1. LABROTORY DIAGNOSIS OF FUNGI PRACTICAL no(3)
DALIA KAMAL ELDIEN

2. Laboratory involvement
Collection of specimen
Direct microscopically examination
Culture of specimen
Colonial morphology& microscopical exam
Identification
Serological test
Reporting of the results

3. Collection of specimen
Skin scrapping:
Ensure that the patient has not used anti-fungal medications for the previous three days.
Prepare the skin for scrapings – remove any traces of skin products or medications with an alcohol wipe
Scrape the skin using a scalpel (held at a blunt angle)
Choose the best area to scrape – if multiple lesions are present choose the most recent for scrapings as old loose scale is often not satisfactory. 
The skin scrapings should then be gently removed from the skin surface and placed into a laboratory specimen container
Skin stripped off with adhesive tape, which is then stuck on a glass slide

4. Skin scrapping

5. Nail cuttings/scrapings
Clean the nail with an alcohol wipe
Use the blunt end of a lancet or other instrument and firmly scrape under the nail plate until the crumbling white degenerating portion is reached
Collect any white keratin debris beneath the nail directly into the specimen container
Nail clippings should also be collected

6. Nail clipping

7. Hair specimens Pluck hairs from the affected area using tweezers
Scrape the affected area using a scalpel (held on a blunt angle), on to a piece of paper
If available, examination of the scalp with a Wood’s lamp can guide the collection of samples from affected areas

8. Hair specimens

9. Other specimen include:
Blood
Biopsy
Pus
Sputum
CSF
urine

10. Macroscopically exam
the specimen is examined macroscopically for caseous, purulent or bloody areas, and necrotic material. Specimens from cases of mycetoma are examined with the dissecting microscope for the presence of granules before proceeding.

11. Direct microscopy
The material is examined by microscopy by one or more of these methods:
Potassium hydroxide (KOH) preparation, stained with blue or black ink
Unstained wet-mount
Stained dried smear
Histopathology of biopsy with special stains, e.g., periodic acid-Schiff (PAS).

12. KOH preparation
Place the material to be examined onto a clean glass microscope slide.
Add a drop of 20% KOH to the material and mix.
Pace a cover glass over the preparation.
Allow the KOH prep to sit at room temperature until the material has been Cleared at 37 C for 30 minutes. The slide may be warmed to speed the clearing process by passing 3 times over Bunsen burner. Or use dimethyl sulpho oxide to accelerate the action of KOH
Observe the preparation by bright field microscopy.

13. Calcofluor White
Potassium Hydroxide - Calcofluor White Solution Mixture
Place the material to be examined onto a clean glass microscope slide.
Add a drop of 15% KOH and a drop of the CFW solution, or mix in equal volumes before processing.
Mix and place a cover glass, examine by florescence microscope

14. India Ink - India ink can be added to specimens such as spinal fluids or exudates to provide a dark background that will highlight hyaline yeast cells and capsular material
Gram Stain for yeast
Modified ZN stain for Nocardia
PAS & H&E for histopathology

15. Macroscopic Examination:
Colonial morphology
Surface pigment on non-blood containing medium
Reverse pigment on non-blood containing medium
Growth on cycloheximide containing medium

16. Indirect microscopical exam
The lactophenol cotton blue (LPCB) wet mount preparation is the most widely used method of staining and observing fungi and is simple to prepare. The preparation has three components: phenol, which will kill any live organisms; lactic acid which preserves fungal structures, and cotton blue which stains the chitin in the fungal cell walls.
Place a drop of 70% alcohol on a microscope slide.
Immerse the specimen/material in the drop of alcohol.
Add one, or at most two drops of the lactophenol/cotton blue mountant/stain before the alcohol dries out.
Holding the coverslip between forefinger and thumb, touch one edge of the drop of mountant with the coverslip edge, and lower gently, avoiding air bubbles. The preparation is now ready for examination.

18. Steps of LPCB

19. Result under the microscope