Staining Procedure for Rapid Identification of Fungi

Scrapings from involved ocular site complemented with the appropriate staining method can offer the ophthalmologist circumstantial as well as definitive information concerning the identity of the invading organism.

STAINING METHODS 

Light Microscopy

• KOH

• Gram’s stain

• Giemsa stain

• Grocott-Gomori Methenamine Silver (GMS) stain.

Fluorescent Microscopy

• Calcofluor white staining

• KOH-Calcofluor white procedure.

KOH mount

Principle: Potassium hydroxide is used as a mounting fluid for visualization of fungal filaments as it helps in the clearance or lyses of all surrounding tissues. In addition, Acanthamoeba cysts and Nocardia filaments can also be visualized.

Preparation of 10% KOH

• Approximately 1 gm (8 pellets) of KOH is weighed

• It is dissolved in 10 ml of distilled water

• One drop of 10% glycerol is added

• Fresh stock should be prepared every week

Can be kept at room temperature in a dropper bottle like a penicillin bottle (Fig. 4.1).

Fig. 4.1: KOH pellets and preparation of 10% KOH

Procedure 

• Take a clean glass slide and place the sample on the center. 

• Add a drop of 10% KOH and place a cover slip. 

• Take special care to avoid any air bubbles. 

• Observe under low and high power. 

Interpretation 

Fungal filaments: Appear as refractile hyphae with septate or aseptate, branching filaments or non-branching filaments. Some filaments look brown in color due to melanin pigments in their cell wall as in some species of dematiaceous fungi. It is however, not possible to identify the species of fungi from the wet mount. 

Different morphological forms of fungal filaments from corneal scraping specimen (Figs 4.2 to 4.8). 

Yeast cells are oval or round and colorless and may at times produce pseudohyphae in the KOH wet mount preparation (Care should be taken to differentiate them from epithelial cells) (Fig. 4.9). Nocardia filaments are slender, branching and much thinner in size when compared to fungal hyphae. 

Acanthamoeba cysts in the KOH wet mount are typical star or hexagonal shaped and double cell walled structures (Care should be taken to differentiate them from epithelial cells).

Fig. 4.2: Hyaline, septate fungal filaments in 10% KOH wet mount of corneal scraping specimen (400 X magnification)

Fig. 4.3: Hyaline, septate, large, branching fungal filaments of corneal scraping in 10% KOH wet mount (400 X magnification)

Fig. 4.4: Hyaline fungal filaments in KOH wet mount (400 X magnification)

Fig. 4.5: Hyaline, septate fungal filament in KOH wet mount of aqueous fluid (400 X magnification)

Fig. 4.6: Pigmented, septate fungal filaments in KOH wet mount of corneal scrapings. Culture grew Curvularia (400 X magnification)

Fig. 4.7: A KOH mount showing hyaline septate fungal filaments and thin Nocardia filaments (isolated from a patient with corneal ulcer caused by mixed infections involving fungi and Nocardia)—(400 X magnification)

Fig. 4.8: Thin broken fungal filaments from a corneal ulcer under antifungal treatment (400 X magnification)

Fig. 4.9: Oval shaped yeast cells in KOH wet mount (400 X magnification)

Gram’s Stain 

Principle: Gram’s staining is employed to distinguish and differentiate the type of infecting organism. The procedure requires application of four reagents in a sequential manner namely, a basic pararosaniline, an aqueous solution of iodine, a decolorizing solvent and pink counter stain. The violet dye and iodine combine to form an insoluble, dark purple compound in the bacterial protoplasm and cell wall. This compound is dissociable by the decolorizer, which dissolves and removes the components from the cell, the removal being much slower from gram-positive than from gram-negative bacteria. Gram-positive have thicker and dense peptidoglycon layers in thin cell walls, which makes them more permeable to the primary stain than the gram-negative bacteria. The iodine has a critical role in enhancing this difference. The organism is considered as gram-positive, if it appears violet under the microscope and gram-negative, if it appears pink under the microscope. 

Procedure 

• Prepare a thin smear of the specimen, dry in air and fix the smear by gentle heating over a Bunsen burner flame. 

• Flood the slide with Gram’s crystal violet for 1 minute. 

• Wash with distilled water and flood the slide with Gram’s iodine for 1 minute. 

• Wash with distilled water 

• The smear is decolorized with acetone – alcohol solution and wash immediately (important to be careful in this step as too much decolorizing will show gram-positive as negative and vice versa ) 

• Wash with distilled water and counter stain with carbol fuchsin for 30 seconds. 

• Wash with water, dry and examine the slide under oil immersion. 

Note: Decolorizer is prepared by adding equal volumes of acetone and isopropanol. Cedar wood oil or liquid paraffin may be used. The components of Gram’s stain are readily available in laboratory supplies stores (Fig. 4.10). 

Interpretation 

Fungus: Filamentous fungi have varying staining response to Gram’s stain They either do not stain at all or may stain gram-positive or gram-negative. However, this method is also useful for identifying any coexisting bacterial infection. Gram-positive bacteria appear purple (blue) in color and may be either cocci or bacilli. Gram-negative bacteria appear pink – red in color and may be either cocci or bacilli. Nocardia is a thin gram positive branching beaded filaments. Yeasts are gram-positive (purple), round or oval shaped (Figs 4.11 to 4.18).

Fig. 4.10: Gram’s stain kit

Fig. 4.11: Gram’s stain showing branching fungal filaments from corneal scraping (1000 X magnification)

Fig. 4.12: Large fungal filaments stained with crystal violet of Gram staining (1000 X magnification)

Fig. 4.13: Long fungal filaments with sickle shaped spores (morphologically resembles Fusarium spp) from corneal ulcer cases (1000 X magnification)

Fig. 4.14: A Gram’s stain slide picture showing fungal filaments (½) and gram-positive bacilli (isolated from a patient with corneal ulcer caused by mixed infections involving fungi and bacteria) (1000 X magnification)

Fig. 4.15: A Gram stain slide picture showing fungal filaments and gram-negative bacilli (¾) ( isolated from

a patient with corneal ulcer  caused by a mixed infections involving fungi and bacteria)  (1000 X magnification)

Fig. 4.16: A Gram stain slide picture showing fungal filaments and gram-positive beaded filaments of Nocardia ( isolated from a patient with corneal ulcer caused by mixed infections involving fungi and Nocardia) (1000 X magnification)

Fig. 4.17: A Gram stain slide picture from corneal scrapping showing gram-positive budding yeast cells of Candida spp (1000 X magnification)

Fig. 4.18: A Gram stain slide picture from corneal scrapping showing clumps of gram-positive yeast cells (100 X magnification)

Grocott-Gomori Methenamine Silver (GMS) Stain

Principle: To detect positive fungal elements, which, after staining take on a silver or black color. The light green counter stain is used to stain the background a contrasting color. Hematoxylin may also be used. 

Preparation 

Materials and reagents for the Gomori methanamine silver stain 

1. Gelatin – coated glass slides (stored at – 20° C to 0 °C) 

2. Chromic acid solution 5% 

3. Methenamine silver nitrate solution: 

a. Methenamine, 3% : 100 ml 

b. Silver nitrate, 0.1% : 7 ml 

4. Gold chloride, 0.1% 

5. Sodium thiosulfate solution, 2% 

6. Stock light green solution: 

a. Light green, SF : 0.2 gm 

b. Distilled water : 100 ml 

c. Glacial acetic acid : 0.2 ml 

7. Absolute methyl alcohol 

8. Distilled water. 

Procedure 

• Fix slide in absolute methyl alcohol for 5 minutes 

• Oxidize in 5% chromic acid for 30 minutes 

• Immerse in preheated methenamine silver nitrate solution for 20 minutes 

• Wash with six changes of distilled water 

• Tone for 2 to 4 minutes in 0.1% gold chloride 

• Rinse with two changes of distilled water 

• Immerse in 2% sodium thiosulfate solution for 2 minutes 

• Wash with tap water

• Counter stain for 1 minute with fresh 1: 5 dilution in distilled water of stock light green solution

• Air dry, clean and mount.

• Examine by light microscopy using 400X and 1000X magnification

Interpretation: Fungal elements stain silver to black, using a low power objective; fungal elements can easily be seen in a tissue section (Fig. 4.19). Acanthamoeba cell walls stain silver, Microsporidia stain primarily silver and then only if the cell wall is intact.

Figs 4.19A and B: Fungal elements stain silver to black in Gomori methenamine silver (GMS) stain – Tissue biopsy (400 X magnification)

Fluorescent Microscopy


Calcofluor White Staining

Purpose: Calcofluor white stain is a rapid staining procedure used to detect fungi, Acanthamoeba cysts, or Microsporidia spores in smears from clinical specimens. 

Principle: Calcofluor white binds to β1 – 3, β1 – 4 polysaccharides such as cellulose, and chitin, and fluoresces bright apple-green when exposed to long-wavelength ultraviolet light or short-wavelength visible light. Cell walls of fungi contain large amounts of chitin, and therefore yeast cells, hyphae, and pseudohyphae are readily visualized. Nonspecific staining of Acanthamoeba cysts and spores of Microsporidia also has been observed. 

Procedure 

1. Fix slide in methanol for 3 to 5 minutes. 

2. Add several drops of solution prepared as follows: combine equal amounts of 0.1% Calcofluor white stain and 0.1% Evans blue dissolved in distilled water 

3. After 5 minutes remove excess stain and apply a cover slip 

4. Examine using a fluorescence microscope for characteristic apple-green (or red if filter is used) chemo fluorescence of fungi and amoebic cysts. We used a Nikon fluorescence microscope with orange-red filter.

Figs 4.20A and B: Blue white fluorescence of fungal hyphae of

corneal scraping in KOH Calcofluor staining (A – 200 X magnification, B – 400 X magnification)

Interpretation 

1. Positive fluorescence is apple-green (or bright orange-red if filter is used) (Fig. 4.20). 

2. Background and other cells stain red-brown. 

Potassium Hydroxide – Calcofluor White Procedure 

Purpose: CFW combining with KOH for enhanced detection of fungi in clinical specimens 

Principle: KOH in solution with Calcofluor white, a fluorescent brightener of textiles, enhances the visualization of fungal elements in microscopic specimens. Calcofluor white binds to both chitin and cellulose in fungal cell walls and fluoresces bright green to blue. 

Preparation 

1. 15% Potassium Hydroxide solution 

Water (distilled) …………..80 ml 

KOH ……………………..15 gm 

Glycerol ………………….20 ml 

Dissolve KOH in water and glycerol. 

Store at 25°C, and re-prepare if a precipitate forms. 

2. 0.1% (wt/vol) Calcofluor white solution 

Water (distilled)………….. 1000 ml 

Calcofluor white stain…… 1 gm 

Mix, heating gently, if precipitate develops (or filter if persistent). Store in the dark at room temperature. 

Procedure 

1. Place specimen on clean glass slide 

2. Add a drop of each solution (15% KOH and Calcofluor white) 

3. Mix and cover with glass cover slip. 

4. Allow to sit at room temperature until material is clear (warming slide will hasten process). 

5. Examine using a fluorescence microscope with 10X and 40X to 50X objective or 100X oil. 

Interpretation: Fungi stain bright green or blue-white, depending on the filter.