Manual of basic techniques for a health laboratory - libdoc.who.int

6. Mycology 2276.2.1 Materials and reagents●●●●●●MicroscopeMicroscope slidesCoverslipsSterile needlesDistilled water70% Methanol● Sodium chloride, 0.85% solution (reagent no. 53)● Potassium hydroxide, 20% solution (reagent no. 45)● Reagents for Gram staining (see section 5.3.1).6.2.2 MethodCollection of specimens1. Use a sterile needle to lift the surface crust over a sinus.2. Carefully remove some of the discharging pus on to a slide.3. Add a drop of saline or water, spread the pus gently and look for granules. Granulesvary in colour, size, shape and degree of hardness.4. Crush a few granules in some distilled water and place on two slides.5. Allow one slide to air-dry, fix with methanol for 2–3 minutes and stain withGram stain (see section 5.3.1).6. Place a few drops of 20% potassium hydroxide on to the second slide and coverwith a coverslip. Leave the specimen to clear for 10 minutes.Microscopic examinationExamine the cleared specimen using the ¥ 10 and ¥ 40 objectives. Adjust the irisdiaphragm of the condenser to give good contrast.Look for branching and twisted hyphae or fragmented threads. Gram-stained granulesmay show thin or fragmented Gram-positive threads.Report as:● “pus from sinus containing granules [specify colour and size] present”;●“Gram staining shows Gram-positive thin hyphae” or “Gram staining does notshow Gram-positive thin hyphae”.6.3 Examination of skin for pityriasis versicolorPityriasis versicolor is a common skin disease in hot climates; it is caused by thefungus Pityrosporum furfur. The face and body are covered with patches, whichappear:— pale and discoloured in dark-skinned patients;— yellowish-brown in white-skinned patients.6.3.1 Materials and reagents●●●●MicroscopeMicroscope slidesAdhesive cellophane tapeTongue depressor or glass rod