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.

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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