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Grocott’s Methenamine Silver Staining Protocol
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Grocott’s Methenamine Silver Staining Protocol

Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011


Chromic acid oxidation forms aldehydes from fungal cell wall polysaccharide components, which are subsequently demonstrated by reduction of an alkaline hexamine-silver complex.  The reaction may be compared to that of the periodic acid Schiff reaction, (see PAS).

Grocott's alkaline hexamine-silver solution represents a vehicle which, upon reduction, precipitates nascent silver ions, thus blackening the site.  This is known as an "argentaffin reaction".

Argentaffin reaction - the ability of a silver complex solution to blacken a tissue element without the need of a reducing bath.  The term is adjectival and is applied to many methods, (eg von Kossa). The term "argentaffin reaction" should therefore not be used as a proper name.

In most developed countries fungal infections are becoming more common in the form of opportunistic infections in patients with suppressed immune systems. Fungi stain weakly with haematoxylin, they are also PAS positive due to carbohydrate found in fungal cell walls.

Technical Points

1.   A known positive control section must be used to ensure correct differentiation has been achieved.

2.   Reagents should be prepared in a fume hood.

3.   Microwave method : Volume of chromic acid must be measured

4.   When micro-waving solutions ensure lids are not put on tightly to allow for expansion.

5.   Prior to leaving slides to stain in micro-waved solutions ensure solutions are thoroughly mixed to ensure even temperature distribution.

6.   Note solutions removed from the microwave oven may spontaneously boil.

7.   Times may vary according to type and power of micro-wave.

8.   A combination of the original method and microwave method can be used ie the microwave technique for silver used with the original method for chromic acid or vice-versa.


1.   Bring sections to distilled water.

2.   Oxidise with 4% aq chromic acid at room temperatur 1 hr

3.   Wash in water for a few seconds.

4.   Treat sections with 1% sodium metabisulphite 1 min

5.   Wash in running tap water 3 mins

6.   Rinse thoroughly in distilled water.

7.   Place in pre-heated working silver solution in a water bath at 60 °C for 15 to 20 mins until section turns yellowish-brown (Check microscopically after washing in distilled water – fungi should be dark brown).

8.   Rinse well in distilled water

9.   Tone sections with 0.2% gold chloride 2 mins

10. Rinse in distilled water

11. Treat sections with 2% sodium thiosulphate 2 mins

12. Wash with running tap water  5 mins

13. Counterstain in working light green 15 sec

14. Rinse excess light green off slide with alcohol

15. Dehydrate, clear and mount.

Microwave Method

1.   Bring sections to distilled water.

2.   Place slides in 40 ml  4% aq chromic acid in a loosely covered plastic coplin jar.

Microwave at 150 Watt  (see technical point 8) 2 min 30 sec

3.   Dip slides up and down in coplin jar and leave to stand for a further 2 min

4.   Wash in running tap water 30 sec

5.   Treat sections with 1% sodium metabisulphite 30 sec

6.   Wash in running tap water 30 sec

7.   Preheat working silver solution (loosely lidded plastic coplin jar) at 450 Watt 60 sec

8.   Rinse slides thoroughly in distilled water then place in hot silver solution.

Microwave at 150 Watt  (see technical point 8) 30 sec

9.   Dip slides up and down. Allow to stand for a further 1 minute (agitating occasionally by dipping up and down)  and then check microscopically after rinsing in distilled water.

If slides are not sufficiently stained return to silver solution and check every minute – fungi should be dark brown.

10.  Tone sections with 0.2% gold chloride 1 min

11.  Rinse in distilled water

12.  Treat sections with 2% sodium thiosulphate 1 min

13.  Wash with running tap water 15 sec

14.  Counterstain in working light green 15 sec

15.  Rinse excess light green off slide with alcohol

16.  Dehydrate, clear and mount.


  • fungi, Pneumocystis carnii, histoplasma spp -------- black
  • inner parts of mycelia and hyphae -------------------- old rose
  • leishmania spp, toxoplasma spp ---------------------- negative
  • mucin ---------------------------------------------------- dark grey
  • background ---------------------------------------------- pale green

Reagent Formulae

Wear protective clothing, gloves and safety glasses when preparing reagents.

1.      4% aq Chromic Acid

Chromium trioxide (analytical) ---- 4 g

Distilled water ----------------------- 100  ml

2.      Silver solution

3% methanamine (= hexamine) ---- 23 ml

5% silver nitrate --------------------- 1.25 ml

5% borax (sodium tetraborate) ---- 3 ml

Distilled water ----------------------- 25 ml

3.      0.2% aq Sodium chloroaurate (yellow gold chloride)

Gold Chloride (analytical) ----------- 1.0 g

Distilled water ------------------------ 500 ml

4.      2% aq Sodium thiosulphate (hypo)

Sodium thiosulphate ----------------- 2.0 g

Distilled water ----------------------- 100 ml

5.      Working light green

1% light green (CI 42095) in 1% acetic acid --- 10 ml

distilled water ------------------------------------ 40 ml


Grocott, R.G.  1955 , A stain for fungi in tissue sections and smears. American Journal of Clinical Pathology, V25, p975

Luna L.G. Histopathological Methods and color atlas of special stains and tissue artefacts, American Histo Labs Inc., Publications Division 1992.

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