Specimen collection
- Hain/nail/sputum/skin scrapings/blood or CSF (depends upon site of infection)
Microscopy
10% glycerol to prevent drying
- 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
- Yeast and yeast-like fungi + gram staining—————————>gram positive budding yeast cells seen
(e.g. Cryptococcus/ candida)
- Demonstration of the capsule of Cryptococcus neoformans by negative stains using India ink and nigrosin stain
- Glycogen/mucoproteins/CHO + periodic acid of PAS stain—–>aldehyde(+ Schiff stain)——->magenta color of live fungal cells
- Polysaccharide of CW+ Gomori methenamine silver(GMS) stain————->bloack colored fungi on green
background tissues Of live and dead fungi
- H and E stain
- Fontana stain
Culture
- SDA agar ————————-> MC used agar consisting of peptone(1%), dextrose (4%) with pH of 5.6
- Neutral SDA———————-> modified SDA agar with neoepitope(1%), dextrose (4%) with pH of 7.2
- Cornmeal agar——————-> stimulate chlamydospore production
- Rice starch agar——————> stimulate chlamydospore production
- Brain Heart Infusion agar——> enriched media for fastidious fungi
- Blood agar———————-.–>enriched media for fastidious fungi
- CHROM agar———————->selective and differential media for candida
- Incubation time ——————> 2-3 weeks
- 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
- Temp——————————–> 25-30°C
- What to look for in colony?
- The rate of growth———————-> slow or rapid
- Pigmentation—————-.——-> pigmented or not
- Texture ——————————> waxy(leathery)/velvety/cottony/granular(powdery)
- Topography————————-.> radial grooves/folded/verrucose/brain-like
- What to do after appreciating the colony morphology?
- Bit of fungal colony——–>LPCB mount——–>view under microscope——–> look for nature of hyphae and type of sporulation
- 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.
- 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
- Take impressions of the colony on the surface of the SDA plate with a cellophane tape
- Germ tube test
- Sugar fermentation test
- Urease test
- Hair perforation test
Immunological methods of identification
- Antibody detection by ELISA, CFT, agglutination test, etc
- Antigen detection by latex agglutination test
- Immunohistochemistry
Molecular methods
- PCR
- Real-time PCR
Skin tests to demonstrate the delayed type of hypersensitivity