Mycology Lecture Notes PDF
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B.P. Koirala Institute of Health Sciences
2024
Dr. Dev Jha
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Summary
These lecture notes provide an overview of mycology, focusing on culture media, stains, and biochemical tests for fungal identification. The document includes various procedures for specimen collection and processing, information about different fungal stains, and details about fungal culturing methods and media.
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CULTURE MEDIAS, STAINS AND BIOCHEMICALS IN DR. DEV MYCOLOGY JHA JR 1 2 oct 2024 CONTENT Sample collection, Handling and Processing of Specimen Culture Medias in Mycology Stains Biochemical Test Collection, Handling and...
CULTURE MEDIAS, STAINS AND BIOCHEMICALS IN DR. DEV MYCOLOGY JHA JR 1 2 oct 2024 CONTENT Sample collection, Handling and Processing of Specimen Culture Medias in Mycology Stains Biochemical Test Collection, Handling and Processing Specimen Skin- Cleaned with alcohol 70% (remove dirt, oil and surface saprophytes) Nails- cleaned same as skin Hair-Edge from infected area of scalp, hair obtained by plucking and brushing Body fluid- Normal sterile collection procedure Skin, Hair and Nail- direct exam flowing KOH preparation CSF- Centrifuged; examine sediment microscopically Pleural fluid, sputum and bronchial must be fresh aspiration- specimen. If delay must be refrigerated up to 2 hours Gastric washing- same as for pleural fluid Genito- urinary specimen- first morning urine Bone marrow- Generally inoculated direct in BHI broth Wound abscess or drainage culture anaerobically Tissue specimen- examine for pus, caseous material or granules Direct Examination of Specimens Direct microscopy required on any material sent for fungus culture. Look for spore, hyphae, mycelial element, budding yeast, mycotic granules (Adv- Rapid, simple and easy method with minimum equipment) Wet mount prep- Good for yeast examination KOH prep- done on skin scrapping, nails, sputum, vaginal specimen etc. KOH clears tissue and mucous so fungal element can be seen ( 10 – 40%) Stains Used in Mycology Lactophenol Cotton Blue(LPCB)- very popular for quick evaluation of fungal structures; will stain the chitin in cell wall of fungi. - To study microscopic appearence of fungal element Phenol- as disinfectant, lactic acid as morphology preservative, glycerol as prevent drying, cotton blue as stains. Periodic Acid-Schiff (PAS)- stains certain polysaccharide in the cell wall of fungi, stain pink-red with blue nuclei Gomori Methenamine Silver Stain- outlines fungi in black due to silver precipitating in cell wall ( background light green) Gridly stain- Hyphae and yeast stain dark blue (tisue deep ) Cont- Fluorescent Antibody stain- simple, sensitive and specific Papanicolaou stain- for differentiation of dimorphic fungi (sputum) Gram Stain – most are Gram positive Modified-Acid Fast Stain – Differentiate acid fast Nocardia from other Giemsa stain- For Blood and Bone marrow India Ink – Demonstrates the capsule Cryptococcus neoformans in CSF Fungal culturing A) Tube Media B) Plate Media Tube media is used rather Plate media because - Less chance for spore release in environment - Less chance for dehydration - Ease to storage Incubation should be in aerobic ( anaerobic – Actinomycetes) Incubate cultures at room temperature also 37´C if Dimorphic fungi suspected Require 10 days to 1 month Media Used in medical Mycology BASAL MEDIA 1 ) Sabouraud Dextrose Agar(SDA) 2) SDA with Antibiotics 3) Sterile Bread NUTRITIONALLY DEFICIENT MEDIA 1) Corn Meal Agar 2) Rice Starch Agar ENRICHED MEDIA 1 ) Brain-Heart Infusion Agar 2) Cysteine-Heart and Hemoglobin Agar 3) Biphasic Medium 4) Blood Agar 5) Lowenstein-Jenson Medium 6) Dermatophyte Test Medium 7) Dermatophyte Identification Medium 8) Czapek-Dox Agar 9) PYG Agar Medium 10) Malt extrat Agar SABOURAUD DEXTROSE AGAR(SDA) 1892 French Dermatologist and Mycologist Raymond Jacques Adrien Sabouraud discover. Type: Selective media due to high sugar concentration Constituents: Peptone - 10 gm Dextrose – 40 gm Agar -20 gm Distilled water - 1000ml pH- 5.6 (Mix – sterilize – cool--poured in sterile tube or plate) Test tubes(25 × 100 mm) Petri dishes (100mm) Test Petri Dish Tube ADVANTAHE Saprotrophic fungi grow ADVANTAGE Adequate surface area fater Minimum aerosol production Acidic pH may inhibit Longer duration due slow significiant pathogenic drying fungal species SDA with ANTIBIOTICS(CSDA) For isolation of pathogenic fungi and avoiding bacterial and Saptotrophic fungal contaminants. Antibiotic added in SDA while it is cooled after autoclave. Ingredients- - Cyclohiximide 500 mg - Chloramphenicol 50 mg - Gentamicin 20 mg Cyclohiximide 0.5/lit dissolve in 2 ml acetone- add to boiling medium and mix properly. Chloramphenicol/ Gentamicin in 10 ml of 95% ethanol and added to boiling medium. Cyclohiximide Inhibit saprophytic fungi( Aspergillus, Fusarium, Cryptococcus , Candida) and antibiotics for bacterial contaminants. NEUTRAL SDA STERILE BREAD Dextrose concentration Inoculation on Sterile lower 40 to 20 g/L( 2% Bread without adding any instead 4%) preservatives Glucose lower Humidified in Petri dish pH neutral If sample from contaminated site then antibacterials before inoculation For diagnostic Mucormycetes grow mycology isolation of rapidly filling the entire opportunistic and Petri Dish Dimorphic fungi (Blastomyces dermatatiditis) Ingredients of medium NUTRITIONALLY DEFICIENT MEDIA CORN MEAL AGAR RICE STARCH AGAR Ingredients Ingredients - Corn meal 8gm -Cream of Rice 2gm - Agar 4 gm - Tween 80 2ml - Tween 80(1%) 2ml - Agar 4 gm - Distilled water 200ml -Distilled water 200 ml Heat corn meal and water at Add cream of rice to boiling water 60´C for 1 hr – pass through and continue boiling for 30 sec. filter paper- add distilled water Filter through cotton tampon and to make 200 ml then sugar add water to make one litr. Add added- autoclave- pour in plates agar and Tween 80 , dissolve white Yeast( C.albicans and heating. Autoclave then pour into C.dubliniensis ) grow to cylinder leave it for overnight in examine morphological details. water bath at 60´c then re-filter Yeast inoculated is streaked through cotton- autoclave acrossed plate containing pH 6.2 medium-coversilp placed to Use for produce chlamydospore of create anaerobic condition C.albicans and C.dubliniesis as compare to Corn Meal Agar ENRICHED AND SELECTED MEDIA Brain-Heart Infusion Agar - Use to grow fastidious and true pathogenic fungi like Histoplasma capsulatum and Blastomyces dermatidis. - Use for isolation as well as in vitro conversion of mycelial to yeast and vice versa. - Ingredients - Brain-Heart infusion 37 gm - Glucose 20gm - L-Cystiene hydrochloride 1 gm - Agar 1gm - Distilled water 900gm - Dissolve ingredients by boiling and dispence into screw cap tube- autoclave-antibiotics added(cyclohiximide, chloramphenical and gentamycin) - pH 6.7 maintain- cool in slant position one inch butt then store in refrigerator Cysteine-Heart and Hemoglobin Agar CHHA may be use in place of BHIA Ingredients- Dehydrated Cysteine -Heart agar 10.2 gm - Hemoglobin 2 gm - Distilled water 100 ml - Antibiotics then added BIPHAGIC MEDIUM Use for isolation of fungi in blood Contain BHIA and broth BHIA sterilized 1st then kept in slanting position– BHI broth added Method is used with or without antibiotics Blood agar Routinely used in bacteriology but also can be used to grow fungus - Ingredients : Agar base 40 gm pH – 7.4 : Sheep Blood 50ml : Distilled water 1000ml - Use for isolation of Histoplasma capsulatum and Blastomyces dermatitidis Lowenstein-Jensen Medium For isolation of Madurella and Nocardia species Contain hens’s egg, mineral salt solution, asparagine and malachite green(inhibit unwanted bacteria) Dermatophyte Test Medium For identification of Dermatophyte (Hair, Nail, Skin) which may be heavily contaminated with bacteria Most produce red colour due to increase in pH Ingredients - Phyton, Dextrose, Phenol red sol, HCL, Acitidione, Gentamicin., Agar, Distilled water pH-5.5 Phenol red solution 0.5 gm in 15 ml of NaOH made up to 100 ml with distilled water – incubated at 25 degree –change in colour within 3-6 days , if no change upto 2 weeks medium descarded. Limitation - It doesn’t inhibit saprotrophic fungi - H.capsulatum and B dermatitidis not inhibited Dermatophyte Identification Medium Use for presumptive identification of dermatophytes Contain-Dextrose, Neopeptone, Cycliheximide, Penicillin, Streptomycin, and Bromocresol purple It is incubated at 37 C If growth than colour change from Greenish blue to purple within 1-2 days. Also use for isolation of Dimorphic fungi Malt Extract Agar - Medium can be used in place of SDA - Can be acidified with hypochloric acid, pH