isolation of fungi from soil sample

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Isolation of Antibiotic ( Secondary metabolites) producing fungi from soil

Purpose :

To isolate secondary metabolites (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 proper development.

These are very important for their proper growth.

2) Secondary metabolites

While secondary metabolites fungi produce for 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) agar2. Czapek Yeast agar (CYS)3. Czapek Yeast broth ( CYB)4. Inhibitory mold agar (IMA)5. Mycosel/Mycobiotic agar6. Potato Dextrose Agar (PDA)7. Sabouraud’s Heart Infusion (SABHI) agar8. Bannerot synthetic media agar ( BSA / BSM )9. Potato flake agar10. 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)

Amjad Khan Afridi 29 /07/ 2016

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Materials:

1) Gloves2) Mask3) Samples (Soil sample) .4) Sterile Plastic bag5) Specula6) Saline solution or Distilled water7) Potato Dextrose Agar (PDA)8) Glass rod ( L- Form)9) Syringe10) Beaker11) Flask12) Graduated cylinder13) Test tubes14) Test tubes stand15) Media plates16) Electric balance17) Aluminum foil18) Cotton19) Micro peptide20) Burner21) Wire loop22) Ethanol23) Para film24) Autoclave25) Incubator26) Shaking incubator27) LFH28) Surgical blade29) Permanent Marker

Sample should be collected from farms , because fertilizer , pesticides fungus are present there.

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 .


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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 for fungi.

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.





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4) Prepared Potato Dextrose Agar (PDA)5) For the purring the sample in the media plates we use Spread Method .6) By using micro peptide We take .5 ml of sample solution from the desire

test tube step by step and dropped on the 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.


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For the isolation of Secondary metabolites 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. SafeullahPrepared By Amjad Khan Afridi

Date: 29/07/ 2016


isolation of fungi from soil sample

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