Isolation of fungi from soil sample
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1. The document describes a procedure for isolating antibiotic-producing fungi from soil samples through serial dilution and culture on various growth media. Key steps include collecting soil, making serial dilutions in test tubes, inoculating prepared potato dextrose agar plates, incubating to obtain isolated fungal cultures, and transferring cultures to broth media to produce secondary metabolites. 2. A variety of media are listed that can be used for fungal growth and isolation. Serial dilution is used to isolate viable fungal colonies from soil samples by diluting the samples across multiple test tubes before culturing. 3. Pure fungal cultures are obtained by sub-culturing isolated colonies onto fresh agar plates. These pure cultures are then used
Isolation of fungi from soil sample
- 1. 1 Amjad Khan Afridi 29 /07/ 2016 Isolation of Antibiotic ( Secondary metabolites) producing fungi from soil Purpose: To isolate secondarymetabolites (antibiotic , enzymes, proteins etc..) producing Fungi from soil samples. Fungi produced two types of metabolites 1) Primary metabolites Fungi produced Primary metabolites for their proper growth, reproduction and their properdevelopment. These are very important for their proper growth. 2) Secondary metabolites While secondary metabolites fungi producefor their own defense , to grow in the same medium and to killed other microbes in the same medium. Names of culture medium used for fungi growth: 1. Brain-heart infusion (BHI) agar 2. Czapek Yeast agar (CYS) 3. Czapek Yeast broth ( CYB) 4. Inhibitory mold agar (IMA) 5. Mycosel/Mycobiotic agar 6. Potato Dextrose Agar (PDA) 7. Sabouraud’s Heart Infusion (SABHI) agar 8. Bannerot synthetic media agar ( BSA / BSM ) 9. Potato flake agar 10. Water Agar ( WA) 11. Antibiotic Agar ( AA) 12. Acidified Cornmeal Agar (ACMA) 13. Potato Carrot Agar (PCA) 14. Malt Agar (MA) 15. Malt Extract Agar (MEA) 16. Potato Dextrose-Yeast Extract Agar (PDYA)
- 2. 2 Amjad Khan Afridi 29 /07/ 2016 Materials: 1) Gloves 2) Mask 3) Samples (Soil sample) . 4) Sterile Plastic bag 5) Specula 6) Saline solution or Distilled water 7) Potato Dextrose Agar (PDA) 8) Glass rod ( L- Form) 9) Syringe 10) Beaker 11) Flask 12) Graduated cylinder 13) Test tubes 14) Test tubes stand 15) Media plates 16) Electric balance 17) Aluminum foil 18) Cotton 19) Micro peptide 20) Burner 21) Wire loop 22) Ethanol 23) Para film 24) Autoclave 25) Incubator 26) Shaking incubator 27) LFH 28) Surgical blade 29) Permanent Marker Sample should be collectedfrom farms , because fertilizer,pesticidesfungusare presentthere. SERIAL DILUTION METHOD Serial dilution method is one of the most old and usable method which is use for the isolation of fungi as well as for the isolation of viable bacterial colony .
- 3. 3 Amjad Khan Afridi 29 /07/ 2016 In this method we collected our desire samples ( soil, slag, water, milk, food ) and make its dilution in the test tubes( Master Test Tube). We inoculated the sample from the diluted test tubes in the prepared medium plates by using Pure Plat Method or spread method and then incubated the inoculated plates in the incubator at 37 C ( for Bacterial growth) for 16 to 24 hours and 28 C (for Fungus growth) for 48 to 72 hours or one week. After the specific growth duration of the microbes ( fungi and bacterial) , we absorb the cultured plates , the growth will appear on each plates. Next we performed sub culturing method for the isolation of pure culture. After isolation of pure culture we perform gram staining method and other Biochemical test for the conformation and identification of bacterial species and also apply staining process forfungi. 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. Take 1 mal of sample solution from 1st test tube ( Master test tube) and put in the 2nd test tube. Take 1mal of sample solution from 2nd test tube and put in the 3rd test tube. Take 1 mal of sample solution from 3rd test tube and put in the 4th test tube. Take 1 mal of sample solution from 4th test tube and put in the 5th test tube. Take 1 mal of sample solution from 5th test tube and put in the 6th test tube. Take 1 mal of sample solution from 6th test tube and put in the 7th test tube. Take 1 mal of sample solution from 7th test tube and put in the 8th test tube.
- 4. 4 Amjad Khan Afridi 29 /07/ 2016 4) Prepared Potato Dextrose Agar (PDA) 5) For the purring the sample in the media plates we use SpreadMethod . 6) By using micro peptide We take .5 ml of sample solution from the desire test tube step by step and dropped onthe prepared media plates and spread through glass rod ( L-Form). 7) Allow the plates to solidified and then we replaced the inoculated media plates in the incubator at 28 C for 48 hours. 8) After 48 hours we absorb the cultured plates, the growth of fungi will be appeared on the media plates. 9) Next we perform sub culturing for the isolation of pure culture. 10) Again we prepare new fresh Potato Dextrose Agar (PDA), purring the media in the plates. Allow the plates to solidified and then we pick a single inoculum from each growth cultured plates and inoculated on each fresh Potato Dextrose Agar (PDA) plates by using syringe. 11) After inoculation replaced the plates in the incubator at 28 C for 48 to 72 hours. 12) After 48 or 72 hours a pure, clear and visible growth will be appeared on each plates. 13) Next we use these pure culture for staining as well as for production of Secondary metabolites.
- 5. 5 Amjad Khan Afridi 29 /07/ 2016 For the isolationof Secondarymetabolites follow the following steps: prepared broth media (Czapek Yeast broth) in the flasks with respect to 400 ml of distilled water. After autoclaving replaced the flasks in the LFH . Cut a piece of fungi inoculum from the growth plate along with media by using surgical blade and put in the flask having broth medium. Shacked each flasks, babbled the flasks and replaced in the shaking incubator for one week After one week or 10 days fungus spores formation will started in the flask . Demonstrated By Dr. Safeullah Prepared By Amjad Khan Afridi Date: 29/07/2016