Fungal Culture PDF: Fungal Culture Media
Discover the secrets of fungal culture with our comprehensive article! Learn about Fungal Culture PDF notes and the vital role of Fungal Culture Media in studying these intriguing organisms. Unravel the complexities of mixed populations and their contributions to ecology as we delve into the process of obtaining single colonies for further study. Explore the fascinating world of fungi through various characterization techniques applied to large homogenous cultures. Don’t miss out on this enriching journey into the realm of fungal culture.
German Botanist Gottileb Haberlandt (1902) developed the concept of in vitro cell culture. He tried to isolate fully differentiated cells on a nutrient medium containing glucose, peptone, and Knop’s salt solution. Culture techniques can be employed in a range of organisms like bacteria, fungi, algae and plants like cereals, legumes, grasses, medicinal plants, vegetable crops, oil-yielding plants, and fruit crops.
FUNGAL CULTURE
Plant diseases can be identified by observation with the naked eye or with a microscope and under such conditions isolation of pathogens is not necessary. If the pathogen is mixed with one or more contaminants and is not producing fruiting bodies or if the disease is caused by a new unknown pathogen the isolation of the pathogen is necessary.
What is fungal culture?
Fungal culture refers to the process of growing and maintaining fungi in controlled laboratory conditions. It involves isolating and cultivating specific fungal species for various purposes, such as research, commercial production, or medical applications. Fungal cultures are typically grown on solid or liquid media, providing a suitable environment for their growth and reproduction.
Process of fungal culture
The process of fungal culture involves several key steps to ensure the successful cultivation and maintenance of fungal species.
Isolation and Selection of Fungi
Fungi can be isolated from various sources, such as soil, plants, or clinical samples. Once isolated, the fungi are carefully selected based on specific characteristics or desired properties. Pure cultures are obtained by transferring individual fungal spores or hyphal fragments onto culture media.
There are different methods of isolation of fungi which are as follows:
1. Isolation of fungi from soil – The method involves the transfer of microbial organisms from the soil to the culture medium to obtain a pure culture. Growth requirements for soil microbes are different and various procedures are developed.
- The soil dilution plate method- It is the classical and most widely used method in soil microbiology and has been adopted from the method which was originally developed for the general isolation of soil bacteria. 10g air-dried soil mixed with 90 ml sterile water and mixed with a magnetic stirrer for 20 to 30 minutes. From the solution, 10ml soil water was taken and added to 90ml sterile water and mixed for 1 minute. from this solution again 10ml suspension was transferred to 90ml sterile water. The procedure is repeated until the desired dilution is obtained. A proper dilution allows 50 to 150 colonies per culture plate.
- Soil plate method- This method is given by Warcup (1950). It is a less complicated method by which more kinds of fungi are obtained. A small amount of soil (0.005 g to 0.15 g) was taken from the soil sample using a sterile needle with a flattened tip. Each sub-sample is placed in sterile water and mixed in Petri plate. 8 to 10 ml molten agar medium is added to each Petri plate and the plate is rotated gently for dispersion of soil particles in a nutrient medium.
- The direct inoculation and soil desiccation method – The lumps of soil having diameter of 1 cm are transferred on the sterile plates containing Czapek’s solution agar. Plates were incubated for 24 hours and hyphal tips are transferred on slant for further growth. Microorganism may be from mycelium which actually found in soil.
2. Isolation by baiting- Microorganisms may be isolated from soil by the use of preferential substrate. Desired organism is able to develop by the exclusion of other organisms.
3. Isolation of pathogen from diseased plant tissues- In this method the tissues which are in active stage of infection are used . They have only those pathogens responsible for infection. Surfaces of tissues are contaminated with saprophytic organisms. The surface disinfestation of tissues which are selected for isolation is essential for successful isolation.
4. Isolation of plant-pathogenic fungi by the tissue-planting method – First the diseased tissue showing symptoms is washed, placed in beaker and covered with cheese cloth and left in running water for 1 to 2hours. Squares of these tissues (0.5cm2) are cut from the advancing margins of lesions and placed in 0.5 to 1.0% Calcium hypochlorite for 3 to 5 minutes, small pieces of tissues are lifted with alcohol flamed forcep and placed on hardened agar medium in Petri plates. The predominated organisms then transferred to fresh agar slants for growth in pure culture.
Fungi which sporulate on diseased tissues are isolated by a dilution method. Bits of diseased tissues are chopped with a flamed scalpel in a few drops of sterilized water and after few minutes a loopful of water which contains fungal spores is transferred to a drop of sterilized water in Petri dish. The serial dilutions are made in several other Petri dishes. Melted agar medium is poured into dishes. Colonies are visible after one day. Single colony can be transferred to
medium in tubes or in other Petri dishes.
Inoculation and Incubation
The isolated fungi are then inoculated onto suitable culture media and placed in controlled incubation conditions. Temperature, humidity, and light conditions are optimized to promote fungal growth. Over time, the fungi multiply and form visible colonies on the culture media.
Maintenance and Preservation
To ensure the long-term viability of fungal cultures, proper maintenance and preservation techniques are employed. Fungi can be stored as freeze-dried spores, in liquid nitrogen, or as actively growing cultures on agar slants. These preservation methods allow for the storage and distribution of fungal cultures for future use.
FUNGAL CULTURE MEDIA
Fungal culture media are nutrient-rich substances used to support the growth, reproduction, and maintenance of fungi in laboratory conditions. These media provide essential nutrients, such as carbohydrates, proteins, vitamins, and minerals, required for fungal metabolism and development. Fungal culture media can be solid (agar-based) or liquid, depending on the specific requirements of the fungi being cultivated.
Fungal culture media can be categorized into various types based on their composition and purpose. Here are some commonly used types of fungal culture media:
Natural Media
Natural media are composed of complex natural materials of unknown composition. Natural substances such as plant parts, malt, yeast, peptone, fruits and vegetables.
- Cooked vegetable agar- Broth of vegetative plant parts is prepared by steaming 10-20% of the tissue in water for 30 minutes. The contents are mashed and squeezed through muslin cloth. Agar (2%) is added and pH is adjusted before autoclaving.
- Lima bean agar- This media is used for Phytophthora species. 50 g of lima bean is blended with 500 ml water which contains 15-20 g melted agar. Final volume is adjusted to 1 liter and autoclaved.
- Oatmeal agar- 60 g of rolled oats blended in 600 ml of water and heated to 45 to 550C. 15-20 g agar dissolved in 400 ml of water and all this autoclaved for 30 minutes.
Semi-Synthetic Media
Semi-synthetic media are made up of natural substances of unknown composition and by adding come chemical compounds of known composition.
- Potato-dextrose agar- 200 g boiled potatoes are peeled and sliced in 1 liter of water until become soft. Mixture is strained through cheese cloth and volume of filtrate is adjusted to 1 liter. 20 g Dextrose and 20 g agar are added and autoclaved.
- Malt extract peptone dextrose agar- It contains 20 g Malt extract, 20 g Dextrose, 1 g Peptone and 20 g Agar and 1000 ml of distilled water.
- Martin-Rose Bengal Streptomycin agar- Contains 10 g Dextrose, 5 g Peptone, 1 g Potassium phosphate, 0.5 g Magnesium Sulphate, 0.05 g Rose Bengal, 0.03 g Streptomycin, 20 g Agar and 1000 ml of Distilled water.
- Nutrient Agar- Contains 3 g Beef extract, 5 g Peptone, 8 g NaCl and 1000 ml Distilled water.
- Soil- extract agar- Soil extract is prepared by autoclaving 500 g soil in water. After cooling it is filtered through filter paper. 0.2 g Potassium phosphate and 20 g Agar are added to 1 liter soil extract. pH is adjusted to 6.8 and autoclaved.
- Yeast extract medium- Contains 1 g Yeast extract, 10 g Potassium phosphate, 1 g Magnesium sulphate, 0.1% Tween 80 and 1000 ml distilled water.
Synthetic Media
Synthetic media are those media in which composition and concentration of each compound is known. Compounds which are used should be chemically pure.
- Brown’s medium- Contains 2 g Glucose, 1.25 g Potassium phosphate, 2 g Asparagine, 0.75 g Magnesium sulphate, 20 g Agar and 1000 ml distilled water.
- Czapek-Dox Agar- Contains 2 g Sodium nitrate, 1 g Potassium phosphate, 0.5 g Magnesium sulphate, 0.5 g Potassium chloride, 0.01 g Ferrous sulphate, 30 g Sucrose, 20 g Agar and 1000 ml distilled water. The medium is autoclaved.
- Galactose-nitrate agar- Contains 10 g Galactose, 2 g Sodium nitrate, 1 g Potassium phosphate, 0.5 g Magnesium sulphate, 0.3 g Potassium metabisulfite, 20 g Agar and 1000 ml distilled water. The medium is autoclaved for 20 minutes.
- V-8 juice agar – It is used to support growth and induce sporulation of many fungi. It can be used in place of PDA.
Composition: V-8 juice-200ml, Calcium carbonate- 3g, Agar-15- 20 g, Distilled water- 1000ml, pH-7.0 to 7.5 (pH can be changed by changing the amount of juice or Calcium carbonate.) - Cassava-dextrose agar- This media is used instead of PDA for isolation and culture of fungi. Peel of Cassava (Manihot utilissima) tubers is removed and it cuts into chips. It is dried over night at 550C and crushed to make powder of 30 mesh. 135 g powder is soaked in 500 ml water for 15 minutes at 600C. It is filtered through cheese cloth. 20 g Glucose and 12 g Agar is added and final volume is adjusted to 1000ml It is autoclaved and pH 5.6 is adjusted.
The composition of fungal culture media can vary depending on the specific fungal species being cultured and the objectives of the experiment or study. Researchers and microbiologists often modify or develop their own media formulations based on the requirements of their research or diagnostic needs.
Can the same fungal culture media be used for all types of fungi?
Different fungi have varying nutritional requirements, and specific culture media are designed to cater to these requirements.
Why is fungal culture important?
Fungal culture allows for the isolation, growth, and study of different fungal species. It is crucial for various purposes, including research, diagnostics, industrial applications, and the development of antifungal treatments.
What is the process of fungal culture?
Fungal culture involves collecting a sample containing fungal spores or mycelium, inoculating it onto a suitable culture medium, providing optimal growth conditions (such as temperature and humidity), and allowing the fungi to grow and form colonies. The colonies can then be studied and further analyzed.
What is the purpose of using agar in fungal culture?
Agar is a solidifying agent commonly used in fungal culture media. It provides a solid surface for fungal growth, allowing the observation and isolation of individual colonies. Agar also provides stability and prevents the diffusion of nutrients, making it an ideal medium for fungal culture.
What are some common media used for fungal culture?
Basic Nutrient Agar, Sabouraud Dextrose Agar, Potato Dextrose Agar, and Yeast Extract Agar are some commonly used media for fungal culture. These media provide the necessary nutrients and growth factors required for the growth of a wide range of fungi.
How do you identify fungi grown in culture?
Fungi can be identified based on their colony morphology, color, texture, and other characteristics observed on culture media. Microscopic examination of fungal structures, such as spores or hyphae, is also crucial for identification. Additionally, biochemical tests, DNA sequencing, and other specialized techniques may be employed for accurate identification.
What are the applications of fungal culture?
Fungal culture has numerous applications. It is used in medical and veterinary diagnostics to identify pathogenic fungi, in industrial settings for the production of enzymes, antibiotics, and other bioactive compounds, in agriculture for the development of biocontrol agents, and in research for studying fungal biology, ecology, and interactions with other organisms.
Can fungal cultures be preserved for future use?
Yes, fungal cultures can be preserved for long-term storage and future use. Common methods of preservation include freezing at low temperatures (-80°C), lyophilization (freeze-drying), and storing in specialized preservation solutions. Preserving fungal cultures ensures their availability for further study, research, and reference purposes.
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