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  Section: Biotechnology Methods » Microbiology
 
 
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The Fungi, Part 2-The Molds

 
     
 
Content
Microbiology
  Introduction
  The Microscopy
  The Bright Field Microscope
  Introduction to the Microscope and Comparison of Sizes and Shapes of Microorganisms
  Cell Size Measurements: Ocular and Stage Micrometers
  Measuring Depth
  Measuring Area
  Cell Count by Hemocytometer or Measuring Volume
  Measurement of Cell Organelles
  Use of Darkfield Illumination
  The Phase Contrast Microscope
  The Inverted Phase Microscope
  Aseptic Technique and Transfer of Microorganisms
  Control of Microorganisms by using Physical Agents
  Control of Microorganisms by using Disinfectants and Antiseptics
  Control of Microorganisms by using Antimicrobial Chemotherapy
  Isolation of Pure Cultures from a Mixed Population
  Bacterial Staining
  Direct Stain and Indirect Stain
  Gram Stain and Capsule Stain
  Endospore Staining and Bacterial Motility
  Enumeration of Microorganisms
  Biochemical Test for Identification of Bacteria
  Triple Sugar Iron Test
  Starch Hydrolysis Test (II Method)
  Gelatin Hydrolysis Test
  Catalase Test
  Oxidase Test
  IMVIC Test
  Extraction of Bacterial DNA
  Medically Significant Gram–Positive Cocci (GPC)
  Protozoans, Fungi, and Animal Parasites
  The Fungi, Part 1–The Yeasts
  Performance Objectives
  The Fungi, Part 2—The Molds
  Viruses: The Bacteriophages
  Serology, Part 1–Direct Serologic Testing
  Serology, Part 2–Indirect Serologic Testing

Introduction
Molds are multinucleated, filamentous fungi composed of hyphae. A hypha is a branching, tubular structure from 2–10 µm in diameter and is usually divided into cell-like units by crosswalls called septa. The total mass of hyphae is termed a mycelium. The portion of the mycelium that anchors the mold and absorbs nutrients is called the vegetative mycelium; the portion that produces asexual reproductive spores is termed the aerial mycelium.

Molds possess a rigid polysaccharide cell wall composed mostly of chitin and, like all fungi, are eukaryotic. Molds reproduce primarily by means of asexual reproductive spores such as conidiospores, sporangiospores, and arthrospores. These spores are disseminated by air, water, animals, or objects. Upon landing on a suitable environment, they germinate and produce new hyphae. Molds may also reproduce by means of sexual spores such as ascospores and zygospores, but this is not common. The form and manner in which the spores are produced, along with the appearance of the hyphae and mycelium, provide the main criteria for identifying and classifying molds.


Nonpathogenic Molds
To illustrate how morphological characteristics, such as the type and form of asexual reproductive spores and the appearance of the mycelium, may be used in identification, we will look at 3 common nonpathogenic molds.

The 2 most common types of asexual reproductive spores produced by molds are conidiospores and sporangiospores. Conidiospores are borne externally in chains on an aerial hypha called a conidiophore; sporangiospores are produced within a sac or sporangium on an aerial hypha called a sporangiophore.

Penicillium and Aspergillus
are examples of molds that produce conidiospores. Penicillium is one of the most common household molds and is a frequent food contaminant. The conidiospores of Penicillium usually appear gray, green, or blue and are produced in chains on finger-like projections called sterigmata. Aspergillus is another common contaminant. Although usually nonpathogenic, it may become opportunistic in the respiratory tract of a compromised host and, in certain foods, can produce mycotoxins. The conidiospores of Aspergillus appear brown to black and are produced in chains on the surface of a ball-like structure called a vesicle.
  • Scanning electron micrograph of the conidiospores of Penicillium.
  • Scanning electron micrograph of the conidiospores of Aspergillus.
  • Rhizopus is an example of a mold that produces sporangiospores. Although usually nonpathogenic, it sometimes causes opportunistic wound and respiratory infections in the compromised host. The sporangiospores of Rhizopus appear brown or black and are found within sacs called sporangia. Anchoring structures called rhizoids are also produced on the vegetative hyphae.
Rhizopus can also reproduce sexually. During sexual reproduction, hyphal tips of a (+) and (–) mating type join together and their nuclei fuse to form a sexual spore called a zygospore. This gives rise to a new sporangium-producing sporangiospore with DNA that is a recombination of the 2 parent strains’ DNA.

Nonpathogenic molds are commonly cultured on fungal-selective or enriched media such as saboraud dextrose agar (SDA), corn meal agar, and potato dextrose agar.


Dermatophytes
The dermatophytes are a group of molds that cause superficial mycoses of the hair, skin, and nails and utilize the protein keratin. Infections are commonly referred to as ringworm or tinea infections and include tinea capitis (head), tinea barbae (face and neck), tinea corporis (body), tinea cruris (groin), tinea unguium (nails), and tinea pedis (athlete’s foot).

The 3 common dermatophytes are Microsporum, Trichophyton, and Epidermophyton. These organisms grow well at 25°C. They may produce large leaf- or club-shaped asexual spores called macroconidia, as well as small spherical asexual spores called microconidia, both from vegetative hyphae.

Microsporum commonly infects the skin and hair, Epidermophyton, the skin and nails, and Trichophyton, the hair, skin, and nails. Dermatophytic infections are acquired by contact with fungal spores from infected humans, animals, or objects. On the skin, the dermatophytes cause reddening, itching, edema, and necrosis of tissue as a result of fungal growth and a hypersensitivity of the host to the fungus and its products. Frequently there is secondary bacterial or Candida invasion of the traumatized tissue.

To diagnose dermatophytic infections, tissue scrapings can be digested with 10% potassium hydroxide (which causes lysis of the human cells but not the fungus) and examined microscopically for the presence of fungal hyphae and spores. To establish the specific cause of the infection, fungi from the affected tissue can be cultured on dermatophyte test medium (DTM) and saboraud dextrose agar (SDA).

DTM has phenol red as a pH indicator with the medium yellow (acid) prior to inoculation. As the dermatophytes utilize the keratin in the medium, they produce alkaline end products, which raise the pH, thus turning the phenol red in the medium from yellow or acid to red or alkaline. On SDA, the types of macroconidia and microconidia can be observed. Many dermatophyte species produce yellow- to red-pigmented colonies on SDA, and the most common species of Microsporum fluoresce under ultraviolet light.


Dimorphic Fungi
Dimorphic fungi may exhibit 2 different growth forms. Outside the body they grow as a mold, producing hyphae and asexual reproductive spores, but inside the body they grow in a nonmycelial form. Dimorphic fungi may cause systemic mycoses, which usually begin by inhaling spores from the mold form. After germination in the lungs, the fungus grows in a nonmycelial form. The infection usually remains localized in the lungs and characteristic lesions called granuloma may be formed in order to wall-off and localize the organism. In rare cases, usually in an immunosuppressed host, the organism may disseminate to other areas of the body and be life-threatening. Examples of dimorphic fungi include Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis.

Coccidioide
s immitis is a dimorphic fungus that causes coccidioidomycosis, a disease endemic to the southwestern United States. The mold form of the fungus grows in arid soil and produces thick-walled, barrel-shaped asexual spores called arthrospores by a fragmentation of its vegetative hyphae. After inhalation, the arthrospores germinate and develop into endosporulating spherules in the lungs. Coccidioidomycosis can be diagnosed by culture, by a coccidioidin skin test, and by indirect serologic tests.

Histoplasma capsulatum is a dimorphic fungus that causes histoplasmosis, a disease commonly found in the Great Lakes region and the Mississippi and Ohio River valleys. The mold form of the fungus often grows in bird or bat droppings, or soil contaminated with these droppings, and produces large tuberculate macroconidia and small microconidia. After inhalation of these spores and their germination in the lungs, the fungus grows as a budding, encapsulated yeast. Histoplasmosis can be diagnosed by culture, by a histoplasmin skin test, and by indirect serologic tests.

Symptomatic and disseminated histoplasmosis and coccidioidomycosis are seen primarily in individuals who are immunosuppressed. Along with a positive HIV antibody test, both are indicator diseases for the diagnosis of AIDS.

Blastomycosis, caused by Blastomyces dermatitidis, produces a mycelium with small conidiospores and grows actively in bird droppings and contaminated soil. When spores are inhaled or enter breaks in the skin, they germinate and the fungus grows as a yeast with a characteristic thick cell wall. Blastomycosis is common around the Great Lakes region and the Mississippi and Ohio River valleys. It is diagnosed by culture and by biopsy examination.


Procedure
Nonpathogenic Molds
  1. Using a dissecting microscope, observe the SDA plate cultures of Penicillium, Aspergillus, and Rhizopus. Note the colony appearance and color and the type and form of the asexual spores produced.
  2. Observe the prepared slides of Penicillium, Aspergillus, and Rhizopus under high magnification. Note the type and form of the asexual spores produced.
  3. Observe the prepared slide showing the zygospore of Rhizopus produced during sexual reproduction.
Dermatophytes  
  1. Observe the dermatophyte Microsporum growing on DTM. Note the red color (from alkaline end products) characteristic of a dermatophyte.
  2. Microscopically observe the SDA culture of Microsporum. Note the macroconidia and microconidia.
  3. Observe the photographs of dermatophytic infections.
Dimorphic Fungi  
  1. Observe the prepared slide of Coccidioides immitis arthrospores.
  2. Observe the pictures showing the mold form and endosporulating spherule form of Coccidioides immitis.
  3. Observe the pictures showing the mold form and yeast form of Histoplasma capsulatum.
  4. Observe the photographs of systemic fungal infections.
Results
Nonpathogenic Molds
Make drawings of the molds as they appear microscopically under high magnification and indicate the type of asexual spore they produce. Also note their color and appearance on SDA.



Dermatophytes
  1. Describe the results of Microsporum growing on DTM:
    • Original color of DTM =

    • Color following growth of Microsporum =

    • Reason for color change =
  2. Draw the macroconidia and microconidia seen on the SDA culture of Microsporum.

Dimorphic Fungi  
  1. Draw the arthrospores of Coccidioides immitis.
  2. Draw the mold form and endosporulating spherule form of Coccidioides immitis.
  3. Draw the mold form and yeast form of Histoplasma capsulatum.
Performance Objectives
Discussion
  1. Define the following: hypha, mycelium, vegetative mycelium, and aerial mycelium.
  2. Describe the principle way molds reproduce asexually.
  3. List the main criteria used in identifying molds.

Nonpathogenic Molds
  1. Describe conidiospores and sporangiospores and name a mold that produces each of these.
  2. Recognize the following genera of molds when given an SDA plate culture and a dissecting microscope and list the type of asexual spore seen:
  3. (a) Penicillium
    (b) Aspergillus
    (c)
    Rhizopus
  4. Recognize the following genera of molds when observing a prepared slide under high magnification and list the type of asexual spore seen:
  5. (a) Penicillium
    (b) Aspergillus
    (c)
    Rhizopus
  6. Recognize Rhizopus zygospores.

Dermatophytes
  1. Define dermatophyte and list 3 common genera of dermatophytes.
  2. Name 4 dermatophytic infections and explain how they are contracted by humans.
  3. Describe macroconidia and microconidia.
  4. Describe how the following may be used to identify dermatophytes: potassium hydroxide
    preparations of tissue scrapings, DTM, and SDA.
  5. Recognize a mold as a dermatophyte and explain how you can tell when given the following:
    1. flask of DTM showing alkaline products.
    2. an SDA culture (under a microscope) or picture showing macroconidia.
  6. Recognize macroconidia and microconidia.

Dimorphic Fungi
  1. Define dimorphic fungi and describe how they are usually contracted by humans.
  2. Name 3 common dimorphic fungal infections found in the United States, explain how they are transmitted to humans, and indicate where they are found geographically.
  3. Describe the mold form and the nonmycelial form of the following:
    (a) Coccidioides immitis
    (b) Histoplasma capsulatum
    (c) Blastomyces dermatitidis
  4. Recognize Coccidioides immitis and its arthrospores when given a prepared slide and a microscope.

 
     
 
 
     




     
 
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