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L1SI/ Practical Sheet/Soil Science/ Page 1 of 14 COLLECTION AND PREPARATION OF SOIL SAMPLES

INTRODUCTION:

Soil sampling includes collection of soil from the field and it's processing for subsequent mechanical and chemical analysis. Results of soil analysis would be misleading if sampling were not done accurately.
OBJECTIVES:

In agricultural point of view soil sampling is done to know the soil type, soil pH, soil CEC, nutrient status and other soil characteristics and ultimately to formulate remedial measures for successful crop production.
MATERIALS REQUIRED:

1. Auger and spade 2. Sieve 3. Polythene bag 4. Plastic or glass bottle 5. Mortar and pestle 6. Thread 7. Brown paper etc.
PROCEDURE:

A. Collection of soil samples: 1. Mark out the spots in a plot of land from which soil sample will be collected. The number of sampling spots may vary from 5 to 10 depending on the size of the plot. 2. Scrap away the surface litter and weeds of the sampling units with the help of a spade. 3. Press an auger vertically into the soil to a depth of 6 inches or 15cm (plough depth) with a little rotatory movement. 4. Lift out the auger and discharge the soil into a polythene bag. Collect few more samples from random spots within the plot. Label the soil samples and tie up the bag with thread. 5. Bring out the samples in the laboratory and mix them up to make a composite sample. B. Preparation of Soil Samples: 1. Spread the soil sample on a sheet of brown paper for air drying; remove stones, brick pieces, crop roots etc. 2. When dry, grind the samples in a mortar with pestle. Pass the soils through a 2 mm (10 mesh) sieve and return the coarse material to the mortar for further crushing. Repeat the crushing and sieving of this residue until all aggregate particles are fine enough to pass through the 2 mm sieve. Mix well the sieved soil. Sub-sampling may be done in the following way. Spread the sieved soil on a brown paper, divide into quarters and discard two portions. Repeat the quartering process until the requisite size is obtained.

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C. Morphological Description of Soil Samples Collection: Location Site General Soil Type Parent material Topography Drainage Previous crops Sampling depth Date of collection Name of collector
PRECAUTIONS:

: : : : : : : : :

Hajee Mohammad Danesh Science and Technology University

Non-Calcareous flood plain Alluvial deposit Flat/Plain Well drained 0-15cm/0-6 inch

(i)
(ii)

Don't collect sample from an unusual area in a plot e.g., old manure piles, wet spots, roadside, tree-side etc. Avoid any metallic utensils during grinding and handling of soil sample.

L1SI/ Practical Sheet/Soil Science/ Page 3 of 14 IDENTIFICATION OF IMPORTANT SOIL FORMING ROCKS AND MINERALS
IDENTIFICATION OF ROCKS:

Rocks may be defined as the mixture of minerals whose physical and chemical properties depend on the characteristics of minerals present in them. On the basis of genesis rocks may be classified into three groupsi) Igneous, ii) Sedimentary, and iii) Metamorphic group Igneous rocks are those rocks, which have been formed by solidification of molten magma from the interior of the earth. The rocks, which have been formed from the consolidation of sediments accumulated through wind or water action on the surface of the earth in past geological ages, are called sedimentary rocks. Metamorphic rocks have been formed from the subsequent transformation of igneous and sedimentary rocks under the influence of heat, pressure and chemically active liquid or gases.
SOME IMPORTANT ROCKS AND THEIR CHARACTERS:
Rocks Group Minerals Granularity Colour Other characters

Granite Gabbro Basalt Diorite *Siltstone Sandstone Shale Limestone Coral limestone Coal Conglomerate Schist Marble Slate

Igneous Igneous Igneous Igneous Sedimentary Sedimentary Sedimentary Sedimentary

Orthoclase and Quartz Plagioclase and Pyroxene " Feldspar, Amphiboles, Biotite, FeO Quartz, calcite Clay minerals Quartz & clay Minerals Calcite, Dolomite, clay, Phosphate Calcite, dolomite Variable Talc Calcite Mica, Quartz

Coarse Coarse Fine Coarse /Medium Fine Medium Fine Fine

Light Dark Black Brownish Yellow Red Dark Dark Grayish White Black Black with mixed brown Dark Grayish Yellow Black

Non-layering Layering visible Layering visible Layering visible Layering visible


Reacts vigorously with HCl, honeycomb structure.

Sedimentary Sedimentary Sedimentary Metamorphic Metamorphic Metamorphic

Medium Fine Fine Fine Fine Fine

Layering visible " Formed from shale Formed from calcite. "

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Gneiss Metamorphic Mica, Quartz, Amphibole Coarse Black "

IDENTIFICATION OF MINERALS: Minerals may be defined as a natural inorganic substance, which has definite physical and chemical properties. Minerals can be classified as primary and secondary. Primary minerals are formed at elevated temperature and inherited from the igneous and metamorphic rocks, and occasionally through a sedimentary cycle. They make up the main part of the sand and silt fractions of the soil. Secondary minerals are formed by low temperature reactions and inherited by soils from sedimentary rocks of formed in soils through weathering processes. They are essentially hydrous aluminum silicates and they constitute the finer fraction of soil. Some Important Minerals and Their Characters:
Minerals Quartz Albite Chemical composition SiO2 NaAlSi3O8 KAl2 (AlSi3)O10 (OH)2 K(Fe,Mg)3 (AlSi3)O10 (OH)2 Fe2O3 2Fe2O3.3H2O Al2O3.2H2O CaSO4.2H2O BaSO4 CaCO3 Ca5 (Cl.F)(PO4)3 KAlSi3O8 KAlSi3O8 Mg3(Si4O10)(OH)2 CuCO3 Cu (OH)2 PbS FeS2 HgS Groups Silicate '' '' '' Oxide '' '' Sulphate '' Carbonate Phosphate Silicate Silicate Silicate Carbonate Sulphide Sulphide Sulphide Colour White Light pink White Black Iron black yellow brown Brick red White White Pinkish white Brick red Black Pinkish white White Bright Green Leadgrey Silver color Red Hard ness Hard Hard Soft Soft hard Hard Hard Soft Hard Soft Hard Soft Hard Softest Hard Soft Hard Soft Transparency Transparent Opaque Opaque Opaque Opaque Opaque Opaque Transparent Opaque Translucent Opaque Opaque Translucent Translucent Opaque Opaque Opaque Opaque Specific gravity 2.65 2.61 2.7-3.1 4.9-5.3 3.6-4.0 3.5 2.3 3.0-3.5 2.71 3.173.23 Other characters Glassy appearance Looks like flesh Thin sheet Thin sheet Luster dull (not bright or clear) Effervesce with HCL Timesheet Scratched by Ginger nail Soluble in Nitric acid -

Muscovite (White mica)


Biotite (black mica)

Hematite Limonite Bauxite Gypsum *Barite Calcite Apatite Orthoclase Microcline Talc Malachite Galena Pyrite Cinnabar

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Orpiment As2 S3 Sulphide Lemon yellow Soft Translucent -

DETERMINATION OF PARTICLE DENSITY OF SOIL BY VOLUMETRIC FLASK METHOD


PARTICLE DENSITY:
The weight per unit volume of the solid portion of soil is called particle density. It depends upon the accumulative of individual inorganic and organic constituents of soil. It is usually expressed in g/cm3 or g/ml. In mineral soil, particle density usually varies from 2.60 to 2.75 g/cm 3 while in organic soil it is 1.2 to 1.7 g/cm 3. The particle density is higher (above 2.75 g/cm3) in soil, where large amount of heavy minerals such as magnetite, limonite zircon etc. are present. With increase in organic matter of soil, particle density decreases. Organic soil (peats, mucks) would have naturally low particle density. Surface soil possesses lower particle density than subsoil. The followings two methods can be used to determine the particle density of soils: 1. Volumetric flask method 2. Pycnometer method MATERIALS REQUIRED: 1. Volumetric flask (100 ml) 2. Beaker 3. Measuring cylinder 4. Balance 5. Hot water bath 6. Thermometer 7. Spatula 8. Ethanol PROCEDURE: 1) Weigh an empty 100 ml volumetric flask. 2) Take some amount (usually 20-30 g) of oven dry soil in the flask and record their weight. 3) Add about 30 ml of distilled water in the flask and boil it in a water bath for 15 minutes to disperse the aggregates. 4) Cool it in room temperature. Add water to bring the volume up to the mark of the flask and take their weight. 5) Record the temperature of water and calculate the volume of water. EXPERIMENTAL DATA/OBSERVATION: No of observation Wt. of empty flask = W g Wt. of flask + soil = W1 g Wt. of flask + soil + water = W2 g Temp. of water = T0C Density of water at T0c = dw g/cm3

CALCULATION: mass of soil (solid) g/cm3 volume of soil (solid) Volume of soil (VS) = 100 Volume of water (VW)
Particle density =

W2 W1 where, dw = density of water dw W2 W1 So, Volume of soil (VS) = 100 __ dw Particle density = g/cm3 W1 W
Volume of water (Vw) =

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100 Vw IMPORTANCE OF PARTICLE DENSITY:
1. Higher particle density retards soil aeration. 2. It is useful for the calculation of soil porosity. RESULT: The particle density of collected soil sample was ............. g/cm3. PRECAUTIONS: 1. Take all the readings carefully. 2. Take proper care to add water in the flask to bring the volume 100 ml.

L1SI/ Practical Sheet/Soil Science/ Page 7 of 14 DETERMINATION OF BULK DENSITY OF SOIL BY CORE SAMPLER METHOD
BULK DENSITY: Bulk density is the mass (weight) per unit bulk volume of soil. Bulk volume indicates the volume of solid soil particles and the volume of pore spaces. It can be expressed by the following equation:
Bulk density =

mass of soil (solid) Total volume of soil (solid)

g/cm3

Bulk density values of clay, clay loam and silt loam soils normally range from 1.0 to 1.6 g/cm3 while sands and sandy loams may range from 1.2 to 1.8 g/cm3. The bulk density of mineral soil is generally 1.0 to 1.8 g/cm3 while organic soils are 0.5 g/cm3. FACTORS AFFECTING BULK DENSITY: Bulk density increase with increasing depth in the soil profile. This is due to lower levels of organic matter, less aggregation and more compaction, ploughing, and the tillage operations usually increase pore space and decrease bulk density. METHODS: The followings two methods can be used to determine the bulk density of soils. 1. Paraffined clod methods 2. Core sampler methods EQUIPMENTS FOR CORE SAMPLER METHODS: 1. Core sampler 2. Balance 3. Oven

4. Slide Calipers
5. Knife 6. Hammer PROCEDURE OF CORE SAMPLER METHODS: 1. Weigh the empty core. 2. Measure the length and internal diameter of the core. 3. Take undisturbed soil by the core from the field. 4. Dry the soil with core in an oven at 105 0c for 24 hours. 5. Weigh the oven dry soil with core. TABLE / OBSERVATION: No. of Wt. of empty observation core,w1(g) 1 2 3 Average

Wt. of core with oven dry soil, w2 (g)

Wt. of oven dry soil,w2-w1 (g)

Volume of soil (cm3)

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CALCULATION: Wt. of the empty core sampler = W1 g Wt. of oven dry soil with core sampler = W2 g Internal diameter of the core sampler = r cm Height of the core sampler = h cm Wt. of soil solid = (W2 W1) g Volume / Bulk volume of soil (cm3) = r2h cm3 So, bulk density = (W2 W1) r2h g/cm3

RESULT: The bulk density of collected soil sample was.......................... g/cm3 PRECAUTIONS: 1. Take undisturbed soil by the core sampler. 2. Take all measurements carefully. 3. Avoid too much wet or dry soil. 4. Remove the sampler carefully, not to disturb the structure and packing of the soil.

L1SI/ Practical Sheet/Soil Science/ Page 9 of 14 TECHNIQUES OF STERILIZATION


INTRODUCTION:

The act or process of destroying all forms of life is known as sterilization. So, a sterile object, in the microbiological sense is free of all living organisms.
AGENTS OF STERILIZATION:

There are many agents of sterilization such as heat, radiation, sound, electricity, pressure, filtration, and chemicals like alcohol, formaldehyde, and acetic acid etc. Heat is the most commonly used sterilizing agent. It is of two types: dry heat and moist heat.
1. DRY HEAT:

Direct heating of the instrument in a flame is an easy way of sterilization. Inoculating needles, scissors, forceps, scalpels etc. are commonly sterilized by direct heat. Specially designed ovens, which are heated by electricity or gas, are commonly used. For all practical purposes, 170 0C (338 0F) temperatures maintained for at least one hour would accomplish sterilization of most laboratory materials subjected to dry heat. Dry heat is used to sterilize materials such as glassware (e.g., pipette, petridish), powders and other materials, which are not injured by high temperature or which may be corroded or deteriorated by moisture.
2. MOIST HEAT:

Sterilization may be accomplished more easily by moist heat than by dry heat. This is due to the more rapid penetration of heat in a moist environment. Moist heat may be applied with hot or boiling water or with steam. With boiling water or with steam (not under pressure) the temperature will be near 100 0C (212 0F) depending upon the atmospheric pressure. Thus, excessively long periods of time may be required to sterilize materials containing spores. For this reason, steam under pressure is used rather than atmospheric steam and it is used solely for the purpose of attaining high temperature. Air mixed with steam will materially lower the temperature attainable at any given pressure.
FUNCTION OF AUTOCLAVE:

Most bacteriological media and solutions are sterilized by moist heat in an apparatus known as autoclave. Here, heat is in the form of saturated steam under pressure and the temperature can be increased or decreased with the increase or decrease of pressure. Usually a temperature of 121 0C (249.8 0F) maintained for 15 to 30 minutes using steam less than 15 PSI (pound per square inch) pressure is kept in autoclave for sterilizing most types of media, dilution water, cloth, rubber etc. Autoclave is equipped with a thermometer, a pressure gauge, and with devices that allow air and condensate to escape and which automatically admit sufficient steam to maintain the desired temperature and pressure in the chamber during operation.

L1SI/ Practical Sheet/Soil Science/ Page 10 of 14 PREPARATION OF BACTERIAL MEDIA


INTRODUCTION:

In microbiology, a "medium" is a substrate in which bacteria can grow. Like all other living forms, microorganisms required suitable nutrients as well as a favourable environment. The culture medium must contain those nutrients essential for growth of a microbial culture. At the same time, the medium must also provide suitable surroundings for growth that is, the proper pH, osmotic pressure, atmospheric oxygen etc. All culture media are of two forms: liquid or broth media and solid media.
LIQUID MEDIA:

There may be numerous recipes for broth or liquid media depending on the kind of bacteria to be grown. All liquid media, however, must provide the proper physical and chemical environment and nutrient substance in water solution. The liquid medium most commonly used the following composition. Yeast Mannitol Broth: Composition for K2HPO4 MgSO4. 7H2O NaCl Mannitol (C6H14O6) Yeast water Distilled water = 0.5 g = 0.2 g = 0.1 g = 10 g = 100 ml = 900 ml

This liquid medium is sterilized in autoclave at 121 oC for 15 minutes. 3 g of CaCO3 per liter can be added when excess neutralizing agent is required. Mannitol may be substituted by cheaper Ccompounds. Fresh yeast extract is prepared from 100 g baker's compressed yeast mixed with one liter of cold water, allowed to stand at room temperature for 1-2 hours, autoclaved for 40-60 minutes, allowed to settle (or centrifuged) and the clear supernatant (adjusted to pH 6.8) used as yeast water.
SOLID MEDIA:

The solid medium contains 15 g agar/litter along with composition of yeast mannitol broth. Such medium is known as YMA (Yeast Mannitol Agar) medium.

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IDENTIFICATION OF BACTERIA BY GRAM STAINING METHOD


INTRODUCTION:

In 1884 Hans Christian Gram" a German bacteriologist developed a special staining procedure known as "Gram staining" to study the bacteria. The staining method is now largely followed in bacteriology. By using this procedure bacteria can be divided into two groups: Gram-positive and Gram-negative. The gram staining requires four different solutions: a basic dye (e.g., crystal violet), a mordant (e.g., iodine), a decolorizing agent (e.g., ethyl alcohol) and a counter stain (e.g., safranin). Bacteria that retain the initial dye when washed with decolorizing agent (alcohol) are termed as Gram-positive; e.g., Corynebacterium, Mycobacterium. Bacteria that fail to retain the initial stain but take the counter stains are called Gram-negative, e.g.; Rhizobium, Chemobacterium, Azotobacter. The difference in staining is believed to be due to the variance in the surface layers or wall of the two types of cells.
APPARATUS REQUIRED:

1. 2. 3. 4. 5. 6. 7.

Watch glass Slide Microscope Cover slip Inoculating needle Bacteria culture/Bacterial suspension Glass rod

REAGENTS REQUIRED:

a. Crystal violet solution: Crystal violet : 10 g NH4- oxalate : 4g Ethanol : 100 ml Distilled water : 400 ml b. Iodine solution: Iodine : 1g KI : 2g Ethanol : 25 ml Distilled water : 100 ml c. Ethyl alcohol (ethanol) - 95% d. Counter stain: 2.5% safranin in ethanol : 10 ml Distilled water : 100 ml
PROCEDURE:

1. Take a little drop of bacterial suspension on a clean slide and spread the drop. (Liquid
culture should be spread without dilution). Allow it to dry in the air when the air-dried suspension appears as a thin film on the slide is termed as smear. The air-dried smear was passed once through the top of a spirit lamp. Allow it to cool. (The air-dried bacterial film fixed in the flame is called smear).

L1SI/ Practical Sheet/Soil Science/ Page 12 of 14 2. Stain the smears with crystal violet solution for one minute. Wash in tap water. 3. Immerse the film for one minute in iodine solution. Wash in tap water, and allow it to dry
in the air. 4. Add ethyl alcohol on the film and after one minute wash it in tap water and allow it to dry in the air. 5. Counter stain the smear with safranin for one minute. Wash it in tap water and dry. 6. Examine under oil immersion objective.
RESULT:

If the color of the smear appears violet, it should be concluded that the bacteria under study were Gram-positive bacteria. On the other hand, if the color appears red the bacteria will be Gramnegative bacteria.

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MOTILITY TEST OF BACTERIA BY HANGING DROP METHOD


INTRODUCTION:

Motile refers capable of motion and motility refers capability of movement of organisms by their on bodily responses that involved muscular rather than audiovisual sensation. Bacteria exhibit two types of motility: 1. Brownian movement and 2. Vital movement
Brownian movement: Brownian movement occurs due to quivering or back and forth motion

exhibited by very small particles suspended in a liquid.


Vital movement: Vital movement is generally believed to be due to the presence of organs of

locomotion known as flagella. Flagellated bacteria are called motile bacteria because they move under their own power. Motile species of bacteria may not exhibit movement at all times, due to loss of the organs of motion or due to physiological conditions. Motility is most readily seen in young cultures, which have not been subjected to mechanical damage. Flagella are too thin to be seen with the ordinary microscope and in order to see them special staining methods are as followed. Example of Motile and Non-motile bacteria: Motile bacteria: Pseudomonas, Rhizobium Non-motile bacteria: Sarcina, Corynebacterium
MATERIALS REQUIRED:

1. 2. 3. 4. 5.

Hollow ground slide Cover slip Inoculating needle Bacteria culture Vaseline etc.

PROCEDURE OF HANGING DROP METHOD:

1. Place a thin rim of Vaseline around the edge of a clean cover slip.
2. Put a drop of water in the centre of the cover slips and emulsifies a tiny bit of culture in it. Don't spread the drop. 3. Invert a hollow ground slides over the cover slip so that it well encloses the drop, press the slide onto the cover slip to seal. 4. Quickly and carefully invert the slide so that the hanging drop is suspended from the cover slip in the well of the slide. 5. Observe, focusing first with low power, and then with the oil immersion objective.
RESULT:

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Bacteria under study were motile or non-motile.