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  Section: Biotechnology Methods » Microbiology
 
 
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The Fungi, Part 1-The Yeasts

 
     
 
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
Fungi are eukaryotic organisms and include yeasts, molds, and fleshy fungi. Yeasts are microscopic, unicellular fungi; molds are multinucleated, filamentous fungi (such as mildews, rusts, and common household molds); the fleshy fungi include mushrooms and puffballs.

All fungi are chemoheterotrophs, requiring organic compounds for both an energy and carbon source, which obtain nutrients by absorbing them from their environment. Most live off of decaying organic material and are termed saprophytes. Some are parasitic, getting their nutrients from living plants or animals.

The study of fungi is termed mycology and the diseases caused by fungi are called mycotic infections or mycoses.

In general, fungi are beneficial to humans. They are involved in the decay of dead plants and animals (resulting in the recycling of nutrients in nature), the manufacturing of various industrial and food products, the production of many common antibiotics, and may be eaten themselves for food. Some fungi, however, damage wood and fabrics, spoil foods, and cause a variety of plant and animal diseases, including human infections.


Discussion
Yeasts are unicellular, oval, or spherical fungi, which increase in number asexually by a process termed budding. A bud forms on the outer surface of a parent cell, the nucleus divides with one nucleus entering the forming bud, and cell wall material is laid down between the parent cell and the bud. Usually the bud breaks away to become a new daughter cell but sometimes, as in the case of the yeast Candida, the buds remain attached, forming fragile branching filaments called pseudohyphae. Because of their unicellular and microscopic nature, yeast colonies appear similar to bacterial colonies on solid media. It should be noted that certain dimorphic fungi can grow as a yeast or as a mold, depending on growth conditions.

Yeasts are facultative anaerobes and can therefore obtain energy by both aerobic respiration and anaerobic fermentation. Most yeasts are nonpathogenic and some are of great value in industrial fermentations. For example, Saccharomyces species are used for both baking and brewing.

The yeast Candida is normal flora of the gastrointestinal tract and is also frequently found on the skin and on the mucus membranes of the mouth and vagina. Candida is normally held in check in the body by normal immune defenses and normal flora bacteria. Therefore, they may become opportunistic pathogens and overgrow an area if the host becomes immunosuppressed or is given broad-spectrum antibiotics that destroy the normal bacterial flora. Any infection caused by the yeast Candida is termed candidiasis. The most common forms of candidiases are oral mucocutaneous candidiasis (thrush), vaginitis, onychomycosis (infection of the nails), and dermatitis (diaper rash and other infections of moist skin). However, antibiotic therapy, cytotoxic and immunosuppressive drugs, and immunosuppressive diseases such as diabetes, leukemias, and AIDS can enable Candida to cause severe opportunistic systemic infections involving the skin, lungs, heart, and other organs. In fact, Candida now accounts for 10% of the cases of septicemia. Candidiasis of the esophagus, trachea, bronchi, or lungs, in conjunction with a positive HIV antibody test, is one of the indicator diseases for AIDS.

The most common Candida species causing human infections is C. albicans. This organism is usually oval and nonencapsulated, but under certain culture conditions may produce pseudohyphae, elongated yeast cells 4–6 mm in diameter that remain attached after budding to produce filament-like structures similar to the hyphae of molds. The pseudohyphae help the yeast invade deeper tissues after it colonizes the epithelium. Asexual spores called blastospores develop in clusters along the pseudohyphae, often at the points of branching. Under certain growth conditions, thick-walled survival spores called chlamydospores may also form at the tips or as a part of the pseudohyphae.

A lesser known but often more serious pathogenic yeast is Cryptococcus neoformans. Like many fungi, this yeast can also reproduce sexually and the name given to the sexual form of the yeast is Filobasidiella neoformans. It appears as an oval yeast 5–6 mm in diameter, forms buds with a thin neck, and is surrounded by a thick capsule. It does not produce pseudohyphae and chlamydospores. The capsule enables the yeast to resist phagocytic engulfment. Cryptococcus infections are usually mild or subclinical but, when symptomatic, usually begin in the lungs after inhalation of the yeast in dried bird feces. It is typically associated with pigeon and chicken droppings and soil contaminated with these droppings. Cryptococcus, found in soil, actively grows in the bird feces but does not grow in the bird itself. Usually the infection does not proceed beyond this pulmonary stage. In the immunosuppressed host, however, it may spread through the blood to the meninges and other body areas, often causing cryptococcal meningoencephalitis. Any disease by this yeast is usually called cryptococcosis.

Dissemination of the pulmonary infection can result in a very severe and often fatal cryptococcal meningoencephalitis. Cutaneous and visceral infections are also found. Although exposure to the organism is probably common, large outbreaks are rare, indicating that an immunosuppressed host is usually required for the development of severe disease. Extrapulmonary cryptococcosis, in conjunction with a positive HIV antibody test, is another indicator disease for AIDS.

Cryptococcus
can be identified by preparing an India ink or nigrosin-negative stain of suspected sputum or cerebral spinal fluid in which the encapsulated, budding, oval yeast cells may be seen. It can be isolated on Saboraud dextrose agar and identified by biochemical testing. Direct and indirect serological tests may also be used in diagnosis.

Pneumocystis carinii, once thought to be a protozoan but now considered a yeast-like fungus belonging to the fungal class Ascomycetes, causes an often lethal disease called pneumocystis carinii pneumonia (PCP). It is seen almost exclusively in highly immunosuppressed individuals such as those with AIDS, late-stage malignancies, or leukemias.

PCP and a positive HIV-antibody test is one of the more common indicators of AIDS.

In biopsies from lung tissue or in tracheobronchial aspirates, both a unicellular organism about 1–3 mm in diameter with a distinct nucleus and a cyst form between 4–7 mm in diameter with 6–8 intracystic bodies, often in rosette formation, can be seen.  
  • P. carinii cysts from bronchoalveolar larvage
  • P. carinii cysts from the lungs
We will use 3 agars to grow our yeast: Saboraud Dextrose agar (SDA), mycosel agar, and rice extract agar. SDA is an agar similar to trypticase soy agar but with a higher sugar concentration and a lower pH, both of which inhibit bacterial growth but promote fungal growth. SDA, therefore, is said to be selective for fungi. Another medium, Mycosel agar, contains chloramphenicol to inhibit bacteria and cycloheximide to inhibit most saprophytic fungi. Mycosel agar, therefore, is said to be selective for pathogenic fungi. Rice extract agar with polysorbate 80 stimulates the formation of pseudohyphae, blastospores, and chlamydospores, structures unique to C. albicans, and may be used in its identification. The speciation of Candida is based on sugar fermentation patterns.

Materials  
  • Coverslips
  • Alcohol
  • Forceps
  • One plate each of Saboraud dextrose agar, mycosel agar, and rice extract agar
Organisms
Trypticase soy broth cultures of Candida albicans and Saccharomyces cerevisiae.


Procedure  
  1. With a wax marker, divide a Saboraud dextrose agar and a Mycosel agar plate in half. Using a sterile swab, inoculate one half of each plate with C. albicans and the other half with S. cerevisiae. Incubate the 2 plates at 37°C until the next lab period.
  2. Using a sterile swab, streak 2 straight lines of C. albicans into a plate of rice extract agar. Pick up a glass coverslip with forceps, dip the coverslip in alcohol, and ignite with the flame of your gas burner. Let the coverslip cool for a few seconds and place it over a portion of the streak line so that the plate can be observed directly under the microscope after incubation. Repeat for the second steak line and incubate the plate at room temperature until the next lab period.
Results  
  1. Describe the appearance of Candida albicans and Saccharomyces cerevisiae on saboraud dextrose agar.
  2. Remove the lid of the rice extract agar plate and put the plate on the stage of the microscope. Using your yellow-striped 10X objective, observe an area under the coverslip that appears “fuzzy” to the naked eye. Reduce the light by moving the iris diaphragm lever almost all the way to the right. Raise the stage all the way up using the coarse focus (large knob) and then lower the stage using the coarse focus until the yeast comes into focus. Draw the pseudohyphae, blastospores, and chlamydospores.
  3. Observe and make drawings of the demonstration yeast slides.

 
     
 
 
     




     
 
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