Mycology: Fungal Culture Methods PDF

MYCOLOGY: FUNGAL CULTURE Compiled by: Angeli Abegail Q. Naranja, MIH 09012024 FUNGAL CULTURE METHOD: ✔ All specimens should be inoculated onto a general purpose fungal...

MYCOLOGY: FUNGAL CULTURE Compiled by: Angeli Abegail Q. Naranja, MIH 09012024 FUNGAL CULTURE METHOD: ✔ All specimens should be inoculated onto a general purpose fungal medium ✔ Fungi will grow very well on culture media used to isolate bacteria ✔ “gold standard” ✔ yield the specific etiological agent ✔ may allow susceptibility testing to be performed ✔ slow - takes one to four weeks ✔ sometimes lack sensitivity Laboratory Safety Consideration: All mold cultures and clinical specimens must be handled in a Class II biologic safety cabinet with no exemptions ✔ Dimorphic fungi especially C. immitis is very hazardous among Reasons why culture cannot always be applied 1. Reliable axenic culture (culture of an isolate free from other associating or contaminating organism) of Pneumocystis jirovecii has not yet been achieved 2. Pathogen is cultivable in only a minority of infected individuals e.g. Candida or Aspergillus 3. Pathogen grows too slowly to provide timely diagnostic information e.g. Histoplasma capsulatum Temperature: 1. Most MOLDS grow best at 25 – 30 OC 2. Most YEAST grow best at 35 – 37 OC 3. Most pathogenic fungi grow best: 30 to 32°C EXCEPT: Sporothrix schenckii : 25 to 27°C than 30°C Incubation period: ✔ 14 days: general incubation period ✔ 7 days: to detect presence of yeast in the mouth, throat, or vagina ✔ 21 days: tissues and sterile body fluids other than blood ✔ 28 days: respiratory, bone marrow, blood specimens, and specimens in which dimorphic fungus are suspected Plates should be checked at least TWICE during the first week, when rapidly growing isolates may appear, weekly hereafter. Importance of culture media selection ✔ General purpose media are Sabouraud dextrose, malt extract and seldom is brain heart infusion medium ✔ Chloramphenicol: added to prevent the growth of bacteria (reduce the yield of Actinomyces) ✔ Cycloheximide: added to reduce the growth of environmental fungi (but reduces the yield of Aspergillus, C. neoformans and Mucorales) Page 1 of 6  MYCOLOGY: FUNGAL CULTURE Compiled by: Angeli Abegail Q. Naranja, MIH 09012024 Media Uses Sabouraud dextrose medium for primary isolation of fungi directly from specimens agar (SDA) Malt Extract agar good medium for fungal detection, isolation and enumeration on yeast and mold Potato Dextrose Agar primary medium for clinically encountered fungi and used for inducing an isolate to exhibit a characteristic pigment and conidia. Recommended for slide cultures. Corn Meal Agar with used in distinguishing the different genera of yeast sand the various species of Tween 80 Candida and can also be useful in slide cultures, as itstimulates conidiation in many fungi Birdseed Agar (Niger For pigment production by C. neoformans and C. gattii Seed Agar; Staib Agar) For differentiation of C. dubliniensis versus Candida albicans Brain Heart Infusion recommended for the cultivation of fastidious pathogenic fungi,such as (BHI) Agar Histoplasma capsulatum and Blastomyces dermatitidis. Dermatophyte Test Specimens from hair, skin, or nails may be inoculated directly onto DTM and Medium (DTM) incu-bated at room temperature with the cap of the culture tube loose. Dermatophytes change the color of the medium from yellow to red within 14 days. (Phenol red solution is the red indicator) SABHI Agar Combination of SDA and BHI CHROMagar Candida Isolation and definitive differentiation of the most common encountered Medium years. Acceptable for use as the sole primary fungal medium for specimens in which yeasts are the primary concern, i.e., throats, urines, and genitals and blood cultures smear-positive for yeast. C. albicans appear as light green colored smooth colonies C. tropicalis appear as blue to metallic blue colored raised colonies. C. glabrata colonies appear as cream to white smooth colonies C. krusei appear as purple fuzzy colonies. Urea Agar Detection of Trichosporon and Trichophyton spp. Rice medium Detection of Microsporum audouinii and M. canis Page 2 of 6  MYCOLOGY: FUNGAL CULTURE Compiled by: Angeli Abegail Q. Naranja, MIH 09012024 General Considerations for Identification of Molds 1. Growth rate a. Dimorphic fungi (Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis) is slow, 1-4 weeks b. Rapid growers: Zygomycetes (24 hours) c. Dematiaceous fungi( “black yeasts” environmental molds): 1-5 days 2. Growth requirements: vary stain to stain however generally requires sources of Carbon, Nitrogen and Vitamins. Glucose (dextrose) is the most commonly used in media while fructose and manose are next, then sometimes sucrose. 3. Colonial morphologic features Different types of fungi will produce different-looking colonies, some colonies may be coloured, some colonies are circular in shape, and others are irregular. Yeast colonies are very similar to bacterial colonies. Moulds often have fuzzy edges a. Form - What is the basic shape of the colony? For example, circular, filamentous, etc. b. Size – The diameter of the colony. Tiny colonies are referred to as punctiform c. Elevation - This describes the side view of a colony. Turn the Petri dish on end. d. Margin/border – The edge of a colony. What is the magnified shape of the edge of the colony? e. Surface Topography (Elevation)- some fungal colonies cover the entire surface of the agar, others in restricted manner (flat, rugose, folded, crateriform, verrucose and cerebriform) f. Texture- cottony or wooly (floccose), granular, chalky, velvety, powdery, silky, glabrous (smooth/creamy), waxy g. Opacity - For example, transparent (clear), opaque, translucent (like looking through frosted glass), etc. h. Colour - (pigmentation) - For example, white, buff, red, purple, etc. i. Obverse: top pigment ii. Reverse: underside pigment 4. Microscopic Morphologic features a. Based on characteristic shape b. Method of reproduction: sexual or asexual c. Arrangement of spores: conidia, blastospore, sporangiospore, ascospores Page 3 of 6 MYCOLOGY: FUNGAL CULTURE Compiled by: Angeli Abegail Q. Naranja, MIH 09012024 Page 4 of 6  MYCOLOGY: FUNGAL CULTURE Compiled by: Angeli Abegail Q. Naranja, MIH 09012024 Microscopic Exam of Fungal Colonies 1. Tease Mount: the colonies are disturbed from their original position but a rapid method for identification of conidia and spores. 2. Cellophane Tape Press the lower, sticky side of the tape very firmly to the surface of the fungal colony, and then pull the tape gently away; aerial hyphae will adhere to the tape. Then, with the tape strip opened, place it on a small drop of LPCB on a glass slide so that the entire sticky side adheres to the slide, and examine it under the microscope. Advantage: Retains the original positions of the characteristic fungal structures Disadvantagr: The organism must be grown on plated medium. 3. Slide culture F i g The best method for preserving and observing the actual structure of a fungus is the slide culture. It is not a rapid u technique, but it is unsurpassed as a routine means rof studying the fine points of the microscopic morphology of fungi. (Always do a tease mount before a slide culture; organisms e suspected of being Histoplasma, Blastomyces ,Coccidioides spp. ornCladophialophora bantian s l i d e c u l t u r Page 5 of 6 e  MYCOLOGY: FUNGAL CULTURE Compiled by: Angeli Abegail Q. Naranja, MIH 09012024 SPECIAL TESTS 1. Germ Tube Test: screening test which is used to differentiate Candida albicans from other yeast. Principle: Formation of germ tube is associated with increased synthesis of protein and ribonucleic acid Procedure: When Candida is grown in human or sheep serum at 37°C for 3 hours, they form a germ tubes. 2. Rapid Urease test On Christensen’s Urea Agar (+) urease: Cryptococcus, Rhodotorulla, Trichosporon spp., Trichophyton mentagrophytes (-) urease: C. albicans 3. Ferric Citrate Test for C. neoformans 4. Hair perforation test Purpose: Differentiate Trichophyton spp. Isolates of T. mentagrophytes produce marked localized areas of pitting and marked erosion whereas those of T. rubrum do not. Page 6 of 6