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1. TUMKUR UNIVERSITY
DEPARTMENT OF STUDIES AND RESEARCH IN
BIOTECHNOLOGY
SEMINAR TOPIC: METHOD OF ISOLATION AND MULTIPLICATION- WET
SIEVING AND DECANTATION METHOD
PRESENTED BY:
Gowthami M
2nd Year MSc
DOSR In Biotechnology

2. CONTENTS
 INTRODUCTION
 WET SIEVING METHOD
 HISTORY
 REQUIREMENTS
 PROCEDURE
 OBSERVATION UNDER MICROSCOPE
 CONCLUSION
 REFERENCES

3. INTRODUCTION:
Arbuscular mycorrhizal fungi (AMF) represent one of the major component of soil microbiota
with a potential to not only aid in the establishment of the host plant, but also to enhance the
overall plant growth. Due to this importance we are going to isolate and culture the arbuscular
mycorrhizal fungi by several methods.
There are some methods for isolation of AMF, such as
1. Wet sieving and decantation method
2. Floatation method
3. Sucrose gradient method

4. WET SIEVING METHOD
 It is also known as wet sieving and decantation method. It was developed by Gerdemann
and Nicolson in the year 1963. Developed to isolate different size of spores.
 The soil near the root system is collected and an aqueous suspension is passed through
different sieves to collect spores of different sizes.
 This method is one of the popular technique when compare to other techniques. This
technique is used for sieving the coarse particles of the soil and retaining AMF spores and
organic particles on the sieves of different sizes.

5.  Earlier, Gerdemann(1955) devised first useful technique for extracting spores from soil. A
soil sample was suspended in four time sits volume of water, heavier particles were
allowed to settle for a few seconds, then the liquid was decanted through a sieve with 1mm
mesh. Whatever passed through this sieve was then poured through another sieve with
0.25mm mesh. Material retained by this sieve was washed and transferred to a petridish,
and the spores picked out by hand under a dissecting microscope.
 This technique slightly refined by Geredemann and Nicolson (1963) who used the
following series of sieves ; 1.0mm; 710µm; 420µm ; 250µm; 150µm; 100µm; 75µm and
45µm.
 They found that most of the desired spores fell in the 420-150µm.

6. Requirements:-
soil sample, 500ml beaker, different size of sieves and Bunsen burner
PROCEDURE:-
 Take 200ml water in 500ml beaker.
 Heat the water to 40-45ºC.
 Add 50g of soil and mix well to form a suspension.
 Allow the heavier particles to settle down.
 Decant most of the suspension through 710µm sieve to remove large organic mater and roots.
 Add 200ml of water to the suspension.
 Decant the suspension through 710µm sieve.
 Decant this through 250µm, 75µm and 45µm sieves consequently.
 Collect the residue on the 45µm sieve.
 Wash the residues well with water and collect the spore.

7. The procedure should be repeated until the upper layer of soil suspension is transparent.
Usually AM fungal spores are collected on 100µm. Some small spores are on 50µm. To collect
large spores such as Gigaspora margarita, 250µm sieve is efficient.

8. Observation of spores under dissecting microscope:-
spores collected from soil are put in a watchglass or a small petri dish, and their shape, colour
and the attachment to spores are observed. Spores should be classified intoeach spore type based
upon morphology.
Here is the microscopic images of morphology of representative genera of arbuscular mycorrhzal fungi

9. CONCLUSION:-
 The sieving method of separation is a valuable technique that allows the separation of
spores based on their size. With its wide range of applications, simplicity, and reliability,
it plays a crucial role in laboratories.
 By following proper procedures and considering the advantages and limitations, accurate
and meaningful results can be obtained through the sieving method.

10. THANK
YOU