All possible techniques for isolation of bacteria and fungi...
- 1. Isolation Techniques for fungi and bacteria MAINLY FROM PLANT SPECIMENS AND SOIL
- 2. Introduction to Fungal and bacterial Isolation Fungal and Bacterial isolation is crucial for understanding plant-pathogen interactions and soil health.This presentation explores comprehensive techniques for isolating fungi and bacteria from plant tissues and soil,highlighting their significance in agriculture and ecology.
- 3. Types of Fungal and bacterial Isolation Techniques Various techniques for fungal isolation include direct plating, dilution plating, and baiting methods. Each method has its advantages and is suited for different types of sample matrices, ensuring a comprehensive understanding of fungal and bacterial diversity.
- 4. Preparation of Artificial media: Potato Dextrose Agar media used for fungi. Materials: Peeled potatoes -200g, Dextrose - 20 g. Agar - 20 g, distilled water 1 L, beaker 1L,knife, muslin cloth, measuring cylinder, cotton. Procedure 1. Take 500 ml of distilled water in IL beaker and add 200g of peeled and sliced potato, boil the potatoes till they become soft. 2. Filter the contents of the beaker through muslin cloth and squeeze out all liquid. 3. Add the dextrose dissolved in water to this extract. 4. Adjust the pH of medium to 6 to 6.5. 5. Add the dissolved agar to dextrose-potato extract and make the volume to 1 liter Nutrient Agar media used for bacteria Materials: Beef extract – 3g,Peptone – 5g,Agar – 20g, distilled water 1 L, beaker 1L,knife, muslin cloth, measuring cylinder, cotton. Procedure 1. Weight all these materials with weighing balance . 2. Filter the contents of the beaker through muslin cloth and squeeze out all liquid 3. Add the dextrose dissolved in water to this extract. 4. Add all weighted materials dissolved in water and make volume of 1 liter. . 5. Adjust the pH of medium to 6 to 6.5
- 5. Sterilization Autoclave All instruments like petri dish,inoculating needle and loop,foreceps should be wraped with news paper flask containing PDA and Nutrient agar in an autoclave at 121°C, 15psi pressure for 20 minutes. • After 20 minutes allow the autoclave to cool down.Remove the conical flask and store at room temperature. Allow the flask to cool up to 45°C or until the flask can be held by hand.
- 6. Isolation involves the following steps: Surface disinfection of sample Materials: Infected plant part (leaves, stem, roots, fruits), sterile Petri-dishes, PDA slants, sodium hypochlorite solution (1 %),ethanol, sterile water, razor blade, forceps, inoculation needle, burner/spirit lamp, spirit, incubator, PDA medium. Procedure 1. Select infected host tissue from the advancing margin of the lesions. 2. Cut into small pieces (2-5 mm) containing both the diseased and healthy tissue and keep in sterile Petri dishes. 3. Dip the pieces into 1 % sodium hypochlorite solution for about one minute. 4. Transfer the pieces to Petri - dishes containing sterile distilled water and wash thoroughly in 2-3 changes of sterile water to free them from the chemicals. 5. Plant parts/sections are taken out aseptically and blot dry on clean, sterile filter paper.
- 7. Isolation of plant pathogenic fungi & bacteria from diseased plant materials: Pouring Add any antibiotic only in PDA flask and mix thoroughly.Lift the lid of Petri dish gently with left hand and pour about 1/3ml of medium.Each petri dish is of 37ml.Close the mouth of the flask with plug near the flame. Inaculation 1. After solidification of the media,place sample on the center of media and slightly press the sample. 2. Cover the petri plate and wrape it with tape and label it with date. 3. Incubate the plate at required conditiods. 4. The pathogen will grow from the sections and the colonies of the pathogen are sub cultured aseptically for further study.
- 8. Isolation of plant pathogenic fungi & bacteria from soil & water: Serial dilution method Serial dilution method is one of the most old and useable method which is use for the isolation of fungi as well as for the isolation of viable bacterial colony.In this method we collected our desire samples (soil, slag, water, milk, food) and make its dilution in the test tubes ( Master Test Tube). Procedure: 1. Dig the earth surface 10 to 12 cm deep and Collect soil sample bellow 12cm. 2. Take 8 test tubes and take 10 ml of distilled water in 1st test tube and then take 9 ml distilled water in each the remaining test tubes and labeled each test tubes. 3. Take 1 gram of soil Sample from the collected soil sample and make a solution in the 1st test tubes ( Master Test Tube) which having 10 ml of distilled water. Distribute the sample solution from the 1st test tube ( Master test tube) in the remaining test tubes. 4. Prepare dilutions by transferring 1ml from previous dilutions to 9ml distilled water.
- 9. Common isolation techniques 1. Streak plate method: Streaking is the process of spreading the microbial culture with an inoculating needle on the surface of the media. Sterilize the inoculating needle by flame to make red hot and allow it to cool for 30 seconds. The sample is streaking such away to provide series of dilution. Purpose-thin out inoculum to get separate colonies. Subculturing can be done by streaking well isolated colonies from streak plate to new plate.1
- 10. Continue… 2. Pour plate method The bacterial culture and liquid agar medium are mixed together. After mixing the medium, the medium containing the culture poured into sterilized petri dishes (petri plates) allowed solidifying and then incubated. After incubation colonies appear on the surface.
- 11. Continue… 3. Spread plate method This is the best method to isolate the pure colonies. In this technique, the culture is not mixed with the agar medium. Insteadit is mixed with normal saline and serially diluted 0.1 ml of sampletaken from diluted mixture, which is placed on the surface of the agarplate and spread evenly over the surface by using L-shaped glass rod called spreader. Incubatethe plates. After incubation, colonies are observed on the agar surface.
- 12. Baiting Techniques Baiting techniques involve using specific substrates (seed,piece of carrot,potato,onion)to attract fungi from the environment. This method is particularly useful for isolating specific substrates to attract fungi from the environment. This method is particularly useful for isolating fastidious fungi that may not grow well on standard media, enhancing diversity recovery. Fill Petri dishes with 10-20 mL of sterile water. Add 5-10 seeds to each dish. Seal dishes with parafilm or plastic wrap. Incubate at 20-25°C for 7-14 days. Observe seeds for fungal growth.
- 13. Presented by: 1). Dua Akram 2). Zeshan Bilal 3). Humayun Hassan 4). Roshan Hafeez 5). Muhammad Jahangir 6). Muhammad Ayyaz THANK YOU