Grocott-Gomori’s Methenamine Silver Stain (GMS) - Principle, Application, Reagents, Procedure, Result
Introduction to Grocott-Gomori’s Methenamine Silver Stain (GMS)
Grocott-Gomori Methenamine Silver Stain (GMS) or Grocott methenamine silver (GMS) is a popular staining method in histology. As it stains carbohydrates, this method is widely used as a screening for the diagnosis of fungi on cytosmears, aspirates, and tissue sections. The cell walls of the organism are stained brown to black.
The background is stained pale green. Pathogenic and non-pathogenic fungi are not differentiated by Grocott-Gomori Methenamine Silver Stain (GMS).
Other structures such as mucins, glycogen, and cytoplasm of neutrophils may also be stained dark brown.
The neutrophils may be mistaken for yeasts (pneumocystis in bronchoalveolar lavage). Careful examination will differentiate the two as lobated nuclei of the neutrophils will remain unstained while organisms will appear as typical “cup and saucer.” Moreover, neutrophils can be used as quality assurance of the staining procedure.
Principle of Grocott-Gomori Methenamine Silver Staining
The silver ions present in the Grocott-Gomori Methenamine Silver Stain will be reduced and color the fungal cell walls, which commonly contain polysaccharides, black.
In Grocott-Gomori Methenamine Silver Staining (GMS), chromic acid first oxidizes polysaccharides of the fungal cell walls resulting in the generation of aldehydes. After Grocott's alkaline hexamine-silver solution is applied, the silver ions are reduced to black amorphous silver.
This ability of cells to reduce the silver solution to metallic silver forming a black tissue element is also known as argentaffin reaction. Argentaffin cells are present in the intestines, melanin, and the epithelial lining of the lungs.
History of Grocott-Gomori’s Methenamine Silver Stain (GMS)
Hungarian physician György Gömöri developed the Grocott-Gomori’s Methenamine Silver Stain (GMS) and it is named after him.
Application of Grocott-Gomori’s Methenamine Silver Stain (GMS)
Grocott-Gomori’s Methenamine Silver Stain (GMS) is used to identify dimorphic fungi Histoplasma spp. and yeast-like fungus Pneumocystis jiroveci, the causative agent of Pneumocystis pneumonia (PCP) or pneumocystosis.
This method is also used to differentiate fungal and bacterial cells
Reagents of Grocott-Gomori’s Methenamine Silver Stain (GMS)
The reagents of Grocott-Gomori’s Methenamine Silver Stain (GMS) are as follows. Most of these ingredients are toxic and/or carcinogenic. Thus proper PPE must be strictly used.
Chromium trioxide solution
Sodium Bisulfite solution
Silver nitrate solution
methenamine solution
Borax solution
Gold chloride solution
Sodium thiosulfate solution
Light green stock solution
Preparation of solution
NOTE: Reagents usually come in Kits for manual preparation while some solutions are already prepared. the solutions must be prepared depending on the desired concentration.
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
Procedure of Grocott-Gomori’s Methenamine Silver Staining
There are two standard procedures for Grocott-Gomori’s Methenamine Silver Staining:
1. The conventional method at room temperature
2. Microwave method
Conventional method
Hydrate tissue sections with distilled water
Add 4% aqueous chromic acid to oxidize the section and leave it at room temperature for 1 hour
Wash in running water for a few seconds
Add 1% sodium metabisulphite to the section for 1 minute
Wash gently in running water for 3 minutes
Rinse well with distilled water
Place the slides in a pre-heated silver solution in a water bath (at 60°C for 15 to 20 minutes) or until the section turns yellowish-brown
Rinse well with distilled water
Add 0.2% gold chloride to the sections for 2 minutes
Rinse well with distilled water
Treat the sections with 2% sodium thiosulphate for 2 mins
Wash in running water for 5 minutes
Add light green counterstain for 15 seconds
Rinse off the excess counterstain with alcohol
Dehydrate the slide
Mount on the microscope
Microwave Procedure
Hydrate tissue sections with distilled water
In a loosely covered plastic Coplin jar containing 40 ml 1. 4% aqueous chromic acid, place the sections
For 2 minutes and 30 sections, microwave at 150 Watt
Dip the slides up and down in the Coplin jar and let it sit for 2 minutes
Washly gently in running water for 30 seconds
Add 3.2% sodium metabisulphite to the sections and leave it for 10 seconds
Wash gently with tap water for 30 seconds
Rinse the slide with distilled water and place it in hot silver solution (pre-heated working silver solution at 450 watts for 60 sec)
Microwave the sections at 150watt for 30 sec
Dip the slides up and down in the Coplin jar and let it sit for another 1 minute
Rinse with distilled water and check under the microscope
If the slides are not sufficiently stained i.e. fungi are not dark brown, dip the slides back in a Coplin jar for 1 minute
Add 3.2% gold chloride for 30 seconds to tone the sections
Rinse gently with distilled water
Add 4.2% of Sodium thiosulphate to the section for 1 min
Wash with running tap water for 15 seconds
Counterstain with the light green solution for 15 seconds
Rinse the counterstain with alcohol
Dry and cover the section
Result of Grocott-Gomori’s Methenamine Silver Stain (GMS)
Fungi, Pneumocystis jirevoci, Histoplasma spp stain black
Inner parts of mycelia and hyphae stain pink-red/ rose
Leishmania spp, Toxoplasma spp – GMS negative
Mucin stains dark grey
The background takes up the counterstain (light green) and stains pale green
Interpretation of Grocott-Gomori’s Methenamine Silver Stain (GMS)
The silver nitrate solution will reduce the presence of polysaccharides in fungal cell walls to form silver ions which are black. These fungal cell walls are stained black. Inner parts of mycelia and hyphae stain pink-red/ rose while mucin stains dark grey.
The background takes up the light green and stains pale green
Advantage of Grocott-Gomori’s Methenamine Silver Stain (GMS)
Higher sensitivity than Periodic Acid-Schiffs stain and Gridley Stain for detection of fungi and other polysaccharide-rich microorganisms in paraffin-prepared sections.
Its aggressive stains are fixed firmly on the sections and thus be used for future reference and observation.
Disadvantage of Grocott-Gomori’s Methenamine Silver Stain (GMS)
Non-specific – can stain non-fungal organisms such as internal organs of Strongiloides stercoralis larvae, in the intranuclear inclusions of Cytomegalovirus infected cells, endospores of Bacillus cereus, and the surface of Nocardia
Reagents may be carcinogenic.
Chemicals can cause irritations of the skin if touched and the gastrointestinal tract if inhaled.
Caution in Grocott-Gomori’s Methenamine Silver Stain (GMS)
Use PPE – gloves, goggles, and laboratory coats
Keep hot uncapped solutions under the fume hood and avoid direct contact and inhalation of chemicals and dyes
Chromic acid is carcinogenic and is corrosive to the skin, and mucous membrane, and highly toxic to kidneys
If ingested, Sodium metabisulfite is toxic and can cause Gastrointestinal distress and irritability. Irritation of skin, eye, and mucous membranes also occurs
Silver nitrate causes irritation of skin and eyes
The oxidizer is a tumorigenic agent (carcinogen) and can cause violent gastrointestinal discomfort if ingested
Sodium thiosulfate is toxic and can cause irritation in the stomach, skin, eyes, and respiratory tract
Light Green SF Yellowish is a possible carcinogen