LABORATORY DIAGNOSIS OF FUNGI – HOW TO DIAGNOSE FUNGI IN LABS ?

LABORATORY DIAGNOSIS OF FUNGI IS CARRIED OUT AS FOLLOWS:

⇒ The laboratory diagnosis of fungi or fungal infections is made by microscopy, culture, serology and skin test (for hypersensitivity).

⇒ Specimens: the specimen is collected from the site of the lesion. In case of disseminated (spreading) infection, blood sample needs to be collected.

⇒ Microscopy: fungal structures can be detected in clinical specimens by direct microscopic examination of material from the lesion and by the morphological study of fungal isolates:

1. Potassium hydroxide (KOH) preparation –
   • Tissue specimens, such as skin scrapings, are generally examined as wet mounts after treatment with 10 % KOH solution.
   • The alkali digests cells and other tissue materials, enabling the fungal elements to e seen clearly.
   • The specimen is placed in a drop of 10% KOH on a microscopic slide and covers it with a coverslip.
   • It is heated gently and examine under the microscope,
   • Yeast cells and Hyphae may be observed; diameter of Hyphae & presence or absence of septae and of special structure helps in the diagnosis.
2. KOH with calcofluor white staining –
   • It is a sensitive staining procedure that provides good visualization of the morphology of the fungus.
   • A drop of calcofluor white solution can be added to the KOH preparation before covering it with the coverslip.
   • Fungal elements fluoresce due to the binding of calcofluor white to the fungus.
3. Gram’s staining –
   • It is useful in identifying yeast and yeast-like fungi.
   • Commonly used to identify Candida species, gram-positive yeast.
4. India Ink Preparation –
   • It is used for negative staining of capsulated yeast; for e.g. – Cryptococcus neoformans.
5. Lactophenol cotton blue (LCB) –
   • It is used for the microscopic study of fungus colonies that are teased on to a slide and mounted.

Check out the General features & Classification of Fungi

GENERAL FEATURES & CLASSIFICATION OF FUNGI

⇒ Culture: some points are considered while attempting to culture fungi in the laboratory as follows –

• Fungi can be grown in media similar to those used for bacteria, usually at a lower pH.
• All fungi are basically aerobic.
• The optimum temperature range for culturing the fungi in the lab is 25-30°C {exception are those causing deep mycoses (grow well at 37°C) and Aspergillus fumigatus (can grow even at 50°C)}.
• The specific media Such as Sabouraud dextrose agar (SDA) and SDA medium with Antibacterials are inoculated and incubated at 25C and 37C for three weeks.
• Chloramphenicol is added to the culture medium to suppress the growth of contaminating bacteria whereas Cycloheximide is incorporated to suppress the contaminating fungi.
• The non-specific media such as Blood agar (BA), Brain heart infusion (BHI) agar or Blood heart infusion (BHI) broth can be made specifically for the isolation of fungi by adding the Antibacterials.
• Slide culture is done for studying the exact morphology of the fungus.

⇒ Identification: The identification of the fungi can be done by observing cultural characteristics and microscopy of the cultured specimens.Growth on the medium is identified by the morphology of the colony and other characteristics. These include –

• The rapidity of growth of fungi in the culture medium.
• Color and morphology of the colony on the obverse/ front side.
• Pigmentation of the colony on reverse/ back side.
• Morphology of Hyphae (diameter, septa), conidia (spores) and other special structures.
• Microscopy is performed from the fungal colony (in teased mounts or Slide culture). Teased mounts are prepared in Lactophenol Cotton blue (LCB) and KOH preparation from the cultured specimen are most commonly used.
• Fungal elements in tissue can be identified by Methenamine Silver Stain and Periodic Acid Schiff (PAS) stain.

⇒ Serology: serological tests can be done to demonstrate the Antigen or Antibody in serum or Body fluids. These includes:

1. Antibody detection
   • Agglutination test
   • Immunodiffusion test
   • Counter Immunoelectrophoresis
   • ELISA
   • Complement fixation test
   • Indirect Fluorescent antibody test
2. Antigen detection
   • Latex Agglutination test
   • Immunohistochemistry – It is useful for a number of important pathogens
3. Skin test – This is used to detect delayed-type hypersensitivity (DTH) for pathogens like Histoplasma or
4. New Rapid Diagnostic Tests for fungal infections includes –
   • Nucleic acid Hybridization
   • Polymerase Chain reaction

SAHIL BATRA

Hi, I’m the Founder and Developer of Paramedics World, a blog truly devoted to Paramedics. I am a Medical Lab Tech, a Web Developer and Bibliophiliac. My greatest hobby is to teach and motivate other peoples to do whatever they wanna do in life.