Grocott-Gomori’s Methenamine Silver Staining: Principle, Procedure & Results

Biopsy specimens are examined for tissue changes and the presence of infectious organisms. Grocott-Gomori’s Methenamine Silver Staining is commonly used in histological examination when fungal elements are suspected in the tissue.

Routine hematoxylin and eosin (H&E) staining is used to examine the tissue architecture and the inflammatory response. But fungal elements may not be clearly visible in every H&E-stained section. They may be present in small numbers or appear faintly stained. Some fungal structures are also difficult to distinguish from the surrounding tissue. In some specimens, inflammatory changes are visible, but the fungal elements are difficult to find.

What is Grocott-Gomori’s Methenamine Silver Staining?

Grocott-Gomori’s Methenamine Silver (GMS) staining is a histochemical silver impregnation technique used mainly to demonstrate fungal organisms in tissue sections and suitable cytological preparations. GMS stands for Grocott-Gomori’s Methenamine Silver. It is also known as Grocott stain, Grocott’s methenamine silver stain, Gomori methenamine silver stain and hexamine silver stain.

The fungal cell wall is the main structure stained by GMS. The staining gives the fungal elements a dark brown to black outline against the background tissue. Some non-fungal organisms and tissue components can also take up the silver stain. GMS staining alone cannot identify the exact fungal species.

The names Grocott and Gomori are related to the same methenamine-silver staining method. Gomori introduced the methenamine-silver reaction, and Grocott later used and modified this technique for the demonstration of fungi in tissue sections and smears.

Objectives of GMS Staining

The objectives of GMS staining are as follows:

• To demonstrate fungal elements present in tissue sections.
• To detect fungi present in small numbers.
• To observe the shape and distribution of fungal elements in infected tissue.
• To detect Pneumocystis jirovecii and other fungi in suitable specimens.
• To provide good contrast between the stained organisms and the surrounding tissue.
• To assist in the histopathological examination of suspected fungal infections.

Principle of Grocott-Gomori’s Methenamine Silver Staining

Grocott-Gomori’s methenamine silver (GMS) staining is based on the oxidation of carbohydrates and the reduction of silver ions. The fungal cell wall contains polysaccharides. Chromic acid oxidises the carbohydrate groups of the polysaccharides to aldehyde groups (some of which are further oxidised to carboxyl groups).

So the aldehyde groups present in the surrounding tissue are all destroyed by further oxidation. The fungal cell walls have a high content of polysaccharide and so retain a sufficient number of aldehyde groups. These aldehyde groups will reduce the silver ions of the methenamine silver reagent to metallic silver which is then deposited on the fungal cell walls resulting in a dark brown to black colour. Some of the other tissue components will also react with the reagent.

Methenamine keeps the Ag+ in a soluble complex. Sodium borate provides the alkaline conditions required for the silver reaction. Sodium bisulphite removes the residual chromic acid and prevents further oxidation.

Gold chloride is used for toning the deposited silver (some of the metallic silver is replaced by gold in the process which gives a better contrast). Sodium thiosulphate is used to remove the residual silver ions. Light green is the counterstain and stains the surrounding tissue pale green, the fungal walls remaining dark brown to black.

The preparation of the methenamine silver solution, the heating, the incubation and the application of the reagents are all part of the staining procedure. They are not part of the chemical principle of GMS staining.

Reagents and Materials Required for GMS Staining

The following are the reagents and materials required for Grocott-Gomori’s methenamine silver (GMS) staining. The concentrations given below are for a method using 4% chromic acid and methenamine-silver treatment at 60°C. Other GMS methods may use different concentrations.

Reagents Required

1. Chromic acid: A 4% aqueous solution is used to oxidise the carbohydrates present in fungal cell walls.
2. Sodium metabisulfite: A 1% aqueous solution removes the residual chromic acid after oxidation. Sodium bisulfite is used in some other GMS methods.
3. Methenamine (hexamethylenetetramine): A 3% aqueous stock solution is required. Methenamine forms a soluble complex with silver ions.
4. Silver nitrate: The 5% aqueous stock solution provides silver ions for staining the fungal cell walls.
5. Borax (sodium tetraborate): A 5% aqueous stock solution maintains the alkaline condition required for the silver reaction.
6. Methenamine-silver working solution: The working solution contains methenamine, silver nitrate and borax stock solutions diluted with distilled water. It is used for silver impregnation. The final concentrations of these chemicals are different from their stock concentrations.
7. Gold chloride: A 0.2% aqueous solution is used for toning the deposited silver. It improves the contrast of the fungal structures.
8. Sodium thiosulfate: A 2% aqueous solution removes the remaining unreduced silver ions after staining.
9. Light green counterstain: The stock solution contains 1% light green in 1% acetic acid. The diluted working solution contains approximately 0.2% light green. It stains the surrounding tissue pale green.
10. Distilled or deionized water: It is used for preparing the staining solutions and washing the sections between treatments.
11. Ethanol: Graded alcohol and absolute alcohol are used for hydration and dehydration of tissue sections.
12. Xylene or a suitable clearing agent: It is used to remove paraffin from the tissue sections and for clearing the stained sections before mounting.

Materials and Equipment

The following are the materials and equipment required for GMS staining:

1. Clean glass slides containing tissue sections or suitable cytological specimens.
2. Coplin jars or staining containers to hold the slides during staining.
3. Acid-cleaned glassware for preparing and handling the methenamine-silver solution.
4. Staining racks and slide holders.
5. A temperature-controlled water bath capable of maintaining 60°C for silver impregnation. Validated microwave equipment may also be used with a suitable microwave staining protocol.
6. Measuring cylinders, pipettes and reagent bottles.
7. A light microscope for examining the stained fungal elements.
8. Coverslips and a permanent mounting medium.
9. Gloves, laboratory coat and safety goggles. A suitable fume hood is required for handling hazardous staining reagents.

Specimen and Control Requirements

1. Formalin-fixed, paraffin-embedded tissue sections: Tissue fixed in 10% neutral buffered formalin is commonly used. The sections are usually cut at a thickness of 4–5 µm and placed on clean glass slides.
2. Cytological preparations: Suitable fixed smears and other cytological specimens may also be stained by GMS. The fixation and staining conditions depend upon the type of specimen and the method used.
3. Preparation of sections: The paraffin sections are properly deparaffinized and brought to water before staining. Cytological specimens require suitable fixation before treatment with the staining reagents.
4. Positive control: A known fungal-positive tissue section is required with each staining run. Tissue containing recognizable Aspergillus hyphae can be used as a positive control. The fungal elements should stain dark brown to black.
5. Control slides: The control and test sections are processed under the same staining conditions. The positive control is examined to check the staining reaction.

Preparation of Reagents for GMS Staining

The preparation of reagents for the conventional water-bath method of Grocott-Gomori’s Methenamine Silver (GMS) staining is given below. This method uses 5% chromic acid, whereas the microwave modification uses 2% chromic acid. The concentrations are expressed as weight/volume (w/v), unless otherwise stated.

Preparation of Methenamine Silver Stock and Working Solutions

1. 3% Methenamine Solution: Dissolve 3 g of methenamine (hexamethylenetetramine) in distilled water and make up the volume to 100 mL. The solution can be stored in an acid-cleaned bottle in the refrigerator for up to 6 months.
2. 5% Silver Nitrate Solution: Take 5 g of silver nitrate and dissolve it in distilled water. Make the final volume 100 mL. Store the solution in an acid-cleaned brown bottle in the refrigerator. It is stable for up to 6 months.
3. Methenamine Silver Stock Solution: Mix 100 mL of 3% methenamine solution with 5 mL of 5% silver nitrate solution. The mixture is stored in an acid-cleaned brown bottle in the refrigerator for up to 3 months. Borax is not added to this stock solution.
4. 5% Borax Solution: Dissolve 5 g of sodium borate (borax) in distilled water and make up the volume to 100 mL. Mix well. The solution can be stored for up to 3 months.
5. Alkaline Methenamine Silver Working Solution: Take 25 mL of methenamine silver stock solution and add 25 mL of distilled water. Then add 2.5 mL of 5% borax solution and mix. The final volume is 52.5 mL. It contains approximately 1.36% methenamine, 0.11% silver nitrate and 0.24% borax. The working solution is prepared fresh before staining and should not be stored for reuse.

The working silver solution should be clear. If the solution becomes cloudy or a silver coating appears on the walls of the staining container, discard the solution and prepare a fresh one.

Preparation of Other Staining Solutions

1. 5% Chromic Acid Solution: Dissolve 5 g of chromium trioxide in distilled water and make up the volume to 100 mL. The 5% solution is used in the conventional water-bath method. Store it in a properly labelled, compatible container. The solution should be replaced if it turns brown. Its storage period depends upon the laboratory’s validated conditions.
2. 1% Sodium Metabisulfite Solution: Take 1 g of sodium metabisulfite and dissolve it in distilled water. Make up the volume to 100 mL. The solution can be stored for up to 6 months under the specified storage conditions. A fresh solution should be used if the staining reaction is unsatisfactory.
3. 0.5% Gold Chloride Solution: Dissolve 0.5 g of gold chloride in distilled water and make up the volume to 100 mL. Store the solution in an acid-cleaned bottle in the refrigerator. It is stable for up to 1 year.
4. 5% Sodium Thiosulfate Solution: Dissolve 5 g of sodium thiosulfate in distilled water and make up the volume to 100 mL. Mix well. This solution is also known as hypo. It is stored in a clean, labelled container and used within the laboratory’s validated storage period.
5. 0.2% Light Green Counterstain: Dissolve 0.2 g of light green SF yellowish in 100 mL of distilled water. Add 0.2 mL of glacial acetic acid and mix well. The prepared solution is used for counterstaining and can be stored for up to 6 months.

Clean glassware and distilled or deionized water are required for preparing the reagents. Acid-cleaned glassware should be used for the methenamine silver solutions. Chromic acid must be handled in a chemical fume hood using suitable protective equipment.

Before staining, check the chromic acid, sodium metabisulfite and borax solutions. Fresh solutions may be required if the fungal elements in the positive-control section fail to stain black.

Procedure of Grocott-Gomori’s Methenamine Silver Staining

Grocott-Gomori’s Methenamine Silver (GMS) staining can be performed by the conventional water-bath method or the microwave method. Both methods use different oxidation and silver impregnation conditions.

Conventional GMS Staining Procedure

The procedure of conventional GMS staining using the water-bath method is given below:

1. Prepare 4–5 µm thick sections of formalin-fixed, paraffin-embedded tissue on clean glass slides. Include a known fungal-positive control section.
2. Deparaffinize the sections in xylene and hydrate through descending grades of alcohol to distilled water.
3. Place the slides in 5% chromic acid at 60°C in a water bath for 1 hour.
4. Wash the sections in tap water and rinse with distilled water.
5. Treat the sections with 1% sodium metabisulfite for 1 minute at room temperature.
6. Wash in tap water and rinse in three changes of distilled water.
7. Place the slides in freshly prepared alkaline methenamine silver working solution at 60°C in a water bath for 1 hour or until the tissue becomes brown, similar to the colour of a brown paper bag.
8. Rinse the slides in two changes of distilled water.
9. Treat the sections with 0.5% gold chloride for 1 minute or until they turn grey.
10. Wash in distilled water.
11. Place the slides in 5% sodium thiosulfate (hypo) for 3 minutes to remove the unreduced silver.
12. Wash in tap water and rinse with distilled water.
13. Counterstain with working light green solution for 1 minute.
14. Rinse the slides in distilled water.
15. Dehydrate the sections through graded alcohol, clear in xylene and mount with a suitable mounting medium and coverslip.
16. Examine the stained sections under a light microscope. The fungal elements appear black against a green background.

Microwave GMS Staining Procedure

The microwave method uses 2% chromic acid and shorter heating times. The procedure of microwave-assisted GMS staining is given below:

1. Prepare 4–5 µm thick sections of formalin-fixed, paraffin-embedded tissue and a fungal-positive control on clean glass slides.
2. Remove the paraffin using xylene and hydrate the sections through graded alcohol to distilled water.
3. Place the slides in 2% chromic acid in a microwave-compatible staining container.
4. Heat in the microwave at high power for 45 seconds. Allow the slides to remain in the solution for another 5 minutes.
5. Wash the sections in tap water and rinse with distilled water.
6. Treat the slides with 1% sodium metabisulfite for 1 minute at room temperature.
7. Wash in tap water and rinse in three changes of distilled water.
8. Place the sections in freshly prepared alkaline methenamine silver working solution.
9. Heat in the microwave at high power for 70 seconds. Agitate the slides in the hot solution. The tissue should become brown, similar to a brown paper bag.
10. Rinse in two changes of distilled water.
11. Treat the sections with 0.5% gold chloride for 1 minute or until they turn grey.
12. Wash in distilled water.
13. Place the slides in 5% sodium thiosulfate for 3 minutes.
14. Wash in tap water and rinse with distilled water.
15. Counterstain with working light green solution for 1 minute.
16. Rinse in distilled water, dehydrate through graded alcohol and clear in xylene.
17. Mount the sections with a suitable mounting medium and coverslip.
18. Examine under a light microscope. Fungal structures are stained black and the surrounding tissue appears green.

The microwave heating times of 45 and 70 seconds are specific to the documented method. Microwave power, heating time and solution temperature must be validated for the equipment used in the laboratory. The 5% chromic acid used in the conventional method should not be substituted for 2% chromic acid in the microwave method because it may cause tissue sections to detach from the slides.

Observation and Results of GMS Staining

Observation

The stained tissue sections are examined under a light microscope. The observations of Grocott-Gomori’s Methenamine Silver (GMS) staining are as follows:

1. The fungal cell walls are stained dark gray to black.
2. Yeast cells are seen as round or oval structures with dark outlines. Budding may also be visible.
3. Pseudohyphae appear as elongated chains of cells with constrictions at their junctions.
4. Fungal hyphae are seen as long, thread-like structures. Branching and septa may be visible.
5. The cyst or ascus walls of Pneumocystis are stained dark gray to black. Some may show a cup-shaped appearance.
6. The surrounding tissue appears pale green when light green is used as the counterstain.
7. The positive-control section shows recognizable fungal structures stained black.
8. Some nonfungal structures may also show gray or black silver deposits.

Results

The results of GMS staining are given below:

The exact colour and background contrast may vary depending upon the staining protocol and the counterstain used.

Interpretation of GMS Staining Results

Positive GMS Staining Result

A positive GMS staining result indicates the presence of dark gray to black fungal or fungus-like structures in the examined specimen. The following are the findings of a positive GMS staining result:

• Fungal elements may be present as budding yeast cells, pseudohyphae or branching hyphae. Their shape, size and arrangement are examined under the microscope.
• The fungal elements may be found on the tissue surface, within a lumen or inside the tissue. Their distribution helps in determining the extent of fungal involvement.
• Fungal elements present within the tissue or blood vessels indicate tissue invasion. Inflammatory changes, necrosis and tissue damage can be examined in the corresponding hematoxylin and eosin (H&E)-stained sections.
• Fungal elements present only on the tissue surface or within a lumen may indicate colonization. Contamination is also possible, particularly when isolated fungal structures are seen without tissue reaction.
• In suspected Aspergillus infection, narrow septate hyphae with acute-angle branching may be observed. Other filamentous fungi can also show similar structures. GMS staining alone cannot confirm Aspergillus infection or identify the species.
• In suspected Candida infection, budding yeast cells and pseudohyphae may be present in the tissue. These structures may suggest Candida, but they are not sufficient for species identification.
• Some nonfungal organisms and tissue structures may also show black or gray staining. Silver precipitates can appear as dark deposits. These should not be confused with fungal elements.
• The GMS findings are examined along with H&E staining and the clinical findings. Fungal culture and suitable molecular tests may be required for identification of the organism.

Negative GMS Staining Result

A negative GMS staining result indicates that no recognizable fungal elements are observed in the examined tissue section. It does not necessarily mean that fungi are absent from the specimen.

The following conditions may be associated with a negative GMS staining result:

• Fungal organisms may be absent from the tissue section examined.
• The fungal elements may be present in very small numbers. They may not be detected in every section.
• Fungal distribution may be uneven within the tissue. A section taken from an area without fungal elements may give a negative result.
• Some fungal structures may stain poorly or remain faint due to differences in their cell-wall properties or the condition of the organisms.
• Improper oxidation, inactive staining reagents or errors during staining may affect the result. The fungal-positive control should show recognizable black-stained fungal structures.
• When the positive control fails to stain properly, the staining result cannot be considered reliable. The staining procedure should be repeated after checking the reagents and staining conditions.
• If fungal infection is still suspected, additional tissue sections may be examined. H&E staining, fungal culture of suitable unfixed specimens and other appropriate tests, including molecular methods, may be used for further examination.

Applications of Grocott-Gomori’s Methenamine Silver Staining

The following are the applications of Grocott-Gomori’s methenamine silver staining:

• GMS staining is used for the detection of fungi in tissue sections.
• It is used for screening tissue samples when a fungal infection is suspected. The fungal cell walls are stained dark brown to black.
• Detection of Pneumocystis jirovecii in lung tissue and other respiratory specimens.
• It helps in the examination of yeast, hyphae and other fungal structures present in infected tissue.
• GMS staining can also demonstrate Actinomyces and Nocardia in tissue sections.
• It is used in the histopathological examination of infectious diseases.
• GMS staining can be combined with H&E staining to examine fungal elements along with the tissue reaction.

Advantages of GMS Staining

The following are the advantages of GMS staining:

• GMS staining is sensitive for the detection of fungi in tissue sections. Fungal elements present in small numbers can also be detected.
• The fungal cell walls are stained dark brown to black. This gives good contrast with the surrounding tissue.
• It is useful for screening tissue sections for fungal elements.
• The shape and distribution of fungal elements can be observed clearly in the stained section.
• Some fungi that do not stain well with PAS can be demonstrated by GMS staining.
• It can be used for the examination of formalin-fixed, paraffin-embedded tissue sections.
• GMS staining can also demonstrate Pneumocystis in tissue sections.
• GMS can be combined with an H&E counterstain when both fungal elements and the tissue reaction need to be examined.

Limitations of GMS Staining

The following are the limitations of GMS staining:

• GMS staining is not specific for fungi. Some bacteria and other non-fungal structures may also take up the silver stain.
• The exact species of fungus cannot be identified by GMS staining. Identification depends upon the morphology of the fungus and other laboratory tests.
• Both living and dead fungi are stained by GMS. The viability of the organism cannot be determined.
• Fungal elements present in very small numbers may be missed during the examination of tissue sections.
• Excess silver deposition or background staining may make the fungal structures difficult to observe.
• Some morphological details of the fungal cells may not be clearly seen due to dark staining.
• The inflammatory response and other tissue changes are not clearly seen with the usual light green counterstain. H&E staining may be required for their examination.
• The staining result depends upon proper oxidation and silver impregnation. Excessive oxidation or improper staining may give weak or uneven results.
• Some staining artifacts and tissue deposits may resemble fungal elements and cause confusion during microscopic examination.

Troubleshooting of GMS Staining

The following are the common problems and troubleshooting of GMS staining:

• If the fungal elements aren’t stained black then check the chromic acid, sodium bisulfite and borax solutions. Use fresh solutions if necessary.
• Get rid of the chromic acid if it goes from orange to brown. Alcohol left in the section following deparaffinisation can reduce the chromic acid.
• If the methenamine silver solution goes cloudy or forms a silver mirror in the staining jar, make a fresh working solution.
• Use chemically clean glassware or clean plasticware for the silver solution. Don’t use metal forceps, racks or any other metal implements with the methenamine-silver solution.
• Weak staining can occur if the silver impregnation is inadequate. Check the temperature and the staining time. The impregnation time will vary depending on the method of heating.
• Check the fungal elements during the silver impregnation when the staining is weak. Carry on with the silver only until you have obtained good dark brown to black staining.
• Don’t over-stain the section with light green as this may mask the fungal elements.
• Background deposits can be easily confused with fungal elements. You need to look at the shape and morphology of the stained elements before reporting them as fungi.

Precautions of GMS Staining

The following are the precautions of GMS staining:

• Wear gloves, goggles and a laboratory coat while handling the staining reagents. Keep hot and uncovered solutions under a fume hood.
• Use freshly prepared methenamine-silver working solution. Filter the solution before use.
• Use fresh chromic acid for proper oxidation. Keep the chromic acid container closed when not in use.
• Wash the sections properly after chromic acid and sodium bisulfite treatment. Rinse with distilled water before the silver treatment.
• Maintain the required temperature during methenamine-silver treatment. Check the control section during silver impregnation.
• Do not continue silver impregnation after proper staining is obtained. Excess silver can increase the background staining.
• Treat the sections with sodium thiosulfate to remove the unreduced silver. Wash the sections properly after treatment.
• Do not overstain the sections with light green. Excess counterstain may mask the fungal elements.
• Keep the staining solutions free from fungal contamination. Contaminated solutions may produce fungus-like structures and give a false-positive observation.

References

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