3.1. Soil texture
Physically, the soil is mixture of mineral matter, organic matter, water and air. The mineral matter is composed of inorganic particles varying in size from stone and gravel to powder. These inorganic particles, separated according to size are referred to as soil separates. The U. S. Department of Agriculture (USDA) recognizes three major groups of soil separates : sand (2.0-0.050 mm), silt (0.050-0.002 mm) and clay ô 0.002 mm).
The three fractions can be subdivided into finer size fractions as follows (73) :
60 Techniques in Mycorrhizal Studies
Very coarse sand Coarse sand Medium sand Fine sand Very fine sand Coarse silt Medium silt
Diameter 2.oo-1.oomm 1.00-0.50 mm 0.SO-O.2!1 mm 0.25-0.10 mm O.IO-O.O!l mm 0.050-0.020 mm 0.020-0.005 mm
Fine silt 0.005-0.002 mm
Coarse clay 2.0-0.2 J1lIl
Medium clay 0.2-0.08 J1lIl
Fine clay < 0.08 J1lIl
Texture is an important characteristic of soil, affecting drainage condition, water-holding capacity, amount and size of po res, and plant root development. Consequently, the rate of water intake, water supplying power, aeration, and soil fertility are all influenced by soil texture.
3.2 Sampling
Before starting soil analysis, it is necessary to procure a test sample that will represent the soil under investigation and to prepare the test sample for analysis. When the sample analyzed is not representative, the result of the soil analysis will yield a value that does not necessarily describe the property of soil as a whole. The analytical value can serve as an accurate description ofthe soil property under investigation only if: *
* *
*
The gross sample accurately represents the whole soil from which it is taken.
No change occur in soil sample prior to analysis.
The subsample analysed represents the gross sample accurately.
The analysis determines a true value of the soil characteristics under investigation.
If the investigation involves small soil volume, it is both admissible and convenient to take samples ofthe whole population. However, with a large soil volume, time and expense can be saved if a representative sample can be drawn and analysed.
3.2.1 Simple Random Sampling: This is a method by which every sampling unit has an equal, independent probability of being drawn. It is a satisfactory method for a highly homogeneous field.
Rupam Kapoor. B. Giri and K.G. Mukerji 61 3.2.2 Systematic Sampling: Systematic sampling provide more accurate results because with this method the samples are distributed more evenly over the population. However, if the soil population contains periodic variation which appear to coincide with interval between successive sampling sites, biased samples may be obtained.
3.2.3 Stratified Sampling: This type of soil sampling is commonly employed in a heterogeneous field. Heterogeneous population is divided into vertical layers called strata each of which is fairly homogenous, more precise sampling can be achieved.
3.2.4 Composite Sampling: Compositing is the mixture of sampling units to form a single sample which is used for chemical analysis. This method offers the advantage of increased accuracy through the use of large number of sampling units per sample (sub-samples). Several subsamples are usually bulked to form a composite sample (77).
3.2.5 Mechanics of Sampling: The tools that can be used to collect the sampling units include soil sampling hibes, augers and till spades or shorels, depending upon the depth and types of mechanics of sampling. Samples can be dug from the soil surface (shallow sampling); or from deeper layers (deep sampling) or from trenches, road banks and soil pits.
3.2.6 Number of Samples : The question is how many samples should be taken as required for accuracy and to make the error as negligible as possible. The objective is to avoid taking too many samples, while on the other hand we must also avoid too small a number of samples so that the results can become too inaccurate.
3.3 Sample Preservation
Air drying is the most accepted procedure of sample preservation as air may reduce the rate of possible reactions in the disturbed soil sample.
Soil aggregates should be broken carefully to accelerate the drying procedure. To accelerate the drying process, samples may be placed in a forced draft of moving air, but not in heated air. At elevated temperature (above 35°C) the physical and chemical characteristic ofthe soil sample drastically change (72).
3.3.1 Sample Storage: Samples should be dried and stored in air-tight containers as soon as possible to avoid adsorption of~, S03 and/or S02 gases in the laboratory. Containers should be clean and should be composed of materials that will not contaminate the sample. Glass jars and or plastic or waxed cardboard containers are suitable for this purpose.
62 Techniques in Mycorrhizal Studies
3.4 Sample Preparation
3.4.1. Grinding and Sieving: Thorough mixing requires that the samples be crushed and ground to particles of unifonn size. Large aggregates are reduced by crushers, and the crushed sample is then further reduced by grinding. The purpose of grinding is to reduce heterogeneity and to provide maximum surface area for physical and chemical reactions. All the particles will not be pulverized in a single grinding operation because some of the very hard particles become coated with powdered material.
The bulk sample is then screened, and the reflected material ground again and rescreened until the whole sample passes through the sieve. The fraction passing a 2-mm sieve is collected and stored as a stock sample.
The soil fraction> 2 mm is usually discarded in soil chemical analysis.
These materials are not soil constituents but are rock fragments which may produce soil constituents after weathering.
3.5 Analysis of Soil Physical Characters
3.5.1 Determination of soil texture : On the basis of percentages of total sand, total silt and total clay United States Department of Agriculture (USDA) has recognized twelve types of soil texture for classifying soils into twelve soil classes. Because of this, the tenus soil texture and soil classes are frequently used interchangeably. However, soil texture is a soil characteristic whereas soil classes are group of soils differentiated by differences in soil texture (Fig. 1).
100
Percent sand
Fig.] : Soil class on basis of texture difference as used by U.S.D.A. Soil Survey.
Rupam Kapoor. B. Giri and K.G. Mukerji 63 An indirect method used in laboratory is conducted through the quantitative determination of soil separates. This type of analysis is called particle size distribution or particle size analysis (50). Two important steps in the analysis are dispersion and sedimentation.
3.5.1.1. Dispersion: In most soil samples, the sand, silt and clay particles are cemented together into aggregates or granules by organic matter, clay and salts.
These aggregates must be disrupted into the individual particles and later suspended in water, a process called "Dispersion". Usually, chemical reagent such as sodium hydroxide or sodium metaphosphate or calgon is added to enhance dispersion. Final dispersion is effected by thorough shaking or mixing mechanically with a blender.
3.5.1.2. Sedimentation: After the dispersion of soil, the different particle size fractions are sorted out by sedimentation techniques using the hydrometer or the pipette method. All these methods of measuring and collection of soil separates are based on the differential rate of setting of particles during sedimentation, as formulated by the law of stokes :
V = [2f (dp-dw)g]/9n
in which V = rate of settling (cm/s) of particles with an effective radius = r (em) and density = dp (g/cm) falling through a liquid medium and density = dw (gI cm3) and viscosity = n (poise, glcm/s) under the acceleration of gravity = g (981/
sec2). Hence, the larger particles the faster the rate of settling. Therefore sand particles will settle out first, followed by silt particles. Clay particles tend to remain in suspension for a longer times.
Since dp' dw (density of water) are constants for a given temperature, 2(dp- dw)g]/9n = K (constant). Consequently, the law of stokes can be reduced into:
V = Kf. The value of K is dependent on the temperature.
3.5.1.2.l. Hydrometer method: This is a simple and rapid method for analysis of soil texture (Fig.2).
at 200e
length
llll approx.
lOl _... ••. 350mm
weighted end
Fig.2 : Hydrometer
64 Techniques in Mycorrhizal Studies
3.5.1.2.1a. Dispersion Reagent : (i) Sodiummetaphosphate, (NaP03)x' Nap, (X- 13). Dissolve 40 g of the reagent in 11 of distilled water, use 10 ml of this solution in the analysis.
(ii) Sodium hydroxide (NaOH) : Instead of sodium metaphosphate, a 2-4 M NaOH solution can also be used, which should be added dropwise, under constant stirring, until the soil suspension has a pH = 10-11.
3.5.1.2.1b. Procedure :
* Weigh 50.0 or 100.0 g of oven dry soil sample and transfer it quantitatively into a blender cup. 50 g is used if the soil is clayey, whereas 100 g is used if the soil is sandy in nature.
* After the soil sample has been transferred, fill the blender cup with distilled water to within 10 cm of the rim and add 10 ml of sodium metaphosphate on sodium hydroxide as directed above.
* Attach the cup to a blender on stirring machine and blend mechanically for 15 minutes.
* Transfer the soil suspension into a soil testing cylinder. Wash the remaining soil residue quantitatively in the cylinder.
* Make up the volume in the cylinder with water to 1130 mllevel if 50 g of soil sample used. If a 100 g sample is used, fill the cylinder to the 1205 mllevel.
* Mix the suspension thoroughly by stirring with a stirring rod so that all sediment disappears from the bottom of the cylinder. Record (clock) the exact time when stirring is stopped.
* Carefully place a hydrometer into the suspension, and exactly 40s after the stirring is stopped, read to the nearest 0.5 scale decision the top of the meniscus on the hydrometer stem.
* Remove and rinse the hydrometer.
* Stir the suspension again and repeat the analysis of the 40s reading. The average of readings equals the amount of silt and clay in grams. Determine and record the temperature of the suspension after hydrometer is removed.
* Stir the suspension thoroughly again. Take a third hydrometer and temperature reading after 120 minutes of settling time. This reading will measure the amount of clay in grams.
3.5.l.2.2. Centrifuge Method: This is an elaborate but rapid method for the determination of soil texture. Organic matter and other cementing agents are removed from the soil sample prior to the analysis.
3.5.1.2.2a. Reagents
(0 30 to 35% Hydrogen peroxide
(ii) Sodium metaphosphate or NaOH to disperse the soil sample;
3.5.1.2.2b. Procedure
* Take 109 of soil sample in 500 ml beaker. Wet the soil with distilled water.
'" Add 25 ml of 1\02 and 10 ml of distilled water and place the beaker on hot plate and cover it with a watchglass.
* After frothing and bubbling have ceased, add an additional 25 ml ~02 and allow the reaction to run again to completion.
*
*
*
Rupam Kapoor. B. Giri and K.G. Mukerji 65 When the organic matter is completely oxidised, heat the mixture gently to remove the excess ".02. Do not heat until dryness.
Wash the soil several times with water to remove the dissolved mineral matter produced by the oxidation of organic matter.
Then transfer the sample quantitatively into a blender cup and disperse the soil mixture as directed in hydrometer method.
3.5.1.3. Separation of Sand: Insert a funnel into a 500 ml beaker and place a 0.050 mm sieve over the funnel. The soil mixture is then poured quantitatively into the sieve.
* Wash the sample on the sieve by gently jet spraying it with distilled water from a water bottle. The material passing through the sieve and collecting in the beaker, contains the silt and clay fractions.
* The total sand fraction remaining on the sieve is dried and weighed.
* Convert the results in percentages on the bases of oven dry weight of soil.
3.5.1.4. Separation of Silt: The suspension in the beaker, containing the silt and clay, is dispersed again, stirred and allowed to stand for 5-10 min. (73).
* The supernatant suspension is carefully decanted and transferred into a 500 ml beaker. The coarse silt fraction sedimented at the bottom is collected, dried and weighed.
* To separate the remaining silt from the clay fraction, the supernatant is centrifuged for 3 min. at 300 rpm.
* The supernatant containing the clay fraction is carefully decanted for further analysis into coarse, medium and fine clay.
* The residue sedimented by centrifugation, composed of the medium and fine silt fractions is dispersed again followed by centrifugation for 3 minutes at 300 rpm; which results in the separation of silt fractions.
* The supernatant (fine silt) and the sediment (medium silt) are collected, dried and weighed. The total silt content, needed for soil texture determination, can be calculated again on a oven dry basis of soil.
3.5.1.5. Separation of Clay: The remaining suspension is dried and weighed which represents the total clay content and should be expressed in tenns of percentages on an oven-dry basis of soil.
3.5.2 Soil pH
Soil reaction is determined by the hydrogen ion (H+) concentration in the soil solution. The W ions may be present in soils as adsorbed H+ ions on the surface of the colloidal complex, or as free H+ ions in the soil solution. The adsorbed W ions create the reserve acidity, also called the potential or exchange acidity of soils. The free W ions represent active acidity of soils. Both taken together constitute the total acidity.
66 Techniques in Mycorrhizal Studies
Based on soil pH values, several types of soil reaction are distinguished as follows (24) :
Slightly acid Moderately acid Strongly acid Very strongly acid
pH 7.0--6.0 6.O-S.0 5.0-4.0 4.0-3.0
Slighlyalkaline Moderately alkaline Strongly alkaline Very strongly alkaline
pH 7.0--8.0 8.0-9.0 9.0-10.0 10.0-11.0 3.5.2.1. Indicator or Colorimetric Method: The method makes use of indicators, and is applied in the field as a rapid test for soil pH. Colour is used to indicate pH levels, the free ion of the indicator has a colour different from the dissociated molecule. The equilibrium concentration between the dissociated molecule and the undissociated indicator governs the colour. Generally, a mixture of selected indicators is used in order to measure the soil pH from 0 tol4.
3.5.2.1.1. Reagent: A mixture indicator (Table 2) assessing pH levels from 2.0 to 10.0 can be prepared by dissolving in 100 ml ethanol:
80mg bromthymol blue 40mg methyl red 60mg methyl yellow 20mg phenolphthalein 100mg thymol blue
Titrate the mixture with 0.1 M NaOH solution until yellow. The pH level corresponds to the 'colour listed in Table 1.
TABLE I
The pH and corresponding color of mixed indicator (73).
Color
pH (British color standard)
2.0 Crimson-red
3.0 Red
4.0 Orange-red
5.0 Orange
6.0 Yellow
7.0 Yellowish-green
8.0 Green
9.0 Bluish-green
10.0 Blue
3.5.2.1.2. Procedure 3.5.2.1.2a. Field Procedure
• Place a small sample of soil in a plastic spoon,
• Wet the sample with distilled water and allow the mixture to react for 5 min.
• Tilt the spoon to separate the liquid from the soil, and add 2 to 3 drops of duplex indicator to the solution.
• Match the colour developed with colour chart to determine the pH.
Rupam Kapoor, B. Giri and K. G. Mukerji 67 3.5.2.1.2b. Laboratory Procedure
* Weigh 15 g of soil in a clean 100 ml centrifuge tube.
* Add 30 ml of water and centrifuge the mixture for 15 minutes at 2500 rpm.
* Filter the supernatant into a beaker and take 10 ml of the above in a test tube.
* Add 10 drops of indicator and mix the two to develop the colour.
* Match the colour with the colour chart to determine pH.
* Prepare a series of standards and match the colour with these standards.
3.5.2.2. Potentiometric Method: Electrodes are used to measure H+ ion concentration in the soil solution. Three types of electrodes are commonly used to determine pH viz. indicator electrode; the calomel electrode, which is the reference electrode; and the combination glass electrode, in which both the indicator and the reference electrode are combined into one electrode. When electrode is placed in a solution, an electrical potential difference develops between the indicator and reference electrodes and the solution. The magnitude of the potential difference is proportional to the W ion concentration, and can be converted into pH units.
TABLE 2
Some pH indicators commonly used in soil analysis (73).
Indicator Critical Color changel )
pH
Thymol blue2) 1.9 R-O-Y
Dinitrophenol 3.1 C-Y-Y
Methyl orange 3.7 R-Y-O
Brorn phenol blue 4.0 Y-PU-V
Brorn cresol green 4.6 Y-G-B
Chlor phenol red 5.6 Y-OP-V
Methyl red 5.7 R-O-Y
Brorn thymol blue 6.9 Y-G-B
Phenol red 7.3 Y-RO-V
Phenolphthalein 8.3 C-P-P
Thymolphtalein 9.4 C-B-B
Alizarin yellow R 10.3 Y-O-R
J) Color at center is at critical pH; B = blue, C = colorless, G = green, 0 =
orange, P = pink, Pu = purple, R = red, V = violet, Y = yellow.
2) Thymol blue has two critical pH values.
68 Techniques in Mycorrhizal Studies 3.5.2.2a. Reagents
Standard buffer solutions of pH 7.0 and pH 4.0 for calibration of the pH meter.
3.5.2.2b. Procedure
•
•
•
•
•
Weigh 25 g of soil in a clean beaker, add 25 ml distilled water and stir or swirl frequently for 15 minutes.
Calibrate the pH meter prior to use dipping the combination glass electrode in a buffer solution of pH 7.0.
Adjust the pH meter to read pH 7.0. Rinse the electrode with distilled water and place it in a buffer solution of pH 4.0 to read pH 4.0. Rinse it again with distilled water.
Place the electrode in soil suspension. Read the pH in the scale of the pH meter and rinse the electrode again with distilled water.
If the electrode is allowed to dry its function is impaired. Therefore, the glass electrode must be soaked in buffer solution of pH 7.0 for storage.
3.6. Soil Moisture
Water is held in the soil by both adhesive and cohesive forces.
Another force affecting retention and movement of water in soil is the capillary force by which water is adsorbed in the micropores or capillaries.
3.6.1 Determination oftota! soil water content 3.6.1.1. Gravimetric method
• Place the soil sample in a clean preweighed stoppered weighing flask or bottle.
* Take approximately 5-10 g of soil sample and put it quickly in the flask.
* Close the flask and carefully weigh it to the nearest I or 0.1 mg, depending upon the accuracy desired.
* Remove the lid from the flask, and dry the flask with its content at 105- 110°C for 24 h in an oven.
* Allow the flask to cool in a dessicator. Place the lid back on the flask and carefully weigh the flask with its content to the nearest 1 or 0.1 mg.
• The amount of water lost, which is the water content of the sample may be calculated as follows :
~O lost = Weight of moist soil - Weight of oven dry soil.
The result is presented in the percentage of water, which can be expressed in a dry mass percentage, a wet mass percentage or a volume percentage.
The dry mass percentage is used in soil science and chemistry. The wet mass percentage is used in the biological sciences and finds practical application in agriculture, horticulture, forestry and nurseries in the weighing of fresh produce and vegetables.
Rupam Kapoor, B. Giri and K. G. Mukerji 69 3.6.2 Determination of Available Water Content: The available water content is the amount of water held by soils between the field capacity and wilting point.
The point at which water is held in soils after excess water has been drained by gravity is called field capacity. As the soil dries the water content decreases.
Eventually a point is reached at which the force exerted by the plant is not sufficient to extract water at a sufficient rate for growth. At this point, the so- called wilting point, the plants start to wilt. The method to find this is as follows:
3.6.2.l. Pressure-Plate Method
* Place a cellulose membrane on the porous porcelain plate in pressure plate or tension plate instrument.
* Fill the retainer rings in the instrument with a known amount of soil sample and fill the plate with water to wet the samples from below.
* Cover with a plastic sheet and allow the sample to soak overnight.
* After the soil sample is saturated with water, remove the excess water.
Close the instrument and tighten it with the screws.
* For field capacity water, apply a pressure of 0.3 bars until no more water is forced out of the sample, which usually takes several days.
* As soon as no more water can be forced out, quickly transfer the moist sample to a preweighed container.
* Record the weight of the container with moist soil.
* Dry the moist soil in the container in a forced-draft oven at 105°C for 24 h, and calculate the moisture content as percentage oven dry weight soil.
* For the determination of water at the wilting point, the analysis can be repeated at a suction force of IS bars. The available water content is then the difference between the water at field capacity and the wilting point.
3.6.3 Soil density and porosity: Soil density is an important physical property that affects both agricultural and engineering operations. The denser the soil, the less permeable the soil. A dense, compacted soil inhibits plant growth. Soil density refers to the soil per unit volume of soil (24).
3.6.3.l. Measurement of Bulk density
Bulk density is defined as the mass of soil per unit volume of undisturbed soil or bulk soil volume. Thus:
Bulk density = Weight of soiVBulk volume.
3.6.3 .l.l. Procedure
* Fill a preweighed 100 ml graduated cylinder with soil.
* Compact the soil by tapping the bottom of the cylinder ten times with the palm of your hand.
* Keep adding soil and tapping the cylinder until a tapped soil volume of 100 ml is obtained.
* Weigh the cylinder containing the soil.
* Determine the moisture content of the soil sample separately and calculate the oven dry weight of the 100 ml soil above.
Calculations: Bulk density = oven-dry weight of 100 ml soil/lOO g (cc).