. .
.
Slide Culture Technique for Fungi
.
.

 

Objectives:

 

  • To become familiar with mycological culture techniques.
  • To visualize and identify the structural components of fungi.

 

Principle:

 

The fungi are a group of eukaryotic protists that lack chlorophyll. We have seen the velvety blue and green growth on rotting oranges and lemons as well as on stale cheeses, the whitish –gray furry outgrowth on bread and jam and the mushrooms in the fields. These are the bodies of various fungi. Thus, fungi have a diversity of morphological appearances, depending on the species. Fungi comprise molds and yeasts. Molds are filamentous and multi cellular whereas yeasts are usually unicellular.

 

 

 

 

 

       

                              Figure 1: (a) Penicillium growing on apple                          Figure 1: (b)  Rhizopus growing on bread

  

The fungi possess rigid cell walls containing chitin, mannan and other polysaccharides. The cytoplasmic membrane contains sterols. They possess true nuclei with nuclear membrane and paired chromosomes. They divide asexually, sexually or by both processes. They reproduce by means of spores. Fungi that reproduce by asexual spores and sexual pores are called perfect fungi and the fungi that reproduce
only by asexual spores are called imperfect fungi (deuteromycetes)
 

Most fungi are saprophytes, securing their nutrients from dead organic materials. Like many bacteria, fungi  release  hydrolytic  exoenzymes  that  digest  external substrates.  They  then  absorb  the  soluble  products.  They  are Most fungi are saprophytes, securing their nutrients from dead organic materials. Like many bacteria, fungi  release  hydrolytic  exoenzymes  that  digest  external substrates.  They  then  absorb  the  soluble  products.  They  are chemoorganoheterotrophs and use organic compounds as a source of carbon, electrons, and energy.

 

Glycogen is the primary storage polysaccharide in fungi. Most fungi use carbohydrates (preferably glucose or maltose) and nitrogenous compounds to synthesize their own amino acids and proteins. Fungi usually are aerobic. Some yeast, however, are facultatively anaerobic and can obtain energy by fermentation, such as in the production of ethyl alcohol from glucose. Obligately anaerobic fungi are found in the rumen of cattle.

 

Distribution:

 

Fungi grow best in dark, moist habitats, but they are found wherever organic material is available. They are  primarily  terrestrial  organisms, although  a  few  are freshwater or marine. Many are pathogenic and infect plants and animals. Fungi also form beneficial relationships with other organisms. For example, the association between  the  roots of vascular plants and fungi called mycorrhizae. Fungi also are found in the upper portions of many plants. These endophytic fungi affect plant reproduction and palatability to herbivores. Lichens are associations of fungi and either algae or Cyanobacteria.

 

 

                                         Figure 2: (a) Mycorrhizal root tip                                         (b) Foliose lichen

 

Importance:

 

Fungi are important to humans in both beneficial and harmful ways. With bacteria and a few other groups of heterotrophic organisms, fungi act as decomposers, which is of great significance. They degrade complex organic materials in the environment to simple organic compounds and inorganic molecules. In this way  carbon, nitrogen, phosphorus, and other  critical  constituents of dead organisms are released and made available for living organisms.

 

As parasites, fungi cause diseases in plants, humans and other animals. Although fungal diseases are less commonly encountered than bacterial or virus disease in humans and other animals, they are of great importance in causing diseases of plants.

 

                                                                               Figure 3: Grey mold disease on tomato

 

Fungi, especially the yeasts, are essential to many industrial processes involving fermentation. Examples include the making of bread, wine, and beer. Fungi also play a major role in the preparation of some cheeses and soy sauce, in the commercial production of many organic acids (citric, gallic) and certain drugs (ergometrine, cortisone); and in the manufacture of many antibiotics (penicillin, griseofulvin) and the immunosuppressive drug cyclosporine.
 

In addition, fungi are important research tools in the study of fundamental biological processes. Cytologists, geneticists, biochemists, biophysicists, and microbiologists regularly use fungi in their research. Based on this research the yeast Saccharomyces cerevisiae is the best understood eucaryotic cell. The branch of science dealing with the study of fungi is known as Mycology.

 

Morphology:

 

The body or vegetative structure of a fungus is called a thallus. It varies in complexity and size, ranging from the single-cell microscopic yeasts to multicellular molds.

 

A yeast is a unicellular fungus that has a single nucleus and reproduces either asexually by budding and transverse division or sexually  through  spore  formation. Each bud  that  separates can grow into a new yeast, and some group together to form colonies. Generally yeast cells are  larger  than bacteria, vary considerably  in size, ranging from 1 to 5µm in width and from 5 to 30µm or more in length. They  are commonly spherical to egg shaped. They have no flagella but do possess most of the other eucaryotic organelles.

 

Eg: Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans (Only pathogenic yeast)

 

                                                                      Figure 4: Oval yeast cells (Saccharomyces cerevisiae)

 

A mold or filamentous fungi consists of long, branched, threadlike filaments of cells called hyphae that form a mycelium, a tangled mass or tissue like aggregation. Vegetative hyphae grow on  the  surface of culture media. They form aerial hyphae, called reproductive hyphae, that bear asexual reproductive spores or conidia. The hyphae  that grow below  the surface of culture media are called rhizoidal hyphae. The hyphal strand of some molds may be separated by a cross-wall called a  septum. Hyphae  that contain septa  are  called  septate hyphae. Molds with hyphae that lack septa are called coenocytic hyphae.

 

Eg: Penicillium sp., Fusarium sp., Aspergillus sp., Alternaria sp.

 


                      Figure 5: (a) Aspergillus niger with conidiophores                                (b) Conidiophores of Penicillium sp.

 

Many fungi, especially those that cause diseases in humans and animals, are dimorphic -that is, they have two forms. Dimorphic fungi can change from the yeast (Y) form in the animal to the mold or mycelial form (M) in the external environment in response to changes in various environmental factors (nutrients, CO2 tension, oxidation-reduction potentials, temperature). This shift  is called  the YM shift. In plant-associated fungi the opposite type of dimorphism exists: the mycelial form occurs in the plant and the yeast form in the external environment

 

Eg: Ustilago maydis , Blastomyces dermatitidis, Histoplasma capsulatum

 

                                                                Figure 6: Histoplasma capsulatum showing dimorphic growth

 

The  isolation, culture, and microscopic examination of molds  require  the use of suitable selective media and  special microscopic  slide  techniques. The process of transferring hyphae to a slide breaks up the  structure of hyphae and sporangiophores so  that identification becomes very difficult. In this method, sporangiophores, the  hyphae,  and the spores remain  intact when stained.

 

When molds are collected from the environment, the Sabouraud’s agar is most frequently used. It is a simple medium constituting 4% glucose, 1% peptone, and 2% agar-agar. The pH of the medium is  then adjusted to 5.6 to inhibit  the growth of other bacteria.

 

For  some  molds  the  pH  of Sabouraud’s agar is too low while the  glucose content of the agar is too high. A better medium for the growth of these organisms is one contributed by C. W. Emmons known as Emmons' medium that contains  2% glucose, with 1% neopeptone, and an adjusted pH of 6.8 to7.0. To  inhibit bacterial growth, 40 mg of chloramphenicol is added to one litre of the medium. In  addition  to  these  two  media,  cornmeal agar, potato dextrose agar, Czapek  solution agar,  are some of the available media for culturing molds.


 

 

Developed under a Research grant from NMEICT, MHRD
by
Amrita CREATE (Center for Research in Advanced Technologies for Education),
VALUE (Virtual Amrita Laboratories Universalizing Education)
Amrita University, India 2009 - 2014
http://www.amrita.edu/create

Downloaded from : http://amrita.vlab.co.in/?sub=3&brch=76&sim=693&cnt=1
.....
..... .....

Copyright @ 2014 Under the NME ICT initiative of MHRD

 Powered by Amrita Virtual Lab Collaborative Platform [ Ver 00.3.2 ]