Specimen collection

1. Hain/nail/sputum/skin scrapings/blood or CSF (depends upon site of infection)

Microscopy                                                                                

                                                                                                10% glycerol to prevent drying

1. Keratinized tissue specimen + 10% KOH————————————————–> keratin digested and

     ( skin scraping or hair)      (20-40% for nails and biopsy tissue)                               clear fungal hyphae seen

Gram staining

2. Yeast and yeast-like fungi + gram staining—————————>gram positive budding yeast cells seen

    (e.g. Cryptococcus/ candida)

3. Demonstration of the capsule of Cryptococcus neoformans by negative stains using India ink and nigrosin stain

4. Glycogen/mucoproteins/CHO + periodic acid of PAS stain—–>aldehyde(+ Schiff stain)——->magenta color of                                                                                                                                                                                        live fungal cells

5. Polysaccharide of CW+ Gomori methenamine silver(GMS) stain————->bloack colored fungi on green

                                                                                                                                         background tissues Of live and dead fungi

6. H and E stain

7. Fontana stain

Culture

1. SDA agar ————————-> MC used agar consisting of peptone(1%), dextrose (4%) with pH of 5.6
2. Neutral SDA———————-> modified SDA agar with neoepitope(1%), dextrose (4%) with pH of 7.2
3. Cornmeal agar——————-> stimulate chlamydospore production
4. Rice starch agar——————> stimulate chlamydospore production
5. Brain Heart Infusion agar——> enriched media for fastidious fungi
6. Blood agar———————-.–>enriched media for fastidious fungi
7. CHROM agar———————->selective and differential media for candida

8. Incubation time ——————> 2-3 weeks
   
   1. Slow growth(1-4 weeks) is seen in agents of systemic and subcutaneous mycoses and rapid growth(>5 weeks) is seen agents of superficial and opportunistic mycoses
9. Temp——————————–> 25-30°C

10. What to look for in colony?
    
    1. The rate of growth———————-> slow or rapid
    2. Pigmentation—————-.——-> pigmented or not
    3. Texture ——————————> waxy(leathery)/velvety/cottony/granular(powdery)
    4. Topography————————-.> radial grooves/folded/verrucose/brain-like

11. What to do after appreciating the colony morphology?

1. Bit of fungal colony——–>LPCB mount——–>view under microscope——–> look for nature of hyphae and type of sporulation
2. LPCB mount is used to study the microscopic features of fungal isolates grown in culture. It contains phenol which acts as a disinfectant, lactic acid which preserves the morphology of fungi, glycerol which prevents drying and cotton blue which stains the fungal elements blue.
3.  Procedure for slide culture

Take a petri dish——–>Place a bent glass rod on it ——–> On this glass rod place a sterile petri dish——–> place 2 agar blocks of 1cm2 on petri dish ——–>innoculate the margins of agar block with bits of fungal colony——–> Place 2 coverslips on the top of the 2 agar blocks——–>innoculate the Petri dish at 25°C——–> make LPCB mounts——–> viewed under microscope

4. Take impressions of the colony on the surface of the SDA plate with a cellophane tape
5. Germ tube test
6. Sugar fermentation test
7. Urease test
8. Hair perforation test

Immunological methods of identification

1. Antibody detection by ELISA, CFT, agglutination test, etc
2. Antigen detection by latex agglutination test
3. Immunohistochemistry

Molecular methods

1. PCR
2. Real-time PCR

Skin tests to demonstrate the delayed type of hypersensitivity