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Presentation on theme: "LABROTORY DIAGNOSIS OF FUNGI PRACTICAL no(3)"— Presentation transcript:


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



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