Gridley Fungus (GF) Stain - Introduction, Reagents, Procedure, Result, Interpretation

Introduction to Gridley Fungus (GF) Stain

Gridley Fungus (GF) Stain is a staining method of fixed tissue sections based on Bauer chromic acid leucofuchsin stain and the addition of Gomori aldehyde fuchsin stain and metanil yellow as counterstains.

After Gridley Fungus (GF) Staining hyphae, conidia, yeast capsules, elastin, and mucin appear in different shades of blue to purple against yellow background. On the other hand, filaments of the Nocardia and actinomyces are not stained by this method.

This method does not stain amoebae differently but does stain ingested erythrocytes (stained by eosin) ingested by pathogenic amoebae.

Reagents of Gridley Fungus (GF) Stain

• Schiff’s reagent

• Aldehyde fuchsin

• Chromic acid

  Chromium trioxide = 5 gm

  Distilled water = 500mL

• Bleach

  Sodium metabisulfite = 5g

  Distilled water = 500mL

• Metanil yellow

  Metanil yellow = 1 g

  Distilled water = 400mL

  Acetic acid, glacial = 2 drops

Procedure of Gridley Fungus (GF) Stain

For the process of Gridley Fungus (GF) Stain, 5µ paraffin sections of neutral buffered formalin-fixed tissue are suitable.

The procedure for Gridley Fungus (GF) Stain is as follows:

1. Bring tissue sections to water via xylene and ethanol.

2. Add chromic acid to the tissue section and let it sit for 1 hour.

3. Wash gently under running tap water.

4. Treat the tissue section with the metabisulfite bleach for 1 minute.

5. Wash well with running tap water.                                        

6. Rinse with distilled water.

7. Place the tissue section in Schiff’s reagent for 20 minutes.

8. Wash well with running tap water.                                        

9. Rinse with 70% ethanol.

10. Place the tissue section in aldehyde fuchsin and let it sit for 30 minutes.

11. Rinse off excess stain with 95% ethanol.

12. Wash well with running tap water.                                        

13. Add counterstain (metanil yellow) and let it stand for 1 minute.

14. Rinse with distilled water.

15. Dehydrate, clear, and mount in a resinous medium.

Result, Interpretation of Gridley Fungus (GF) Stain

After Gridley Fungus (GF) Staining, hyphae, conidia, yeast capsules, conidia, elastin, and mucin appear in different shades of blue to purple while the background stains are yellow.

Amoebae stain blue-green, nuclei of amoebae stain deeper blue-green while ingested erythrocytes stain deep rose.

Caution for Gridley Fungus (GF) Stain

• 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.

• Eosin Y is a possible carcinogen.

• Aniline is a severe eye irritant, toxic by skin absorption, a possible carcinogen, and a combustible liquid.

• Glacial acetic acid is a severe irritant to the skin and eyes and is corrosive. It targets organs and also affects the respiratory system.