ISOLATION OF FUNGI FROM CLINICAL SAMPLES Fungi are significant, sometimes overlooked, human pathogens. Infection caused by the fungus ranges from mild to life threatening. Diagnosis is based on a combination of clinical and laboratory investigations. Laboratory procedure includes, Demonstration of fungi by microscopy, Identification by culture, Detection of specific humoral response and Detection of fungal antigens and metabolites in body fluids. Successful of laboratory fungal diagnosis depends on specimen selection, Specimen collection, Specimen transport, Processing, Microscopic examination, Culturing. Specimen Collect ion Specimen collection for the detection of an etiologic agent of mycoses is very similar to specimen collection for bacteria. But fungal detection requires large quantity of specimen than bacterial identification. Fingernails and toenails suspected of Dermatophytosis should be cleaned extensively with 70% ethanol. Swabs are not optimal specimen collection for fungal identification. Depends on the site of infection, sample collection method and specimen type may vary. For dermatophytic infection : Skin scrapings, Pus, Nail clippings, Hair plug not cut. Subcutaneuous infection: Pus, biopsy tissues, Aspirated fluid, Skin scrapings Systemic mycoses : Sputum, Bronchial washings, Exudates from cutaneous lesion, CSF, Urine, Tissue biopsy, Vaginal swab, Blood. Specimen Processing Liquid specimens may have low concentration of fungus and will require centrifugation to increase fungal cell concentration. Hard specimens should be mined before inoculation. Concent rat ion Large volume of fluid should be concentrated by centrifugation (2000 rpm for 10 min). Use the resulting pellet for culture and KOH examination. Body fluids, Urine, CSF, Sputum are used after digestion with N-acetyl L-cysteine. Mincing or homogenizat ion Biopsy samples, tissues and nails must be processed to increase the recovery. With sterile scalpels, mince specimens into small pieces in a petriplate with a few drops of sterile distilled water. If Histoplasma is suspected, specimen is homogenize by using tissue grinder after mincing. Media select ion Media selection depends on body site, nature of infection and nature of contamination. To prevent bacterial pathogens antibiotics should be used or reduce the pH of the medium Brain heart infusion agar with antibiotics, Potato Dextrose Agar, Mold agar, Sabouraud Glucose Agar, Niger seed agar, Yeast extract phosphate medium. , Buffered charcoal yeast extract agar, Rose Bengal chloramphenical agar. From the above list of mycotic media, any one or two media selected for cultivation. Inoculat ion Concentrated aspirated body fluids are directly inoculated on to the medium by making use of inoculation loop. Swabs are vortexed in distilled water before inoculation. Vortexed specimens are inoculated on medium after concentration. After inoculation hair and skin scrapings are pressed firmly on the surface of the medium. Nail should be pulverized using scalpels and press fragments firmly onto medium surface. Incubat ion All fungal media were incubated at 28 to 30°C for 4 weeks, because most fungi will produce colonies by the end of the 3rd Week.. Vaginal cultures may be incubated for only 7 days. Observe the media once in two days. Histoplasma capsulatum and Blastomyces dermatidis may require 4-8 weeks. Aerobic non clinical fungus may produce fruiting bodies within 72 hours. Examinat ion of fungal growth on primary media Read primary plates daily for the first week, every other day for the second week and twice weekly for the remaining two weeks. When growth appears, differentiate yeast from mold by microscopic examination. Once colonies are formed on the media, the organism should be viewed microscopically. Zygomycetes are observed under dissecting microscope. Yeast is observed by Wet preparation, Lacto phenol cotton blue staining and Indian ink preparation. Mold is observed by Wet preparation, Scotch tape , Tease preparation , Slide cultures, Lactophenol cotton blue staining , Ascospore. KOH Mount KOH may be used to examine hair, nails, skin scrapings, fluids, exudates or biopsies. The fungal structures such as hyphae, large yeast (Blastomyces), spherules, and sporangia may be distinguished. Examine slides with reduced light (narrow the iris diaphragm) and examine negative smears on several consecutive days. The fungal structures may be enhanced by using a phase-contrast microscope. Specimens placed in a drop of 15% KOH will dissolve at a greater rate than fungi because fungi have chitinous cell walls. The clearing effect throughout the clinical specimen can be accelerated by gently heating the KOH preparation. Visualization of fungi can be further enhanced by the addition of Parker Superquink permanent black ink to the preparation. Mycelium and spore were observed within tissue. KOH clears hard tissue and facilitates easy observation. KOH mount provides first hand information about fungal infection in tissues. KOH-DMSO-INK mount / stain Specimens with tissue placed in a drop of 15% KOH will dissolve at a greater rate than fungi because fungi have chitinous cell walls. The clearing effect throughout the clinical specimen can be accelerated by gently heating the KOH preparation. Addition of Ink to KOH enhances contrast. This method is most useful for detecting Malassezia furfur in skin scrapings. DMSO present in staining reagent eliminates the need of heat during staining Mycelial elements clearly demonstrated, which indicates the availability of fungal infection in particular tissue. Spore along with conidium describes mould species. KOH-Calcoflour fluorescent -stain Calcoflour white stain may be used for direct examination of most specimens using fluorescent microscopy. The cell walls of the fungi bind the stain and fluoresce blue-white or apple-green depending on the filter combination used. The use of calcofluor white (CFW), a fluorescent brightener used in the textile industry, with the addition of potassium hydroxide (KOH) will enhance the visualization of fungal elements in specimens for microscopic examination. The CFW nonspecifically binds to the chitin and cellulose in the fungal cell wall and fluoresces a bright green to blue. A substantial amount of non-specific fluorescence from human cellular materials and natural and synthetic fibers should be expected. The CFW highlights suspicious structures but the interpretation of the structures relies on traditional fungal morphologic features. India ink Preparations An India ink preparation can be used for the rapid detection of the encapsulated yeasts based on negative staining. The capsule repels the carbon particles of the Indian ink, giving a clear well-demonstrated halo around each encapsulated stain. Giemsa Stain for Histoplasma capsulatum Giemsa stain is used for examining intracellular structures and is applied to primary specimens of bone marrow tissue and WBC’s in which H.capsulatum is suspected. Necrotic cells in the specimen will have pink cytoplasm. Normal cell- light blue - violet lavender cytoplasm. Phagocytised yeast cells will stain light to dark blue, and each will have a clear halo around it. Look for purple pseudoencapsulated yeast forms of H.capsulatum inside PMN cells and monocytes. IDENTIFICATION OF YEAST Yeasts are a heterogenous group of fungi that superficially appear to be homogeneous. Yeasts grow in a conspicuous unicellular form that reproduces by fission, budding, or a combination of both. True yeasts reproduce sexually, developing ascospores or basidiospores under favorable conditions. Yeast-like fungi (imperfect yeasts) reproduce only by asexual means. The identification of these fungi is based upon a combination of morphological and biochemical criteria. Morphology is primarily used to establish the genera, whereas biochemical assimilations are used to differentiate the various species. Principal Criteria and Test s for Identifying Yeast s 1. Culture characteristics - Colony colour, shape, texture 2. Asexual structures: a. Shape and size of cells; b. Bipolar, fission, multipolar or unipolar “budding”; c. Absence or presence of arthroconidia, ballistoconidia, blastoconidia, clamp connections, endoconidia, germ tubes, hyphae, pseudohyphae, or sporangia and sporgangiospores. 3. Sexual structures - Arrangement, cell wall ornamentation, number, shape and size of ascospores or basidiospores 4. Physiological studies: a. Assimilation; c. Fermentation d. Nitrogen utilization e. Urea hydrolysis Cultural characters Clinical sample is streaked on Sabourad dextrose agar or Rose Bengal chloramphenicol agar plates. Plates were incubated at 25-30°C for 48 hours and observed for colony morphology, colour, shape and texture. Record the result and interpretate the results. CHROM agar test CHROM agar contains enzymatic substrates that are linked to chromogenic compounds. When specific enzymes cleave the substrates, the chromogenic substrates produce colour. The action of different enzymes produced by yeast species results in colour variations useful for the presumptive identification of yeasts. The test provides only presumptive identification of yeast eg: C.kruses, C.tropicalis, C.albicans. Cultures are streaked on the medium and incubate at 35°C in humidified dark chamber for 48-72 hours. After 72 hours observe colour change and interpretate the results. C.albicans: A medium sized, green, smooth matte colony with a very slight green halo ion the surrounding medium. C.tropicalois : Smooth medium sized matte colony, which is blue to blue