Biology 250. Laboratory Exercise in Medical Mycology

Fungi are eukaryotic organisms, and are much larger in size and are more structurally complex than prokaryotic bacteria. Microscopically, fungi exhibit one of two basic growth forms. Molds produce threadlike filamentous structures called hyphae, while yeasts are typically single-celled organisms which reproduce by budding, although if the buds fail to disarticulate from the parent cell, a hypha-like pseudo-hyphal strand may be produced. Most fungi produce only filamentous or yeastlike growth, but some species can produce either filamentous or yeastlike growth, usually in response to culture conditions. This phenomenon is referred to as dimorphism. The usual conditions favorable for yeastlike growth by a dimorphic organism are growth on a nutrient-rich medium and incubation at 37 C, while growth on a less nutrient-rich medium at 25 C favors filamentous growth.

Approximately 150 fungus species can cause disease in human beings, with involvement ranging from superficial to systemic infection. Laboratory identification of fungi, especially filamentous molds, is based to a large extent on morphological characteristics, while identification of morphologically similar yeasts is based on physiological characteristics such as carbohydrate assimilation and extracellular enzyme production.

The objectives of this exercise are for you to be able to:

  1. Recognize yeast and filamentous fungi.
  2. Interpret laboratory tests used to identify fungus pathogens, including:
    1. Germ tube screening test for Candida albicans
    2. India ink preparation for visualizing Cryptococcus neoformans from spinal fluid (not available).
  3. Prepare scotch tape mounts for identification of dermatophytes and other fungi.

I. Morphological Characteristics of Fungi

Microscopic slide preparations for this exercise will be made in lactophenol-cotton blue (LPCB), a mounting medium which kills and preserves the fungi, and provides for staining of the fungal elements by the cotton blue dye. Handle these organisms with standard sterile techniques.

  1. Saccharomyces cerevisiae is the yeast widely used in the baking and brewing industries.  Make a mount of the yeast culture provided. Place a drop of LPCB on a clean microscope slide, remove a very small amount of the yeast colony from the plate with a wire loop or needle, then touch the loop to the drop of LPCB and place a coverslip over the preparation. (LPCB initially dehydrates the specimen, but this effect may be overcome by gently warming the slide over your burner's flame). Observe the preparation with your microscope (10x and 40x). Note the size of the cells and the asexual budding reproductive process. Various stages of bud formation should be present.
  2. One simple technique for preparing microscopic mounts of filamentous fungi is the scotch tape mount, where a piece of clear tape is gently pressed down on the surface of a fungus colony, and then placed in a drop of LPCB on a microscope slide. Make a tape mount slide of the mold provided. (Mucor alternans). Observe the preparation (10x) and note the filamentous growth of this organism. M. alternans produces spores (asexual reproductive structures) in culture, and they are formed in sac-like structures (sporangia) borne terminally on hyphal branches called sporangiophores. Many of the sporangia will have their internal contents cleaved up into spores.
  3. Mucor alternans is an interesting fungus, in that it exhibits dimorphic growth (growth as either a mold or a yeast). Under reduced oxygen conditions, its spores will begin to bud and grow as yeasts. Make a LPCB mount of a Sabouraud's broth culture containing Mucor alternans spores with a Pasteur pipette, and note the yeast-like growth (10x). Look for "Mickey Mouse ear" budding.
  4. Penicillium. Note the many chains of spores and how they are produced. Looking down on the spore chains with a dissecting microscope would suggest the appearance of a paintbrush for which this fungus is named. The antibiotic penicillin is obtained from many mamber of this genus. Do the hyphae branch?

II. Interpretation of Diagnostic Laboratory Tests Used for the Identification of Fungi

  1. Two hours ago, a small amount of Candida albicans and Candida tropicalis were placed in a test tube containing human serum, and incubated at 37 C. Place a drop of each yeast suspension on a slide, add a coverslip, and observe them with your microscope (10x). Note that germ tubes (hyphal extensions) are formed from some of the Candida albicans yeast cells, while no germ tubes are formed by Candida tropicalis. Since Candida albicans is the only pathogenic yeast for human beings which forms germ tubes in serum after 2 hours at 37 oC, this procedure allows for inexpensive and rapid identification of Candida albicans, which accounts for about 90% of all yeasts isolated by the diagnostic laboratory.  Candida albicans is part of human normal flora, and can cause many sorts of opportunistic infections. 
  2. When a fungus infection of the central nervous system (CNS) is suspected, often a spinal tap will be performed, and the cerebrospinal fluid (CSF) not only cultured for fungi, but also directly observed for the presence of fungal forms. An etiological agent with a predilection for the CNS is Cryptococcus neoformans, a yeast which usually forms a large polysaccharide capsule in tissue. To see Cryptococcus neoformans in CSF, a small amount of a concentrated specimen of CSF is added to a drop of India ink. The particulate carbon in the ink provides a negative staining effect, highlighting the presence of any encapsulated yeasts. [You will not be working with Cryptococcus neoformans this semester.]

III. Identification of the Dermatophytes

The dermatophytes are fungi which grow on the keratin of skin, hair, and nails. Diagnosis of infections caused by these organisms is done based on the clinical presentation of the patient, as well as the isolation and identification of the fungus. Infected tissues are plated on antibiotic-containing medium (with chloramphenicol and cyclohexamide), which is available commercially under the trade names of Mycosel agar (BBL) or Mycobiotic agar (Difco). Once isolated, the fungus is identified based on its colony morphology and pigmentation, microscopic appearance, and in the absence of sporulation, on additional biochemical tests.

Microsporum gypseum is related to the athlete’s foot fungi, but it is isolated from soil and not infectious.  Prepare a scotch tape mount from the dermatophyte culture provided, and observe the hyphae and spores produced by this fungus. Fresh isolates of M. gypseum are characterized by the production of tan, powdery colonies and numerous multi-celled macroconidia with thick and roughened outer walls. An advantage of the tape mount slide for viewing this fungus is that the microscopic structure of the organism remains intact, providing an easily seen picture of the morphology. Look for flame-shaped cells (macroconidia) and bullet-shaped cells (microconidia).