Isolation of AM Fungi by Wet Sieving and Sucrose Gradient Methods
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I
SOLATION
OF
AM
FUNGI
BY
WET SEIVING AND SUCROSE
GRADIENT METHODS
Wet
sieving
method
•
Also
known
as wet
sieving
and
decanting
method
(Gerdemann and
Nicolson,
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.
•
The
wet
sieving and decanting
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
sieves
of
different
sizes.10
g
of
soil was
mixed
with
100ml
of water in the
500
ml
conical flask. The
soil mixture was
agitated vigorously to free the
AMF spores
from
soil
and allowed to
settle
for
15-45
minutes
and the
supernatant was decanted through
standard
sieves.
By using
a dissecting
microscope,
spores
were picked
by
means
of
pipette
or needle.
•
Earlier,
Gerdemann
(1955)
devised
the
first useful
technique
f
or
ex
t
rac
t
i
n
g
s
por
e
s
f
rom
s
oi
l
.
A
s
oil
sample
was
s
u
s
pen
d
ed in
four times
its
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 0.25
mm
mesh.
Material
retained
by
this
sieve
was
washed
and transferred to a
petridish, and the spores picked out by
hand
under
a dissecting
microscope.
•
Technique
given
by
Gerdemann
(1955)
was
slightly
refined
by
Gerdemann
and
Nicolson (1963)
who
used
the
following
series of sieves: 1.0
mm
; 710
µm;
420
µm;
250
µm;
149
µm;
105
µm;
74
µm;
and
44
µm.
•
They
found
that
most
of
the
desired
spores
fell
in the
420-
149
µm
range, and
they used
this
fraction
for
their
study.
Source:
http
s://w
ww.s
lideshare.net/NawabKhatoon/mycorrhyzae-association
Soil
sample
+
sterile
water
Hot
water
Filter
and
sieve
(
719μm
→
250μm
→
50μm
→
45μm
)
Spores
separated
from
soil
particles
Mix
with carrier
material
Use
when
required
as
biofertilizer
S
IEVING
METHOD
F
LOATATION
METHOD
Soil
sample
+
sterile
water
Separate
the
soil
particles
using
membrane
filter
Centrifuge
(
Density
gradient
centrifuge
=
at
3000rpm
for
30
min
)
Spores
separated
from
soil particles
Mix
with
carrier
material
Use
when
required
as
biofertilizer
I
SOLATION
OF
V
ESICULAR
-A
RBUSCULAR
M
YCORRHIZAL
(VAM)
SPORES
FROM
THE
SOIL
Sieving
method
Requirements
Soil
sample
500
ml
beaker
Sieves of
710
µm,
250
µm,
75
µm
and
45
µm.
Bunsen
burner
P
ROCEDURE
•
Take
200
ml
water
in
500
ml
beaker.
•
Heat
the
water
to
40-50˚
C.
•
Add
50
g
of
soil
and
mix
well
to
form
a
suspension.
•
Allow
the
heavier
particles
to
settle
down.
•
Decant
most
of
the
suspension
through
a 710 µm
sieve
to
remove
large
organic
matter
and
roots.
•
Add
200
ml
of water
to the
suspension.
•
Decant
the
suspension
through
710
µm sieve.
•
Decant
this through
250
µm,
75
µm
and
45
µm
sieves
consequently.
•
Collect
the
residue
on
the 45
µm
sieve.
•
Wash
the
residues
well with water and
collect
the
spore.
F
LOATATION
METHOD
Requirements
•
Soil
sample
•
Sucrose
solutions
(20,
40
and
60
%)
•
Blender
•
Fine
sieve
•
Centrifuge
•
Centrifuge
tube
(50
ml)
P
ROCEDURE
•
Collect
fresh
soil samples
from
the
field,
mix
them
well
and
weigh
20
g soil.
•
Transfer
the
soil
into
a
blender.
•
Blend it
at
high speed
for
1-2
minutes so that
the
spores attached
to
the
soil
particles
or
roots
may
become
free.
•
Fi
l
ter the
con
t
ents thr
o
u
g
h
a
fi
n
e
sieve and wash with
str
o
ng
stream
of
water.
•
Pour
10
ml
of
20%
sucrose
into
a
centrifuge
tube
followed
by
the
same
amount
of
40%
and
60%
sucrose
into
the
bottom
of
the
tube.
(Cont….)
•
Take
10-15
ml
of
blended
sieving
and add
onto
the
surface
of
20%
sucrose
layer.
•
Centrifuge
the
contents
for
3 minutes at 3000 rpm.
Thereafter,
remove
the debris
which accumulate at the interfaces of
20-40%
and
40-60%
of
sucrose.
•
Gently
wash
the
spores present
on
fine
sieve with
a
strong
stream
of
water
so
that
sucrose
should
be removed.
•
Collect
the
spores
and
observe
under
microscope.
S
UCROSE
GRADIENT
METHOD
•
Developed
by
Daniel
and
Skipper
(1982);
commonly
used
technique
for
AM
spore
extraction.
Requires
prior
sieving
and
decanting.
•
This
gradient
centrifugation
method
is
result
of
many
modifications,
right
from
Ohms
(1957),
Mosse
and
Jones
(1968),
Mertz
et
al.
(1979)
etc.
•
Spores
were
purified
by
re-suspending
the
sieving
in
the
40%
sucrose
solution
and
centrifugation
was
carried
out.
Centrifugation
was carried
out
at
1750
rpm
for 5
minutes. The
supernatant
was
removed
and
poured
into
the
sieves.
The
spores
that
hold
on
the
sieves
are
carefully
rinsed
with
tap
water.
The
spores
were
collected
by
using
dissecting
microscope.
•
Mertz
et
al.
(1979)
protocol
needs
a
special
mention.
•
They
used
discontinuous
sucrose
gradients
to
recover
large
number
of
spores
from
massive
soil
samples.
They decanted and
wet
sieved 18
kg of soil with cold
water,
and
found
that
most
spores
were
present in
the
425-250
µm
fraction.
•
Spores
were separated from
most
of the
remaining debris
using
discontinuous 30% (w/v) aqueous sucrose
gradients.
The sieved
material was
layered on 600
ml
water over 200
ml
sucrose in 1 L
beake
r
.
A
f
ter
se
tt
l
ing,
the
spor
es
and debris
that
col
l
ected
at
the
interface were
removed
by vacuum aspiration, rinsed in cold
water,
and centrifuged
for
1- 5
min
at 1600 X g in clinical centrifuge on a
second
gradient
(15
ml
water
over 20
ml
sucrose
in a
50
ml
tube),
the
duration
of
centrifugation
being determined by the kind and amount of
debris
present.
THANK YOU
Wet sieving is a popular technique to isolate different sizes of spores from soil samples. Developed by Gerdemann and Nicolson in 1963, this method involves passing an aqueous suspension through different sieves to collect spores of varying sizes. The process includes agitating the soil-water mixture in a conical flask, allowing it to settle, and decanting the supernatant through sieves. The isolated spores are then picked out using a dissecting microscope. The technique has been refined over the years to effectively extract AM fungi spores for further study and application as biofertilizers.
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Presentation Transcript
ISOLATION OFAM FUNGI BY WET SEIVING AND SUCROSE GRADIENT METHODS
Wet sieving method Also known as wet sieving and decanting method (Gerdemann and Nicolson, 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. The wet sieving and decanting 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 sieves of different sizes.10 g of soil was mixed with 100ml of water in the 500 ml conical flask. The soil mixture was agitated vigorously to free the AMF spores from soil and allowed to settle for 15-45 minutes and the supernatant was decanted through standard sieves. By using a dissecting microscope, spores were picked by means of pipette or needle.
Earlier, Gerdemann (1955) devised the first useful technique for extracting spores from soil. A soil sample was suspended in four times its 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 0.25 mm mesh. Material retained by this sieve was washed and transferred to a petridish, and the spores picked out by hand under a dissecting microscope. Technique given by Gerdemann (1955) was slightly refined by Gerdemann and Nicolson (1963) who used the following series of sieves: 1.0 mm ; 710 m; 420 m; 250 m; 149 m; 105 m; 74 m; and 44 m. They found that most of the desired spores fell in the 420- 149 m range, and they used this fraction for their study.
Source: https://www.slideshare.net/NawabKhatoon/mycorrhyzae-association
SIEVING METHOD Soil sample + sterile water Hot water Filter and sieve ( 719 m 250 m 50 m 45 m) Spores separated from soil particles Mix with carrier material Use when required as biofertilizer
FLOATATION METHOD Soil sample + sterile water Separate the soil particles using membrane filter Centrifuge ( Density gradient centrifuge = at 3000rpm for 30 min ) Spores separated from soil particles Mix with carrier material Use when required as biofertilizer
ISOLATIONOF VESICULAR-ARBUSCULAR MYCORRHIZAL (VAM) SPORESFROMTHESOIL Sieving method Requirements Soil sample 500 ml beaker Sieves of 710 m, 250 m, 75 m and 45 m. Bunsen burner
PROCEDURE Take 200 ml water in 500 ml beaker. Heat the water to 40-50 C. Add 50 g of soil and mix well to form a suspension. Allow the heavier particles to settle down. Decant most of the suspension through a 710 m sieve to remove large organic matter and roots. Add 200 ml of water to the suspension. Decant the suspension through 710 m sieve. Decant this through 250 m, 75 m and 45 m sieves consequently. Collect the residue on the 45 m sieve. Wash the residues well with water and collect the spore.
FLOATATIONMETHOD Requirements Soil sample Sucrose solutions (20, 40 and 60 %) Blender Fine sieve Centrifuge Centrifuge tube (50 ml)
PROCEDURE Collect fresh soil samples from the field, mix them well and weigh 20 g soil. Transfer the soil into a blender. Blend it at high speed for 1-2 minutes so that the spores attached to the soil particles or roots may become free. Filter the contents through a fine sieve and wash with strong stream of water. Pour 10 ml of 20% sucrose into a centrifuge tube followed by the same amount of 40% and 60% sucrose into the bottom of the tube. (Cont .)
Take 10-15 ml of blended sieving and add onto the surface of 20% sucrose layer. Centrifuge the contents for 3 minutes at 3000 rpm. Thereafter, remove the debris which accumulate at the interfaces of 20-40% and 40-60% of sucrose. Gently wash the spores present on fine sieve with a strong stream of water so that sucrose should be removed. Collect the spores and observe under microscope.
SUCROSEGRADIENTMETHOD Developed by Daniel and Skipper (1982); commonly used technique for AM spore extraction. Requires prior sieving and decanting. This modifications, right from Ohms (1957), Mosse and Jones (1968), Mertz et al. (1979) etc. gradient centrifugation method is result of many Spores were purified by re-suspending the sieving in the 40% sucrose solution and centrifugation was carried out. Centrifugation was carried out at 1750 rpm for 5 minutes. The supernatant was removed and poured into the sieves. The spores that hold on the sieves are carefully rinsed with tap water. The spores were collected by using dissecting microscope.
Mertz et al. (1979) protocol needs a special mention. They used discontinuous sucrose gradients to recover large number of spores from massive soil samples. They decanted and wet sieved 18 kg of soil with cold water, and found that most spores were present in the 425-250 m fraction. Spores were separated from most of the remaining debris using discontinuous 30% (w/v) aqueous sucrose gradients. The sieved material was layered on 600 ml water over 200 ml sucrose in 1 L beaker.After settling, the spores and debris that collected at the interface were removed by vacuum aspiration, rinsed in cold water, and centrifuged for 1- 5 min at 1600 X g in clinical centrifuge on a second gradient (15 ml water over 20 ml sucrose in a 50 ml tube), the duration of centrifugation being determined by the kind and amount of debris present.
