6. Again add another portion of phosphate reagent no.

2 and is mixed as before and

observe the color and match with the color chart.
7. Deep blue color indicates the abundant phosphate but light green color indicates
smaller amount of phosphate.

III. Potash test for plants

1. ½ teaspoonful of finely cut plant material is taken in a vial.

2. Fill up to the (10cc) with potash reagent no. 1.
3. Shake the vial vigorously for 1 minute.
4. Add 5 cc of (to the upper mark) of potash reagent no. 3 with pipette carefully.
5. Mix the contents and after 3-5 minutes note the color developed.
6. Compare the color of the vial with the potash determining color chart.

Advantages:
1. Rapid, simple and easy
2. Anyone can apply it anywhere
3. Cheaper and less time consuming
4. Portable
5. Does not require any heavy instrument

Disadvantages:
1. It gives approximate result, so that result is not accurate.

DETERMINATION OF TOTAL NITROGEN IN SOIL BY SEMI-MICRO

KJHELDAHL METHOD

Introduction
The total N content of soils ranges from <0.02% in subsoils to >2.5% in peats; the surface
layer of most cultivated soils contains between 0.06% and 0.05% N.
Two methods have gained general acceptance for determination of total N: the kjeldahl
(1383) method, which is essentially a wet oxidation procedure and the Dumas (1831) method,
which is fundamentally a dry oxidation (i.e., combustion) technique.
In the kjeldahl method, organic N in the sample under analysis in converted to NH +4 – N
by digestion with concentrated H2SO4 containing substances that promote this conversion, and
the NH+4.N is determined from the amount of NH3 liberated by distillation of the digest with
alkali. In the original kjeldahl procedure, H2SO4 alone was employed for digestion, KMnO4
being used to complete the oxidation of organic matter. However, numerous investigation have
shown that both the speed and the completeness of conversion of organic N to NH+4.N by
digestion with H2SO4 can be increased by adding soils to raise the temperature of digestion or
by adding catalysts to promote oxidation of organic matter. In practically all kjeldahl methods
now employed, K2SO4 is used to raise the temperature of digestion, and catalysts such as Se,
Hg or Cu are used to promote oxidation of organic matter.

Reagents:
1. Sulphuric acid (H2SO4) – concentrated
COPY BY: MD.MAHDI HASAN, BSc IN AGRICULTURE, SYLHET AGRICULTURAL UNIVERSITY

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 2. Sodium hydroxide (NaOH) 10 N (approx.): Place 4.2 kg of NaOH in a heavy
walled 10 liter flask; and 4 liters of water and swirl the flask until the alkali is
dissolved. Cool the solution with a rubber stopper in the neck of the flask, allow it
to stand for several days. Siphon the clear supernatant liquid into a large Pyrex
bottle which contains about 1.5 liters of CO2 free water and marked to indicate a
volume of 10 liters and make the solution to 10 liters by addition of CO2 free water.
3. Boric acid indicator solution: Place 0 gm of pure boric acid (H3BO2) in a 5 liter
flask marked to indicate a volume of 4 liter. Add about 3800 ml of water and heat
and swirl the flask until the H3BO3 is dissolved. Cool the solution and add 80 ml of
mixed indicator solution prepare by dissolving 0.099 gm of Bromocresol green and
0.065 gm of methyl red in 100 ml of ethanol. Then add 0.1 N NaOH continuously
until the solution assumes a reddish purple tint (pH-5.0) and volume to 4 liter by
addition of water. Mix the solution thoroughly before use.

4. Potassium Sulphate Catalyst mixture: Prepare an intimate mixture of 100 g of

K2SO4, 10 gm of copper sulphate (CuSO4.H2O) and 1 gm of selenium powder.
Prepare the reagents separately before mixing and grind the mixture in a mortar to
powder.

Procedure:
Take 1 gm soil sample in a microkjeldahl flask, add 2 ml of H 2O2 and after swirling the
flask for a few minutes allow it to stand for a further 30 minutes. Then add 1.1 gm of K 2SO4
catalyst mixture, add 3 ml of the concentration Sulphuric acid and heat the flask continuously
on the digestion stand, when the water has been removed and frothing has ceased, increase the
heat until the contents become colorless. Regulate the beating during this boiling, so that the
H2SO4 condenses to about 1/3 of the way up the neck of the digestion flask.
After completion of digestion, allow the flask to cool and add about 20 ml of water (slowly
and with shaking). Then swirl the flask to bring any insoluble material into suspension and
transfer the contents to the distillation chamber of the Hoskins apparatus through the funnel of
the apparatus. Rinse the kjeldahl flask 3 times with about 9 ml of water to complete the transfer.
Add enough water to the distillation chamber to bring to bring the level of the solution to mark
made previously to indicate a volume of 50 ml and close the cock connection of the funnel of
distillation chamber. Add 10 ml of H3BO3 indicator solution to a 50 ml Erlenmeyer flask which
is marked to indicate a volume of 35 ml and place the flask under the condenser of the
distillation apparatus so that the end of the condenser is about 4 cm above the surface of the
H3BO3. Then add 20 ml of 10N- NaOH to the funnel of the apparatus and run the alkali slowly
into the distillation chamber by opening the funnel stop cork. When about 1 ml of the alkali
remains in the funnel, rinse the funnel rapidly with about 15 ml of water and after allowing
enough of this water to run into the distillation chamber to bring the level of the solution to a
mark made previously to indicate a volume of 80 ml, close the funnel stop cork and
immediately commence distillation by closing the steam jacked of the distillation chamber.
When the distillation reaches the 35 ml mark on the receiver flask, stop the distillation by
opening the steam by pass tube, rains the end of the condenser and determine NH4-N in the
distillation by titration with 0.01N H2SO4 using a 10 ml burette gradually at 0.01 ml interval.
The color change at the end point from green to pink.

(volume of 0.01N H2SO4 required in titration- blank) × 0.00014 × 100

%N =
Weight of soil taken

COPY BY: MD.MAHDI HASAN, BSc IN AGRICULTURE, SYLHET AGRICULTURAL UNIVERSITY

15
 DETERMINATION OF AVAILABLE PHOSPHORUS IN SOIL BY OLSEN
METHOD

Introduction:
There are many methods for determining the soil phosphorus that vary in principle and
technical details. The selection of suitable method requires a clear understanding of objectives
that necessitate the measurement of soil phosphorus. Other considerations are the properties of
the soil involved, accuracy and reproducibility needed, and the facilities and personnel
available.
Most soil phosphorus determinations have two distinct phase-first the preparation of a
solution containing the soil P or fraction thereof, and second, the quantitative determination of
the P in this solution. The choice of a colorimetric method for determining P depends on the
concentration of P in the solution, the concentration of interfering substances in the solution to
be analyzed, and the particular acid system involved in the analytical procedure.
The molybdenum blue methods are the most sensitive and as a result are widely used for
soil extracts containing small amounts of P as well as for total P in the soils. These methods
are based on the principle that in an acid molybdate solution containing orthophosphate ions, a
phosphomolybdate complex forms that can be reduced by stannous chloride and other reducing
agents to a molybdenum blue color. The intensity of the blue color is determined by
colorimeter.

Principles:
Phosphorus is extracted from the soil with 0.5 M NaHCO 3 at a nearly constant pH 8.5. In
calcareous, alkaline or neutral soils containing calcium phosphates, this extractant decrease the
concentration of Ca in solution by causing precipitation of Ca as CaCO 3. As a result, the
concentration of P in solution increases. In acid soils containing Al and Fe phosphates such as
variscite and srengite, P concentration in solution increases as the pH rises. Secondary
precipitation reactions in acid and calcareous soils are reduces to a minimum because the
concentration of Al, Ca, and Fe remains at a low level in this extractant.
Apparatus required:
1. Erlenmeyer flask (125ml)
2. Volumetric flask (50ml)
3. Funnel, filter paper (whatman No. 40)
4. Colorimeter and
5. Pipette.
Reagents required:
1. 0.5 M sodium bicarbonate solution: Dissolve 42 gm of NaHCO3 in distilled water,
make the volume to about 900 ml. adjust the pH of this solution to 8.5 with 1 M
NaOH and volume to 1 liter with water. Add mineral oil to avoid exposure of the
solution before use if it has been standing over 1 month in a glass container.
2. Carbon black: Use carbon black G. or activated charcoal.
3. Ammonium molybdate (NH4)6 MO7 O24 4H2O solution: Dissolve 15 g of (NH4)6
MO7 O24 4H2O in 300 ml. of warm distilled water. Filter the mixture if necessary,
and allow it to cool. Then add 342 ml of conc. HCl gradually with mixing. Dilute
the contents to 1000 ml with distilled water.

COPY BY: MD.MAHDI HASAN, BSc IN AGRICULTURE, SYLHET AGRICULTURAL UNIVERSITY

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 4. Stannous chloride (SnCl2.2H2O), concentrated solution: Dissolve 10gm of
SnCl2.2H2O in 25 ml of conc. HCl. Prepare a fresh solution every 2 months or less.
Store the solution in a refrigerator.
5. Stannous chloride, dilute solution: Add 0.5 ml of conc. SnCl2 solution to 66 ml
of distilled water. Prepare the dilute solution for each set of determinations.
6. Standard P solution: Weigh 0.4393 gm of monobasic potassium phosphate
(KH2PO4) into a 1 liter volumetric flask. Add 500 ml of distilled water, and shake
the contents until the salt dissolves. Dilute the solution to 1 liter with distilled water.
Add 5 drops of toluene to diminish microbial activity. This solution contains 0.1
mg of P per ml.
7. Dilute phosphorus solution: Dilute 20 ml of the standard P solution to 1 liter with
distilled water. This solution contains 2 µg of P per ml.

Procedure:
Add 5 gm of soil, 1 teaspoon of carbon black, and 100 ml of the extracting solution to a
250 ml Erlenmeyer flask. Shake the flask for 30 minutes with a suitable shaker. Filter the
suspension through the whatman no. 40 filter paper.
Place a 10 ml aliquot of the extract in a 50 ml volumetric flask. Slowly add 10 ml of the
ammonium molybdate solution into the flask. After rapid evolution of CO2 has ceased, shake
the flask gently to mix the contents. Wash down the neck of the flask with distilled water to
avoid direct contact of the SnCl2 solution with the conc. ammonium molybdate solution. Dilute
the contents to about 44 ml with distilled water. Add 2 ml of dilute SnCl2 solution. Make to
volume with water and mix the contents immediately. Measure the absorbance of the solution
in the colorimeter 10 minutes after addition of the SnCl2 solution. Use 660 mµ incident light in
the colorimeter.
Prepare a standard curve as follows: pipette aliquots of the dilute P solution containing from
2 to 25 µg. of P (1, 2.5, 5, 7.5 and 10 ml of dilute P solution) into 50 ml volumetric flasks and
10 ml of the NaHCO3 extracting solution to each flask. Develop the color as stated above. Plot
the per cent absorbance against P concentration on a graph paper. Express the results as P in
ppm of soil.

Comments:
1. Possible source of variation in the results in this method are associated with the
temperature of the extracting solution and the shaking speed.
2. The extractable P increased approximately by 0.43 ppm P of soil for each degree rise
in temperature between 20 and 30, for testing between 5 and 40 ppm of P.
3. Higher results will be obtained when the speed increase greatly.

COPY BY: MD.MAHDI HASAN, BSc IN AGRICULTURE, SYLHET AGRICULTURAL UNIVERSITY

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 DETERMINATION OF AVAILABLE POTASSIUM IN SOIL BY AMMONIUM
ACETATE EXTRACTION METHOD

Probably the most universally employed index to K availability in soil is the sum of the
exchangeable and water-soluble K, i.e.; the total K extracted by neutral, 1 N NH 4OAc.
As an index to K availability for rapidly growing annual and perennial agronomic and
vegetable crops. The exchangeable K is perhaps best expressed as a quantity, such as ppm,
pounds per 2000,000 pounds, or meq. per 100 gm. However , with some other crops, such as
tree crops, the exchangeable K is perhaps best calculated as a percentage of the cation exchange
capacity.
Reagents:
1. Hydrochloric acid (HCl), 2 N
2. Hydrochloric acid (HCl), 0.1 N
3. Ammonium acetate (NH4OAc), 1 N: adjusted to pH 7.0 for each liter of solution
desired, add 58 ml of glacial acetic acid to about 600 ml of water and then add 70 ml
of concentrated NH4OH, specific gravity 0.90. The NH4OH is best added under a fume
hood through a long-stemmed glass funnel so that it is introduced into the bottom of
the acid or NH4OH using a pH meter or bromothymol blue indicator. Dilute the solution
to volume, mix it and store it in a pyrex-bottle.
4. Standard potassium solution: Dissolve 0.9533 g of dried KCl in water, dilute the
solution of 500 ml and mix it. This solution contains 1000 ppm K.
Procedure:
Place 10 g of soil (use 5 g if the soil contains 500 ppm extractable K) in a 50 ml centrifuge
tube. Add 25 ml of NH4OAc, and shake the tube for 10 minutes. Centrifuge the tube until the
supernatant liquid is clear. Decant the supernatant liquid into a 100 ml volumetric flask. Make
three additional extractions in the same manner. Dilute the combined extracts to 100 ml with
NH4OAc. Mix the solution, and determine K on a flame photometer by comparing the emission
with standard made in NH4OAc containing 2, 4, 6, 8 and 10 ppm K. the NH4OAc extract can
be out directly into most flame photometers if care is taken to avoid solid particle that may clod
the atomizer. If solid particles appear in the extract, filter a small portion of the extract using
whatman No. 40 or equivalent quality filer paper before putting the solution into the flame
photometer.
Precaution:
Do not use soil that has been oven-dried. If water content is to be determined, use a separate
sample for this.
In most soils the exchangeable K changes with drying. When the exchangeable K is
relatively high following applications of K fertilizers, there is likely to be fixation or a decrease
with drying, and when a given soil is relatively low in exchangeable K there is usually a release
of K from non-exchangeable forms during drying. Thus, if an accurate estimate of
exchangeable K is to be made for determining the water content, s that the results can be
calculated air-dry basis or oven-dry basis.

COPY BY: MD.MAHDI HASAN, BSc IN AGRICULTURE, SYLHET AGRICULTURAL UNIVERSITY

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 DETERMINATION OF AVAILABLE SULPHUR IN SOIL BY CALCIUM
CHLORIDE EXTRACTION METHOD

Introduction:
The specific forms of sulphur (S) that are available to plants are incomplete understood.
Although it has been demonstrated in nutrient cultures that inorganic S compounds may be
utilized by plants, it is generally accepted that most of the S in the soils absorbed by plants as
SO42-. However both organic S and insoluble inorganic S in soils may contribute to plant
nutrition depending on the soils particulars cropping conditions. It is generally accepted that
organic S becomes available to plants primarily through microbial conversion to SO42-. Since
sulphate is readily available to plants, it represents S supply for immediate use and particularly
important for seeding plants. S deficiency symptoms are frequently observed in early growth
stage of crops.
Reagents:
1. Extracting solutions: 1.5 g CaCl2 was dissolved in 1 L distilled water.
2. Acid seed solution: 6 N HCl containing 20 ppm S as K2SO4.
3. Barium chloride (BaCl2.2H2O): 20-60 mesh crystals.
4. Whatman no. 42 filter paper: If the paper is not free of sulphate, it should be washed
with extracting solution.
5. Potassium sulphate, reagent grade.
Procedure:
Shake 10 of 20 mesh soils with 50 ml extracting solution in a 100 ml Erlenmeyer flask for
30 minutes. Filter the soil suspension on a sulphate free whatman no. 42 filter paper. Pipette
10 ml filtrate to a 50 ml Erlenmeyer flask and then add 1 ml of acid seed solution. The solution
should be swirled and 0.5 g of BaCl2 crystals should be added. Allow the mixture to stand for
1 minute and the solution should be swirled in the flask frequently until the crystals were
dissolved. The light absorbance of a suitable colorimeter at a wave length of 420 mµ should be
read after the crystals get dissolved and the concentration of sulphate (SO42-) should find out
from the curve.
Preparation of a standard curve:
To prepare a calibration curve the following procedures should be maintained. Dissolve
0.5424 g of reagent grade K2SO4 in extracting solution in a 1 L volumetric flask. The resulting
solution will contain 100 ppm sulphate. Prepare different standard solution such as 0, 2, 5, 10,
15, 20 ppm S by using 100 ppm stock solution. For this operation the volumetric flasks of a
least 25 ml size can be used.
Pipette 10 ml of each solution into a separate 50 ml Erlenmeyer flask. Add 1 ml of seed
solution. Allow the flask to stand for 1 minute and then swirl the contents of the flask frequently
until the crystals get dissolved. The absorbance should read within 2 to 8 minutes on a
colorimeter at a wave length of 420 mµ. Plot percent light absorbance for different respective
concentration of S of the standard solutions on a graph paper.

COPY BY: MD.MAHDI HASAN, BSc IN AGRICULTURE, SYLHET AGRICULTURAL UNIVERSITY

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 ISOLATION AND IDENTIFICATION OF AZOTOBACTER

For many years, Azotobacter spp. have been considered to be important in the nitrogen
economy of field soils. Such evaluation has been based largely on extrapolation to field
conditions of their nitrogen-fixing proficiency in laboratory culture.
Effective culture of Azotobacter is based on their ability to grow on carbohydrate-rich
substances containing no added nitrogen. This deficiency does not restrict growth of
Azotobacter in as much as they are able to us atmospheric nitrogen, but it does restrict the
growth of commonly occurring heterotrophic bacteria unable to use atmospheric nitrogen. The
fact that Azotobacter colonies develop rapidly and present a rather characteristic appearance
also facilitates the determination of this group in soil.

Materials required:
1. Mineral – salt solution: Add 5 g K2HO4; 2 g MgSo4.1H2O; 1 g CaSO4; 0.2 g MnSO4;
0.1 g of MoO3.H2O; and 0.1 g KI to 1000 ml of distilled water, sterilize the solution by
autoclaving at 15 pounds pressure for 15 minutes.
2. Sterile petridishes, pipettes, glass microscope slides, small spatula etc.

Procedure: Soil plaque method or mud pie method

Air dry the soil to be examined, and pulverize it to pass through a 20 mesh screen.
Determine the soil pH. If the p/h is less than 6.8 mix 5 g of precipitated CaCO 3 with 50 g of
soil. Place 50 g of soil in a glass beaker and mix 2.5 g of powdered cornstarch. Add slowly and
with stirring, sufficient mineral – salt solution to form a plastic mass with the consistency of
modeling clay. Transfer the wetted soil to the bottom half of a petridish, and spread it carefully
into a mud pie, leaving the top slightly convex. Smooth the surface with a sterile glass
microscope slide or small spatula moistened in sterile water.
Incubate the plaque, uninverted, in of an incubator for 3 to 4 days at 30˚C. Then examine
the surface of the plaque for raised, moist, glistening, circular colonies, easily visible to the
naked – eyes.

Identification: The need of microscopic examination of colonies from the plaques is

imperative for those unfamiliar with Azotobacter. Colonies of Azotobacter are flat on top,
sometimes slightly depressed in the center, and milky or turbid in appearance.

Azotobacter chroococcum:
Usually develops brownish or black pigmentation. Gram staining and motility tests may be
carried out for further observations.

COPY BY: MD.MAHDI HASAN, BSc IN AGRICULTURE, SYLHET AGRICULTURAL UNIVERSITY

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 ISOLATION OF RHIZOBIUM FROM ROOT NODULES

Isolation of Rhizobium from legumes of interest is the most practical method of obtaining
different strains for a culture collection. Strains isolated through this method provide one with
a large number of strains usually varying in nitrogen-fixing potential. There is a distinct
methodology which should be observed when isolating Rhizobia from nodules.
Materials required:
1. Sets of mall petri dishes,
2. Spatula, forceps, blade etc.
3. YMA plates,
4. Sterile water,
5. 0.1% acidified mercuric chloride
6. Ethanol
7. Inoculation needle,
8. Spirit lamp.
Preparation of YMA plates:
Mix the following:
Mannitol (C6H14O6) 10 g
Yeast extract 1g
K2HPO4 0.5g
MgSO4.7H2O 0.2 g
NaCl 0.1g
Distilled water 1000 ml
Agar 15 g
Adjust pH at 7.0

The medium should be autoclaved for 30 minutes at 15 PSI and poured to the sterile
petridish before cooling.
Procedure:
A general method representing as easy but accurate method of isolation from legume root
nodules is detailed below (Vincent, 1970).
1. Wash roots and nodules in tap water to remove gross soil contamination.
2. Sample nodules (preferably as many as 10) from the root by cutting the root about 0.5
cm on either side of the site of the nodule attachment. These root tails can then be used
to manipulate the nodules with forceps, thereby reducing the risk of damage to the
nodule itself.
3. Immerse the nodules briefly (1-5 seconds) in 95% ethanol and pass across to the next
container of 0.1% acidified mercuric chloride and leave for 3-4 minutes.
4. Rinse in six changes in sterile water, making transfers with alcohol flamed forceps. In
case of dried nodules, these should be allowed to dehydrate in sterile water for 10
minutes after the final rinse.
5. Nodules are then transferred to a sterile petri plate and crushed individually with fine
pointed forceps to give turbid suspension. Forceps should be flamed in alcohol for each
nodule.
6. Streak out one loopful of the suspension on yeast-mannitol agar plates.
COPY BY: MD.MAHDI HASAN, BSc IN AGRICULTURE, SYLHET AGRICULTURAL UNIVERSITY

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 7. Incubate the plates at 28˚C and check for growth typical of Rhizobium along the streak
lines. Well isolated single colonies should be picked off and restreaked on clean plates
to obtain pure rhizobia cultures. It is possible that more than one typical colony type
may appear on the plate streaked from a single nodule and each of these should be taken
to pure culture and held for characterization.
Precautions:
1. Care should be taken in removing the root system such that the nodules are not
damaged.
2. The undamaged nodules should be cleansed and surface sterilizes to prevent confusion
from rhizobia on the outside of the nodule and those on the inside.
3. The healthy nodules should be used as soon as possible or tore at low temperature if
they are not used at that time.

AUTHENTICATION OF RHIZOBIUM CULTURE

No fresh isolate should be put into a collection or regarded as a Rhizobium until it has been
authenticated. This is usually dependent on a demonstration of its capacity to infect a suitable
host under bacteriologically controlled conditions. The same precaution should be observed
when a culture is being brought back into use after long storage, and in the case of cultures
supplied from other collections. Rhizobium have various characteristics which are important in
distinguishing them from other organisms. There are tests which utilize these characteristics
and help determine whether Rhizobium or contaminations are present in the isolates.
A. Gram staining: This is a stain which divides all bacteria into two classes, they either
react positively or negatively. Rhizobia are gram negative and appear red color.
1. Make a thin film of bacteria on a clean glass slide.
2. Add a few drops of crystal violet solution. Wait for 1 minute, wash in tap water.
3. Add iodine solution, wait for 1 minute, and wash in tap water.
4. Wash with alcohol for 1 minute, wash in tap water.
5. Add saffranine, wait for 1 minute, wash in tap water, air dry and observe under oil
immersion using a compound microscope.
6. Red colored cells are gram negative but violet colored ones are gram positive.
B. Growth and pH change in yeast mannitol agar: Freshly prepared YMA tubes
containing bromothymol blue have a pH of 6.8 and are green. As a rule of thumb rule
slow-growing rhizobia produce alkali in the medium, turning the dye blue, whereas fast
growing rhizobia produce acid, turning medium yellow. Non-conforming
microorganisms are probably no Rhizobium.
Procedure:
1. Prepare an 0.5 % alcoholic solution of bromothymol blue.
2. Add 5 ml of the solution to 1 liter of YMA media and adjust the pH at 6.6
3. Sterilize the media and prepare YMA plates.
4. Grow the cultures on the YMA plates and examine.

C. Growth on YMA with Congo red: In general the rhizobia absorb the dye weakly
whereas many other bacteria take it up strongly.
Procedure:
COPY BY: MD.MAHDI HASAN, BSc IN AGRICULTURE, SYLHET AGRICULTURAL UNIVERSITY

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 1. Prepare an 0.2% aqueous solution of congo red.
2. Sterilize the solution separately.
3. Add 10 ml of the solution to 1 liter sterile melted YMA media.
4. Mix thoroughly and prepare plates with the media.
5. Grow the culture on the media of the plates and examine.

A. Nodulation test: The only absolute identification of Rhizobium is through nodulation

tests. If the unknown bacterium does no nodulate its host legume then it is not
Rhizobium.

PREPARATION OF RHIZOBIUM INOCULANTS

Introduction:
Nitrogen is the most deficient nutrient in Bangladesh soils. Legume crop have unique
capability of fixing and utilizing atmospheric dinitrogen through formation of nodules on their
roots by N2-fixing bacteria such as Rhizobium / Bradyrhizobium. Many soils do not have
effective N2-fixing nodule bacteria in adequate number and so it I necessary to use bacterial
inoculants for successful cultivation of food legume crops. Thus, legume inoculants may be
used to supplement or replace urea-N fertilizer.
Materials required:
1. Peat soil.
2. Stock culture of effective Rhizobium / Bradyrhizobium strain
3. Yeast-Mannitol Broth cultures
4. YMA plate, conical flask, inoculating needle, poly bag, scotch tape, CaCO 3
5. Autoclave
6. Incubator.
Procedure:
The bacterial or legume inoculants can be prepared as follows:
a. Preparation and processing of carrier material:
Peat is the most suitable carrier material for use in the preparation of legume inoculants.
Collect, dry and grind the peat material. Sieve the finely ground peat material and then
mix with required amount of CaCO3 (if the peat soil is acidic). Moist the peat material
and sterilize it by autoclave at 121˚C and 15 PSI for 2-3 hours. Put the sterilize peat
into 15cm × 10 cm size poly bags of desired thickness. The poly bags are ready for
inoculation with bacterial broth culture.
b. Preparation of broth culture with desired bacterial strain:
At first, select the test tube slant with desired bacterial strain from the stock. Prepare a
fresh culture with this strain in YMA plate. Prepare broth culture medium in a conical
flask. Sterilize the medium by autoclaving at 121˚C and 15 PSI for 20-30 minutes. After
cooling the medium is inoculated with a loop of the fresh bacterial culture using a flame
inoculating needle. Shake the flask in a rotary shaker. Within 2-3 days, the medium in
the flask will become turbid due to growth of bacteria. This broth culture is now ready
for inoculating the processed peat.
c. Inoculation of processed peat with broth culture:
Inoculate the processed peat contained in the poly bags with the broth culture using a
sterilized syringe. The amount of broth culture for inoculation depends on the amount
of peat material taken into the poly bags.
d. Incubation of the inoculated peat:
COPY BY: MD.MAHDI HASAN, BSc IN AGRICULTURE, SYLHET AGRICULTURAL UNIVERSITY

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 Incubate the inoculated peat contain din the poly bags in an incubator at 28˚C for 2
weeks. The inoculated peat culture is then ready for use in the field with legume seeds.
The required number of bacterial cells should be at least 109 g of the peat inoculant.
Use of inoculants:
Weighed quantity of legume seeds is taken in a plastic pot or poly bag. Sufficient amount
of gum acacia (40% solution) is added so that the seeds are just soaked and become sticky.
Then measured quantity of the inoculants (20 g/ kg seed) is added to the soaked seeds in several
installments. Each time the inoculant is mixed with the seeds by stirring with a stick or by
twisting the poly nag containing the seeds. The well coated seeds look blackish due to sticking
of the black powder i.e. the inoculant on the surface of the seeds. The seed thus coated, are
spread over a polyethylene sheet to break any crumb and the seeds are then ready for sowing
into the field.

TOTAL COUNT OF BACTERIA IN THE BACTERIAL INOCULANT BY DROP

PLATE COUNT METHOD

The method involves the dispersion of the soil or other material in an agar medium to such
an extent that individual bacterial cells or spores, have reasonable opportunity, when exposed
to suitable conditions, to develop into discrete and macroscopically visible colonies. The
necessary degree of dispersion is usually achieved by making successive dilution of given
sample.
A primary assumption in the method is that each viable micro-organism present in the
suspension develops into a visible colony following its seeding into agar and exposure to
incubation, any such assumption is, of course, highly idealistic. This method commonly gives
total counts of order of 1-10 % of those obtained by direct staining methods. Various factors
are responsible for the wide discrepancy between the two counts.

Materials:
1. Inoculum
2. YMA plates: 6 (prepare YMA Plates 3-4 days before use).
3. Water blanks: 400 ml sterile water in 500 ml bottle. Five 9 ml sterile water in 15 ml
screw cap test tubes.
4. 1 ml pipette: 7 Nos.
Procedure:
1. Preparation of dilution series:
Transfer 4 g of inoculum to a water blank containing 400 ml of sterile water. Tightly
cap the bottle and shake it for 10 minutes. Within 10 minutes after removing the bottle
from the shaker, shake the bottle vigorously by hand for few seconds and immediately
transfer 1 ml from the centre of the suspension to a 9 ml water blank using a sterile 1ml
to further water blanks of 9 ml to get dilution series upto 10-7
2. Inoculation:
Transfer 4 drops in each YMA plate from one dilution using standard 1 ml pipette. Use
three dilutions of 10-5, 10-6 and 10-7. Keep the plates undisturbed for 24 hours. Then
insert them and place them in an incubator at 28˚C.
3. Counting of plates:
After 6-10 days incubation, count the colonies develop in the drop of the plate. Count
the plates containing 10-30 colonies in each drop.
Calculation:
COPY BY: MD.MAHDI HASAN, BSc IN AGRICULTURE, SYLHET AGRICULTURAL UNIVERSITY

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If 10-7 dilution develops 12, 15, 12 and 13 colonies in one plate for example then,
No. of bacteria = (1215+12+13) ÷ 4 = 52 ÷ 4 = 13/drop.
13 × 20 × 107 = 2.6 × 109 /g inoculant.
PREPARATION OF BLUE-GREEN ALGAL INOCULUM

Blue-green algae are a self-sufficient system of fixing nitrogen from the air into forms
easily available to growing plants. These algae are greenish brown in color, slimy to touch and
are commonly found floating on stagnant water. The algae meet up their nitrogen requirement
by fixing atmospheric nitrogen and other nutrient requirement from water.
If the algae decompose in the soil, the nitrogen becomes available to other plants
growing in the soil and so, as the algae rapidly grow on water, they form a way of providing
nitrogen to rice.
Procedure:
a) Tray method:

i. Use galvanized iron trays of 1m × 2m × 0.2m.

ii. Spread 8-10 kg fertile soil in the tray and mix it with 200 g super phosphate or
equivalent amount of TSP.
iii. Fill the trays with water to a depth of 5-15 cm depending upon local rates evaporation.
iv. If the soil is acidic, add 100 g lime.
v. When the soil has settled, sprinkle 150-200 g dry algal flakes on the surface of the
water.
vi. Keep the trays in the open air completely exposed to the sun. The algae will begin to
grow.
vii. During the hot summer season, the algae will grow rapidly add will form a thick mat
on the water within one week. If evaporation is high, occasionally add more water until
the algal mat has been formed.
viii. Allow the water to evaporate in the sunshine and the algal matter will dry into flakes
on the surface of the soil.
ix. Collect the algal flakes and store in the plastic bags until needed for their application
to rice fields. The material can be kept in this way for several years.

b) Pit method:
The pit method needs no capital expenditure by the farmer.

Dig shallow pits in the soil of any desired area and line them with sheets polythene.
These pits take the place of the trays and the procedure to follow is exactly the same as
described above.

As the pits will normally be larger than the trays, the quantities of fertilizer, pesticide
and algal material must be raised as necessary.

COPY BY: MD.MAHDI HASAN, BSc IN AGRICULTURE, SYLHET AGRICULTURAL UNIVERSITY

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