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MICROBIOLOGY PRAX REVIEWER: Exercise 10: KOH MOUNT KOH (potassium hydroxide) Mount - is a rapid method for the

demonstration of fungal forms in clinical material - facilitates the clearing of specimen for enhanced microscopic observation without altering the fungal elements. - when a specimen if placed in KOH, the specimen will dissolve faster than the fungi since the chitinous cell walls of the fungi will protect it from disintegration A. Collection of Specimen 1. Clean the area with 70% alcohol and allow to air dry 2. With a sharp scalpel, scrape the epidermal scales at the active edge of the skin or nail lesion. B. Microscopic Examination 1. Place specimen on the glass slide 2. Add 1 2 drops of 10% KOH for skin scrapings and 20% KOH for nails and hair specimen 3. Put a cover slip and let the mount set for 10 30mins. If material is hard (nail or tissue), heat the slide gently by passing it over the flame once of twice. Do not overheat or KOH will crystallize. 4. Examine the mount under the microscope. Note the presence of granules, hypal elements, spores or yeast cells. Demo Slide:

Two advantages of KOH preparation: 1. Facilitates clearing of specimens for enhanced microscopic observation without altering fungal elements 2. Simple, cheap and rapid Two disadvantages of KOH preparation: 1. Gives an idea about the presence of hypal elements, but cannot distinguish different fungi 2. Preparation cannot be kept for too long Three factors that may affect the results of a KOH preparation: 1. Pressure application 2. KOH droplet size 3. KOH indication Exercise 11: Microscopic Morphology of Fungal Culture Identification of fungi is often accomplished by isolation organisms on appropriate culture media and observing their morphological characters Accurate Identification of filamentous fungi is based on the microscopic examination of sporulating parts of a colony, since each specie has a characteristic morphology and arrangement of its spores and fruiting bodies

Laboratory Techniques: Tease Mount Preparation - technique traditionally used by most laboratories - primary purpose is to demonstrate conidia or other reproductive structures or morphological forms which might give information toward the identification of the organism Procedure: 1. Using an inoculating needle, pick a small portion of each fungus growth and place on separate slides containing a drop of LPCB (Lacto-phenol cotton blue) 2. Tease with inoculating needle. Emulsify the growth of the yeast and yeast-like fungi 3. Put a cover slip and examine under low and high power objectives.

4. Observe record and draw the following important morphological features of the various fungi. a. Nature and type of mycelium (presence or absence of crosswalls) b. Characteristic arrangement, size, shape, and type of spores produced (blastospores, arthrospores, chlamydospores, sporangiospores, macro/microconidia, etc) and some special characteristic c. Nature of growth (fluffy, velvety, powdery, dry, moist) d. Color of growth on surface and reverse side. 5. Label all drawings accordingly. 6. Observe and characterize the different fungal colonies Demo Slides: I. Trichophyton Mentagrophyte

E. Floccosum

Candida Albicans

Microsporum Gypseum

Slide Culture or van Tieghem Cell - Considered the best method for preserving and observing the actual structure of a fungus. - in an undisturbed state, important microscopic structures and morphologic details are demonstrated Procedure: 1. Inoculate the microculture plates separately with each of the given fungus. 2. Hold a microscope coverslip with a pair of forceps and put it on top of the agar inside the plates.

3. Check the culture periodically for growth and sporulation. When the fungus has grown onto the square of the Sabourauds dextrose agar and out onto the small part of the slide and coverslip, proceed with the next step. Incubation time varies with each fungus, usually 5 10 days for contaminants and 1 3 weeks for pathogens. 4. Remove the slide from the petri dish. Take a new clean slide, and place a drop of LPCB on it near the center and put the coverslip that has fungus growth from the other slide. Label the slide. 5. Examine the slide for reproductive structures and notice the undisturbed morphology. Demo Slides: Penicillium sp.
a. b. c. d. Small, ovoid structures dividing by transverse fission Flat, powdery to velvety, tan to reddish yellow colonies Conidiosphore bearing short broad metullae At 37C colonies are soft and yeast-like, round or ovoid cells with central septum are seen

Aspergillus sp
a. b. c. d. Mycelia Fruiting heads Septate, monomorphic fungi Forms branches at acute angles

Disadvantages of slide Culture: Waiting period for incubation of slide culture Advantages of Tease Mount Preparation: This can be done easily and quickly Disadvantages of Tease Mount Preparation: The characteristic arrangement of conidia may be disrupted when pressure is applied to coverslip. Advantages of Scotch Tape Lactophenol Mount: Easy and fast procedure that is used for identification of filamentous fungi since most structures will be intact for observation. Disadvantages of Scotch Tape Lactophenol Mount: Tape will dissolve eventually so that it is not used for permanent mounts, the procedure can only be performed on moulds growing on plates.

Lactophenol Cotton Blue (LPCB) - is very popular for quick evaluation of fungal structures - 3 components: lactic acid which preserves the fungal structures cotton blue which stains the chitin present in the cell walls of fungi phenol which kills any live organisms suspended in the stain Advantages of Slide Culture: Fungal elements are grown and maintained in their original juxtaposition, thus making it easier to morphologically identify the organism 2 mounts obtained from one culture Simple and relatively inexpensive

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