Oplo

CENTRE OF ADVANCED FACULTY TRAINING
Laboratory Manual
Course Programme on
Management of Soil Health: Challenges and
Opportunities
(29th September to 19th October, 2014)
R.K. Thakur
S.S. Baghel
G.D. Sharma
P.C. Amule
N. Chouhan
Sponsored by
Indian Council of Agricultural Research
Organized by
Department of Soil Science and Agril. Chemistry
Jawaharlal Nehru Krishi Vishwavidyalaya
Jabalpur – 482 004 (M.P.)
INDEX
S. No. Title Page No.
01 Importance of Soil Testing, Collection of Soil Samples, its 1-4

Processing and Handling in Laboratory
02 Sampling, Processing and Storage of Plant Samples for Analysis 5-7
03 Profile Studies of Deep Black Soils (Vertisols) 8-11
04 Simultaneous Measurement of Bulk Density and Water Content 12-13

in Soil by Nuclear Methods
05 Estimation of Soil Moisture Content by Different Methods 14-16
06 Determination of pH and Electrical Conductivity in Soil Samples 17-18
07 Determination of Organic Carbon Content in Soil 19
08 Determination of Available Nitrogen in Soil by Alkaline 20-21

Permanganate Method
09 Determination of Total Nitrogen in Soil and Plant Samples 22-24
10 Determination of Phosphorous in Soil and Plant Samples 25-27
11 Determination of Potassium in Soil and Plant Samples 28-29
12 Determination of Sulphur Content in Soil and Plant 30-31
13 Determination of Micronutrients in Soil and Plant Samples by 32-35

Atomic Absorption Spectrophotometer
14 Estimation of Boron in Soils, Plants and Water 36-37
15 Estimation of Microbial Biomass Carbon in Soil 38
16 Preparation of thematic maps of land use/land cover through GIS 39-40

techniques
17 Gamma Irradiation and its Importance for Food Preservation 41-46
18 Plant Tissue Culture Techniques for Mass Propagation of Banana 47-51
Appendix - I to VI 52-59
CAFT on Management of Soil Health: Challenges and Opportunities” 29 Sept. to 19 Oct. 2014
Importance of Soil Testing, Collection of Soil Sample, its Processing and

Handling in Laboratory
P.S. Kulhare
Department of Soil Science and Agricultural Chemistry
College of Agriculture, J.N.K.V.V., Jabalpur
Assessment of a soils fertility status involves (ii) To determine the specific soil problem
such as an acidity, alkalinity and sodicity
an estimation of its available nutrient status
if exist. Subsequently giving
i.e. the portion or amount of nutrient directly
recommendation for their correction
available in soil for subsequent uptake by
(Lime/Gypsum requirement etc.)
crop plant. This exercise commonly referred
(iii) To predict the probability of getting
to as soil testing and is used to arrive at
maximum response of crops to
optimum fertilizer application ratio. The need
fertilizers.
for estimation of available nutrient arises
because only a small fraction of what the soil Procedure for soil testing
contains is the total nutrient content of the
The procedure for testing the soil to
soil. Soil test are calibrated by correlating
meet these objectives is divided into the
them with crop response and the result from
following phases:
the basis for making fertilizer
(i) Collection of soil samples and its
recommendations.
preparation
Estimation of nutrient contents and
(ii) Extraction and determination of nutrients
forms in materials that are involved in
and physico-chemical properties of the
nutrient supply and dynamics is a conical step
soil.
towards planning scientific nutrient
(iii) Interpretation of analytical results.
management. In this content, both soil and
(iv) Recommendation and follow up of
plant testing information comes out of the
results and evaluation of
interpretation of analysis assumes a greater
recommendations.
value when their concentrations and amounts
can relate to soil fertility, nutrient availability, Soil testing is a chemical method for
plant growth, yield and quality of the crop estimation of nutrient supplying power of a
produce. soil/ soil fertility evaluation.
Why soil testing ? Soil fertility may be defined as the
• Soil fertility status assessment involves an capacity of soil to furnish available plant
estimation of its available nutrient status. nutrients to the plants in proper amount and
It gives the amount of nutrient directly appropriate balance, under ideal condition of
available in soil for subsequent uptake by plant growth. Whereas, Soil productivity is
crop plant. the capacity of soil to produce under specific
condition of crop production.
• Guides to arrive at optimum fertilizer
application. Advantages of soil testing :
• It is a method of evaluating nutrient status  More rapid method as compare to
(physico-chemical properties) of the soil biological or deficiency symptoms/ plant
i.e. the assessment of the fertility of the analysis.
soil to determine nutrient deficiencies.  One may determine the need of the soil
• It is also concerned with environmental before the planting of crop.
quality for the community hazards.  To determine the suitability of the soil
Objectives for laying gardens.
To evaluate soil fertility and its productivity  Lime problems.
by the estimation of level of nutrient (Low,  Soil survey.
Medium, High). The error in soil sampling in a field is
(i) Grouping of soil for their classification generally greater than the error in laboratory
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 1

CAFT on Management of Soi l Health: Challenges and Opportunities” 29 Sept. to 19 Oct. 2014
analysis. The most recommendation call for  Depending on field conditions and the
soil testing of each field is about every three objective of sampling, select proper
years with more frequent testing on lighter sampling tool (s).
soils. Therefore, it is necessary that the soil  Based on difference in soil type, colour,
sample should be representative of the area. crop growth or slope, divide the area in
Further, the subsequent handling operation in different homogenous units.
the laboratory should be carefully performed
because a minute quantity (1 – 10g) of the
large soil mass of the field is actually used for
the analysis in the laboratory. Unless one is
sure of representative and proper sampling,
the results obtained in the laboratory analysis
will be of no use under the field conditions.
Apparatus and materials : DIVISION OF AREA
 Khurpi
 In the uniform field, demarket the
 Spade
sampling points in a zig zag fashion or
 Augers
randomly in such a way that the whole
 Plastic bowl
field should be covered i.e. about 30-35
 Scale
sample per ha.
 Rack
 Wooden roller
 Mortar and pestle
 Sieve
 Polythene/paper/cloth bags
 Labels
 Card board cartons
 Aluminium boxes
SELECTION OF SAMPLING POINTS
 At the sampling site, remove the surface

litter with Khurpi or spade. With the
help of the sampling tool (Auger) collect
a sample in a plastic bowl.
 If the soils is hard, make a ‘V’ shape cut
upto 15 cm depth. Remove the soil of
the pit. Now scrap or remove 1 cm / 1”
soil from the surface upto 15 cm depth
from both the side with the help of
khurpi. This scraped soil is collected in a
Screw Auger
plastic bowl. This sample is known as
‘primary’ sample. Such primary samples
should be approximately the same
weight.
Parts of an Auger COLLECTION OF SOIL SAMPLE BY

KHURPI
Collection of representative soil sample :

After collecting at least 30 – 35 primary When labels are hand written special
samples, mix all the samples in plastic bowl care should be taken to prevent ambiguity.
thoroughly and draw about ½ to 1 kg For arabic numbers “6” and “9” and
composite sample by quartering method. combination like “69” and “96” should be
Label the sample in the bowl and divide the under line, and ‘1’ and ‘7’ should be clearly
sample approximately 4 equal part. Discard distinguished. Where letters & numbers are
the 2 opposite portions of the samples and used ‘5’ may be easily confused with ‘S’.
remaining 2 portions are again thoroughly This is why printed labels are always better.
mixed and again divided in to 4 equal parts Field samplers have their own
and 2 opposite parts again discarded. This numbering system and may have record book
procedure is continue until ½ to 1 kg sample containing printed forms for entry of
remain in the bowl. This is known as information serially numbered. These ‘sample
composite sample which is true representative number’ are main identification of soil for
of the area. most purposes but the relevant form should
 The most suitable containers for soil also have a record of the ‘bag number’ and
samples are polythene bags 6x9’’ made subsequent ‘laboratory number’.
of film about 0.13 mm thick, which may When a box of samples is dispatched
be sealed by twisting or tying the neck or to the laboratory, it should contain a packing
by mean of rubber bands or adhesive note giving the total number of sample,
tape. ‘sample number’ of each sample and its
 If the soil is to be kept in moist condition corresponding ‘bag number’, the depth of soil
for moisture determination, bacterial sample from profile pit and other information
count and nitrate estimation etc. air tight needed by the laboratory staff for registration
containers are preferred. purposes, particularly on the analysis
If the soil to be used for the estimation of required. A duplicate packing note should be
micronutrients like Zn, Cu, Fe, Mn. Use of sent separately so that missing boxes can be
metallic tools should be avoided. Use sharp investigated.
stick or stainless steel. Brass sieve should be On arrival of the soil samples at the
avoided. Nylon net or aluminum sieve should laboratory, the content of a box should be
be used. checked against the packing note if any
In majority of cases, large stones and discrepancies should be reported to the
pieces of gravel (7.6 – 2mm) can be sampler. The samples are register in
discarded. Soil is broken up and spread out in laboratory giving each sample a ‘laboratory
a thin layer on strong paper or polythene film, number’ for particular analysis.
preferably on a rack of wire mesh to allow air Small laboratory simply the
to circulate. The drying area protected from numbered serially as they arrive. Larger
direct sun and wind. laboratory may have 2 or more numbering
Soil samples should be labelled/ system, using a prefixed letter (group of
numbered by field staff with water proof ink letter) to distinguish them. This procedure
or paint. These bag numbers being entered in helping to channel samples into various
the sampler’s record books, as each sample is analytical stream. Larger laboratory may need
taken together with other information. He to ‘punched card system’ or other means of
needs to identify and describe the samples. storing complete information on all samples.
If labels are used, they should either So that can be recovered quickly.
printed numbers on them already or water It is essential to keep a record of the
proof ink should be used to write information date of arrival and the source of all samples.
on them (this excludes pencil, washable ink A table can be drawn up for each month.
pens and ball point pens). Duplicate labels Information sheet: The soil sample thus
should be placed between the two bags, never collected must be furnished important
in the bag with the soil. The information on a information like –
label should be kept to a minimum preferably 1. Sample number
a number which may be either a ‘bag number’ 2. Name and address of the farmers.
or ‘sample number’. Depth may be given 3. Details of the field and site. Local name
usefully for soil profile samples. of field, Khasra no etc.
4. Date of sampling 4. In row crop take sample in between rows.

5. Name of crop and variety to be sown 5. Keep the sample in a clean bag.
6. Source of irrigation 6. A sample should not be taken from large
7. Whether the crop in the subsequent area (more than 1-2 ha).
season will be irrigated or un-irrigated. 7. Sample for micronutrient analysis must
8. Name of crops and fertilizer used in be collected by steel or rust free
previous years. khurpi/auger and kept in clean polythene
9. Date of harvest of the previous crop. bag.
10. Any other problem observed in the field. 8. Avoid sampling from low – lying spots,
manure dumping sites, near trees and
Preparation of soil sample for testing
from fertilizer placed zones.
1. Spread sample for drying on clean cloth, 9. Use clean bags for sample collection. Do
plastic or brown paper sheet. not use bags which had earlier contained
2. Remove the stone pieces, roots, leaves & fertilizer, manure or plant protection
other un-decomposed organic residues chemicals etc.
from the samples. STORAGE :-
3. Large lumps of moist soils should be  The registered and labelled samples in
broken. laboratory are finally placed in a
4. After air drying the samples should be cardboard carton. Label the carton
crushed gently and sieved through a 2 properly with the details of soil sample
mm sieve. and store in a separate room. The room
5. About 250 g of sieved sample should be should be away from direct sunlight/wind
kept in properly labeled sample bag for or dampness.
testing.  The room exposed to heat or cold or
Appropriate time for soil sampling dampness in not advisable.
An ideal time for soil sampling is just REFERENCES:-

after harvest of the Rabi crops,
 Physical and chemical methods of soil
Precautions to be taken during collection and water analysis FAO soils bulletin
of soil sampling 10. Food and Agricultural Organisation
of United Nations.
1. Remove all debris from surface before  Soil Fertility and Fertilizers: Samuel
collection of soil sample. Tisdale and Warner Nelson, Machmillan
2. Avoid taking sample from upland and Pub. Newyork.
low land areas in the same field.  Analytical Agricultural Chemistry: S.L.
3. Take separate sample from the areas of Chopra and J.S. Kanwar.
different appearances.

Sampling, Processing and Storage of Plant Samples for Analysis

P.S. Kulhare
Sampling thorough rinsing twice with deionized

A sample must be a true water or distilled water.
representative of the crop under field
conditions. The best way to collect a Drying
representative plant sample is to traverse After washing, the excess water in
the field diagonally and collect the the sample may be soaked by placing
samples from the deficient and normal them between the folds of filter paper
plants. These samples should be sheets. All the samples should dry as
composited separately representing poor rapidly as possible so as to reduce
growth and normal growth. Plants which chemical and biological degradation if
are infected with disease or attack of any. Sample should be dried in a hot air
insects, which are under stress due to oven at 700C. The material to be dried
excess water or drought and those which should be loosely packed so as to allow
are heavily coated with dust should not the free movement of moisture laiden air.
form a part of the sample. Extraordinary Oven made of stainless steel shelves
care must be taken to avoid contamination should be used. Plant samples have been
resulting from dust, soil, fertilizer and dried generally for 24 to 36 hours. Care
spray residues etc. should be taken to insure that the plant
The samples so collected should sample is not bunched together in isolated
be transferred to paper bags indicating the area of the oven, as large losses of N any
sample number and/or the field number or occur. Plant samples may also be dried in
other details. In case the sampling site is a microwave oven. This procedure is
far away from laboratory and requires rapid and can dry individual samples to a
longer travel period, the sample may be brittle state in as little as 5 minutes.
placed in polyethylene bag and Grinding and storage
transported in an ice chest. Low The oven dried samples should be
temperature will minimize the group in a grinder, fitted with stainless
physiological activity and also check the steel blades so as to pass the samples
spoilage of sample due to higher through a 40- mesh sieve. After grinding,
temperature and high humidity. the plant sample should be mixed
Processing and storage of plant samples thoroughly and transferred to
polyethylene or paper bags, labeled
Washing clearly and then stored in the room meant
The fresh plant samples brought to for plant samples. Avoid regular type of
the laboratory should be immediately grinder as it normally provided
washed in order to make them free from contamination. The sample grinder
dust or any other adhering substance. consisting of hard plastic can be used for
Samples should first be washed under the grinding (such type of grinder is
running tap water and if necessary. developed by tecator).
Subsequently, these samples should be Precautions
washed with acidified distilled water (1
ml concentrated HCl/litre) followed by 1. Avoid sampling the plants which are
infected with disease and/or insects

or those suffering from effects of Wet digestion of plant sample with

excess or scarcity of water. diacid mixture
2. Before subjecting the samples for The plant sample is diacid with
testing, decontaminate the collected mineral acid mixture (conc. HNO3, &
samples by thorough washing, and HClO4) and heated for more rapid
rinsing. decomposition. The volatile constituents
3. Washed samples should be dried as disappear and non-volatile mineral
rapidly as possible. The samples elements enter into solution.
should not be packed tightly during
drying in an oven and the The heating is continued until
temperature should not exceed 700C. digest is reduced to few ml of clear white
4. The grinder to be used for grinding residue. The residue is dissolved in HCl.
the samples should have stainless The test solution is prepared by dilution
steel blades or hard plastic to avoid and filtration.
contamination and the ground Reagents
samples must be passed through a
40- mesh sieve to ensure uniform  Diacid mixture. Mix 100 ml of conc.
fineness. For iron determination, It HNO3 and 40 ml of 60% HClO4 or
is advisable to grind the sample by else 30 ml of 72% HClO4.
hand in an agate or porcelain mortar Procedure
or in a mill made of non ferrous
alloy if hard plastic grinder is not Digestion with diacid mixture: Add 5 ml
available. of diacid mixture into the beaker in which
5. The processed samples should be 1.0 g plant sample has been transferred in
kept in polyethylene or paper bags the beaker and place on a hot plate for
and stored in room free of dust soil brisk heating. Keep a portion of the
and smoke etc. beaker mouth open to permit the gases to
6. Care should be taken not to rub the escape. Continue heating until evolution
plant tissue acid mixture like any of of copious dense white fumes subsides
the washing the samples. leaving about 3 ml of colourless solution
Methods of ashing of plant tissues in the beaker which on cooling gives a
Wet ashing is accomplished by whitish residue. If the residue is not white
digestion with HNO3, HClO4 and H2SO4 and signs of beginning of charring are
or else with HNO3 and HClO4 especially seen, remove the beaker from the hot
for S determination. The method is plate and let it cool. Then add 2 ml of
unsuited for B determination as the diacid mixture and place the beaker again
element is volatilized during wet ashing. on hot plate. Continue heating until the
Dry ashing is done in muffle contents is reduced to about 2-3 ml of
furnace at temperature varying from 400 colourless clear solution. In order to avoid
to 5000C for 2-8 hours. The plant sample excessive heating, a layer of sand may be
taken in platinum or vitreous silica spread over the hot plate. If white residue
crucible is treated with Mg (NO3)2 or is not obtained, the residue is treated
Na2CO3 prior to placing in a muffle again with 2ml of the diacid mixture and
furnace. Dry ashing at 5000C is the heating be continued till the contents
considered satisfactory for the is reduced to about 2-3ml. thereafter, the
determination of Fe, Mn, Cu in biological beaker be removed from the hot plate or
materials. The use of stainless steel lined sand bath. Allow the beaker to cool.
muffle furnaces eliminate contamination Preparation of plant test solution: Add
from Al and Zn if any during ashing. 5ml of glass distilled water into the

beaker containing digested residue. Heat heat it to warm. Take a clean 100 ml
the contents gently on a hot plate until volumetric flask fitted with a funnel lined
white fumes appear. Remove the beaker with Whatman No. 1 filter paper. Transfer
and let it cool. Then, add 10ml of glass the contents of the beaker into the funnel
distilled water and heat the contents to and collect the filtrate in the volumetric
dissolve the residue. flask. Then, add another 5ml of conc. HCl
Take a 100ml clean volumetric into the beaker to dissolve the remaining
flask fitted with a funnel lined with residue in the beaker. Transfer the
Whatman No. 1 paper. Transfer the solution into the same funnel, collecting
contents of the beaker into the funnel the filtrate in the same volumetric flask.
using a glass rod collecting the filtrate in Repeat subsequent washing of the
the volumetric flask. Rinse the beaker contents of the beaker with small portions
with about 15ml portions of glass distilled of 6N HCl and transfer the washing into
water and transfer each rinsing into the the same funnel until the filtrate reaches
funnel in order to transfer the digested 100ml mark. Similarly run a blank
residue quantitatively. Collect the filtrate digestion and then prepare test solution
from each washing into the same using conc. And 6N HCl instead of glass
volumetric flask. Then wash the residue distilled water as described above.
on filter paper with small portions of glass Reference :
distilled water and collect the washing
Amma, M.K. 1990. Plant and Soil
until the volume of filtrate reaches 100ml
Analysis. Rubber Research
mark. Stopper the flask, label it and then
Institute, Rubber Board,
preserve it for the elemental analysis other
Kottayam-686009 (Keral).
than Ca, Mg and S. similarly run a blank
Jones (Jr) J. Benton. 1972. Plant Tissue
digestion and subsequently prepare the
Analysis for Micronutrients pp.
blank test solution following the above
319-348. In j.J. Mortvedt et. al.
mentioned procedure.
(ed.) Micronutrients in
If Ca and Mg is to be determined, add 5ml Agriculture. Soil Sci. Soc. Am.,
of conc. HCl instead of glass distilled Madison, Wisconsin, USA.
water into the beaker containing the plant
digested residue. Swirl the contents and

Profile Studies of Deep Black Soils (Vertisols)

G.P. Gupta
Vertisoils or deep black soils occur unique morphological properties such as

globally under various parent materials the presence of slickensides, wedge-
and environmental conditions (Table 1). shaped aggregates, diapir (mukara), and
gilgai. Shrink-swell phenomena are the
Table 1. Distribution of Vertisols and dominant pedogenic processes in
associated soils Vertisols and are attributed to changes in
Juris- Location Area % of
Diction (m ha) Gross
interparticle and intraparticle porosity
Black with changes in moisture content.
soils Definition of Vertisols :
Continent Africa 105.0 38.7
Asia & far 70.3 25.9 Taxonomically for defining
East (Mainly Vertisols, there must be
India) 1. A layer 25 cm or more thick with an
Australia 48.8 17.7
Latin 27.0 9.9
upper boundary within 100 cm of the
America mineral soil surface, that has either
North 10.0 3.7 SLICKENSIDES or WEDGE
America SHAPED PEDS that have their long
Near & 5.7 2.1 axes tilted 10 to 60o from the
Middle East
horizontal; and
Europe 5.4 2.0
TOTAL 271.4 100
2. A weighted average of 30 % or more
Country India 70.3 25.9 clay in fine earth fraction either
Australia 48.8 17.7 between the mineral soil surface and a
Sudan 43.4 16.6 depth of 18 cm or in Ap horizon,
USA 18.1 6.7 whichever is thicker and
CHAD 15.5 5.7 3. 30 % or more clay in fine earth
China 11.6 4.3
fraction of all horizons between a
Others 64.5 23.7
( in parts) depth of 18 cm and either a depth of
TOTAL 271.4 100 50 cm or a densic, lithic or paralithic
India MS 24.2 34.4 contact, a duripan, or a petrocalcic
MP 21.2 30.1 horizon if shallower and
GUJ. 4.9 7.0 4. Cracks that open and close
AP 9.4 13.4
periodically.
KTK. 5.8 8.2
TN 2.6 3.7
RAJ. 1.1 1.6 Vertisols are significant global
UP 1.1 1.6 resources that serve as the lifeline in
TOTAL 70.3 100 subsistence agriculture due to their high
MP Vertisols 8.0 37.7 productivity.
Inceptisols 8.6 40.6 Efforts towards comprehension and
Entisols 4.2 19.8
successful utilization are imperative for
Alfisols 0.4 1.9
TOTAL 21.2 100 continued productivity and long term
They clayey soils that shrink and sustainability of these resources for
swell extensively upon changing soil current and future civilizations.
moisture conditions. Vertisols exhibit

Morphology of a soil is best evaluated %) and predominance of montmorillonite

from the in situ examination of the soil (2:1 expanding clay). Other important
profile. A recently dug pit large enough parameters for the development of
for observation of a pedon is desirable. Vertisols are:
Old exposures such as road banks and (i) Parent material having basalt,
ditches are acceptable only for argillaceous limestone, marine clays
preliminary studies because and shale
morphological features often become (ii) Weathering period must be
altered after prolong exposure. extensive for the development of
Normally the size of profile pit is solum with 2:1 expanding clays
kept 1.8 m long, 1.2 m wide and 1.8 m (iii)Weathering environment should be
deep but for the study of black soils, the such that no further weathering of 2:1
width of pit varies from place to place expanding clays takes place. Even no
depending on its cyclic wave length of inter-layering exists to the extent the
puffs and shelves. It should be kept in properties are destroyed
mind that atleast half wave length (iv)Sequence of events should continue
covering both, puff and shelve is like churning/mixing, development of
considered while exposing profile pit in argilli-pedoturbation, development of
order to study the pattern of cracks and slickensides and formation of wedge
slickensides perfectly. shaped structures
The profile examination begins Pedogenesis of Vertisols :
with a first approximation and marking of
soil horizon boundaries on the profile. 1. Separation of blocks :
Each horizon is then carefully observed Deep wide cracks separate the soil
and described. Horizon boundaries are into strong and massive prism like blocks
relocated as required by the detailed study in the upper part of the pedon that break
(Buol et al. 1998). Th description sheet into angular blocky peds of hard and firm
containing the columns of site and soil consistence.
characteristics is filled up by the profile (a) Cracking of soil : During dry season,
study group during pedon studies. the soil cracks to the surface due to
Vertisols are relatively shrinkage of 2:1 expanding clays that
homogneous in their morphology. may extend to a depth of 1 metre or
Although horizonation is not distinct yet a more.
few horizons above the parent material (b) Falling of surface soil material :
may be identified as self mulching surface While cracks are open, surface soil
(Ap), blocky subsurface (A12), material falls into them by several
slickensided horizon and wedge shaped mechanisms such as animal activity,
subsoil (Bss). wind or at the onset of rainy season by
The depth of these soils may vary water.
from shallow to very deep. Previously the 2. Hydration of clays : The clay
black soils were grouped as shallow (<30 hydrate and due to their high coefficient
cm) medium (30-100 cm) and deep (>100 of expansion and contraction, expand 3
cm) but later on Sehgal (2008) modified dimensionally on wetting.
the depth of shallow soil as less than 50 (a) Expansion of clays : Cracks close on
cm. the surface but because of the extra
Requirement of Vertisols : material now present in the lower part
of the profile, a greater volume is
Main requirements of Vertisols are attained and the expanding material
the presence of high content of clay (>30 presses and slides the aggregates

against each other, developing a "lentil" approaches or reaches the surface. If diapir
angular blocky structure with and gilgai occur, the mound in gilgai is
slickenside features on the pad surfaces. always developed over the diapir.
(b) Shear stress development : The Hallsworth and Beckman (1969)
slipping occurs where shear strength is classified gilgai into 6 types i.e. normal or
surpassed by shear stress acting upon a round, melon hole, Lattice, Linear or wavy,
soil mass. The shear stress is a major tank or stony but later on Paton (1974)
force caused by swelling and develops suggested only two types of gilgai i.e. linear
when volume expansion results during and circular (Nuram or Pockmarked) each
the wet cycle. of which were grouped into 4 types.
(c) Formation of slickensides : The  type - Mound and depression equally
slickensides, intersecting or close developed (No shelf present)
enough to intersect, also result in wedge  type - Mound of much greater
shaped structural aggregates, the most extent than depression (No shelf present)
characteristics feature of Vertisols  type - Depression of much greater
which develop with their longitudinal extent than mound (No shelf present)
axes inclined at 30 to 60° from  type - Mound, shelf and depression all
horizontal (Sehgal and Bhattacharjee, present
1988). 2. Size of cyclic pedons : Half cycle
(d) Buckeling of land space : This linear distance (HCLD) measures the lateral
expansion buckles the land scape, dimension of a cyclic pedon. It may be
forming the micro relief called gilgai. small, medium or large i.e. below 1, 1 to 2
The micro basins contain more organic or above 2 to 3.5 meter, respectively.
matter than the micro ridges and 3. Horizon sequence : In Vertisols,
probably it results from admixtures of the horizon sequence has been suggested to
subsurface material into micro ridge be A1-Bss-BC-C where "ss" indicates about
area and slight erosion of organic rich the presence of slickensides.
fines from the ridges to the basins. 4. Thickness of horizon : Thickness
3. Incomplete leaching : In most of A1 in Vertisols varies with the linear
shrink swell soils, the temperature being frequencies of puffs and shelves of gilgai
high, the potential evapotranspiration micro relief.
suggesting incomplete leaching and 5. Horizon boundary (Amplitude): It
inducing the process of calcification in is the difference between vertical distance
these soils. from the surface of pedon to the lower
Cyclic movement of soil material : boundary of crest of cycle and the lowest
Amongst several processes acting in point of trough of cycle in same pedon. The
the formation of Vertisols, the predominant amplitudes are grouped as low, medium of
process seems to be haploidization i.e. high according to the vertical distance as
mixing by argilli pedoturbation. The below 25, 25 to 75 or above 75 cm,
specific features of such soils are : respectively. Shape of apparent topography
1. Gilgai micro relief : The term of the intermittent horizon is also graded as
gilgai is an Australian aboriginal term tongued (vertical extent > horizontal
meaning small water hole. distance), wavy (vertical extent
Pedogenic micro topographical approximating the horizontal distance) and
features like puffs (microknolls) and smooth (vertical extent < horizontal
shelves (micro basins) develop that remain distances) as suggested by Bartelli (1971).
intimately associated with one another Age of Vertisols :
(Bhattacharjee et al. 1977), Columbe et al. It is difficult to assign the Vertisols
(1996) introduced a term "diapir" meaning a place in the genetic scheme of soil
a protusion of subjacent soil material which classification as there are greater
penetrates to the overlying horizons and differences of opinion whether they are old,

young or remain in equilibrium with the would dominate. This interpretation

environment. suggests that Vertisols are relatively young
1. Views as Vertisols are old : The soils.
end product of a development sequence 3. View as Vertisols are in
involves the soils whose B horizon has equilibrium : Vertisols remain in
become so clayey that shrink-swell cycles equilibrium with their environment and that
developed and eventually "swallowed" the the 2:1 expanding lattice clays are stable
A horizon. It is possible because high and will persist, barring a climate change.
content of fine clay and high fc/cc ratio may Vertisols then can be considered diagnostic
be produced by lessivage on a large scale. of environments in which the parent
2. Views as Vertisols are young : material is basic and gives rise to the
The fate of Vertisol may be to formation of 2:1 expanding lattice silicates
undergo alteration of 2:1 clays to non under the influence of wet dry climate.
expanding type of clay. The profile would
then cease to churn and eluviation process
Table 2 : Range in characteristics of Vertisols and Vertic Inceptisols
Horizon Soil colour Texture Structure Special features Width of
(10 YR) cracks (cm)
A. Typic Haplustert (10 YR - 2.5 YR)
Ap/A11 4/2, 3/3, 3/2, 3/1 C 1f/1m sbk 1c/2c pr-3c pr 2-5
A12 3/3, 3/2, 3/1 C 2m/2c abk 2c pr - 3c pr 2-5
Bss 3/3, 3,2, 3/1, 2/1 C 2m/3c abk Intersecting lickensides* 1-2
BC 4/4, 3/4 C 2m/2c abk ----do---- 0.5-1
C 5/4, 4/4, 4/3 c-gc 2msbk/ massive - -
B. Vertic Haplustept (10 YR - 7.5 YR)
Ap/A 5/2, 4/3, 4/2, 3/2 Cl gr/1m sbk 1c pr-2c pr 2-2.5
AB 4/3, 3/3 cl-c 1m/2m sbk ----do---- 2-2.5
B21 4/3, 3/3, 3/2 cl-c 2m sbk-3m/3c 2c pr - 3c pr or pressure 1.5
faces/abk slickensides
B22 6/3, 5/3, 4/4, 4/3 gscl-cl ----do---- - -
C 7/6, 6/3, 5/3, 4/4 gsl-gscl 1f sbk/ massive - -
*or parallelepipeds with long axes tilted from 35° to 55° from horizontal
References : Paton, T.R. (1974). Origin and terminology for
gilgai in Australia. Geoderma. 11 : 221-242.
Bartelli, L.J. (1971). Soil taxonomy, correlation Sehgal, J. (2008) Pedology – concepts and
and Interpretation. Proceedings of application, 2nd edition Kalyani Publication,
Workshop organized by All India Soil and Ludhiana P. 416.
Land Use Survey, New Delhi. Sehgal, J., and Bhattacharjee, J.C. (1988). Typical
Bhattacharjee, J.C., Landey, R.J. and Kalbande, Vertisol of India and Iraq-their
A.R. (1977). A new approach in the study of characterization and classification.
Vertisol morphology. J. Indian Soc. Soil Sci. Pedologie. 38 : 67-95.
25 : 221-232. Sehgal, J., Saxena, R.K. and Vadivelu, S. (1987).
Buol, S.W., Hole, F.D. , McCracken, R.J. and Field Manual for Soil Resource Mapping of
Southard R.J. (1998). Soil Genesis and different states. NBSS & LUP, Nagpur, pp.
classification. Panima Publication 73.
Corporation, New Delhi. Soil Survey Staff (1999). Keys to Soil Taxonomy.
Coulombe, C.E., Wilding, L.P. and Dixon, J.B. 9th Ed. CSC, SMSS, United States
(1996). An Overview of Vertisols: Department of Agriculture (USDA)
Characteristics and Impact on Society. Adv. Washington, D.C.
Agron. 57 : 289-375.

Simultaneous Measurement of Bulk Density and Water Content in Soil by

Nuclear Methods
G.P. Tembe
A recent advance is nuclear technology content of three soils. The samples were
which has evolved methods for the non placed between a cesium source and
destructive determination of the soil water detector and then between americium to
content. Two methods have been used using a combined source but neither
with success. One the neutron scattering Soanenor other workers (Gardner and
method and the other is the y-radiation Calissendoff, 1967; Gardner, Campbell
method. and Calissendaff, 1969) have attempted to
Neutron method : In this method combine the sources in a single collimator
measurement is made of the number of because the higher energy gamma
hydrogen nuclear that are present per unit photons produce Compton scatter through
volume of soil and therefore, water interaction with the sample and some of
content by volume θ, is measured. this Compton scatter will be counted as
Neutrons are uncharged particles having gamma rays from the lower energy
almost the same mass as that of protons or source. The error can be eliminated by
that of hydrogen nuclei. A radium- with equipment and method evolved by
beryllium mixture in form of pellets is Corey, Peterson and Wakat, 1971). This
used as source of fast neutrons. The method is feasible for measuring the
measurement of soil water by the neutron water content and soil density of soil
method is essentially a measure of the columns simultaneously when two
density of the slowed neutron cloud sources are combined in a single
developing when Ra-Ba source is inserted collimator. Measurement of attenuation of
137
in the soil, the density of the slowed cesium and 241americium is done in this
neutrons being a measure of the soil water method. Radiation intensity (counts/min)
content by volume θ. after passage through the soil, container
and container alone is calculated by the
Simultaneous determination of bulk equation (Corey et al.) given in method.
density and water content : To obtain values of intensity the
With the increased interest in the equipment is required as shown in Fig. 1.
241
behavior of water in swelling soils and Am emits a large number of
resulting theoretical studies (Smile and gamma rays of various energies but the
Rosenthal, 1968; Philip, 1969) on the major energy is 59.6 KeV 241Am has a
subject, a method was required for half life of 458 years eliminating the need
measuring changes in both the water for decay corrections 137CS decays with a
content and soil density in order to gamma ray of 662 KeV and has a half life
experimentally evaluate these theoretical of 30 years.
analyses. Numerous authors have Detector and pulse height
proposed measuring the attenuation of analyzers are used in this system.
gamma rays of two different energies to Two methods were compared and
non destructively determine both water dual source method evaluated. Known
content and soil density in the same water content and soil densities were
sample. Soane (1967) illustrated the measured. The two soils were Houston
effectiveness of the dual gamma method black, a soil containing montmorillonitic
for measuring bulk density and water clay and cecil a soil containing kaolinitic

clay. The soil containing different amount Houston Black soil at the end of 24 hour
of water was packed to varying densities period but had infiltrated completely with
into plastic boxes 7.5 x 4.46 x 4.95 cm. the Cecil soil. The two soils responded
These boxes were placed between sources quite differently to the addition of water.
and detector with long axis parallel to Neither the soil density nor lengths of
beam. Comparisons between the known, Cecil soil column changed. The column
soil density. of Hoston black soil increased 2 cm in
Water content and values length and the soil density decreased in
determined by two methods for four cecil top 4 cm.
soil samples and five Hoston Black soil The combined source method is
samples are given in table 1. applicable to the study of swelling soils,
Table 1: Water content and values the phenomena of freezing and thawing
determined by twi methods. and measurement of water content and
density of the soil inside core barrels.
Water content g/cm3 Soil density g/cm3
References :
Known Calculated Known Calculated
Cecil soil Cerey, J.C., Peterson, S.F. and Wakat, M.A.
(1971). Measurement of attenuation
0.001 0.000 0.00 1.831 1.830 1.830
of 137CS and 241Am gamma rays for
0.044 0.081 0.073 1.712 1.711 1.720 soil density and water content
0.120 0.124 0.143 1.999 2.001 1.980 determinations. Soil Sci. Soc. Am.
0.175 0.118 0.151 2.050 2.067 2.081
Proc. Vol. 35 : 215-19.
Gardner, W.H. and Callissendortt, C.
Houston Black Soil (1967). Gamma rays and neutron
0.010 0.000 0.000 1.504 1.504 1.504 attenuation in measurement of soil
0.141 0.147 0.139 1.297 1.281 1.290 bulk density and water content. Pp
101-113. In Isotope and radiation
0.270 0.284 0.281 1.343 1.367 1.371
techniques in soil physics and
0.438 0.472 0.446 1.369 1.349 1.378 irrigation studies. International
0.495 0.532 0.514 1.297 1.234 1.255 atomic agency, Vienna.
Gaedner, W.H., Campbell, G.S. and
There is little difference between Calissendorff, C. (1969). Water
the results obtained by the two correction content and soil bulk density
techniques and method best suited will be measured concurrent using two
determined by the instrumentation gamma photon energies US AEC
available. Report, RLO 1543-6 Washington
Following satisfactory evaluation State University. Pullman Wash 42
of the dual source method for determining p.
the known water content and soil density Philip, J.R. (1969). Moisture equilibrium in
of soils, the method was used to the vertical in swelling soil. I Basic
determine water content and soil densities theory. Anst. J. Soil Res. 7 : 99-120.
of Houstan Black and Cecil soil columns Smiles, D.E. and Rosenthal, M.J. (1968).
following infiltration. More water was The movement of water in swelling
added to Houston soil because its greater material. Aust. J. Soil Res. 6 : 237-
water holding capacity. Twenty four 248.
hours following the addition of water the Soane, B.D. (1967). Dual energy gamma
transmission measurements were repeated ray transmission for coincident
measurement of water content and
and the water content and density was
dry bulk density of soil. Nature 214
calculated water remained ponded on
: 1273-1274.
Estimation of Soil Moisture Content by Different Methods

G.P. Tembe
Direct method (Gravimetric method) : alcohol or spirit. The process is repeated

to get constant weight of ignited soil.
This is the simplest and most Equipments and reagents :
widely used method for measuring soil
moisture. (1) Moisture cans
(2) Glass rod
Equipments :
(3) Ethyl, methyl or propyl alcohol
Sampling tube/auger (4) Match box
Moisture cans (numbered) (5) Balance with weights
Balance with weights/automatic (6) Measuring cylinder
Drying oven (7) Desiccator.
Desiccator Procedure :
Procedure :
Weigh about 20-25 gm of wet soil
Collect soil samples by tube or in moisture can add about 5ml alcohol or
auger from a number of points within the spirit drop to fully saturate the soil and
experimental site and mix thoroughly. ignite. Cool the cans in a desiccator and
Place composite sub samples of about 50 record the weight. Repeat the process
gm to 100 gm in soil moisture cans with several times till a constant weight of the
tight fitting lids. Take at least three sub- ignited soil is recorded. Compute the
samples. The moist samples are weighed percentage moisture on the basis of the
immediately, dried to constant weight in constant dry weight of the burnt soil.
an oven at 105°C (for about 24 hrs) and This method is rapid, reproducible
reweighed after cooling in a desiccator. and more suited for field soil moisture
Determine the tare weight of moisture determinations where laboratory facilities
cans. Calculate the soil moisture content are not adequate.
by determining the loss in weight on Burning of the soil results in loss
drying and the weight of the oven dry soil of organic matter. This method needs to
as follows : be calibrated against oven dry method for
Soil (wt. of wet soil + tare) each soil type.
moisture - (wt. of dry soil + tare) In direct method (By use of neutron
=
content by (wt. of dry soil + tare) moisture probe) :
weight (%) - (tare)
Rather than taking a sample of
Loss of wt. on drying soil, it is often desired to measure the soil
Mw (%) = X 100
Wt. of oven dry soil moisture status in situ without disturbing
the system. The neutron moisture meter is
Percentage of moisture on volume basis
such a device which is much used in the
= Percentage of moisture on weight basis
field for measuring water content
x bulk density.
(Belcher, 1952, Gardner and Kirkhana,
Gravimetry with drying by burning 1952, Holmes, 1956 and Van Bavel et al.,
Alcohol or spirit (Bouyoucos, 1937) : 1956). The neutron method is a measure
In this method, the soil moisture is of the number of hydrogen nuclei that are
evaporated by igniting the soil mass with present per unit volume of soil. Therefore,

it is a means of directly observing the nitrogen in soil is associated with water

moisture content by volume. and lesser amounts with organic matter.
Principle : Thus, the measurement of soil water by
neutron method is essentially a
Hydrogen nuclei have a marked measurement of the density of the slow
property for scattering and slowing neutron cloud developing when the Ra-Be
neutrons. This property is exploited in the or Americium-Beryllium source is
neutron method for measuring soil water inserted into the soil.
content. High energy neutrons (0.1 to 10
Equipments and material :
MeV) emitted from a radioactive
1. Small fast neutron source such as
substance such as radium beryllium
radium-beryllium (Mc) or americium-
(5Mc) or americium-beryllium (30Mc)
beryllium (30 MC)
neutron source are called fast neutrons
2. Shield for storage of the neutron
and travel with high speed of the order of
source. Shielding commonly used
100 miles per second.
consists of lead, paraffin or
When such a fast neutron source is
polyethylene in a cylindrical shaped
placed in a moist soil, the emitted
unit with a cylindrical hole through
neutrons interact with the surrounding
the axis to accommodate the probe.
medium. They collide with the nuclei of
3. Detector of slow neutron - most
the soil in a billiard ball fashion, changing
commonly used for soil moisture
direction as a result and loosing energy.
measurement is a Bf3-enriched
With the energy loses, the speed
proportional counter mounted in a
diminishes until it approaches the speed
cylindrical arrangement with
characteristics for molecules at the
transistorized preamplifier mounted in
prevailing temperature. Such neutrons are
the cable end.
called thermal or slow neutrons. The slow
4. Counting device (scalar) - the density
neutrons are finally absorbed by other
of slow neutrons in the soil and in the
nuclei + and thus their existence ends. In
counting tube is evident in the form of
a material containing appreciable
a given counting rate. This counting
hydrogen a neutron after the first the
rate may be determined by a rate
collision with a hydrogen nucleus is not
meter or by a scalar. The first
likely to travel much farther consequently
indicates the count rate directly and
if the neutron source is enclosed in a
the second register the total number of
material that is rich in hydrogen, it will be
counts over a given time period. In
enveloped in a dense spherical cloud of
either case identical results are
slow neutrons. The density of this cloud
obtained.
represents equilibrium between the rates
5. Access tubing and soil auger sted or
of thermalization and absorbed by the
aluminum tubing is most commonly
medium and the rate of production by the
used, but other materials such as
source. This equilibrium is reached in a
plastic have been used. Two sizes of
fraction of micro second after the
access tubes are in common use. 20-
insertion of source. If the medium
gauge steel or aluminum tubings
surrounding the source contains less
1.625 inches (4.13) or 2 inches (5cm)
hydrogen, the cloud of slow neutrons will
outer diameter or 1.9 inches (4.83 cm)
be less dense and extend farther from the
of inner diameter of aluminum
source. This is so because a fast neutron
irrigation tubing. Aluminum is
has to travel further, on an average, to in
practically transparent for fast and
counter a hydrogen nucleus and start
slow neutrons and does not corrode
becoming thermalized. Most of the

seriously. Tube once installed may provide an index of equipment condition.

give years of service. After determining the standard count, take
A soil auger slightly smaller the the reading at successive depth intervals
tubing should be available for drilling starting at least 18 to 25 cm from the soil
the access holes. Moisture probe surface. Approximately, 9-15 cm soil
should be calibrated for particular layer is characterized by a single
material and size of access tubing to measurement.
be used.
6. Cable - A 10 meter electric cable Divide readings by standard to
connects detector and scalar. All obtain a count ratio (referred to as relative
possible care should be taken to keep count ratio to standard or per cent of
the connections and firm and the standard etc.) and refer to instrument
connectors free of dirt and moisture. calibration curve to obtain water content
Procedure : by volume at various depths. The
calibration curve supplied with instrument
The access tube is first inserted usually may be used. But, since wide
into the soil after drilling a hole with the differences of soils are known to exist, the
help of auger taking care that no bend in calibration checks, should be made to
the access tube is created. The access tube each soil type as guided by experience in
is kept few inches above the soil and area. It is very important to realize that
covered with an inverted can to prevent the potential precision of the neutron
entrance of trash. method is greater than that of any other
The neutron probe must be method under field condition. Therefore,
inserted in the access tube and held at the calibration should be carried out with
desired depth. Holding the probe may be large homogeneous masses of soil of
done by various means, but a simple and which the bulk density and moisture
effective method is to use an ordinary content (by gravimetric method) are
marking tape, coloured cloth adhesive accurately known.
tape or surgical tape. While making a
References :
measurement turn on the scalar a few
minutes earlier to warm up (transistorized Belchev, D.J. (1952). The measurement
units require no warm up). Make several of soil moisture and density by
standardization counts with the probe neutron and gammaray scattering.
each time. The normal counting time is Highway Res. Board Spl. Rpt. 2 :
minute. The background "thus obtained 98-110.
should not be much more than 100 counts Bouyoucos, G.J. (1937). Evaporation
per minute. As measurements are being water with burning alcohol as a
taken, the standard count is determined rapid means of determining
again from time to time. The frequency moisture content of soils. Soil Sci.
can depend on convenience, plolayout and 44 : 337-383.
experience. It is often convenient to make VanBavel, C.H.M. (1958). Measurement
a standard count at the start and end a of soil moisture content by the
series of readings in each access tube. neutron method. Agr. Res. Serv.
Keep a record of standard count to ARS. 41-24.

Determination of pH and Electrical Conductivity in Soil Samples

Narendra Chouhan
Determination of pH is actually a 4.0 (Dissolve 10.21 g. of A.R. grade

measurement of hydrogen ions activity in potassium hydrogen phthalate in distilled
soil – water system. It is defined as water and dilute to 1 litre. Add 1 ml of
negative logarithm of the hydrogen ion chloroform or a crystal of thymol per litre
activity. Mathematically, it is expressed as a preservative).
as: Procedure :
pH = - log a H+ (a) Soil to water ratio of 1:2 (pH2)
The pH value of a soil is an Take 20 g soil in 100 ml beaker and add
indication of soil reaction i.e. acidic, 40 ml. of distilled water to it. The
neutral or alkaline. The nutrient suspension is stirred at a regular interval for
availability is governed by soil reaction. It 30 minutes. Determine the pH by
is maximum at neutral pH and decreases immersing electrodes in suspension. For
with increase in acidity or alkalinity. soils containing high salts, the pH should be
Thus, pH value gives an idea about the determined by using 0.01M calcium
availability of nutrients to plants. chloride solution. (Dissolve 0.110 g of
CaCl2 in water and dilute to 1 litre).
Principle :
(b) Saturates soil paste (pHs)
The pH is usually measured by pH Add small amount of distilled water
meter, in which the potential of hydrogen to 250g of air dried soil. Stir the mixture
ion indicating electrode (glass electrode) with a spatula. At saturation, the soil paste
is measured potentiometrically against glistens and flows slightly when the
calomel saturated reference electrode container is tapped it slides freely and
which also serves as salt bridge. Now a ensures cleanly off the spatula. After
day, most of the pH meters have single mixing, allow the sample to stand for an
combined electrode. Before measuring the hour. If the paste has stiffened markedly or
pH of the soil, the instrument has to be lost its glistening, add more water or if free
calibrated with standard buffer solution of water has collected on the surface of the
known pH. Since, the pH is also affected paste, add an additional weighed quantity of
by the temperature, hence, the pH meter dry soil and mix it again. Then insert the
should be adjusted to the temperature of electrode carefully in the paste and measure
the solution by temperature correction the pH.
knob. (c) Saturation extract (pHe)
The soil is extracted using vacuum
Reagents : extractor and the pH is measured in the
Standard buffer solutions: These saturation extract.
may be of pH 4.0, 7.0 or 9.2 and are Categories of soil pH values :
prepared by dissolving one standard Soil pH : Interpretaion
buffer tablet in 100 ml distilled water, It is < 5.0 : Strongly Acidic
necessary to prepare fresh buffer solution 5.1 – 6-5 : Slightly Acidic
after few days. In absence of buffer tablet, 6.6 – 7.5 : Neutral
a 0.05 M potassium hydrogen phthalate 7.6 – 8.0 : Mild Alkaline
solution can be used which gives a pH of > 8.0 : Strongly Alkaline

Electrical Conductivity : Reagents :

Amount of soluble salts in a Potassium chloride: Dissolve
sample is expressed in terms of the 0.7456g dry potassium chloride (AR) in
electrical conductivity (EC) and measured distilled water and make up the volume to
by a conductivity meter. The instrument one litre.
consists of an AC solubridge or electrical Procedure :
resistance bridge and conductivity cell
having electrodes coated with platinum Take 20 g of soil in 100 ml
black. The Instrument is also available as beaker, add 40 ml of distilled water and
an already calibrated assembly shake intermittently for 30 minutes.
(Solubridge) for representing the Determine the conductivity of the
conductivity of solutions in dSm-1 (deci supernatant liquid with the help of
Siemen per meter) at 250C. conductivity meter. The electrical
Principle conductivity of saturation extract (E.C.) is
also determined for salinity ratings.
A simple wheatstone bride circuit
is used to measure EC by null method. EC Effect
The bridge consists of two known and (dS m-1)
fixed resistance r1, r2, one variable- <1 - No deleterious effect on crop
standard resistance r4 and the unknown r3. 1-2 - Critical for salt sensitive crops
The variable resistance r4 is adjusted until 2-3 - Critical for salt tolerant crops
a minimum or zero current flows through >3 - Injurious to most crops
the AC galvanometer. At equilibrium.
pH Meter :-
r1 r3 r1
= or = r3 X r4
r2 r4 r2
Since conductivity is reciprocal of

receptivity, it is measured with the help of
r3.
Electrical Conductivity Meter :-

Determination of Organic Carbon Content in Soil

F.C. Amule and N. Chouhan
The majority of mineral surface soils Apparatus and Reagents :

range from 1.2 to 3.5% organic carbon. 1. 500 ml conical flasks.
Since soil organic matter averages about 2. Pipette
58% carbon, it follows that soils generally 3. Burette
range from about 2 to 6 % organic matter 4. Phosphoric acid 85%.
(% O.M. = %C x 1,724. The factor 1.724 5. Sodium fluoride 2%.
= 100/58). There is also a close 6. Sulphuric acid 96 % containing 1.25
relationship between carbon and nitrogen % Ag2SO4.
in soils. Most organic matter average 7. Standard 1N K2Cr2O7 – 49.04 g/liter.
about 5% nitrogen so that the N : C ratio 8. Standard 0.5 N Fe (NH4)2 (SO4)2.
is 1:11.6. Therefore by multiplying the 6H2O 196 g in 800 ml water
soil organic matter percentage by 0.05 an containing 20 cc H2SO4 and diluted
approximate value for the soil nitrogen, to1 litre.
percentage is obtained. 9. Diphenylamine – 0.5g in 20cc water
In soil the chief source of some of and add 100 ml conc. H2SO4.
the nutrients essential for plant growth is
organic matter, such nutrients are N, S Procedure :
and boron is also largely derived from 1. Weigh 1g soil sample in 500 ml
organic matter. conical flask. Add 10 ml of 1 N
K2Cr2O7 and 20 ml conc. H2SO4
Principle : (containing Ag2SO4). Mix thoroughly
A suitable quantity of the soil is and allow reaction to proceed for 30
digested with chromic acid and Sulphuric minutes.
acid making the use of heat of dilution of 2. Dilute the reaction mixture with 200
Sulphuric acid soil is digested and organic ml water and 10 H3PO4 add 10 ml of
matter of the soil is oxidized. Excess of NaF solution and 2 ml of
chromic acid left over unreduced by the diphenylamine.
organic matter of the soil is determined by 3. Titrate the solution with standard FAS
a titration with standard Ferrous to a brilliant green colour. A blank
Ammonium sulphate solution using without soil should be run
diphenylamine as indicator. simultaneously.
In this exercise, chromic acid in Observations & Results :
the presence of excess H2SO4 is to be Weight of sample - 1g
used as an oxidizing agent for oxidizable Normality of K2Cr2O7 used - 1N
organic matter of the soil. The heat of Vol. of K2Cr2O7 - 10 ml
dilution of H2SO4 works as a standardized Normality of FAS - 0.5 N
ferrous sulphate solution. 10 0.003 x 100
OC (%) = Blank (Blank - Reading) X Wt. of soil
4 Cr6+ + 3C 4Cr+++ + 3C4+
2H2Cr2O7+ 3C+6H2SO4 2Cr2 (SO4)3+ 3CO2+8H2O Limits :
K2Cr2O7 + 4 H2SO4 K2SO4+ 2Cr2 (SO4)3+ H2O+3O
Low : < 5.0 g OC kg-1
2FeSO4 + H2SO4+O Fe2 (SO4)3 + H2O
Medium : 5.0 to 7.5 g OC kg-1
High : > 7.5 g OC kg-1

CAFT on Management of Soil Health: Challenges and Opportunities” 29 Sept . to 19 Oct. 2014
Determination of Available Nitrogen in Soil by Alkaline

Permanganate Method
F.C. Amule
liberated by potassium permanganate, in

Available Nitrogen in Soil (Alkaline
the presence of sodium hydroxide and the
Permanganate Method) :
released ammonia is condensed and
Principle : absorbed in known volume of a boric acid
A known weight of the soil is with mix indicator to form ammonium
mixed with alkaline potassium borate, the excess of which is titrated with
permanganate (KMnO4) solution and a standard sulphuric acid.
distilled. The organic matter present in
soil is oxidized by the nascent oxygen,
Reactions involved:
I. Distillation :
Alkaline
2 KMnO4 + H2O 2 MnO4 + 2KOH + 3O-
medium (Nascent oxygen)
Oxidation
R.CHNH2COOH + O- R.CO.COOH + NH3
Organic- N fraction (Ammonia)
Distillation
NH3 + H2O NH4OH
Absorption
3NH4OH + H3BO3 (NH4)3BO3 + 3H2O
(Green colour)
II. Titration
2(NH4)3BO3 + 3H2SO4 2(NH4)3SO4 + 2 H3BO3
(Pink colour and original)
Equipment and apparatus : with automatic dilution and addition of
1. KEL PLUS Automatic Nitrogen boric acid, NaOH and KMnO4. Both
Estimation System modes (auto and manual) are available
The said instrument is used for for distillation reagents addition.
determination of available nitrogen in soil.  Refrigerated Water Cooling System
It consists of the following: for Condenser (Model Kel Freeze): It
 Automatic Distillation System is refrigerated water cooling system for
(Model Classic DX): It is fully distillation and condensing system with
automatic distillation system with inbuilt compressor and recirculate
programmable auto run digital features, pump.

2. Electronic balance  25 ml each of potassium

3. Burette permanganate (0.32 %) and sodium
4. Conical flask hydroxide (2.5 %) solution is
5. Distilled water automatically added by distillation
Reagents : unit programme.
 The sample is heated by passing
1. 0.32 % potassium permanganate steam at a steady rate and the
(KMnO4) solution. liberated ammonia absorbed in 20 ml
2. 2.5 % sodium hydroxide (NaOH). of 2 % boric acid containing mixed
3. 2 % boric acid solution containing 20 indicator solution kept in a 250 ml
- 25 ml of mixed indicator / liter. conical flask.
4. Mixed indicator: 0.066g methyl red +  With the absorption of ammonia, the
0.099g bromocresol green dissolve in pinkish colour turns to green.
100 ml of 95 % alcohol.  Nearly 150 ml of distillate is
5. 0.02 N sulphuric acid (H2SO4). collected in about 10 minutes.
 The green colour distillate is titrating
Procedure :
with 0.02N sulphuric acid and the
 Weigh 5 g of prepared soil sample colour changes to original shade
and transfer it to the digestion tube. (pinkish color).
 Load the tube in distillation unit and  Simultaneously, blank sample
other sides of hose keep 20 ml of 2 % (without soil) is to be run.
boric acid with mixed indicator in  Note the blank & sample titer reading
250 ml conical flask. (ml) and calculate the available
nitrogen in soil.
Calculations :
R (Titer reading - Blank reading) × Normality of acid
× Atomic weight of nitrogen × Weight of one hectare of soil
Available N (kg ha-1) =
Sample weight (g) x 1000
R × 0.02 × 14 × 2.24 × 106
=
5 x 1000
Factor = R x 125.44
Interpretation of results :
Available N (kg ha-1) Soil rating
< 280 : Low
280-560 : Medium
> 560 : High

Determination of Total Nitrogen in Soil and Plant Samples

F.C. Amule
Total nitrogen is estimated by the micro- of which is titrated with a standard

Kjeldahl method as per procedure sulphuric acid.
suggested by AOAC (1995). The micro-Kjeldahl method consists of
Preparation of plant and soil samples : the three steps;
1. Digestion
The plant analysis has been 2. Distillation and
considered as a superior diagnostic 3. Titration.
technique for mineral content. Whole Equipment and apparatus :
plant is dried in open air for few days 1. KEL PLUS Automatic Nitrogen
after that it was further dried in hot air Estimation System :
oven at about 60 ± 2o C for eight to ten
hours per day to achieve complete drying. The said instrument is used for
After drying, whole plant is powdered determination of nitrogen. It consists of
with the help of a grinder, passed through the following :
2 mm stainless steel sieve and used for  Macro Block Digestion System
chemical assay. The soil sample from (Model KES 12L): This digestion system
definite depth was randomly collected is suitable for soil, plant, water,
from the field with the help of screw pesticides, fertilizers, food and feed
auger. All the possible technical samples. It is microprocessor based
precautions as prescribed for standard soil automatic twelve place macro block
sampling were also taken. Samples were digestion system with temperature
brought to the laboratory, air-dried in the controller fitted with sensor break
shade and grounded by wooden roller, protection (Microprocessor based) feature
thereafter sieved through 2 mm stainless and temperature range from 50-450 0C.
steel sieve and stored in polythene bags  Acid Neutralizer Scrubber (Model
and used for chemical assay. KEL VAC): It is used to neutralize the
acid fumes, which are absorbed in 15%
Principle : sodium hydroxide and dissolved in water
Nitrogen in samples like plant and stored in the system tank. After every 2
soil exists in a very complicated bonding cycles of digestion, the 15% sodium
structure. During digestion, a known hydroxide solution is replaced and after 3
weight of the plant/soil samples in the cycles of digestion, acid fumes dissolved
presence of sulphuric acid with catalyst in water tank is drained off and refilled
mixture under high temperature is with fresh water in the system tank.
digested where complicated structures are  Automatic Distillation System
broken to simple structure, thereby (Model Classic DX): It is fully automatic
releasing nitrogen in the form of distillation system with programmable
ammonium radical (NH4+). During auto run digital features, with automatic
distillation in presence of sodium dilution and addition of boric acid and
hydroxide, the released ammonia is NaOH. Both modes (auto and manual) are
condensed and absorbed in known available for distillation reagents addition.
volume of a boric acid with mix indicator  Refrigerated Water Cooling
to form ammonium borate, the excess System for Condenser (Model Kel

Freeze): It is refrigerated water cooling  Then block temperature is raised to

system for distillation and condensing 400 0C. The effective digestion starts
system with inbuilt compressor and re- only at 360 0C and beyond 410 0C.
circulator pump.  The sample turns light green colour
2. Electronic balance or colorless at the end of the
3. Burette digestion process.
4. Pipette
II. Distillation :
5. Conical flask
6. Measuring cylinder  After cooling the digestion tube, load
7. Distilled water the tube in distillation unit and other
Reagents : side of hose keep 20 ml of 4 % boric
acid with mixed indicator in 250 ml
1. Concentrated sulphuric acid (H2SO4). conical flask.
2. Catalyst mixture: Mix with 250 g  40 ml NaOH (40 %) is automatically
potassium sulphate (K2SO4), 50 g added by distillation unit programme.
cupric sulphate (CuSO4. 5 H2O) and  The digested sample is heated by
5 g metallic selenium powder in the passing steam at a steady rate and the
ratio of 50:10:1. liberated ammonia absorbed in 20 ml
3. 40 % sodium hydroxide (NaOH). of 4 % boric acid containing mixed
4. 4 % boric acid containing 20 - 25 ml indicator solution kept in a 250 ml
mixed indicator /liter. conical flask.
5. Mixed indicator: 0.066 g methyl red  With the absorption of ammonia, the
+ 0.099 g bromocresol green dissolve pinkish colour turns to green.
in 100 ml of 95 % alcohol.  Nearly 150 ml of distillate is
6. 0.02N sulphuric acid (H2SO4). collected in about 8 minutes.
Procedure :  Simultaneously, blank sample
I. Digestion : (without plant/soil) is to be run.
 Weigh 0.5 g of prepared plant sample III. Titration :
or 1 g of soil sample and transfer it to  The green colour distillate is titrating
the digestion tube. with 0.02N sulphuric acid and the
 Add 10 ml of concentrated sulphuric colour changes to original shade
acid and 5 g of catalyst mixture to the (pinkish color).
sample.  Note the blank & sample titer reading
 Load the digestion tubes in to the (ml) and calculate the total nitrogen
digester and heat the digestion block. content present in plant/soil samples.
 Switch on the digestion unit and set
the initial temperature 100 0C till
frothing is over.
Calculations :
R (sample titer-blank titer) x Normality of acid x
Atomic weight of nitrogen x 100
Nitrogen content in plant (%) =
Sample weight (g) x 1000
R x 0.1 x 14 x 100
= 0.5 x 1000
Factor = R x 0.28
R (sample titer-blank titer) x Normality of acid x
Atomic weight of nitrogen x 100
Nitrogen content in soil (%) = Sample weight (g) x 1000

R x 0.1 x 14 x 100
= 1 x 1000
Factor = R x 0.14
Crude protein content : References :
The total nitrogen is estimated by Subbiah, B.V. and Asija, G. L. (1956).
micro-Kjeldahl method as per procedure A rapid procedure for the
suggested by AOAC (1995) and the crude estimation of nitrogen in soils.
protein is calculated by the following Curr. Sci., 25: 259-260.
formula:
Crude protein content (%) = micro- AOAC, (1995). Official Methods of
Kjeldahl nitrogen content (%) x 6.25 Analysis. 16th edn. Association of
(based on the assumptions that nitrogen Official Analytical Chemists,
constitutes 16 % of protein). Washington, DC.
NITROGEN ANALYZER

Determination of Phosphorous in Soil and Plant Samples

R.K. Thakur
The phosphorus is an essential plant  Above both solution mix

nutrient and it occurs in many different thoroughly and made one litre in
forms. Therefore, a reliable procedure for volumetric flask with the help of
measuring the amount both in soil as well distilled water.
as in plant is needed. There are many  Add these dissolved reagents to
methods available for the determination, one litre of 5N H2SO4.
however, colorimetric measurement is
5. Ascorbic acid working solution
presented here:
(Reagent B): Dissolve 1.056 g of
Principle : ascorbic acid in 200 ml of reagent A
and mix. This ascorbic acid (reagent
Phosphorus is extracted from the B) should be prepared as required
soil with 0.5 m NaHCO3 at a nearly because it does not keep more than
constant pH of 8.5. The phosphate ion in 24 hours.
solution treated with ascorbic acid in an
acidic medium provides a blue colour 6. Standard phosphate solution:
complex. Measurement of the quantitative Weigh 0.4393 g of potassium
determination of phosphorous in soil dihydrogen phosphate (KH2PO4) into
(Olsen’s et al., 1954) one litre volumetric flask. Add 500
ml of distilled water and shake the
Reagents : contents until the salt dissolves.
Dilute the solution to one litre with
1. 0.5 M Sodium bicarbonate distilled water to get 100 ppm P
(NaHCO3) solution: Dissolve 42 g solution. Dilute 20 ml of 100 ppm P
of NaHCO3 in distilled water to get solution to one litre to get form-
one litre solution and adjust the pH of working solution of 2 ppm.
the solution to 8.5 by small quantity
of NaOH. Preparation of standard curve :
2. Activated Charcoal: Darco G-60 (P-  Take different concentration of P (0,

Free) 1, 2, 3, 4, 5, etc ml of 2 ppm standard
P Solution) in 25 ml volumetric
3. 5 N Sulphuric acid (H2SO4) flasks.
Solution: Add 141 ml of con. H2SO4
to 800 ml of distilled water. Cool the  Add 5 ml of the 0.5M NaHCO3
solution and dilute to one litre with extracting solution to each flask, and
distilled water. acidify with 5N H2SO4 drop by drop.
4. Reagent A:  Add about 10 ml distilled water and 4

ml of reagent ‘B’, then shake the
 Dissolve 12.00 g of ammonium solution.
paramolybdate in 250 ml of
distilled water.  Make the volume 25 ml by distilled
water.
 Dissolve 0.2908 g of potassium
antimony tartrate (KSbO.C4H4O6)  The intensity of blue colour is read
in 100 ml distilled water. on spectrophotometer at 660 nm
wavelengths after 10 minutes.
 Plot the curve by taking P Limits of available P in soil :

concentration on X axis and Very low : Less than 5 P kg ha-1
colorimeter reading on Y axis. Repeat Low : 5-10 P kg ha-1
the process till you get straight line Medium : 10-20 P kg ha-1
relationship.
High : 20-40 P kg ha-1
 Calculate the factor i.e. 1 colorimeter Very high : More than 40 P kg ha-1
reading is equal to how much ppm of
phosphorus? Determination of total phosphorus in
plant :
Procedure :
Principle : Vanadate molybdate and
 Take 2.5 g of soil sample in 150 ml orthophosphates react to give a yellow
conical flask and 0.5 g Darco G-60 colour complex in acidic medium. The
activated charcoal. intensity of colour provides the basis of
 Then add 50 ml of 0.5 M NaHCO3 quantitative measurement of total P in
solution and shake the solution for 30 plant (Koenig and Johnson, 1942).
minute in a shaker. Similar processes Apparatus and reagents :
run for a blank without soil.
 Colourimeter/spectrophotometer
 Filter the suspension through the
Whatman no. 40 paper.  50 ml volumetric flask
 Take 5 ml aliquot of the extract in a  ammonium molybdate ammonium
25 ml volumetric flask, and acidify vanadate (in NHO3) solution :
with 5N H2SO4. Dissolve 2.5 g (NH4)6 Mo7O2.4 4H2O
in 400 ml distilled water. Dissolve
 Add small quantity of distilled water, 1.25 g of ammonium vanadate in 300
and then add 4 ml of reagent B. ml boiling water. Add the ammonium
 The intensity of blue colour is read vanadate solution to the ammonium
on spectrophotometer at 660 nm molybdate solution and cool to room
wavelengths after 10 minutes. temperature. Add 250 ml conc. NHO3
and dilute to 1 lit.
Observations :
 Phosphate standard solution : Dissolve
1. Weight of soil sample : 2.5 g
0.2195 g KH2PO4 and dilute to 12%.
2. Volume of extractant used : 50 ml This solution contains 50µg P/ml.
3. Volume of filtrate used : 5 ml

Procedure :
Preparation of standard curve :
4. Absorbency : R
 Transfer 0, 1, 2, 3, 4 and 5 ml of 50
5. Absorbency from standard curve : A ppm P solution to 50 ml volumetric
flasks in order to get 0, 50, 100, 150,
6. Concentration of P for absorbency A : B ppm
200 and 250 µg P.
Calculation :  Add 10 ml vanadomolybdate reagent
R x F x 50 x 2.24 make up the volume and mix the
Available P (kg ha-1) = content thoroughly.
5 x 2.5
 Read the transmittance/absorbance at
Where, F (factor) = B / A 420 mµ (blue filter).
 Plot the reading against µg P and
calculate the factor (F).

Digestion of plant material : Calculation :

Take one gram of plant material in 50 µg = R
digestion flask. Add 10-15 ml of Diacid 1R = 50/R µg (Factor)
(3:1: Nitric acid : Perchloric acid) mixture Factor (F) x Reading x 100x100
and swirl the content in 150 ml Total (%) sample
volumetric flask. Place the content on hot P = ------------------------------------------
plate till the digestion is over. Filter the 10000 x 1000 x 10 x 1
solution in 100 ml conical flask, wash the Reference :
residue on filter paper several times with Koenig, R.A. and Johnson, C.R. (1942).
the hot water. Make up the volume with Colorimetric determination of
distilled water, store the solution in air biological materials Ind. Eng. Chem.
tight container. Analyt. Edn. 14 : 155-156.
Olsen, S.R., Cole, C.V., Watanabe, F.S. and
Estimation : Dean, L.A. (1954). Estimation of
 Transfer 10 ml dilute in 50 ml available phosphorus in soils by
volumetric flask. extraction with sodium bicarbonate.
 Add 10 ml ammonium molybdate Circ. U.S. Dept. Agric. 939 : 1-19.
vanadate solution shake the content.
 Make up the volume and record the
reading as per the procedure under
preparation of standard curve.
Spectrophotometer

Determination of Potassium in Soil and Plant Samples

S.S. Baghel
The available potassium i.e. (CH3COONH4) (77.08 eq.wt.) directly in

exchangeable and water soluble H2O and volume to be made one litre and
potassium is determined by extracting soil then adjust the pH 7.0 .
with neutral normal ammonium acetate (b) Standard Potassium Solution :
solution. The estimation of potassium is Dissolve 1.9066 g of dried KCl
carried out by flame photometer. (AR) in distilled water and dilute to one
1. Principle : litre. This is 1000 mg kg-1 K solution.
100 ml of this solution diluted to 1 lit. to
The principle underlying this is make 100 ppm K solution.
that a large number of elements when
excited in a flame, emit radiation of 4. Preparation of the standard curve :
characteristic wave length. The excitation Take 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and
cause one of the outer electron of neutral 10 ml of 100 mg kg-1 K solution in
atoms to move to an outer orbit of higher different 25 ml volumetric flasks. Make
energy level or the atoms may be excited up the volume with 1N NH4OAc Soln.
sufficiently to loose an electron Adjust the flame photometer reading at
completely from the attractive force of the zero with blank (zero K) solution and at
nucleus where excited atom return to 100 for 40 mg kg-1 K solution. Take the
lower energy level, light at characteristic flame photometer readings for every
wave length is emitted. Excited atoms or dilution. Plot the standard curve on graph
ions give line radiation at very definite paper by taking K concentration on X axis
wave length and thus K gives at 404.4 and and flame photometer reading an y axis.
767 mµ. The flame photometer This will give a factor (F) of 1 flame
employees a relatively low temperature photometer reading = 0.4 mg kg-1 K.
excitation and measures with a photocell
5. Procedure :
the emission intensity which is
proportional and to concentration in Take 5g soil in 100 ml conical
selected wave length (767 mµ) and for flask and add 25 ml of 1N NH4OAc Soln.
this red filter is used. Shake the content for 5 minutes and then
filter through Whatman No.1 filter paper.
2. Apparatus and reagents :
Potassium extract is measured by flame
a) Flame photometer with red filter, photometer after calibration.
b) Pipette, volumetric flasks and conical 6. Calculation :
flask (100 ml).
RxFx25x100x20x1.121
3. Reagents : Available K (kg ha-1) =
5 x 1000
(a) Neutral Normal Ammonium Acetate : = R x F x 11.217.
Add 58 ml of glacial acetic acid to
Limits of available K in soil :
about 600 ml H2O and then add 70 ml of
Very low : Less than 200 K kg ha-1
concentrated ammonia (sp. gr 0.90) Dilute
the solution to one litre. Then adjust pH Low : 200 – 250 K kg ha-1
of solution at 7.0 with the help of Medium : 250 – 400 K kg ha-1
ammonia or acetic Acid or this can be High : 400 – 600 K kg ha-1
prepared by dissolving ammo. Acetate Very high : More than 600 k kg ha-1
CAFT on Management of Soil Health: Challenges and Opportuni ties” 29 Sept. to 19 Oct. 2014
8. Precaution : (b) Determination of K :

a) These should not be any turbidity or Take the aliquot and get the
suspended particles in extract, it will reading of K through flame photometer
chock the capillary feeding tube . using red filter and calculate the amount
b) The gas and air pressure should be of K in the plant sample on the oven dry
constant. matter basis.
c) If sample reading goes beyond 100
then dilute the extract. K (%) in plant sample = X x 4 x 10-3
9. Determination of k in plant sample : References :
(a) Wet digestion : Black, C.A. (1965) Methods of soil
Place 1-2g of ground plant sample analysis Part I Am. Soc. Agron.
in 100ml digestion flask. Add 20-25 ml of Inc. Publi. Madison Wisconsin
acid mixture Acid mixture 750 ml conc. USA.
HNO3 + 150 ml conc H2SO4 + 300 ml of
HClO4 and mix the contents of the flask Flame Photometer :-
by swirling well. Heat the flask at a low
temp and then slowly increase the flame
or temp. of hot plate. Completion of
digestion is confirmed when liquid is
colorless. After cooling, add 20-25 ml
H2O and filter through whatman No.40
into a 100 ml/250 ml volume flask and
make up the volume.

CAFT on Management of Soil Health: Challenges and Opportuni ties” 29 Sept. to 19 Oct. 2014
Determination of Sulphur Content in Soil and Plant

N. Chouhan
Principle : and filtered in hot condition through

Whatman No. 42 filter paper. Cool
Besides some amount in the soil
solution, available sulphur in mineral soils
occurs mainly as adsorbed SO4= ions.
and dilute to one litre with dilute
Phosphate ions (as monocalcium
acetic acid.
phosphate) are generally preferred for
3. Barium chloride: Pass AR grade
replacement of the adsorbed SO4= ions.
BaCl2 salt through 1 mm sieve and
The extraction is also carried out using
store for use.
CaCl2 solution. However, the former is
4. Standard stock solution (2000 mg S
considered to be better for more efficient
L-1): Dissolve 1.089 g of oven dried
replacement of SO4= ions. Use of Ca salts
AR grade potassium sulphate per 100
have a distinct advantage over and leads
mL.
to easy filtration SO4= in the extract can
5. Working standard solution (10 mg S
be estimated turbid metrically using a
L-1): Measure exactly 2.5 mL of the
colorimeter/spectrophotometer.
stock solution and dilute to 500 mL.
A major problem arises when the
6. Barium sulphate seed suspension:
amount of extracted sulphur is too low to
Dissolve18 g of AR grade BaCl2 in 44
be measured precisely.; To overcome this
mL of hot water and add 0.5 mL of the
problem, addition of seed solution of
standard stock solution (given above).
known S concentration is added to the
Heat the contents to boiling and then
extract to raise concentration to easily
cool quickly. Add 4 mL of gum acacia-
detectable level. Sulphur in the extract
acetic acid solution to it. Prepare a
can also be estimated by a colorimetric
fresh seed suspension for each
method using barium chromate (Nemeth
estimation every day.
1964; Palaskar et al. 1981), but the
7. Dilute nitric acid (approx 25%):
turbidimetric method (Chesnin and Yien
Dilute 250 mL of AR grade conc.
1950) given below is mainly used in the
HNO3 to one litre.
soil testing laboratories.
8. Acetic-phosphoric acid: Mix 900 mL
Instruments : of AR grade glacial acetic acid with
300 mL of H3PO4 (AR grade).
(i) Colorimeter or spectrophotometer or
autoanalyzer. Procedure :
(ii) Mechanical shaker
1. Weight 20 g of soil sample in a 250
Reagents mL conical flask.
2. Add 100 mL of the monocalcium
1. Mono-calcium phosphate extracting phosphate extracting solution (500 mg
solution (500 mg P L-1): Dissolve 2.035 P L-1) and shake for one hour. Filter
g of Ca(H2PO4)2.H2O per litre. through Whatman No. 42 filter Paper.
2. Gum acacia-acetic acid solution: 3. Measure 10 mL of the clear filtrate into
Dissolve 5 g of chemically pure gum a 25 mL volumetric flask.
acacia powder in 500 mL of hot water 4. Add 2.5 mL of 25% HNO3 and 2 mL
of acetic-phosphoric acid. Dilute to

about 22 mL, stopper the flask and Determination of total sulphur in plant:
shake well. Sulphur is an essential plant
5. Shake the BaSO4 seed suspension and nutrient and occurs in many different
then add 0.5 mL of it and 0.2 g of forms. The procedure for total sulphur
BaCl2 crystals. Stopper the flask and estimation is as follows :
invert three times and keep.
6. After 10 minutes, invert 10 times and Digestion of plant material :
keep. After another 5 minutes, invert 5 Take one gram of plant material in
times. digestion flask. Add 10-15 ml of Diacid
7. Allow to stand for 15 minutes and (3:1: Nitric acid : Perchloric acid) mixture
then add 1 mL of gum acacia-acetic and swirl the content in 150 ml
acid solution. volumetric flask. Place the content on hot
8. Make up the volume, invert three plate till the digestion is over. Filter the
times and keep aside for 90 minutes. solution in 100 ml conical flask, wash the
9. Invert 10 times and measure the residue on filter paper several times with
colour intensity at 440 nm (blue the hot water. Make up the volume with
filter). distilled water, store the solution in air
10. Run a blank side by side. tight container.
Preparation of standard curve for S : Estimation :
1. Place 2.5,5.0,7.5,10.0,12.5 and 15.0 Take 10 ml aliquot from extract
mL portions of the working standard and proceed as per the method described
solution (10 mg S L-1) into a series of under preparation of standard curve
25 mL volumetric flasks to obtain (Bardsley and Lancaster, 1960).
25,50,75,100,125 and 150 µg S.
2. Proceed to develop turbidity as Calculation :
described above for sample aliquots.
3. Read the colour intensity and prepare 5 µg = R
the curve by plotting readings against 1R = R/5 µg (Factor)
sulphur concentration (In µg in the
final volume of 25 mL) Total S (%) =
Factor x Sample R x1000 x 100 x 100
Calculation : -----------------------------------------------
1000 x 10 x 1
R x 100
Available S in soil (mg kg-1) = References :
10 x 20
Where, r stands for the quantity of S in Arora, C.L. and Bajwa, M.S. (1994).
mg as obtained on X-axis against a Curr. Sci. 66 : 314-316.
reading. Bardsley, C.S. and Lancaster, J.P. (1960).
Proc. Soil Sci. Soc. Am. 24 : 265.
Chesnin, L. and Yien, C.N. (1951). Proc.
Soil Sci. Soc. Am. 15 : 149.

Determination of Micronutrients in Soil and Plant Samples

by Atomic Absorption Spectrophotometer
G.D. Sharma and R.K. Thakur
All atoms can absorb light at certain thickness of the medium absorbs an equal
discrete wavelengths corresponding to the fraction of the light passing through it.
energy requirement of the particular atom. Beer’s Law : Light absorption is
When at ground state the atom absorbs proportional to the number of absorbing
light it is transformed into the excited atoms in the sample.
state. It is the same atom containing more The combined Beer - Lambert law may
energy. This energy is measured in be given as :
relation to the ground state and a It = Io – (abc)
particular excited state say for example in Io
case of Na may be 2.2 eV (electron volts) thus, log10 --- = abc = absorbance
above the ground state. It
Each transition between different Where,Io = incident radiation power
electronic energy states is characterized It = transmitted radiation power
by a different energy and by a different a = absorption coefficient
wavelength. These wavelengths are b = length of absorption path
sharply defined and when a range of c = concentration of absorbing
wavelengths is surveyed, each wavelength atoms
shows as a sharp energy maximum (a i.e. the absorbance is proportional
spectronic line). These characteristic lines to the concentration of the elements for a
distinguish atomic spectra. The lines, given absorption path length at any given
which originate in the ground state of wave length.
atom, are most often of interest in atomic In principle, it might be possible
absorption spectroscopy. These are called to calculate the concentration directly
the resonance lines. The atomic spectrum, from the above equation. In practice,
characteristic of each element, then however, the a and b are constants hence
comprises a number of discrete lines, the variation of results is directly related
some of which are resonance lines. Most the concentration of atoms. For analysis,
of the other lines arise from excited states the absorbance of different concentration
rather than the ground state. The lines of of standard solution is first measured with
excited states are not useful generally in the help of atomic absorption
atomic absorption analysis as most of the spectrophotometer and then the results of
atoms in a practical atomizer are found in unknown samples are compared with the
the ground state. standards and thus concentration of
The relationship of light absorbed unknown sample is calculated.
by the atom in ground state and their Atomic absorption spectrophotometer :
concentration in the solution is defined in Atomic absorption spectro-photo-
the fundamental laws of light absorptions. meter is based on the principle that when
Lambert’s Law : The portion of light atomic vapours of an element are
absorption by a transparent medium is irradiated by the radiation of a
independent of the intensity of the characteristic wavelength (i.e. the light
incidence light and each successive unit from a source whose emission lines are

those of the element in question), they It measures the absorbance in

absorb in direct proportion to the volts. It is normally a strip chart recorder,
concentration of the element being a digital display, a meter or printer. The
determined. presently available AAS have features
Instrument features : like automatic calibration with one or
more standards, automatic curve
A wide range of atomic absorption
corrections, automatic and foolproof gas
spectrophotometer is available today, all
switching and calculation of average and
of them have the basic features in
standard deviations in repetitive runs.
common and consist of the following
components: Collection and preparation of soil and
(a) A Light source : plant samples : To avoid contamination,
soil samples are to be collected in plastic
A Light source emits the spectrum tub, using rust free instrument or wood
of the element to be determined. The most and kept in polythene lined cloth bags.
widely used light source is hollow Samples are prepared with the help of
cathode lamp which is designed and wooden mortar and pestle and sieved
operated in such a way that the lines to be through 2mm nylon screen/mosquito net
measured are sharp, of stable intensity cloth or stainless steel sieve.
and free from background. Similarly plant samples (leaves,
(b) Atomizer-Burner assembly : grains or straw) should be washed with
A means of producing atomic 0.01N HCl, rinsed with glass distilled
vapours of the element to be analyzed. water dried in oven at 65°C and crushed
The solution to be analyzed is drawn by with the help of stainless steel scissors.
capillary and converted into stream of Soil extraction : DTPA offers the most
compressed air to a fine spray which after favorable combination of stability
condensation of larger droplets is mixed constants for the simultaneous
with the fuel gas acetylene and burnt in a complexing of Zn, Cu, Fe and Mn, Cd,
long flame (at 2100-2400oC) in a stainless Co, Ni and Pb (Lindsay and Norvell,
steel burner. 1978). Buffering of extractant in a slightly
(c) A Monochromator : alkaline pH range (7.3) by including
It isolates the absorbing soluble Ca2+, avoids the dissolution of
resonance lines from other non absorbing CaCO3 with the release of occluded
lines. When the light coming from the micronutrients due to CO2 partial pressure
HCL, after traversing the flam, enters the of approximately 10 times that in
monochromator which is already set at the atmosphere, as the soil contains slightly
wavelength of the resonance lines of the higher CO2 levels than found in the
desired element, the monochromator atmosphere.
performs its function. (a) Extracting solution : (0.005 M
(d) A Detector : DTPA) Dissolve 1.9679g of DTPA
(Diethylene tri amine penta acetic
It measures the magnitude of acid) + 13.3 ml TEA (Triethanol
absorption of the characteristic radiation. amine) + 1.47g CaCl2 .2H2O in 200
(e) A Photomultiplier Tube : ml distilled water, dilute to 900 ml,
It amplifies the absorption signal adjust pH 7.3 with 6N HCl while
and converts the light radiation into stirring and then make upto 1 liter
electrical energy. and mix thoroughly.
(f) A readout system : (b) Apparatus required : Shaker
(Horizontal or Rotatory), iodine value

flasks (100 ml capacity) or conical used in the lamp because a continuum is

flasks with glass stoppers, funnels, produced rather than a line spectrum.
filter paper whatman No.1, plastic The deuterium lamp is different from
storage bottles and Atomic absorption a hollow cathode lamp in construction and
spectrophotometer. operation. The lamp incorporates a
(c) Stock Standard Solutions : The heated, electron-emitting cathode, a metal
standard solutions of different micro- anode and a restrictive aperture between
nutrients should preferably be the two. A discharge current of several
prepared by using their wires. hundred milli amperes excites the
Dissolve 1g wire in a minimum deuterium gas. The discharge is forced to
volume of 1:1 nitric acid and dilute to pass through the small aperture, forming a
1000ml with distilled water to obtain defined area of high excitation and hence
1000 µg/ml solution of micro-nutrient, high light emission. A suitable window
or take salts of metals as follows: transmits the light to the spectrometer's
optical system.
Zn- 4.398g l-1 ZnSO4,7H2O To obtain successful background
Cu- 3.929g l-1 CuSO4,5H2O correction the deuterium lamp must be
Fe- 4.977g l-1 FeSO4,7H2O correctly aligned, and its intensity must be
Mn- 3.598g l-1 MnSO4,H2O. matched to that of the hollow cathode
The prepared standards are also lamp.
available in the market. Out of these It is important that both the
standards, prepare working solution of deuterium source and the hollow cathode
50 ppm. Then a series of standard source are aligned to follow the same
solution of 0.5, 1.0, 1.5, 2.0 and 2.5 optical path. If they are not, then the two
ppm may be prepared for each metal. measurements may not be made on the
(d) Background correction : The same atom population and significant
reading of a spectral line always errors may occur.
includes any contribution from the In order to balance the intensity of
flame and sample matrix. Failure to the deuterium lamp with the hollow
correct properly for the background cathode lamp, it may be necessary to
reading can be a source of serious change the hollow cathode lamp current
error. Although the need for fast to a higher or lower value depending on
background correction is most the relative intensities of the lamps.
obvious with graphite furnace work, Although most modern AA spectro-
it is also a consideration with flame photometers incorporate so called
atomic absorption. "simultaneous" background correction, they
The most common method of rely on two measurements separated slight
background correction in atomic in time. One measurement is of the total
absorption spectrometry involves the use absorbance (atomic plus background) and
of a continuum source such as a the other is of the background only. The
deuterium lamp to measure the background is electronically subtracted
background. The source used is a from the absorbance to give the background
deuterium filled discharge lamp, which corrected atomic absorbance with the
emits an intense continuum spectrum continuum source method of background
from 190 nm to about 400 nm. This is the correction, the total absorbance is measured
during the hollow cathode lamp pulse and
region where most atomic absorption
the background during the deuterium lamp
lines occur and where the effects of
pulse. With the Zeeman method using a
background absorption are most modulated magnetic field, the total
pronounced. The poly-atomic gas D2, is

absorbance is measured with the magnetic hot plate till the residue is colour less.
field off and the background with the field on. Now take off, cool dilute with
distilled water and filter through
(e) Soil analysis : Weigh 12.5g soil whatman No.1 filter paper. Make up
sample in 100 ml iodine value flasks. the volume of digestate to 50 ml.
Add 25 ml DTPA solution. Shake Read for micronutrient content on
this mixture for 2 hours on shaker at atomic absorption spectro-
70 to 80 oscillation per minute, filter photometer.
through acid washed distilled water
Factors : For soil multiply the
rinsed, whatman No.1 filter paper and
concentration read on AAS computer
collect the filtrate in plastic bottles.
sheet by “2”. Similarly for plants the
Determine the content of
multiplying factor will be 100 to get
micronutrients on atomic absorption
concentration in mg kg-1.
spectrophotometer.
(i) Plant analysis : Weigh 0.5g plant Reference :
sample in a conical flask (corning,
100 ml capacity). Add 10 to 12 ml of Lindsay, W.L. and Norvell, W.A. (1978).
di acid mixture (1 perchloric + 4 Proc. Soil Sci. Soc. Am. 42 :
nitric acid) and digest the mixture on 421-428.
Atomic Absorption Spectrophotometer

Estimation of Boron in Soils, Plants and Water

G.D. Sharma
Boron occurs as anion in soils and is  In alkaline soils the availability of B is

required by plants in very small quantity. high and may be even toxic for plant
Water soluble B makes the estimate of its growth.
availability to plants. Total boron in soils Besides this the low moisture
varies from 20 to 200 mg kg-1 and availability also causes B deficiency.
available (water soluble) boron in soils Irrigation water containing Boron
ranges from 0.03 to 12 mg kg-1 between 0.3 to 0.6 mg kg-1 can be used
respectively. The threshhold value safely, whereas, irrigating soils with water
ranging from 0.1 to 0.5 mg kg-1 (water containing 1 to 3 mg kg-1 B causes
soluble B) depends upon the soil type, toxicity of B in plants.
crops, and other factors, below which the Boron determination (Azomethine H
response to applied boron may be Method) :
expected. Some sensitive crops to boron Azomethine H forms coloured
deficiency are listed in table 1. Its complex with H3BO3 in aqueous media.
availability is affected by soil pH as Over a concentration range of 0.5 to 10
under: µg B/ml the complex is stable at pH 5.1.
 Deficiency of B is generally observed Maximum absorbance occur at 420 nm
in old acid leached soils. with little or no interference from a wide
 Availability increased with the rise in variety of salts. This technique is rapid,
soil pH having significant positive reliable and more convenient to use than
correlation with pH rising from 4.7 to traditional procedures employing carmin,
6.7. curcumin or quinalizarin (John et al.,
 In neutral, saline and calcareous soils 1975).
the B availability again decreases with
the rise in soil pH having significant Apparatus :
negative correlation with the rise in pH (1) Spectrophotometer
from 7.1 to 8.1. In calcareous soils B (2) Poly-propylene tubes 10 ml capacity.
fixation occurs with the condensation
of borate radical into long chains in the Reagents :
presence of Ca. 1. Distilled water
Table 1 : Sensitivity of crop to B deficiency 2. Buffer solution : Dissolve 250 g of
ammonium acetate (NH4OAc) and 15 g
Sensitive Medium Low of ethylenediaminetetracetic acid
Alfalfa Apple Barley (EDTA disodium salt) in 400 ml of
Cauliflower Cabbage Beans distilled water. Slowly add 125 ml of
Rape seed Carrot Corn glacial acetic acid and mix.
Conifers Clover Grasses 3. Azomethine H reagent : Dissolve 0.45 g
Peanuts Cotton Oat of azomethine H in 100 ml of 1% L
Sugarbeet Onion ascorbic acid solution. Fresh reagent
Turnip Pea should be prepared weekly and stored in
Potato a refrigerator.
Soybean 4. Calcium hydroxide suspension : Add
Wheat 0.4g Ca(OH)2 to 100 ml distilled water.
Rice

5. 0.1 N HCl : Add 8.3 ml conc. HCl to Transfer 20 ml aliquot to

900 ml distilled water, mix, cool to evaporating dish, add 2 ml Ca(OH)2)
room temperature and make up the suspension and evaporate the solution to
volume to 1000 ml. dryness. Heat the evaporating dishes gently
6. Calcium chloride 0.01 M Dissolve 1.11 to destroy organic matter, cool to room
g of anhydrous CaCl2 in 900 ml temperature, add 5 ml 0.1N HCl. Triturate
distilled water and make up the volume the residue with rubber policeman to ensure
to 1000 ml. the complete dissolution of the residue
7. Boron standard solution : Dissolve (Bingham, 1982).
0.114g of Boric acid (H3BO3) in For analysis of B pipette 1 ml of the
distilled water and adjust the volume to aliquot and proceed as for the standard
1000 ml. Each ml contains 20 µg B. curve.
Dilute 10, 20, 30, 40 and 50ml of the 2. Plant digest : Take 0.5 g plant
stock solution to 100 ml with distilled sample in porcelain/platinum dishes Add
water to have solution with B 0.5 g Ca(OH)2. Ignite the sample in the
concentration of 2,4,6,8 and 10 µg of muffle furnace at 550°C for 4 hours to
B/ml respectively. Include a distilled obtain white grey ash. Cool the dishes and
water sample for the 0.0 µg of B/ml moist the ash carefully with little distilled
standard solution. water and then add 5 ml 0.1N HCl. Transfer
the content in to 25 ml volumetric flask mix
Procedure :
and make up the volume to 25 ml with
Take 1 ml of aliquot of blank and
distilled water. For analysis of B take 1 ml
diluted B standards into a 10 ml
of the aliquot and proceed as for the
polypropylene tube, add 2 ml of buffer
standard curve.
solution and mix. Add 2 ml of azomethine
H reagent, mix and after 30 minutes read 3. Water analysis : Take suitable
the absorbance at 420 nm on quantity of water sample (containing 0.2 to
spetrophotometer. With the help of 5.0 µg B) in porcelain dishes add 2 ml
absorbance readings of standard solutions Ca(OH)2 and proceed as described for soil
of different concentration of B the standard extract. It is important to keep a definite
curve is drawn and a factor for volume of aliquot i.e. 1 ml of either soil,
concentration of B for 1 absorbance is plant or water in final step of B
calculated which is utilized to calculate B in determination.
the soils, plant or water sample.
References :
Preparation of Extracts :
Berger, K.C. and Truog, E. (1939). Boron
1. Soil extracts : The hot water determination in soils and plants.
soluble extraction procedure of Berger and Ind. Eng. Chem. Anal. Ed. 11 : 540-
Truog (1939) is being used widely with 545.
slight modification of adding dilute Bighman, F.T. (1982). Boron p. 501-508. In
electrolyte (0.01 M CaCl2) instead of water A.L. Page (ed.) Methods of Soil
only. This provides clear, colourless extract Analysis. Part II Agron. Monogr. 9
which eliminates the need of charcoal for ASA and SSSA, Mandison, W.I.
decolourzation. Beside this a negative error, Jackson, M.L. (Ed.) (1958). Boron
associated with B adsorption by charcoal, is determination for soil and plant
also removed. tissues. In Soil Chemical Analysis
Place 20 g air dry soil in 250 ml low page 370-387.
B flat bottom flasks and add 40 ml of 0.01 John, M.K., Chuah, H.H. and Neufld, J.H.
M CaCl2 solution. Attach water cooled (1975). Application of improved
reflux condenser to the flask. Heat the azomethine H method for the
flasks for 5 minutes and then cool and filter determination of boron in soil and
the suspension in plastic bottles. plants. Anal. Lett. 8 : 559-568.

Estimation of Microbial Biomass Carbon in Soil

R.K. Sahu
Microbial biomass carbon : same volume of distilled water similarly to

make the chloroform free of ethanol and the
It was estimated by chloroform bottom whitish phase was collected.
fumigation extraction method (Brookes et 4.One set of soil samples were taken in
al., 1985). crucible and fumigated with ethanol free
Principle: chloroform in a vacuum desiccator. For the
Overnight fumigation of chloroform purpose 20 ml ethanol free chloroform was
is done to kill al the organisms in soil taken in petridish and was placed inside the
samples; after which the amount of the desiccator the bottom portion inside the
organic C in the sample can be measured by vacuum desiccators. It was attached to the
fumigation- extraction method. vacuum pump and the air was evacuated
until the chloroform starts boiling to
Fumigation extraction method:-
The microbial biomass constituents saturate the desiccators with chloroform
released by CHCl3 fumigation treatment can fumes. Then the vacuum desiccator was
be extracted directly through chemical kept in dark room for overnight.
extractants and the readily oxidisable C 5.Next day the vacuum was released and
contained in the extract can be measured chloroform was removed from the
desiccator.
through standard chemical procedures.
6.10 g each of fumigated and non fumigated
Reagents soil samples were weighed in 150 ml
1. Ethanol free chloroform conical flask and 40 ml of K2SO4 (0.5 M)
Conc. H2SO4 was added to each flask. Samples were
2.
3. 0.5M K2SO4: 43.563 g K2SO4 was shaken for 30 minutes on a rotary shaker.
dissolved in distilled water and diluted 7.Both the samples were filtered with
with 500 ml. Whatman no. 42 filter paper, labelled and
4. 0.2 N K2Cr2O7: 0.9808 g K2Cr2O7 was freezed until digestion.
dissolved in 100 ml of distilled water. 8.10 ml of the filtrate was taken in 100 ml
5. Orthophosphoric acid conical flask and 2 M of K2Cr2O7 (0.2 N)
6. 0.005 N Ferrous Ammonium Sulphate and 10 ml of con. H2SO4 was added to it.
(FAS): 3.92 g of ferrous ammonium The contents of the flask were allowed to
sulphate was dissolved in 0.15 ml conc. cool for half an hour then 5 ml
H2SO4 and then diluted to 2 liters by orthophosphoric acid was added along with
distilled water. 200 ml distilled water. Minimum two blanks
7. Ferroin/Diphenylamine indictor were also run with 10 ml distilled water.
9.Few drops of diphenylamine indicator were
Procedure
added and titrated against ferrous
1.10g of soil (three sets of each soil sample) ammonium sulphate (0.2 M) till the colour
was weighed and kept in to 100 ml changed from violet to green.
beakers each.
Calculation:
2.8 ml of distilled water was added to both
beakers and were incubated for seven days Microbial carbon Fumigated C – Unfumigated C
at 250C in an incubator. (ppm) = 0.44
3.20 ml of chloroform was taken in a Reference:
separating funnel. It was washed two times Brookes, P.C., Kragt, J.F., Powlson, D.S. and
with conc. H2SO4 (with half of the volume Jenkinson D.S. (1985). Chloroform
of chloroform) and the acid (bottom phase) fumigated and the release of soil nitrogen.
was discarded. It was washed twice with the The effect of fumigation and temperature.
Soil Bio. Biochem., (17): 831-835.

Preparation of thematic maps of land use/land cover through GIS techniques

G.S. Tagore and F.C. Amule
Geographical information system (GIS) achieved, but it requires too much time
is an effective tool and a decision support and labour. To overcome such difficulty,
system involving the integration of GIS technique comes as a helping tool in
spatially referenced data in solving order to generate such comprehensive
environmental problem. With the help of maps and that too in a precise manner.
conventional methods it could be
Fig 1. Flowchart showing the conceptual view of data stream in GIS

CAFT on Management of Soil Health: Challenges an d Opportunities” 29 Sept. to 19 Oct. 2014
Methodology adopted for generation of thematic maps

a) Collection of data like survey of India Toposheet etc.
b) Thematic maps like land use or land cover, Geo
morphology maps have been collected and Geo referenced.
c) Collect the Ground truth data and check the delineated units
of the maps.
d) All the thematic maps prepared integrated and analysed to
get statistical findings.
LU LC Maps of Sanwer tehsil of Indore district (Madhya Pradesh)

Land is become scarce resource due to consuming and also not available on real-
immense agricultural and demographic time basis. Application of remotely
pressure. Hence, information on land use sensed data made possible to study the
and land cover and possibilities for their changes in land cover in less time, at
optimal use is essential for the selection, low cost and with better accuracy in
planning and implementation of land use association with Geographical
schemes to meet the increasing demands Information System (GIS) that provide
for the basic human needs and welfare. suitable platform for data analysis,
This could be achieved through update and retrieval according to many
conventional methods of surveying and researchers.
mapping which were costly, time
Fig- 2 sampling sites selected Fig-3 land use land cover map of sanwer
The number, types of applications and existing databases. Once information

analyses that can be performed by GIS are system they are created, any information
large and diverse based on available required for user or client would be
geographic data sets. The Geographical available at one place, updated,
Information System could be used to manipulated, retrieved and it easy to make
reduce data collection demands by decisions.
extracting valuable information from

Gamma Irradiation and its Importance for Food Preservation

R.K. Thakur and G.D. Shrma
Gamma irradiation has been extensively producer of fruits and vegetables live stock
used for food irradiation and sterilisation, and marine products. India has a
killing of fungus and micro organisms, tremendous potential as the world largest
sterilisation of medical accessories and food factory.
surgical equipments, high energy radiation It has been estimated that about 30-
chemistry, seed irradiation and 35% of fruit and vegetables of worth Rs
semiconductor irradiation. Gamma 3000/- corers are perished every year. The
reasons for such losses are seasonal nature
Chamber can also be used in many other
of fruits and vegetables production. The
research applications which require
long distance between production and
irradiation of materials with ionizing consumption centers and also rising gap
radiations to varying doses. between demand and supply. The hot and
The radiation processing of food humid climate in the country is also quite
involve the controlled application of energy favorable for the growth of numerous
from ionizing radiation such as gamma insects and micro organisms that destroy
rays, electrons and X rays for food stored crops and cause spoilage of food
preservation. The gamma rays and X-rays every year. The spoilage also occurs due to
are short wavelength radiation of chemical and physiological changes in
electromagnetic spectrum, which includes stored foods. To preserve the food and food
radiowaves, microwaves, infrared, visible, products, technologies such as freezing
and a violet light. Radioisotopes such as caning sun drying pickling fermentation
cobalt 60 and caesium-137 emit the gamma have been recommended by researchers but,
rays, while machines using electricity each of these methods have its own merits
generate electrons and X-rays. The gamma and limitation. The search for an alternative
rays and electrons are distinguished from newer economical methods to preserve food
other form of radiation by their ionizing and causes least changes in sensory quality
ability. (That they are able to break have been under taken since long back, and
chemical bond when absorbed by material). has been observed that radiation processing
The product of ionizing radiation may be of food is one of the latest method
electrically charged ions) or neutral (free developed for food preservation.
radicals). These there further react to cause
change in an irradiated material known as Irradiation by Gamma Chamber 5000 :
the process of radiolysis. It is this reaction Gamma Chamber 5000 is a
that causes the death of micro- organism, compact self shielded cobalt-60 gamma
insect and parasites during food irradiation.
irradiator providing an irradiation volume
The conservation and preservation
of approximately 5000cc. The material for
of food is a prerequisite for food security.
It provides self-reliance to nation. The
irradiation is placed in an irradiation
Indian Food Industries contributes about chamber located in the vertical drawer
25-28% towards GDP. The food processing inside the lead flask. This drawer can be
sector provides 60-65% employment with a moved up and down with the help of a
turnover of in US$ 36.1 billion of which system of motorised drive which enables
US$ 27.8 billion in organized sector, any precise positioning of the irradiation
change or any stagnation in technology will chamber at the centre of the radiation
inevitably have very large impact field.
throughout the economy. India is a potential
Radiation field is provided by a set Features :

of stationary cobalt-60 sources placed in a  Safe and self-shielded: The shielding
cylindrical cage. The sources are doubly provided is adequate to limit the
encapsulated in corrosion resistant radiation field on the external surface of
stainless steel pencils and are tested in the unit, well within the permissible
accordance with international standards. levels. No additional shielding is
Two access holes of 8 mm diameter are required for its installation and use.
provided in the vertical drawer for  Automatic control of irradiation time:
introduction of service sleeves for gases, Built-in timer provides accurate control
thermocouple, etc. A mechanism for of irradiation time from 6 seconds
rotating/stirring samples during onwards. The unit can also be operated
manually. Solid state programmable
irradiation is also incorporated. The lead
controls have been provided. In the
shield provided around the source is
event of power failure battery backup
adequate to keep the external radiation displays the programmes.
field well within permissible limits. The  Manual control of irradiation
Gamma Chamber 5000 unit can be temperature: It is possible to irradiate
installed in a room measuring 4 metres x samples at low or high temperature by
4 metres x 4 metres. circulating liquid nitrogen or hot air.
GAMMA CHAMBER 5000 These can be introduced through the
service sleeves provided in the vertical
drawer. The irradiation temperature is
sensed by a thermocouple and displayed
on the panel.
 Remote operation: An additional table
top control panel is provided for remote
operation in addition to the normal one
provided on the unit.
 Dose uniformity: Stationary source
pencils, symmetrically placed in a
cylindrical cage ensure good uniformity
of radiation field in the sample chamber.
In addition a mechanism is also provided
for rotating/stirring samples during
irradiation.
 Easy loading and unloading of
samples: Sample chamber extends to a
convenient height for easy loading and
unloading of samples.
 Safety assurance: The design of Gamm
Chamber conforms to American
National Standards, ANSI-N433.1-1977
for safe design and use of self-contained
dry source storage gamma irradiators
(Category I). It also meets the
requirements of type B(U) package for
safety in transport of radioactive
materials as per AERB code No.SC/TR-
1, 1986 of Atomic Energy Regulatory
Board of INDIA.

Applications : photographic film, Perspex and cobalt

glasses. The poly vinyl chloride (PVC)
Gamma Chamber is a versatile
dosimeters are impregnated with a dye. The
equipment for research studies in many
Hydrogen chloride is released from the
fields such as:
PVC by irradiation and it produces a
 Radiobiology
qualitative or quantitative change in the
 Preservation of tissue grafts
colour of the dye to indicate the dose
 Mutation breeding
received.
 Food preservation
 Sterile male insect technique Dose distribution :
 Biological and genetic effects of The penetration of gamma radiation
radiations depends on the density of the food as well
 Radiation chemistry as the energy of the ray. At a density of
 Radiation effects on materials 1000 kg m-3 half of the rays are absorbed in
 Radiation sterilization 11 cm. Halving the density approximately
 Modification of properties of double the depth of penetration. The
materials uniformity of dose distribution can be
expressed as a ratio of D max : D min. For
Food Preservation by Gamma Radiation
sensitive food such as chicken the ratio
The radiation processing of food is should be as low as possible1.5.
carried out inside an irradiation chamber
Potential Applications of Gamma Radiation:
shielded by 1.5 - 1.8 meter thick concrete
walls. Food either pre-packed or in bulk The radiation dose administrated to a
placed in suitable containers is sent into the food depends on the resistance of the
irradiation chamber with the help of an organisms present and the objective of the
automatic conveyor. The conveyor goes treatment. The maximum recommended
through a concrete wall labyrinth, which dose is 15 kGy, with average dose not
prevents radiation from reaching the work exceeding 100 kGy. Various application of
area and operator room. When the facility is this technology are as under:
not in use the radiation source is stored 1. Sterilisation ( or radappertisation) :
under 6 meter deep water. The water shield It is possible to sterilize meat and
does not allow radiation to escape in to the other product, the dose required exceed the
irradiation chamber, thus permitting free current limit of 10 kGy. A dose of 48 kGy
access to personnel to carry out plant is needed for 12 D reduction of Cl.
maintenance. For treating food, the source butulinum. High dose makes the product
is brought to the irradiation position above organoleptically un acceptable.
the water level after activation of all safety
devices. The goods in aluminum carriers or 2. Reduction of pathogens (radicidation):
tote boxes are mechanically positioned Food poisoning bacteria such as
around the source rack and are turned round salmonella typhimurium are less resistant to
their own axis, so that contents are radiation than Cl. Botulinum, and doses of
irradiated on both the sides. The absorbed 3-10 kGy are sufficient for destruction.
dose is determined by the residence time of
the carrier or tote box in irradiation 3. Prolonging shelf life (or radurisation) :
position. Relatively low doses are needed to
Measurement of radiation dose : destroy yeast, moulds and non-spore
forming bacteria. This process is used to
Placing dosimeters at various increase shelf life by an overall reduction of
positions in a tote box or carrier we can vegetative cells.
check the absorbed dose. The dosimeters
are made from a material including

Table 1: List of radiation processing facilities available in the world :

S. No. Country No. of Food Commodities
irradiators
1. Algeria 1 Potato
2. Argentina 1 Spices, spinach, cocoa powder
3. Bangladesh 1 Spices, onion, dried fish
4. Belgium 1 Spices, dehydrasted vegetables, deep frozen foods
5. Brazil 3 Spices, dehydrasted vegetables, fruits, vegetables, grain
6. Canada 1 Spices
7. Chile 1 Spices, dehydrasted vegetables, onion, potato, poultry meat
8. China 11 Spices and vegetable seasonings, Chinese sausage, garlic,
apple, potato, onion, dehydrated vegetables, sauses, rice,
tomatoes
9. Croatia 1 Spices, food ingredients, dried beef noodles
10. Czech. 1 Spices, dry food ingredients
Repulic
11. Cuba 1 Potato, onion, beans
12. Denmark 1 Spices
13. Finland 1 Spices
14. France 5 Spices, vegetable seasonings, herbs, poultry (frozen
boneless chicken, dried fruit, frozen frog legs, shrimp)
15. Hungary 1 Spices, onion, wine cork, enzyme
16. India 2 Spices, onion, potato
17. Indonesia 2 Spices, rice
18. Iran 1 Spices,
19. Israel 1 Spices, condiments, dry ingredients
20. Japan 1 Potato
21. Korea 1 Garlic powder, spices, condiments
Republic
22. Mexico 1 Spices, dry food ingredients
23. Netherland 1 Spices, frozen products, poultry dehydrated vegetables, egg
powder, packaging material
24. Norway 1 Spices
25. Poland 3 Not specified
26. Peru 1 Spices, food additives, animal feed
27. South Africa 4 Spices, shelf-stable food, fruits
28. Thailand 1 Spices, fermented pork sausages, enzymes
29. Ukraine 1 Grain
30. UK 1 Spices
31. USA 10 Spices, poultry, fruits, vegetables
32. Vietnam 1 Onion
33. Yugosla 1 Spices
(Source : ICGFI, Food & Environmental Protection Section, Update, 1997)

4. Control of ripening : 4. Amenability of a particular food

Fruits and vegetables can be commodity to radiation processing has to
irradiated to extend their shelf life about 2-3 be tested in a laboratory
time when stored at 100C The ripening and
maturation of fruits are arrested by inhibition
Plant Mutation Breeding by Gamma
of hormone production and interupting the Radiation
biochemical process of cell division. Plant mutation breeding by
5. Disinfestations : radiation has been investigated for long
Grain and tropical fruits may be time in many countries. New mutant
infested with insect and larvae, they reduces varieties give us useful gene resources for
export potential. A low dose below 1 kGy is
the security of food resources, the
effective for disifestation
conservation of our ecosystem, and the
6. Inhibition of Sprouting:
The technology is effective in
promotion of new industries. Using
inhibiting sprouting of potatoes, A dose of radiation technique (gamma-rays, X-rays
about 150 Gy has been recommended. and EB) 128 varieties were developed in
Similar doses are also effective in preventing Japan. Many new species were developed
sprouting of onion and garlic. for disease resistant crops, i.e. 55 species
Benefits and limitation of gamma of rice, 10 of barley and 2 of wheat. Other
radiation processing: species of beans, fruits including pears
Benefits : resistant for black spot decease, grass,
vegetables, etc, were also developed.
1. Radiation processing is a cold process Recently, a lot of fascinating new
and therefore, unlike heat, it can be used mutants are generated by ion beams. Ion
on agricultural commodities without beams can frequently cause large DNA
changing their fresh-like character
alterations such as inversion, translocation
2. Radiation processing does not alter
significantly nutritional value, flavour, and large deletion rather than point
texture and appearance of food mutation, which result in producing
3. Radiation using Cobalt-60 cannot induce characteristic mutants otherwise
any radioactivity in food and does not attainable. Ion-beam irradiation of
leave any harmful or toxic radioactive Arabidopsis seeds has produced the UV-
residues on foods as is the case with B-resistant, the frilled-petal, and other
chemical fumigants novel mutants. The features of ion beams
4. Due to the highly penetrating nature of in the mutation induction seem 1) to
the radiation energy, it is a very effective induce mutants with high frequency, 2) to
method. show a broad mutation spectrum, and
5. Prepackaged foods can be treated for
therefore, 3) to produce novel mutants.
hygienization and improving shelf-life
6. The radiation processing facilities are New mutants of chrysanthemum and
environment friendly and are safe to carnation with complex and striped
workers and public around. flower-color, and new flower-shape have
been produced and commercialized.
Limitations: Nuclear techniques, in contrast to
1. Radiation processing is a need based conventional breeding techniques, are
technology and cannot be applied to all widely applied in agriculture for
kinds of foods improving genetically diversity. Unlike
2. Radiation processing cannot make a bad conventional breeding procedures which
or spoiled food look good involve, the production of new genetic
3. It cannot destroy already present combinations from already existing
pesticides and toxins in foods parental genes, nuclear technology causes
exclusively new gene combinations with

high mutation frequency. Basic tool of Becquerel (Bq) : The SI unit of activity is the
nuclear technology for crop improvement becquerel (Bq). One becquerel is 1
is the use of ionizing radiation which disintegration per second. The common
causes induced mutations in plants. These multiple is the megabecquerel (1 mCi = 37
MBq).
mutations might be beneficial and have
higher economical values. Half –life: The time (t) taken for the
radioactivity of a sample to fall to half its
Measures of activity (A) : initial value.
The number of disintegrations, or t1/2= 0.693 / k
decay events, or nuclear transformations, in a Electron volt (eV): energy of radiation
sample per unit time is its activity A. Two (usually as mega electron volts (MeV).
common informal units are disintegrations per
leV=1.602x 10-19 J
second and disintegrations per minute.
Curie (Ci) : The US unit of activity is the Grays (Gy): Absorbed dose (where 1 Gy
curie (Ci). 3.7x1010 disintegrations per is the absorption of 1 J of energy per
second. Common multiples are the millicurie kilogram of food)
and microcurie. Previously rods (radiological unit) were
used. 1 rad = 10-2 J kg-1

Plant Tissue Culture Techniques for Mass Propagation of Banana

Sharad Tiwari
Biotechnology Centre, J.N.K.V.V., Jabalpur
Ex 1. Aseptic culture techniques for A. Steam or Wet sterilization

establishment and maintenance of (Autoclaving): This relies on the
cultures sterilization effect of super-heated
steam under pressure as in a domestic
Principle : pressure cooker. Most instruments/
Maintenance of aseptic environment: nutrient media are sterilized with the
use of an autoclave. The standard
All culture vessels, media and
conditions for autoclaving have a
instruments used in handling tissues as
temperature of 121ºC and a pressure
well as the explants must be sterilized.
of 15 psi (Pounds per square inch) for
The importance is to keep the air surface
15 minutes to achieve sterility. This
and floor free of dust. All operations are
figure is based on the conditions
carried out in laminar air-flow, a sterile
necessary to kill thermophilic
cabinet. Infection can be classified in
microorganisms. The time taken for
three ways:
liquids to reach this temperature
1. The air contains a large quantity of depends on their volume. It may also
suspended microorganisms in the depend on the thickness of the vessel.
form of fungal and bacterial spores.
2. The plant tissue is covered with Precautions:
pathogens on its surface. 1. Excessive autoclaving should be
3. The human body (a skin, breathe etc) avoided as it will degrade some
carries several microorganisms. medium components, particularly
4. In general, the methods of sucrose and agar breakdown under
elimination of these sources of prolonged heating.
infection can be grouped under 2. At the bottom of the autoclave the
different categories of sterilization level of water should be verified.
procedures: 3. To ensure that the lid of the autoclave
5. Preparation of sterile media, culture is properly closed.
vessels and instruments (sterilization 4. To ensure that the air- exhaust is
is done in autoclave) functioning normally.
6. Preparation of sterile plant growth 5. Not to accelerate the reduction of
regulators stocks (by filter pressure after the required time of
sterilization) autoclaving. If the temperature is not
7. Aseptic working condition reduced slowly, the media begin to
8. Explants (isolated tissues) are boil again. Also the medium in the
sterilized using chemical sterilants, containers might burst out from their
e.g. HgCl2 and NaOCl. closures because of the fast and
forced release of pressure.
Sterilization: It follows that all the 6. Bottles, when being autoclaved,
articles used in the plant cell culture must should not be tightly screwed and
be sterilized to kill the microorganisms their tops should be loose. After
that are present. autoclaving these bottles are kept in

the laminar air-flow and the tops of concentrations. Vitamins are organic
these bottles are tightened on cooling. substances that are parts of enzymes or
B. Filter sterilization: Some growth cofactors for essential metabolic
regulators like amino acids and functions. Sugar is essential for in vitro
vitamins are heat labile and get growth and development as most plant
destroyed on autoclaving with the rest cultures are unable to photosynthesize
of the nutrient medium. Therefore, it effectively for a variety of reasons.
Murashige & Skoog (1962) medium (MS)
is sterilized by filtration through a
is the most suitable and commonly used
sieve or a filtration assembly using
basic tissue culture medium for plant
filter membranes of 0.22 μm to regeneration.
0.45μm size. Plant growth regulators (PGRs) at
C. Laminar Airflow Cabinet: This is the a very low concentration (0.1 to 100 μM)
primary equipment used for aseptic regulate the initiation and development of
manipulation. This cabinet is used for shoots and roots on explants on semisolid
horizontal air-flow from the back to or in liquid medium cultures. The auxins
the front, Air is drawn in electric fans and cytokinins are the two most important
and passed through the coarse filter classes of PGRs used in tissue culture.
and then through the fine bacterial The relative effects of auxin and cytokinin
filter (HEPA). HEPA or High ratio determine the morphogenesis of
Efficiency Particulate Air Filter is an cultured tissues.
apparatus designed such that the air- Materials: Amber bottles, Plastic beakers
flow through the working place flows (100 ml, 500 ml and 1000 ml), Measuring
in direct lines (i.e. laminar flow). cylinders (500 ml), Glass beakers (50 ml),
Before commencing any experiment it Disposable syringes (5 ml), Disposable
is desirable to clean the working syringe filter (0.22 μm), Autoclaved
surface with 70% alcohol. eppendorf tubes (2 ml), Eppendorf stand,
Ex 2. Preparation of stock solutions of Benzyl-aminopurine (BAP), Naphthalene
MS (Murashige & Skoog, 1962) acetic acid (NAA)
basal medium and plant growth MS Nutrients Stock Solutions: Nutrient
regulator stocks. salts and vitamins are prepared as stock
Principle: solutions (20X or 200 X concentrations of
that required in the medium) as specified.
The basal medium is formulated
The stocks are stored at 4ºC. The desired
so that it provides all of the compounds
amount of concentrated stocks is mixed to
needed for plant growth and development,
prepare 1 liter of medium.
including certain compounds that can be
made by an intact plant, but not by an MS major salts mg/L 500 ml stock
isolated piece of plant tissue. The tissue medium (20X)
culture medium consists of 95% water,
1. NH4NO3 1650 16.5 gm
macro- and micronutrients, vitamins, mg
aminoacids, sugars. The nutrients in the
media are used by the plant cells as 2. KNO 1900 19 gm
3
building blocks for the synthesis of mg
organic molecules, or as catalysts in 3. Cacl .2H O
2 2
440 mg 4.4 gm
enzymatic reactions. The macronutrients
are required in millimolar (mM) 4. MgSO4.7H2O 370 mg 3.7 gm
quantities while micronutrients are needed 5. KH2PO4 170 mg 1.7 gm
in much lower (micromolar, μM)

MS minor salts mg/L 500 ml stock The desired amount of plant

medium (200X) growth regulators is dissolved as above
1. H BO 6.2 mg 620 mg and the volume is raised with double
3 3
distilled water. The solutions are passed
2. MnSO .4H O 22.3 mg 2230 mg through disposable syringe filter (0.22
4 2
3. ZnSO 4H O 8.6 mg 860 mg

μm). The stocks are stored at –20ºC.
4. 2
Ex 3. Micropropagation of Banana
4. KI 0.83 mg 83 mg
through shoot tip culture
5. Na MoO 2H O 0.25 mg 25 mg
2 4. 2 Principle: Plant cells and tissues are
6. CoCl 6H O
2. 2
0.025 mg 2.5 mg totipotent in nature i.e., every individual
plant cell or tissue has the same genetic
7. CuSO 5H O 0.025 mg 2.5 mg
4. 2 makeup and capable of developing along
a "programmed" pathway leading to the
MS Vitamins mg/L 500 ml formation of an entire plant that is
medium stock
(200X)
identical to the plant from which it was
derived. The totipotency of the plant cells
1. Thiamine (HCl) 0.1 mg 10 mg and tissues is the basis for in vitro cloning
2. Niacine 0.5 mg 50 mg i.e., generation or multiplication of
genetically identical plants in in vitro
3. Glycine 2.0 mg 200 mg culture.
4. Pyrodoxine (HCl) 0.5 mg 50 mg Micropropagation is used
commercially to asexually propagate
Iron, 500ml Stock (200X) plants. Using micropropagation, millions
Dissolve 3.725gm of Na2EDTA (Ethylene of new plants can be derived from a single
diamine tetra acetic acid, disodium salt) plant. This rapid multiplication allows
in 250ml dH2O. Dissolve 2.785gm of breeders and growers to introduce new
FeSO4.7H2O in 250 ml ddH2O cultivars much earlier than they could by
Boil Na2EDTA solution and add to it, using conventional propagation
techniques, such as cuttings.
FeSO solution gently by stirring.
4 Micropropagation also can be used to
Plant Growth Regulator Stock: The establish and maintain virus-free plant
heat-labile plant growth regulators are stock. This is done by culturing the plant's
filtered through a bacteria-proof apical meristem, which typically is not
membrane (0.22 μm) and added to virus-infected, even though the remainder
autoclaved medium after it has cooled of the plant may be. Once new plants are
enough (less than 60ºC). The stocks of developed from the apical meristem, they
plant growth regulators are prepared as can be maintained and sold as virus-free
mentioned below. plants.
Plant Nature Mol. Stock Soluble Micropropagation differs from all
in
Growth Wt. (1mM) other conventional propagation methods
Regulator
in that aseptic conditions are essential to
Benzyl Autoclavable 225.2 mg/ml 1N
aminopurine NaOH achieve success. The process of
(BAP) micropropagation can be divided into four
Naphtalene Heat labile 186.2 mg/ml Ethanol stages:
acetic acid
(NAA)
1. Initiation stage: A piece of plant
Indole-3- Heat labile 203.2 mg/ml EtOH/
butyric 1N tissue (called an explant) is (a) cut
acid (IBA) NaOH from the plant, (b) disinfested

(removal of surface contaminants), • Myoinositol 100 mg

and (c) placed on a medium. A • Sucrose 30 gm (3%)
medium typically contains mineral 1. Add BAP at this stage (Calculate, how
salts, sucrose, and a solidifying agent much to add)
such as agar. The objective of this 2. Make final volume to 1000 ml by
stage is to achieve an aseptic culture. double distilled water
An aseptic culture is one without 3. Set pH at 5.8
contaminating bacteria or fungi. 4. Add agar agar 8 gm/L (0.8%), melt the
agar agar in microwave oven
2. Multiplication stage: A growing 0
explant can be induced to produce 5. Sterilize the media at 15 psi/121 C for
vegetative shoots by including a 15 minutes
cytokinin in the medium. A cytokinin 6. After autoclaving, gently swirl the
is a plant growth regulator that medium to mix the agar. When the agar
is completely dissolved and mixed, the
promotes shoot formation from
medium should appear clear and not
growing plant cells.
turbid.
3. Rooting or preplant stage: Growing 7. Add filter sterilized IBA (desired
shoots can be induced to produce amount, calculate) once the temperature
0
adventitious roots by including an of the medium cools down to 60 C.
auxin in the medium. Auxins are plant 8. Dispense the medium to sterilized
growth regulators that promote root Petridishes (25 ml medium/plate)
formation. For easily rooted plants, an
auxin is usually not necessary and Preparation and inoculation of explant:
The 2-3 months old young, healthy
many commercial labs will skip this
suckers are selected for shoot tip explants.
step.
The adhering soil and dirt is removed.
4. Acclimatization: A growing, rooted Remove roots and prepare rhizome of 3-5
shoot can be removed from tissue cm with 2-4 inches of suckers, than wash
culture and placed in soil. When this thoroughly under tap water with Tween
is done, the humidity must be 20 for 10-15 min. There after dip the plant
gradually reduced over time because material in a solution of ascorbic acid 100
tissue-cultured plants are extremely mg/lit and citric acid 150 mg /lit for one
susceptible to wilting. hour. Sterilize the explants using 0.1%
Materials: Beakers, Measuring cylinders, HgCl2 for 7 min. subsequently the
Conical flasks, Cotton plugs, Myoinositol, explants are washed gently three times
Sucrose, BAP (1mM stock), Agar Agar, with sterile DDH2O (double distilled
Forceps, Blade Holder (No.3), Sterilzed water) in aseptic condition under laminar
blades (No.11), NAA (1 mM stock), flow. Shoot tips of 1-2 cm are excised and
Micropipettes, sterilized microtips, placed the rhizome pieces on MS
petridishes. medium, and incubated it at 25±2°C
temperature and 65-70 % RH in dark for
The shoot multiplication medium for 7-10 days, for plant regenerations.
Banana is MS basal + BAP (3 mg/l) +
NAA (0.5 mg/l) Multiplication of shoots: Shoot
multiplication is carried out on MS
Preparation of MS medium (1000 ml) medium containing 3-8mg/l of BAP
• MS Major (20X) 50 ml combined with 0.2-0.5mg/l of IBA. All
• MS Minor (200X) 5 ml cultures are maintained at temperature of
25±2°C under 16h photoperiod regime at
• MS Vitamin (200X) 5 ml
3000 lux and 70% RH.
• Iron (200X) 5 ml
Hardening of regenerated plantlets: weeks transfer polybags to open for field

Transfer the rooted plantlets to mixture of plantation.
sand and FYM (3:1)/ Vermiculite to a
References :
poly tunnel under poly house at 25-30°C
and 85-90% RH for 21-25 days. Plant the Murashige T & Skoog F (1962) A revised
acclimatized plantlets in to polybags medium for rapid growth and
containing sand, soil and FYM (1:1:1) bioassays with tobacco tissue
under net house conditions, after 3-4 cultures. Physiol. Plant 15: 473-497.

Appendix I
Conversion factors :
me. mg/eq.wt
ppm mg/l or µg/ml
1 ppm 2.25 kg/ha
10,000 ppm 1 per cent
1 mg/100 g 22.5 kg/ha
1 kg/ha 0.1 g /sq m
1 hectare 2.471 acres
1 acre 0.405 hectare
1 kg /ha 1.12 lbs/ acres
Mesh number 16/ mm
Normality Specific gravity X Purity X 10
(Liquid) Equivalent weight
Normality Wt of salt per liter X Purity
(Solid) Equivalent weight
Grams per liter Normality X Eq. Wt.
Organic matter Organic carbon X 1.724
Optical density 2 – log T (T= transmission)
Per cent by Wt. Grams of solute
100 g of solution
1 Angstrom (Å) 10-8cm or 10-10 m
10 (Å) 1 nanometer or 1 millimicrometer
Temperature conversion :
o
C/5 (oF-32) / 9
P X 2.29 P2O5
P2O5 0.44 X P
K X 1.20 K2O
K2O X 0.83 K
SX3 SO4
N X 1.12 NH3
Protein (%) Nitrogen (%) X 6.25
Velocity of light 3 X 10-10 cm / sec.
Velocity of sound 332 m / sec.

Appendix II
Percentage composition of manures and fertilizers
Material N P2O5 K2O Others

FYM 0.5-1.5 0.4-0.8 0.5-1.9 -
Compost (urban) 1.0-2.0 1.0 1.5 -
Green manure 0.5-0.7 0.1-0.2 0.8-1.6 -
Bone meal(steamed) 1.0-2.0 25-30 - -
Anhydrous ammonia 82 - - -
Ammonium chloride 24 - - -
Ammonium nitrate 33 - - -
Ammonium sulphate 20.6 - - 24 (S)
Ammonium phosphate 11 48 - -
Diammonium phosphate 21 53 - -
Calcium cynamide 20 - - -
Calcium nitrate 16 - - -
Sodium nitrate 16 - - -
Urea 46 - - -
Super phosphate - 16 - 12 (S)
Dicalcium phosphate - 23 - -
Tricalcium phosphate - 45 - -
Rock phosphate - 11-17 - -
Basic slag - 3.5-8 - -
Muriate of potash - - 60 -
Sulphate of potash - - 48 18 (S)
Zinc sulphate - - - 35 (Zn); 18 (S)
Zinc chelate - - - 14 (Zn)
Copper sulphate - - - 25 (Cu); 13 (S)
Ferrous sulphate - - - 19 (Fe); 19 (S)
Iron chelate - - - 5-9 (Fe)
Borax - - - 11 (B)
Sodium tetra borate - - - 14 (B)
Manganese sulphate - - - 26 (Mn)
Manganese chelate - - - 10-12 (Mn)
Calcium sulphate (Gypsum) - - - 18 (S); 33 (Ca)

Appendix III
Ready reckoner of fertilizer schedule at varying soil test values for different crops (kg ha-1)
Fertilizer Name Fertilizers Recommendations
Very Low Low Medium High Very High
PADDY (80:50:30)
Urea 260 215 175 130 85
Super Phosphate 470 390 310 235 155
Muriate of Potash 75 65 50 40 25
SOYBEAN (20:80:20)
Urea 65 55 45 35 20
WHEAT IRRIGATED (100:50:30)
Urea 325 270 220 165 110
WHEAT UNIRRIGATED (30:40:20)
Urea 80 65 30 15 -
Muriate of Potash 50 30 15 10 -
GRAM (30:60:30)
Urea 100 80 65 50 35
MOONG / URID / LENTIL / ARHAR (20:50:20)
Urea 65 55 45 35 20
PEA (35-75-30)
Urea 100 80 65 50 35
MUSTARD (60-30-20)
Urea 195 165 130 100 65
SUNFLOWER / SAFFLOWER (40:40:30)
Urea 130 110 90 65 45
MAIZE (120:80:60)
Urea 390 325 260 195 130
VEGETABLES (100:50:30)
Urea 325 270 220 165 110
JWAR (50:35:25)
Urea 190 160 120 90 60
Note: In wheat, paddy, mustard and maize the 50% of urea to be applied as basal doses. Rest 50% may be
applies in 2 to 3 split doses as top dressing.

Appendix IV
General recommended doses of micronutrient fertilizer
Micronutrient Material and doses for application

Zinc Zinc sulphate (25-50 kg ha-1) (25 kg 0.5% zinc sulphate +
ha-1 for light soils and 50 kg for 0.25%lime
heavy soils)
Iron Ferrous sulphate (25-50 kg ha-1) 1-2% ferrous sulphate + half
of lime
Copper Copper sulphate (10 kg ha-1) 0.1% copper sulphate +
0.05% lime
Manganese Manganese suphate (10 kg ha-1) 1% manganese sulphate +
0.25% lime
Boron Borax (10 kg ha-1) 0.2% borax
Appendix V
Rating of Nutrients:
Rating Organic carbon Available N Available P Available K

(%)
(kg ha-1)
Very Low < 0.3 150 <5 < 200
Low 0.3 – 0.5 150 – 250 5 – 10 200 – 250
Medium 0.5 – 0.75 250 – 400 10 – 20 250 – 400
High 0.75 –1.0 400 – 600 20 – 40 400 – 600
Very High > 1.0 > 600 > 40 > 600

Appendix VI
Performa for Soil Health Card
In-situ information
To be recorded while collection of sample Sample No………….
I. General Information :
Farmers Name
Age
Male/Female
Education
Address
II. Land details :
Name of the Area (ha) Survey Owned Leased Irrigated/ Soil

field No in/out rainfed type
III. Cropping details :
Survey Kharif Rabi

number
Crop/ Yield q/ha Crop/ Crop/ Yield Crop/
variety Variety variety q/ha Variety
proposed proposed

IV. Farmers Self Assessment-Score Card :

S. Parameters Ratings Details Year
No.
I II III
I Soil health
Biological activity (Deep Medium Shallow)
1 Earthworms Good Many holses and casts >10

worms
Fair Few holes and casts >7-5
Poor Little sign of worm activity 0 -
3 worms
2 Birds Good Many birds follow plough /
following tractor during ploughing
plough
Fair Some birds
Poor Very few birds /sometimes no
birds at all
Plant growth and yield
3 Seed Good Seed come up quickly, and
germination even emergence
Fair Germination is uneven, takes
one or two days more for
emergence
Poor Germination is very poor with
high degree of unevenness
4 Uniformity in Good Even stand in growth, uniform
growth green colour
Fair Slight variation in crop height,
moderate growth and
differences in colour
Poor Uneven stand, stunted growth
and stressed
5 Grain yield Good Good yield, and quality
Fair Average crop in the region,
Poor Poor crop in the area and yield
is very poor

Soil Analysis Report by laboratory Incharge/Technical Person
Name of laboratory :
Date of Sampling :
S. No. Soil properties Kharif Rabi

I II III I II III
1. Soil pH
2. EC (dsm-1)
3. Organic Carbon (%)
4. Available N kg/ha
5. Available P kg/ha
6. Available K kg/ha
7. Available S (mg kg-1)
8. Zinc (mg kg-1)
9. Iron (mg kg-1)
10. Manganese (mg kg-1)
11. Copper (mg kg-1)
Irrigation facility and water quality
Date of Sampling :
Irrigated/Un-irrigated/ Open well/ Kharif Rabi

Source of irrigation borewell /tank
I II III I II III
Average depth of ground

water
Annual average rainfall

(mm)
Normal onset of rainfall

(week/month)
Quality of irrigation water Poor/medium/

good

Recommendation :
Soil Testing Sample No. ………………..

Kharif Rabi
Details Manure/Fertilizer Manure/Fertilizer
Recommended Applied Recommended Applied
-1
FYM(tha )
Green manure(tha-1)
Nitrogen(kg ha-1)
 Urea
 DAP
 Complex
Phosphorus (kg ha-1)
 Super phosphate
 DAP
 Complex
Potash(kg ha-1)
Micronutrients(kg ha-1)
 Zinc sulphate
 Micronutrient
mixture
Biofertilizers
 Azosprillum
 Rhizobium
 Phospho bacteria
Other details :
Nearest Agriculture Department Office

Address and Phone number
Input dealers
Address and Phone number
Name of the bank
Account Number
Nearest KVK
Address and phone number
Soil Testing lab
Address and Phone Number

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CENTRE OF ADVANCED FACULTY TRAINING

S. No. Title Page No.

01 Importance of Soil Testing, Collection of Soil Samples, its 1-4

02 Sampling, Processing and Storage of Plant Samples for Analysis 5-7

03 Profile Studies of Deep Black Soils (Vertisols) 8-11

04 Simultaneous Measurement of Bulk Density and Water Content 12-13

05 Estimation of Soil Moisture Content by Different Methods 14-16

06 Determination of pH and Electrical Conductivity in Soil Samples 17-18

07 Determination of Organic Carbon Content in Soil 19

08 Determination of Available Nitrogen in Soil by Alkaline 20-21

09 Determination of Total Nitrogen in Soil and Plant Samples 22-24

10 Determination of Phosphorous in Soil and Plant Samples 25-27

11 Determination of Potassium in Soil and Plant Samples 28-29

12 Determination of Sulphur Content in Soil and Plant 30-31

13 Determination of Micronutrients in Soil and Plant Samples by 32-35

14 Estimation of Boron in Soils, Plants and Water 36-37

15 Estimation of Microbial Biomass Carbon in Soil 38

16 Preparation of thematic maps of land use/land cover through GIS 39-40

17 Gamma Irradiation and its Importance for Food Preservation 41-46

18 Plant Tissue Culture Techniques for Mass Propagation of Banana 47-51

Importance of Soil Testing, Collection of Soil Sample, its Processing and

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 1

 At the sampling site, remove the surface

Parts of an Auger COLLECTION OF SOIL SAMPLE BY

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 2

4. Date of sampling 4. In row crop take sample in between rows.

An ideal time for soil sampling is just REFERENCES:-

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 4

Sampling, Processing and Storage of Plant Samples for Analysis

Sampling thorough rinsing twice with deionized

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 5

or those suffering from effects of Wet digestion of plant sample with

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 6

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 7

Profile Studies of Deep Black Soils (Vertisols)

Vertisoils or deep black soils occur unique morphological properties such as

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 8

Morphology of a soil is best evaluated %) and predominance of montmorillonite

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 9

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 10

young or remain in equilibrium with the would dominate. This interpretation

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 11

Simultaneous Measurement of Bulk Density and Water Content in Soil by

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 12

Estimation of Soil Moisture Content by Different Methods

Direct method (Gravimetric method) : alcohol or spirit. The process is repeated

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 14

it is a means of directly observing the nitrogen in soil is associated with water

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 15

seriously. Tube once installed may provide an index of equipment condition.

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 16

Determination of pH and Electrical Conductivity in Soil Samples

Determination of pH is actually a 4.0 (Dissolve 10.21 g. of A.R. grade

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 17

Electrical Conductivity : Reagents :

Since conductivity is reciprocal of

Electrical Conductivity Meter :-

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 18

Determination of Organic Carbon Content in Soil

The majority of mineral surface soils Apparatus and Reagents :

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) 19

Determination of Available Nitrogen in Soil by Alkaline

liberated by potassium permanganate, in