Laboratory Diagnosis of Fungal Infections - Microscopy, KOH, Calcofluor white stain

Microscopy

Microscopy (direct microscopy) is performed following specific preparation to demonstrate fungal particles in the specimen. Although fungal culture is a gold standard for diagnosis, direct microscopic examination is one of the important and most rapid diagnostic methods.

Numerous methods implied include:

• KOH preparation

• Calcofluor white stain

• Gram stain

• Negative staining

• Wright stain

• Periodic acid-schiff (PAS) staining

• Grocott-Gomori’s Methenamine Silver Stain (GMS)

• Tissue stains

KOH preparation

KOH preparation is a traditionally recommended method.

Samples such as keratinized tissues or nail materials are digested by KOH and wet preparations. As KOH clears the tissue and cellular debris without impacting fungal cells, the KOH preparation is examined under the microscope for the presence of fungal particles.

Calcofluor White Stain

Calcofluor White (CFW) is a non-specific fluorescent blue dye used in biology as well as textiles. The dye binds to 1-3 beta and 1-4 beta polysaccharides of chitin and cellulose which are present in the cell walls of fungi, algae, and plants.

CFW can be mixed with potassium hydroxide for clearing up specimens and aid in the visualization of fungal elements.

Depending upon the combination of filters used, the fungal elements appear as apple green or blue-white. Evans blue is also used as counterstain while using blue light excitation (instead of UV) as it reduces the background fluorescence of tissues and cells.

Fungal or parasitic elements may appear bright apple-green while other biological agents give reddish-orange fluoresce.

Gram Staining

Gram staining is a differential staining technique used for the classification and differentiation of microorganisms.

Candida spp. can also be detected by gram staining.

Advantages

• Rapid diagnostic procedure

• readily available or accessible

Disadvantages

• although some fungi stain well, some other species such as Cryptococcus neoformans are stained weakly

Negative Staining (India ink staining)

Negative Staining is a type of staining technique used to study the morphology and arrangement of microorganisms. It engages the use of acidic stains such as Indian ink or nigrosin, yielding a clear cell with a dark background.

Heat fixation is not required, and the cells are not subjected to the distorting effects of heat or chemicals.

In mycology, negative staining is used for the detection of encapsulated yeast Cryptococcus neoformans in CSF and the detection of spherules of Coccidiodes immitis in CSF.

Wright Stain

Commonly used in hematology, Wright Stain is a type of Romanowsky stain used for the differentiation of blood cell types. It is mostly used to stain peripheral blood smears, bone marrow aspirates, urine samples, and demonstrate malarial parasites in blood smears.

Wright Stain is also used in cytogenetics to stain chromosomes for diagnosis of syndromes and diseases.

Stains similar to the Wright Stain are also known as the buffered Wright stain, the Wright-Giemsa stain, buffered Wright-Giemsa stain. These may include eosin Y, azure B, methylene blue,  May–Grünwald stain.

It is used in the detection of Histoplasma capsulatum and Cryptococcus neoformans.

Periodic acid Schiff (PAS) stain

The staining method Periodic acid–Schiff (PAS) is used to detect the presence of carbohydrates and carbohydrate compounds such as polysaccharides (glycogen) and mucosubstances (glycoproteins, glycolipids, mucin) in connective tissues, mucus, the glycocalyx, basal laminae, skeletal muscles, the liver, kidney, and the cardiac muscles.

Capsule and cell walls of fungi such as Candida albicans, Histoplasma capsulatum, Cryptococcus neoformans, Aspergillus fumigatus, and Blastomycosis contain polysaccharides (Glycogen, cellulose, and starch). Thus PAS staining procedure is also used to demonstrate hyphae and yeast forms of fungi in tissue samples due to the high carbohydrate content of the organism's cell walls.

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 for the staining procedure.

Tissue Staining

Tissue staining mostly used in mycology includes:

• Haematoxylin and eosin (H&E) stain

• Gridley Fungus (GF) Stain

• Mayer’s mucicaramine and alcian blue stain

Haematoxylin and eosin (H&E) stain

Haematoxylin and eosin (H&E) stain or HE stain is a widely used staining technique in medical diagnosis and is often called the gold standard. It is mostly used in histo-pathology to book biopsy and histological sections.

Haematoxylin and eosin (H&E) stain is a combination of two histological stains – hematoxylin (cationic/positively charged stain) and eosin (anionic/negatively charged stain). Hematoxylin has a deep blue-purple color and stains nucleic acids while Eosin is pink and stains proteins nonspecifically.

Many fungi are stained by Haematoxylin and eosin (H&E) stain while some fungi do not stain with Haematoxylin and eosin (H&E) stain.

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.

Mayer’s mucicaramine and alcian blue stain

Mayer’s mucicaramine and alcian blue stain stains the mucopolysaccharide capsule of Cryptococcus spp. Red and fungal cell blue.