Med Tech Mycology Notes

FUNGI

CHARACTERISTICS:

• Eukaryotic
• Thallophytes
  • have true nuclei
  • heterotrophic members of the plant family that lack stems and roots
• Lack chlorophyll
• Larger and with more complex morphology than the bacteria
• Chitin in the cell wall
• Ergosterol in the cell membrane
• Saprophytic nature (derive nutrition from organic materials)
• Lack of susceptibility to antibacterial antibiotics

TWO PHASES:

• Multicellular MOLD – fluffy, cottony, woolly, or powdery mycelial mass, grows at 25°C
• Unicellular YEAST – moist, creamy, opaque or pasty, resembling bacterial colony, grows from 35°C to 37°C

DIMORPHIC FUNGI – capable of two phases Mold at 25°C to 30°C – INFECTIVE TO MAN Yeast at 37°C – TISSUE/IN VIVO/INVASIVE

PARTS:

• MYCELIUM – intertwining structure composed of tubular filaments known as HYPHAE
  • Vegetative portion or thallus – grows in or on a substrate and absorbs water and nutrients
  • Reproductive or aerial part – contains fruiting bodies that produce reproductive structures (conidia or spores); extends above the agar surface

• HYPHAE – microscopic unit of fungi
  • Septate – contain cross-walls
    • All fungi except Zygomycetes
  • Aseptate/Coenocytic – continuous, without cross-walls
    • ZYGOMYCETES (Rhizopus, Mucor)

FUNGAL REPRODUCTION:

• SEXUAL – meiosis (reduction division of two fertile cells) followed by merging of the cells and nuclear fusion occurs
  • PERFECT FUNGI – fungi that exhibit sexual phase
  • ASCOSPORES – contained in a saclike ASCUS
    • CLEISTOTHECIUM – large, round, multicellular structure that surrounds the asci until it ruptures, releasing ascospores
  • BASIDIOSPORES – contained in a club-shaped BASIDIUM
  • OOSPORES – fusion of cells from two separate, nonidentical hyphae
  • ZYGOSPORES – fusion of two identical cells arising from the same hypha
    
• ASEXUAL – involves only mitosis with nuclear and cytoplasmic division
  • IMPERFECT FUNGI – do not exhibit sexual phase
  • SPORANGIOSPORES – asexual spores contained in sporangia (sacs) and produced terminally on sporangiophores or aseptate hyphae
    • UNIQUE TO THE ZYGOMYCETES
  • CONIDIA – asexual spores produced either singly or multiply in long chains or clusters by specialized vegetative hyphae (conidiophores)
    • MACROCONIDIA – large, usually septate
      • Club, oval, or spindle shaped
      • Thick or thin walled
      • Spiny (echinulate) or smooth surface
    • MICROCONIDIA – small, unicellular
      • Round, elliptical, or pyriform (pear) shape
  • BLASTOCONIDIA or BLASTOSPORES
    • Develop as daughter cell buds off the mother cell and is pinched off
    • Blastoconidia of yeasts (including Candida) may elongate to form pseudohyphae
  • CHLAMYDOCONIDIA or CHLAMYDOSPORES
    • Thick-walled, resistant, resting spores produced by “rounding up” and enlargement of the terminal hyphal cells
    • Germinate into a new organism when favorable environmental conditions exist
      • Terminal – form at the hyphal tip
      • Sessile – form on the hyphal sides
      • Intercalary – form within the hyphal strand
  • ARTHROCONIDIA or ARTHROSPORES
    • Simple fragmentation of the mycelium at the septum into rectangular-, cylinder-, or cask-shaped spores
    • Thick walled spores which may be adjacent or alternate (empty spaces or disjunctor cells in between each arthrospores) in arrangement
    • Useful identification characteristic of Coccidioides immitis and Geotrichum candidum

CLASSIFICATION

Botanical Taxonomy

• Zygomycota –Mucor, Absidia, Rhizopus
• Ascomycota
• Basidomycota
• Deuteromycota – most medically important fungi
  • With septate hyphae
  • Asexual reproduction

Type of Mycoses

• Superficial and cutaneous mycoses
• Subcutaneous mycoses
• Systemic mycoses
• Opportunistic mycoses

IDENTIFICATION METHODS

MICROSCOPIC

• SALINE MOUNT – quick and simple method to observe fungal elements (budding yeast, hyphae, pseudohyphae)
  • Major disadvantage: Lack of contrast

• 10% KOH PREPARATION – rapid and simple method to examine hyphae, budding yeast and spherules
  • KOH dissolves keratin in skin, hair, or nail
  • Chitin is resistant to effects of KOH
  • Hair can be examined to determine type of infection
    • ENDOTHRIX – fungal invasion within the hair shaft
    • ECTOTHRIX – infection outside the hair shaft
  • Disadvantage: Lack of contrast

• CELLUFLUOR – chemofluorescent brightening agent
  • Can be added to the KOH solution
  • Binds to the chitin in fungal cell wall
  • Provides excellent contrast when examined with a fluorescent microscope
  • Fungi fluoresce intense apple green

• INDIA INK/NIGROSIN – used to identify hyaline capsule of the yeast Cryptococcus neoformans
  • Capsules do not stain with India ink and appear as clear halos against a dark background
  • May be difficult to interpret; WBCs and artifacts can be mistaken for yeast or capsules
  • Cryptococcus may be capsule negative in immunodeficiency
  • Replaced by direct antigen testing for the cryptococcal capsular protein

• LACTOPHENOL COTTON BLUE (LPCB) (AMAN) – imparts blue color to the fungal cell wall
  • Slides can be permanently sealed for later study with either Permount or clear nail polish
  • Can also be used in the tease preparation (wet mount) and slide cultures

• HUCKER MODIFICATION OF GRAM STAIN – recommended for mycology
  • Fungi generally stain gram positive
  • Oval or budding yeast, hyphae, arthrospores generally stain well
  • C. neoformans may appear pale lavender with blue inclusions (capsule prevents adequate staining)
  • Fungi are 2 to 3x the size of gram-positive cocci
  • Hyphae are 2 to 3x wider than gram-positive bacilli

• GIEMSA or WRIGHT’S STAIN – used for the detection of intracellular Histoplasma capsulatum in blood smears, lymph nodes, lung, liver, or bone marrow
  • H. capsulatum appears as small, oval yeast cell staining light to dark blue
  • C. neoformans also stain well

• METHENAMINE SLIVER NITRATE STAIN – useful for screening of clinical specimens
  • Provides good contrast and staining for fungal elements
  • Fungi appear outlined in black, with an inner dark rose to black color against a pale green background
  • Viable and nonviable fungi are stained using this method
  • GOMORI METHENAMINE SILVER (GMS) NITRATE MODIFICATION – used in histology to detect fungi in specimens

• PERIODIC ACID SCHIFF (PAS) – stains hyphae of molds and yeast
  • Periodic acid oxidizes the hydroxyl in the carbohydrates of the cell walls to form aldehydes which react with basic fuchsin dye to form a pink-purple complex
  • Counterstain of fast green can be used to provide contrast
  • Useful in staining tissue in histology

CULTURE

• Must include a source of nitrogen (nitrite, nitrate, amino acids, or urea) and a carbon source (usually glucose)
• Incubated at 30°C or RT (25-30°C)
• Chloramphenicol – inhibits bacteria
• Cycloheximide – inhibits saprophytic, contaminating fungi

PRIMARY ISOLATION MEDIA

SABOURAUD DEXTROSE AGAR (SDA) Main general isolation medium Primary recovery of saprobic and pathogenic fungi Primary agar for initial culture Contains peptone and glucose Inhibitor for bacteria: ACIDIC pH (5.6)

SDA WITH CYCLOHEXIMIDE AND CHLORAMPHENICOL (SDA-CC) Recovery of pathogenic fungi Bacteria and saprophytic fungi inhibited Available commercially as Mycosel or Mycobiotic medium

MYCOSEL OR MYCOBIOTIC AGAR Isolation of dermatophytes from hair, skin, and nail specimens Contains the inhibitory agents, cycloheximide and chloramphenicol Similar to DTM

DERMATOPHYTE TEST MEDIUM (DTM) Can be substituted for SDA-CC for the recovery of dermatophytes from specimens contaminated with fungi or bacteria Isolation of dermatophytes from hair, skin, and nail specimens Dermatophytes produce alkaline metabolites, which raise the pH and change the color of the indicator from yellow to red Indicator: PHENOL RED Antibiotics inhibit saprophytic fungi and bacteria

BRAIN-HEART INFUSION (BHI) AGAR Isolation of saprophytic and pathogenic fungi from sterile sites Bacteria also grown in BHI Can be supplemented with blood

BHI AGAR WITH ANTIBIOTICS, CYCLOHEXIMIDE AND CHLORAMPHENICOL Isolation of pathogenic fungi exclusive of dermatophytes; useful for specimens that may be contaminated with bacteria or saprophytic fungi

BHI BIPHASIC BLOOD CULTURE BOTTLES Recovery of fungi from blood or bone marrow

DIFFERENTIAL MEDIA

BIRDSEED (NIGER SEED) AGAR/ STAIB’S MEDIUM Isolation of Cryptococcus neoformans: brown to black colonies in 4 to 7 days C. neoformans produces phenol oxidase which breaks down the medium resulting in the production of melanin Similar to caffeic acid agar

CORNMEAL AGAR WITH TWEEN 80 Stimulation of conidiation and chlamydospore production in Candida species; useful for species differentiation of Candida Cornmeal agar + 1% glucose: differentiates T. rubrum from T. mentagrophytes based on PIGMENTATION

COTTONSEED AGAR Conversion of mold phase of Blastomyces dermatitidis to its yeast phase

NITRATE REDUCTION MEDIUM Confirmation of nitrate reduction in C. neoformans

POTATO DEXTROSE AGAR Stimulation of conidia production in fungi Useful in slide culture Also demonstrates pigment production of Trichophyton rubrum

RICE MEDIUM Identification of Microsporum audouinii

TRICHOPHYTON AGARS Nutritional requirement tests for the differentiation of Trichophyton #1: casein agar base (vitamin free) #2: casein agar base and inositol #3: casein agar base, inositol, and thiamine #4: casein agar base and thiamine #5: casein agar base and nicotinic acid #6: ammonium nitrate agar base #7: ammonium nitrate agar base and histidine

UREA AGAR Detection of urease production by C. neoformans and differentiation of Trichophyton mentagrophytes from T. rubrum

YEAST ASSIMILATION MEDIA (CARBON OR NITROGEN) Detection of carbohydrate assimilation through utilization of carbon (or nitrogen) by yeast in the presence of oxygen

YEAST FERMENTATION BROTH Identification of yeasts by fermentation reactions with various carbohydrates

ORGANISMS AND THEIR DESCRIPTION TO FOLLOW 🙂