NU LiPa Lesson 3: Culture Media & Fungal Culture Techniques PDF

LESSON #3: CULTURE MEDIA & FUNGAL CULTURE TECHNIQUES  INTENDED LEARNING OUTCOMES: 1. Understand the role and composition of Fungal Culture Media 2. Identify and differentiate between types of culture media used for cultivating fungi and selecting the appropriate culture media for the isolat...

LESSON #3: CULTURE MEDIA & FUNGAL CULTURE TECHNIQUES  INTENDED LEARNING OUTCOMES: 1. Understand the role and composition of Fungal Culture Media 2. Identify and differentiate between types of culture media used for cultivating fungi and selecting the appropriate culture media for the isolation of fungi from different sources, including clinical, environmental, or industrial samples. 3. Collection and processing clinical specimens (e.g., skin scrapings, blood, sputum, urine, etc.) for fungal culture using proper techniques to prevent contamination and ensure fungal viability. 4. Understand the role of clinical fungal culture in diagnosing systemic or localized fungal infections in immunocompromised patients, including understanding the time required for fungal cultures to grow.  FUNGAL CULTURE MEDIA Most fungi usually grow best on media high in Carbohydrates, with a pH 5.0-6.0. Sterilization of Media- Autoclave 15-20 minutes at 120 ̊C at 15-psi TYPES OF MEDIA: NATURAL MEDIA SYNTHETIC MEDIA CULTURE METHODS SELECTIVE MEDIA -incorporates Chloramphenicol & Cycloheximide Most laboratories incubate at room temperature: 25-30 ̊C If dimorphic fungi is suspected, cultures are incubated at 37 ̊C  FUNGAL CULTURE MEDIA CHLAMYDOSPORE or CORN MEAL AGAR (CMA) Satisfactory for the growth and sporulation of fungi Addition of Glucose (Dextrose)- T. rubrum produces RED pigment POTATO DEXTROSE AGAR (PDA) Includes spore (conidia) formation and pigmentation Demonstration of pigment production by T. rubrum POTATO CARROT AGAR (PCA) conservation of fungi For the reproduction of Pyronema domesticum MALT EXTRACT AGAR (MEA) For the growth of wood destroying fungi, yeasts and molds SABORAUD DEXTROSE AGAR (SDA) Most commonly used for fungal cultures Most fungi will grow on the medium SDA WITH ANTIBIOTICS (MYCOSEL OR Used for Isolation of Dermatophytes and most MYCOBIOTIC) pathogenic fungi  FUNGAL CULTURE MEDIA BRAIN HEART INFUSION AGAR (BHI) Routinely used to culture yeast phase at 35 ̊C- 37 C̊ and also at room temp. Primary recovery of saprobic and pathogenic fungi Useful in fastidious fungi BRAIN-HEART INFUSION AGAR WITH Primary recovery of pathogenic fungi ANTIBIOTICS exclusive of dermatophytes BRAIN–HEART INFUSION BIPHASIC BLOOD Recovery of fungi from blood CULTURE BOTTLES BRAIN- HEART INFUSION WITH BLOOD Enhances the growth of yeasts at 37 ̊C SABHI agar (SDA+BHI) Primary recovery of saprobic and pathogenic fungi CZAPEK DOX BROTH For identification of Apergillus and Penicillum spp.  FUNGAL CULTURE MEDIA RICE MEDIUM Used to distinguish Microsporum audouinii from Microsporum canis BIRD SEED AGAR/NIGER SEED AGAR/ CAFFEIN For cultivation of Cryptococcus neoformans AGAR (produce BROWN pigment) INHIBITORY MOLD AGAR Primary recovery of pathogenic fungi exclusive of dermatophytes POTATO FLAKE AGAR Primary recovery of saprobic and pathogenic fungi MYCOSEL Primary recovery of dermatophytes YEAST-EXTRACT AND PHOSPHATE AGAR Primary recovery of pathogenic fungi exclusive of dermatophytes  FUNGAL CULTURE MEDIA ASCOSPORE AGAR Detection of ascospores in Ascosporogenous yeasts such as Saccharomyces spp. CORNMEAL AGAR WITH TWEEN 80 AND Identification of C. albicans by chlamydospore TRYPAN BLUE production; identification of Candida by microscopic morphology COTTONSEED CONVERSION AGAR Conversion of dimorphic fungus B. dermatitidis from mold to yeast form NITRATE REDUCTION MEDIUM Detection of nitrate reduction in confirmation of Cryptococcus spp. TRICHOPHYTON AGARS 1 – 7 Identification of members of Trichophyton genus  FUNGAL CULTURE MEDIA UREA AGAR Detection of Cryptococcus spp. Differentiate T. mentagrophytes from T. rubrum Detection of Trichosporon spp. YEAST FERMENTATION BROTH Identification of yeasts by determining fermentation YEAST NITROGEN BASE AGAR Identification of yeasts by determining carbohydrate assimilation TYPES OF CULTURE AGAR SLOPES (SLANT) Care should be taken not to permit the molten agar to come in contact with the cotton plug. Agar slopes (in tubes or bottles) are usually used for keeping culture for some time or which are to be stored in a culture collection. AGAR PLATES Fungi are usually grown in Petri dishes as well as on slopes for morphological studies. Useful for determining rate of growth and colony characteristics. CULTURE TECHNIQUES MICROSLIDE/ SLIDE CULTURE TECHNIQUE These are used to study fungal species that produce very small and delicate sporophores. Also known ass Riddell's method (1950)  RIDDELL’S TECHNIQUE One plate of Nutrient Agar; Potato Dextrose is recommended. Using a sterile blade cut out an agar block (7 x 7 mm) small enough to fit under a coverslip. 1. Flip the block up onto the surface of the agar plate. 2. Inoculate the four sides of the agar block with spores or mycelial fragments of the fungus to be grown. 3. Place a flamed coverslip centrally upon the agar block. 4. Incubate the plate at 26 degree Celsius until growth and sporulation have occurred. 5. Remove the coverslip from the agar block. Note: Removing the cover slip must be done carefully to keep the reproductive structures intact. 6. Gently lower the coverslip onto a small drop of Lactophenol cotton blue on a clean glass slide. 7. Examine under the microscope CULTURE TECHNIQUES HANGING DROP CULTURES These are especially useful for studying spore germination and for checking spores for appressoria formation. 1. Prepare a suitable agar medium (e.g. MEA, SDA, CMA, and PDA), and pour into a plate to a depth of about 2-mm. 2. When it has set completely, cut out a small block, about 1- cm square using a sterile scalpel and transfer to the center of a sterile slide. 3. Inoculate the block with the fungus on all four edges. 4. Place a sterile coverslip on top of the block of agar.  HANGING DROP CULTURES 5. Incubate the slide in a moist chamber. This may be made from a sterile Petri dish with filter paper in the bottom. Two sterile glass rods (or a bent glass rod) are placed on the filter paper to function as supports for the slide. About 10 ml of a sterile 20 % solution of glycerol is poured into the dish. This will keep the agar moist. The dish with the inoculated slide is incubated until the growth reaches a desired stage (until the mycelium formed from each edge of the block has reached the edge of the coverglass). 6. The fungus usually spreads out from the agar block and tends to attach itself to the two glass surfaces. When the desired stage of fungal growth has been reached, lift off the coverslip and carefully lower fungus side down onto a drop of mounting fluid such as lactophenol (with or without stain) on a clean slide. 7. Carefully remove the agar block, add a drop of mountant (e.g., lactophenol) to the growth adhering to the slide, and apply a clean coverslip. Then seal the slides using nail varnish. SLIDE CULTURE TECHNIQUE MICROSCOPIC EXAMINATION OF CULTURES TEASE MOUNT Uses Lactophenol Blue (LPCB) for staining Teasing often disrupts delicate fruiting structures CELLOPHANE TAPE PREPARATION Uses unfrosted, clear cellophane tape (Scotch tape) The tape is pressed firmly to the structure of the colony and serves as a coverslip Uses LPCB Delicate structures are preserved better GROSS EXAMINATION OF CULTURES: 1. APPEARANCE OF GROWTH Dimorphic fungi (pathogens) and Dermatophytes produce delicate hyphae or cobweb or hair-like colonies of not more than 1-2 mm. Jet black reverse surface of colonies help distinguish dematiaceous fungi 2. RATE OF GROWTH Saprobic molds produce mature colonies within 3-5 days. (GENERAL RULE) Dimorphic Fungi grow much slower Used only as a guide and not a criterion GROSS EXAMINATION OF CULTURES: 3. COLONY PIGMENTATION Some fungi produce characteristic pigmentation 4. GROWTH ON SELECTIVE MEDIA Only allows growth of limited number of microorganism 5. DIMORPHIC GROWTH Ability to grow in both yeast and mold form