Presentation on theme: "LABROTORY DIAGNOSIS OF FUNGI PRACTICAL no(3)"— Presentation transcript:
1 LABROTORY DIAGNOSIS OF FUNGI PRACTICAL no(3) DALIA KAMAL ELDIEN
2 Laboratory involvement Collection of specimenDirect microscopically examinationCulture of specimenColonial morphology& microscopical examIdentificationSerological testReporting 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 wipeScrape 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 containerSkin stripped off with adhesive tape, which is then stuck on a glass slide
5 Nail cuttings/scrapings Clean the nail with an alcohol wipeUse the blunt end of a lancet or other instrument and firmly scrape under the nail plate until the crumbling white degenerating portion is reachedCollect any white keratin debris beneath the nail directly into the specimen containerNail clippings should also be collected
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 paperIf available, examination of the scalp with a Wood’s lamp can guide the collection of samples from affected areas
9 Other specimen include: BloodBiopsyPusSputumCSFurine
10 Macroscopically examthe 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 microscopyThe material is examined by microscopy by one or more of these methods:Potassium hydroxide (KOH) preparation, stained with blue or black inkUnstained wet-mountStained dried smearHistopathology of biopsy with special stains, e.g., periodic acid-Schiff (PAS).
12 KOH preparationPlace 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 KOHObserve the preparation by bright field microscopy.
13 Calcofluor WhitePotassium Hydroxide - Calcofluor White Solution MixturePlace 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 materialGram Stain for yeastModified ZN stain for NocardiaPAS & H&E for histopathology
15 Macroscopic Examination: Colonial morphologySurface pigment on non-blood containing mediumReverse pigment on non-blood containing mediumGrowth 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.