Cultivation of Fungi: Natural and synthetic types of fungal culture media are commonly used for the growth and cultivation of fungi. Natural media are composed of natural substrates, such as herbaceous or woody stems, seeds, leaves, corn meal, wheat germ, oatmeal, etc. Natural media are usually easy to prepare but they have the disadvantage of their unknown composition e.g. corn meal agar and potato dextrose agar. Synthetic media contain ingredients of known composition. These types of media contain defined amounts of carbohydrates, nitrogen, and vitamin sources e. g. Czapek-Dox medium, glucose-asparagine, and Neurosporacrassa minimal medium. General purpose media, which are commonly used for fungal culture are Sabouraud dextrose agar (SDA). Selective media, like inhibitory mold agar and dermatophyte test media, are important in the isolation of fungal pathogens such as Cryptococcus neoformans and dermatophytes. Agar supplemented by rice, casein, and other nutrients like corn meal agar with tween 80 have been used to differentiate Candida species and Trichophytan species. Potato dextrose agar is used to enhance the conidia and pigment development of Trichophytan rubrum.
Sabouraud agar is a type of agar growth medium containing peptones. It is used to cultivate dermatophytes and other types of fungi, and can also grow filamentous bacteria
such as Nocardia. It has utility for research and clinical care. It was developed by Raymond Sabouraud in 1892. The acidic pH (5.6) of traditional Sabouraud agar inhibits bacterial growth.
For optimal recovery of the fungal pathogen, a modified media should be used such as media with or without cycloheximide and media with or without an antibacterial agent (Chloramphenicol, Gentamicin, and Ciprofloxacin). Antibacterial agents are used to killing the contaminating bacterial species. If the sample is taken from a sterile site, it is not necessary to use media containing antibacterial agents.
The standard temperature for incubation of fungi is 30 ºC and cultures should be incubated in a humidified environment for 21 days. They should be inspected daily for at least a week, and at least 3 times weekly thereafter. Some fungi are very slow to grow and may need longer incubation times. Different types of media used for the growth of fungi are as follows.
Brain-heart infusion (BHI) agar: It is a non-selective fungal culture medium that permits the growth of all clinically relevant fungi. It is used for the primary recovery of saprophytic and dimorphic fungi.
Czapek’s agar: It is used for the subculture of Aspergillus species for their differential diagnosis.
Inhibitory mold agar (IMA): Primary recovery of dimorphic pathogenic fungi. Saprophytic fungi and dermatophytes will not be recovered.
Mycobiotic agar: It is generally Sabouraud’s dextrose agar with cycloheximide and chloramphenicol, and is used for the primary recovery of dermatophytes.
Potato dextrose agar (PDA): It is a relatively rich medium for growing a wide range of fungi.
Sabouraud’s heart infusion (SABHI) agar: Primary recovery of saprophytic and dimorphic fungi, particularly fastidious strains.
Sabouraud’s dextrose agar (SDA): Sabouraud’s agar is sufficient for the recovery of dermatophytes from cutaneous samples and yeasts from vaginal cultures. Not recommended as a primary isolation medium because it is insufficiently rich to recover certain fastidious pathogenic species, particularly most of the dimorphic fungi. Sabouraud’s dextrose agar (2%) is most useful as a medium for the subculture of fungi recovered on enriched medium to enhance typical sporulation and provide more characteristic colony morphology.
Potato flake agar: Primary recovery of saprophytic and dimorphic fungi, particularly fastidious and slow-growing strains.
Cornmeal agar (CMA): It is used for growing a wide range of fungi, particularly members of the Fungi imperfecti; provides a good balance of mycelial growth and sporulation.
Malt extract agar (MEA): It is a good growth medium for soil fungi, isolated from wood, basidiomycetes, etc.
Potato dextrose-yeast extract agar (PDYA): This media is good for growing cultures derived from mushrooms.
Saccharomyces cells are elliptical, measuring about 6 to 8 µm. They multiply asexually through the budding process. A raised scar remains present on the cell, where a bud has formed (Fig.1). During budding, the nucleus divides by constriction and a portion of it enters the bud along with other organelles. Hyphae are absent.
The inability to utilize nitrate and the ability to ferment various carbohydrates are typical characteristics of Saccharomyces. Saccharomyces colonies grow rapidly and mature within 2 to 3 days. They are flat, smooth, moist, and cream to tannish cream in color.
There are more than thirty species of Saccharomyces. The best known is S. cerevisiae, strains of which are used in the fermentation of beer and wine and the production of bakery products. It is found in nature on ripe fruit. Grape wines are often made by spontaneous fermentation by yeasts growing on the surface of the fruit. Thus, S. cerevisiae is a yeast of great importance in fermentation. Other members of this genus include Saccharomyces bayanus, used in making wine, and Saccharomyces boulardii, used in medicine. Genetically engineered strain is also used to produce different types of proteins
There are more than 150 known species of the genus Penicillium. They occur as saprophytes in soil and decomposing organic debris.
On Sabouraud’s dextrose agar at 20 oC for 1 to 4 days, the colonies become velvety, white, and later become blue-green. Microscopic examination of these colonies shows brush like arrangement of conidia, sterigmata, and conidiophores (Fig.2). Penicillium has septate vegetative mycelia which penetrate the substrate and then produce aerial hyphae on which develop conidiophores. Conidiophores may be branched and have brushlike heads bearing spores. Clusters of sterigmata are usually in one plan and form a chain of conidia. The color of the mature fungi is useful in helping to identify species. They grow best at temperatures ranging from 15 to 30 oC.
Some species of Penicillum cause spoilage of fruits, vegetables, grains, and grasses. Penicillium species cause different infections such as mycotic keratitis, penicilliosis, otomycosis, onychomycosis, and deep infections. In otomycosis, a 5% aluminum acetate solution may be used to reduce edema and remove epithelial debris. In mycotic keratitis, topical application of amphotericin B may be used.
Many species of Penicillium are used in the ripening of cheese. Some are used in industrial fermentations for the production of antibiotics e.g. Penicillin is produced by Penicillium notatum and Penicillium chrysogenum.
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