaurate (Yellow gold chloride): Gold chloride 1.0g + 500ml Distilled water
• 4.2 % Sodium thiosulphate (Hypo): Sodium thiosulphate 2.0g + 100 ml Distilled water
• Working Light Green Stock Solution: 10ml of 1% light Green in 1% Acetic Acid + 40ml Distilled water

Note: Prepare the solutions in accordance with the staining concentrations that you require.

Procedure of Grocott-Gomori’s Methenamine Silver Staining

Conventional Procedure

1. Hydrate sections with distilled water.
2. After that, oxidise the portion for an hour at room temperature with 4% aqueous chromic acid.
3. Wash for a few seconds in the water
4. Use 1% sodium metabisulphite on the portions for one minute.
5. Wash for three minutes under a tap with a steady flow
6. Thoroughly rinse with distilled water.
7. Slides should be placed in a water bath with working silver solution that has been preheated for 15 to 20 minutes at 60 °C, or until the section turns yellowish-brown (check microscopically to observe fungi that have gone dark brown).
8. Well-rinse with distilled water.
9. Add 0.2% gold chloride to the sections and let sit for two minutes.
10. Well-rinse with distilled water.
11. For two minutes, apply 2% sodium thiosulfate to the portions.
12. Wash for five minutes under gently running tap water.
13. 15 seconds after adding the counterstain to the light green
14. Alcohol should be used to clean the slide of any excess bright green solution.
15. Dehydrate, clear and mount

Microwave Procedure

1. Sections should be hydrated with pure water.
2. Slides should be placed in a plastic Coplin jar with a loose lid containing 40 ml of 1.4% aqueous chromic acid.
3. 2 minutes, 30 seconds of 150 watts in the microwave
4. Slides should be dipped in the Coplin jar several times before standing for a further two minutes.
5. 30 seconds of washing with steadily running tap water
6. Sections should be treated with 3.2% sodium metabisulphite, left on for 10 seconds, and then thoroughly washed with tap water.
7. Warm up the working silver solution for 60 seconds at 450 watts.
8. Then thoroughly rinse the slide with distilled water before submerging the individual slides in the hot silver solution.
9. Microwave the sections at 150watt for 30 sec
10. In the Coplin jar, the dip moves up and down as it is left to stand for an additional minute. Use distilled water to rinse, then examine under a microscope. Note: If the slides are not adequately stained, dip them again into the silver solution, and examine them every minute until the fungus stains are dark brown.
11. After 30 seconds of 3.2% gold chloride treatment, clean the areas with distilled water.
12. For one minute, apply 4.2% sodium thiosulfate to the area, and then rinse it with tap water for 15 seconds.
13. Counterstain the area for 15 seconds with the light green solution.
14. Use alcohol to rinse the pale green solution off.
15. Cover and dry.

Results

Grocott-Gomori's Methenamine Silver Staining

Source: DOI: 10.1177/1040638716640313

Figure: Fungal organisms are highlighted by the Grocott methenamine silver special stain. Hyphae are 4-6 um wide; septate fungal hyphae with nonparallel walls, non-dichotomous branching, and terminal bulbous dilations.

• Histoplasma spp., Pneumocystis jirevoci, and fungi all produce black stains.
• Mycelia and hyphae inner portions have a pink-red or rose stain.
• Negative for toxoplasma and leishmania species
• Dark grey streaks from mucin
• The backdrop absorbs and stains the bright green.

Interpretation

Due to the silver nitrate solution's reduction process, the fungi will tint black. A black stain for fungus cells is produced by reducing silver nitrate solution to produce silver ions that have a black colour.

The conversion of silver nitrate to silver ions results in the mycelium and hyphae of the fungus staining rose pink or pink-red, while the mucin also stains dark grey. By absorbing the light green solution, the backdrop will seem a pale green colour.

CAUTION

• Put on gloves, safety glasses, and lab coats. Under the fume hood, keep hot, uncapped solutions. Avoid coming into touch with and inhaling chemicals and dyes.
• Chromic acid is very poisonous to the kidneys, caustic to skin and mucous membranes, and extremely carcinogenic.
• Ingestion of sodium metabisulfite can be toxic and result in gastrointestinal discomfort and irritation. Additionally, it irritates the mucous membrane, skin, and eyes.
• The skin and eyes might get irritated by silver nitrate.
• The oxidizer is a tumorigenic substance that, when consumed, produces a severe gastrointestinal pain and may be carcinogenic.
• Ingestion of sodium thiosulfate is hazardous and can irritate the stomach, skin, eyes, and respiratory tract.
• Green Light SF Yellowish is thought to be a potential cancer-causing agent.

Applications of Grocott-Gomori’s Methenamine Silver Staining

• used to distinguish fungi in tissue slices
• used to distinguish bacterial cells from fungal cells
• also used to distinguish fungi that resemble yeast, such as Pneumocystis jiroveci

Advantages

• It is an aggressive stain that securely fixes the stained areas so they may be utilised for future observation and reference.
• Tt is able to distinguish between distinct fungi's morphologies.

Limitations

• If breathed, the chemical reagents might irritate the skin and digestive system.
• The agents also have cancer-causing properties.