Reading Assignment:|Mahon, Chapter 10, pgs 215-219, Chapter 27, pgs 626-629, 634-636, Appendix B Lecture Notes: Medical Mycology| |U of W Tutorial on Mycology (organisms listed in objectives), www.medtraining.org[->0]| _____________________________________________________________________ 1.Discuss the difference between yeasts and molds.
Fungi seen in the clinical laboratory can be generally separated into two groups based on the appearance of the colonies formed: Yeasts: Moist, creamy bacteria-like, opaque, or pasty colonies on media. They reproduce by budding. (when they start budding, they cause infections) Molds (filamentous fungi): Fluffy cottony, woolly or powdery colonies on medium. They reproduce by sporulation. 2.Describe or diagram the following types of structures:
a.Blastoconidia- Asexual yeast reproduction by blastoconidia formation. A daughter cell buds off from mother cell. A septum is created and daughter cell detaches. b.Pseudohyphae- Elongated blastoconidia. May align end-to-end like links of sausage. Associated with yeasts. True septae are not present. (Characteristic of Candida albicans) c.Chlamydoconidia- Thick-walled resistant asexual spores produced by “rounding up” and enlargement of terminal hyphal cells. d.True hyphae- Fundamental microscopic units of fungus, tube-like projections with no constictions at the cell wall. The cell walls remain parallel with no indentation. 3.Describe the appropriate specimen collection procedures, staining methods, and culture techniques used for isolation of yeast. Collection procedures
Specimen of choice include respiratory secretions, hair, skin, nails, tissue blood or bone marrow and CSF. Swabs are inadequate. CSF: Concentrate by centrifugation before inoculation. Make India ink preparation or latex agglutination for Crytococcus neoformans with part of the specimen and then inoculate the remainder onto media. Either culture plates or screw-capped culture tubes are used. Tubes are more convenient. Fungi grow optimally at 30OC. Fungal cultures should be incubated for 14-30 days, periodically observing for growth. Plates should be sealed to prevent loss of moisture. Staining Methods
1.Saline mount: Provides a quick simple quick method to observe fungal elements and budding. Method lacks contrast. 2.KOH preparation: Dissolves tissue which may obscure fungal elements. Method also lacks contrast ( for skin, hair, nails) 3.Calcofluor white or cellufluor stain: Superior to KOH. Binds to polysaccharide in cellulose and chitin in fungal cell wall. Under UV fluoresces, enhancing detection of fungi. Requires fluorescent microscope. 4.India ink (nigrosin): used to visualize the capsule of certain yeasts (ex., Cryptococcus neoformans) Capsule appears as clear halos against a dark background. 5.Lactophenol cotton blue (LPCB): Stains hyphae cell walls blue. 6.Giemsa or Wright’s stain: Stains mycelium and spores dark blue. C. neoformans and H. capsulatum stain well 7. Methenamine-silver nitrate stain: Good contrast for fungal elements. 8.Periodic acid-Schiff (PAS): Stains hyphae purple. Green counterstain used to provide contrast. Culture techniques
Primary isolation media should include media with and without blood, and with and without antibiotics. Primary media
Sabouraud agar(Sabouraud dextrose agar, [SDA]) General isolation media. Contains glucose and peptones, has a pH of 5.6 to inhibit bacterial growth. May be modified by addition of cycloheximide and chloramphenicol to inhibit saprobic fungi and bacteria capable of growing at lowered pH. Dermatophyte agar(Dermatophyte test medium [DTM]) Isolation of dermatophytes. Uses cycloheximide to inhibit saprophytic fungi, chloramphenicol to inhibit bacteria. Color change (pH change, yellow to red) indicates growth. MycoselSimilar to DTM
BHI medium(Brain-heart infusion medium) Useful for isolation of systemic mycoses....