eChapters

The articulations of the neck are between the bones of the cervical vertebrae. The atlanto occipital articulation and the atlanto axial articulation are the special vertebral articulations while the articulations between the rest of the cervical vertebrae are common vertebral articulations.

Atlanto-occipital articulation

Type of joint: This is a condyloid type of hinge joint (Ginglymus).

Articular surfaces: The atlanto-occipital joint (Figure 2-3 B) is formed between the condyles of the occipital bone and the condyloid cavities of the atlas vertebra.

Most of the bones of the skull are joined (articulated) with the adjacent bones by sutures. These are immovable joints (synarthroses). The uniting medium is a fibrous tissue and is called the sutural ligament. Most of these joints exist in the early life of the animals and as the age of the animal advances the adjacent bones fuse with each other.

Horse

The first cervical vertebra (atlas) of horse is more or less similar to that of ox with a few variations as below

The wings of atlas are curved ventrad and laterad. These are thinner than in ox but their borders are thick.

The wings present an additional foramen, the transverse foramen in the caudal part.

A faint groove on the ventral surface of the wing connects the transverse foramen to the intervertebral foramen.

Cervical vertebrae

The skeleton of neck (Fig 2-1) is formed by the cervical vertebrae. These are seven in number. Of these, the first and second cervical vertebrae are highly modified and deviate greatly from the general architectural plan of the vertebrae. The third, fourth and fifth cervical vertebrae are similar and confirm the general plan of the vertebrae. The sixth and seventh vertebrae have some special characteristics but do not deviate much from the general plan. The general plan of vertebrae and the vertebral column has been described in the chapter on skeletal system under Part I on systemic anatomy. Hence, only regional specific characters of the cervical vertebrae are described hereunder.

First cervical vertebra (Atlas)

The first cervical vertebra (Figure 2-2 A) is called the atlas vertebra because in man it supports the globe of the head. This is an atypical vertebra and it deviates greatly from the general plan of vertebrae. The body and the spinous process are absent. The arch forms a bony ring from which two large curved plates, the wings project laterally. The ring encloses the vertebral foramen. It forms a part of the vertebral canal through which passes the spinal cord.

Horse

The skull of horse is also pyramidal in shape like that of ox but the base of the pyramid is less wide than that of the ox. It is shorter in length and narrower in width than that of the ox. The cranium is smaller externally, however, the cavity of cranium is larger than that of the ox. The nasal cavity is divided into two chambers by nasal septum. The floor of the nasal cavity is shorter and less concave than that of the ox. The left and the right nasal chambers are completely separated and do not communicate with each other. The horn core is absent.

The occipital bone is placed at the extreme caudal part of the skull, caudal condyloid foramen is absent, the basi occipital is longer and prismatic and the squamous occipital is crossed by a nuchal crest. The sphenoid bone has two parts, the pre and post sphenoid, as that in case of ox. The wing of the post sphenoid presents three notches, carotid, oval and spinosal for internal carotid artery, ventral maxillary nerve and spheno-spinous artery respectively. The foramen orbitale and the foramen rotundum are not united. There are no major differences in the ethmoid bone of ox and horse. The frontal bones are less expansive than those of ox. These are situated between parietals caudally and nasals rostrally. The supraorbital foramen perforates the root of the supra orbital process that joins the zygomatic process of the squamous temporal. The horn core is absent. The ventral borders of the frontal bones of the opposite sides form a projection which fits into the notch formed by the two nasal bones. The temporal bone in horse has three parts, the squamous, petrous and tympanic as against two parts, the squamous and petrous of the ox. The squamous part is much more developed than that of ox. It has three parts, the body, zygomatic process and the occipital (pyramidal) process. The later one arises from the caudal part of the squamous. The stylomastoid foramen is between the hyoid and the mastoid processes and the auditory bulla is smaller. The interparietal is a single bone as that of ox.

The bones of skull (Fig 2-1) are divided into two groups, as cranial group of bones and facial group of bones. Most of the bones of the skull are flat bones and a few are irregular bones.

Cranial bones

The cranial bones form the cranium that encloses and protects the brain and organs of special senses e.g. eye and ear. These bones include the occipital, sphenoid, ethmoid, inter-parietal, parietal, temporal and frontal. The first four of these bones are single and the rests are paired.

Occipital

The occipital is a single bone (Fig 2 -1C) and is situated on the caudo- dorsal aspect of the skull. It consists of three parts, the lateral parts are called lateral occipital, the basal part is called the basi occipital and the dorsal (squamous) part is called the squamous occipital.

The sense organs are the organs of the body that respond to external stimuli by conveying impulses to the nervous system. The sense organs allow animals to sense changes in the environment around them and in their bodies so that they can respond properly. Animals can sense a wide range of stimuli that include the sense of vision, the sense of hearing, the sense of olfaction (smell), the sense of taste and the sense of touch including pain and pressure. In animals, there are two major groups of senses, the general senses and the special senses.

Peripheral nerves

The peripheral nervous system (PNS) is the part of the nervous system that lies outside the brain and spinal cord. It includes cranial nerves, spinal nerves and their roots and branches, peripheral nerves and neuromuscular junctions. In the peripheral nervous system, bundles of nerve fibres or axons conduct information to and from the central nervous system. The primary role of the peripheral nervous system is to connect the central nervous system to the bones, muscles and skin of the body trunk and the limbs and the organs to allow the complex movements and behaviour.

The peripheral nervous system is divided into the somatic nervous system and the autonomic (visceral) nervous system.

The somatic nervous system is associated with skeletal muscle voluntary control of body movents. It consists of afferent and efferent nerves. Afferent nerves are concerned with relaying sensation from the body to the central nervous system. The efferent nerves are responsible for sending out commands from central nervous system to the body, stimulating muscle contraction. This system is made up of nerves that connect to the skin, sensory organs and all skeletal muscles.

The autonomic nervous system is the part of peripheral nervous system that controls the visceral functions which occur below the level of consciousness. It consists of two divisions, the sympathetic nervous system and the parasympathetic nervous system.

Brain

The brain, a command centre of the body, is a complex organ. The substance of the brain is made of the grey matter and the white matter. The grey matter is often identified as cerebral cortex but other parts of the brain also contain the grey matter. Inside the brain, the grey matter is organized into large accumulations of neurons which are called the nuclei of the brain. Each nucleus is associated with a specific function. In addition to neurons, there is other type of cells the glial cells called neuroglia. The neurons are responsible for sending and receiving impulses or signals. The glial cells are nonneuronal and provide support and nutrition to neuronal cells, form myelin and facilitate signal transmission in the nervous system. The white matter is made of millions of nerve fibres that are coming from neurons and are going to establish communications with different parts of the brain and spinal cord. Since these nerve fibres are coated with myelin sheath around them these appear white and the area occupied by these nerve fibres become the white matter of the brain.

The brain has three basic components, the forebrain, midbrain and the hindbrain.

Fore brain: The forebrain (Prosencephalon) is the rostral portion of the brain. It consists of cerebrum (Telencephalon) and the thalamus, hypothalamus and pineal gland together constituting the interbrain (Diencephalon).

Mid brain: The midbrain (Mesencephalon) is the middle portion of the brain. It is in the centre lying between the interbrain and hindbrain. It consists of a cranial part of brain stem.

Hind brain: The hindbrain (Rhombencephalon) is the caudal most portion of the brain. It consists of remaining (caudal) part of the brain stem, the medulla oblongata (Myelencephalon) and the cerebellum and pons (Metencephalon).

The lymphatic system is a part of circulatory system and a vital part of the body’s immune system. It is comprised of lymph vessels, lymph ducts, and lymph nodes that move the lymph from tissues to the blood stream and the organs such as the spleen, thymus and bone marrow that produce the lymphocytes and white blood cells. Lymph is very similar to blood plasma. During circulation through the body, the blood reaches to the capillaries at the interstitial spaces. The plasma of the blood is filtered through the capillary wall. The fresh oxygen along with nutrients, hormones and other necessary fluid matters from this plasma are released in to the interstitial fluid and about 85 per cent of the plasma along with the carbon dioxide and metabolic wastes, return into the circulation through the capillary walls. The remaining 15 per cent of the plasma along with the debris of other waste products and the lymphocytes constitutes the lymph which is returned to the circulation through an accessory route of lymphatic vessels and lymph nodes constituting the lymphatic system.

Lymph Vessels: The lymph vessels start as finger-like blind ending thin walled lymph capillaries in the interstitial connective tissue. These capillaries form a network and drain into the thicker walled collecting lymph vessels, lymph ducts and lymphatic trunks. Finally the larger lymph trunks and ducts empty their content the lymph into the cranial vena cava at the thoracic inlet. The lymph vessels contain numerous valves with one or two leaflets preventing the back flow of lymph through the lymph vessels. During its course in a centripetal direction through the lymph vessel, the lymph of each lymph vessel has to pass through at least one lymph node prior to its entry into the blood circulation. As a result there lies a chain of lymph nodes on the pathway of lymph vessels in the body.

The great vessels that leave and enter the chambers of the heart include the Aorta, the Pulmonary Artery and the Pulmonary Veins.

Aorta

The Aorta is the main systemic arterial trunk. It begins as ascending aorta at the base of the left ventricle, passes craniad and dorsad between the pulmonary artery on the left and the right atrium on the right and then turns caudad inclining slightly to the left, thus describing a curve called the aortic arch with its convexity directed upward and forward and the concavity facing downward and backward. From here it proceeds caudad on the ventrolateral surface of the thoracic vertebrae and the longus colli muscle as the descending aorta. At its origin from the base of left ventricle, the caliber of the aorta is greatest forming the bulbus aorta and here it presents three dilatations called the aortic sinuses. As the descending aorta reaches the level of the seventh thoracic vertebra it continues behind as thoracic aorta running through the middle and caudal mediastinum between the left and right lungs. The thoracic aorta first lies to the left of the median plane and then gradually inclines inwards medially and becomes median in position when it reaches to the diaphragm. It then passes through the hiatus aorticus of the diaphragm and enter the abdominal cavity as abdominal aorta. From here it passes caudad below the bodies of the lumbar vertebrae and the psoas major muscle. After reaching at the level of the sixth lumbar vertebra, the abdominal aorta terminates in the iliac quadrification by dividing into two external and two internal iliac arteries.

Heart

The heart is a cone shaped thick walled and four chambered muscular organ. It lies obliquely craniocaudal in the middle mediastinum in between the left and right lungs and is covered by pericardium inside the thoracic cavity. The major part of heart (about 60 per cent) lies to the left of the middle mediastinum. The broad upper end is the base of the heart. It lies at the junction of the dorsal and middle third of the dorsoventral diameter of thorax at the level of about third to sixth rib and it faces craniodorsally. The narrow lower end is the apex. It lies just dorsal to the sternum at the level of about sixth rib and it faces caudoventrally. The surfaces, right and left are convex. They meet cranially at the right ventricular border and caudally at the left ventricular border. The right (cranial) border is strongly convex. It runs ventrally and caudally. The greater part of this border lies parallel to the sternum. The left (caudal) border is much shorter and nearly vertical. The surfaces of the heart are marked by grooves which indicate the division of the heart into four chambers the two atria dorsally and the two ventricles ventrally. The atria are separated internally by the interatrial septum. The ventricles are separated by interventricular septum. The circular coronary groove indicates the division of the heart in to atria dorsally and ventricles ventrally. It encircles the heart in its upper third part except at the level of the origin of pulmonary artery. The coronary groove is quite deep and wide. It provides passage for the coronary artery and vein. Apart from these, it is deeply filled with fat. The right (subsinuosal) interventricular groove and left (paraconal) interventricular groove indicates the line of separation between the two (right and left) ventricles by the interventricular septum. Both of these grooves run downward from the coronary groove towards the apex parallel to the respective borders and terminate just above the apex. The right and left ventricular grooves are occupied by the respective coronary vessels along with the fat.

Pituitary gland

The pituitary gland (hypophysis) lies at the base of the skull and is housed within a bony structure called the sella turcica in the body of the basisphenoid bone. It is a small pea shaped gland which is attached to the hypothalamus by the pituitary stalk. The pituitary gland has two distinct parts called the lobes. These are the anterior lobe (adenohypophysis) and the posterior lobe (neurohypophysis). The anterior lobe which is also called as anterior pituitary consists of five types of cells, each of which secretes different hormone or hormones. The types of cells, the hormones secreted by each type and the functions performed by them are narrated below.

Reproduction is the physiological process by which organisms produce offspring. Sexual reproduction involves production of gametes. The gametes are produced by the gonads. The rest of the process of converting the gametes into the offspring involves other reproductive organs. The male and female animals have different genital organs that contribute to reproduction. The organs of reproduction (genital organs) in male include a pair of male gonads the testes (sing.-testis), the epididymis, ductus deferens, the seminal vesicles, a prostate gland, a pair of bulbourethral glands (Cowper’s glands), the male urethra, and the penis. In female the genital organs include a pair of gonads the ovaries, the fallopian tubes, the uterus, the vagina, the vulva and the mammary glands.

Male genital system

The overall function of the male genital organs is to continuously produce and store the male gametes the sperms, to maintain the continuous supply of semen, to detect estrus in female, and to inseminate and fertilize the female gametes. While the testes are the site for production of sperms, the epididymis, vas deferens and the accessory glands like prostate, seminal vesicles, and bulbourethral glands are responsible for the production of different secretions which mix together and are known as seminal plasma or seminal fluid. The seminal fluid provides a source of energy, buffers, antioxidants and other compounds needed for sperm survivability, maturation and motility. Once spermatozoa are mixed with the seminal fluid, the combined product is called the semen.

Urinary system is relatively simple and consists of a pair of kidneys, a pair of ureters, the bladder and the urethra. The kidneys are the main organs that filter the blood and produce the urine while the rests of the organs of this system are simply accessory structures that are used for transport and storage of urine. For proper functioning of the metabolic processes of the body, it is essential to constantly maintain the chemical composition and volume of the tissue fluid. This is called the homeostasis. This particular task is performed by the urinary system and in particular by the kidneys.

Kidneys

The kidneys right and left, are bean shaped red brown relatively large glands situated in the abdominal cavity. These are placed retroperitoneal against the dorsal wall of the abdomen on either side of the spine. The right kidney is about half of its length ahead of the left kidney and it fits into the renal impression on the caudate lobe of the liver. An indentation at about the middle of the medial border is the hilus of the kidney through which pass the renal vessels, nerves and ureters. Both the kidneys are covered by a thin capsule formed of fibrous tissue which surrounds the parenchyma of the kidneys. The capsule can be easily striped away from the kidney. The function of the kidneys is to maintain the volume and composition of the plasma,regulate water, ion, and pH levels, retain nutrients and excrete fluid wastes, toxins and excess electrolytes. The kidneys achieve these functions through glomerular filtration, solute reabsorption, tubular secretion, water balance and acid-base regulation.

The renal capsule provides a stiff outer shell to maintain the shape and protect the inner soft parenchyma. Deep to the capsule is the soft, dense vascular cortex. Several cone shaped pyramids form the medulla deep to the cortex. The pyramids are aligned with their bases facing outward towards the cortex and their apices point inward towards the centre of the kidney. Each apex connects to a minor calyx, a small hollow tube that collects urine. The minor calyces merge to form three major calyces, which further merge to form the hollow renal pelvis at the centre of the kidney. The urine collected into the pelvis drains into the ureter that exits through the hilus of the kidney.

The respiratory system is a complex biological system comprised of several organs that facilitate the inhalation of oxygen and exhalation of carbon dioxide (breathing) in living organisms. The major functions of the respiratory system is providing oxygen to the body cells and removing carbon dioxide from the body. In addition to this, the respiratory system helps in controlling body temperature (especially in dogs), eliminating water (as vapour) and assisting in voice production. The organs of respiratory system extend from nose to lungs. These consist of two major parts, the upper respiratory tract and the lower respiratory tract. The upper respiratory tract is comprised of nose, nasal cavity, the part of mouth, and the part of pharynx. The lower respiratory tract is comprised of the larynx, trachea, bronchi and lungs.

Nose

The nose is the externally visible part of the respiratory system. It forms the part of the face of the animal extending from the transverse level of eyes to the rostral end of the head. Exteriorly it is formed by the bones, muscles, cartilages and the skin. Internally it encloses the nasal cavity which is lined by the mucous membrane. The dorsal part of the nose (roof of the nasal cavity) is termed as dorsum nasi. It is formed by the nasal and frontal bones of the skull. The lateral parts are formed by the incisive, maxillary, lacrimal and zygomatic bones. Covering these bones externally is a muscular layer formed by the facial muscles running over these bones and acting on the upper lip and nostrils. The muscular layer is externally covered by the skin. The skin on the nose bears fine hairs except at the tip of the nose that is formed by the nostrils. The rostral end of the nose is formed by nostrils which provide entrance to the nasal cavity. The nostrils are supported by cartilages which are the dorsal and ventral extensions of the cartilage of the nasal septum. The main aim of the nose is to support and protect the nasal cavity.

The digestive system consists of organs concerned with prehension and digestion of food, absorption of digested food and expulsion of unabsorbed food. The digestive system extends from lips to the anus and consists of the primary and accessory organs of digestion. The primary organs include the mouth, pharynx and the alimentary canal (gastrointestinal tube) in that order of sequence. The accessory organs include the tongue, teeth, salivary glands, liver and pancreas. The alimentary canal extends from pharynx to anus and consists of the oesophagus, the stomach, the small intestine and the large intestine. The wall of the alimentary canal consists of, from within outward, the mucous membrane, the muscular layer and the serous layer forming the visceral peritoneum.

Mouth

The mouth is bounded dorsally (roof) by hard palate and behind that the soft palate, ventrally by the body of the mandible along with the mylohyoideus muscles and laterally by the cheeks. The entrance of the mouth cavity is bounded by the lips and it is called as rima oris, while its caudal opening into the pharynx is called as aditus pharyngis. The cavity of the mouth (cavum oris) is subdivided into two parts by the teeth and alveolar processes (the alveoli) of the mandible. The inner part i.e. the space within the teeth and alveoli is termed as mouth cavity proper, while the space between the cheeks and lips and the teeth and alveoli is called the vestibule of the mouth. The mucous membrane of the mouth cavity is continuous with the margin of the lips and the common integument and caudally it continues with the mucous membrane of the pharynx.

A branch of anatomy that deals with muscles and their accessory structures is called as myology and the body system concerned with myology is called muscular system. The muscular system is composed of specialized cells called muscle fibres because of their shape in the form of fibre or spindle. The plasma of these cells contains myofibrils which are contractile elements that cause contraction of muscles in a definite manner under the influence of a stimulus which is a unique property of muscles. These cells together constitute the distinctive muscular tissue.

Classification of muscles

Based on their morphological and functional characteristics, there are three types of muscles in the body.

Smooth muscle: The muscular tissue in this type of muscles is constituted by masses of spindle shaped cells and they occur mainly in the walls of hollow visceral organs including glands, the vessels, eye ball and hair follicles. These are nonstriated muscles. Contraction in these muscles is weak but sustained and mostly consistent and rhythmic. The action of this muscle is involuntary i.e. not under the will.

Cardiac muscle: The muscle fibres of this muscle are arranged in irregular masses and they form cross striations. The ends of some fibres come in contact with the sides of others forming a kind of syncytium. This is a striated muscle and its action is involuntary. This muscle occurs only in the wall of the heart and the immediate proximal ends of the major blood vessels like aorta, venacava and the pulmonary veins.

A branch of anatomy that deals with joints or articulations in the body is termed as arthrology or syndesmology. A joint is an articulation or union formed by two or more bones or cartilages with the help of connective tissue. To form a joint, the long bones meet at their extremities e.g. joints of fore limb and hind limb. Joints of flat bones are formed by their borders or edges e.g. joint of bones of skull, the cranial and facial bones. The short bones and irregular bones form a joint by uniting at any of their parts or surfaces e.g. intervertebral joints.

Classification of joints

Joints are classified on the basis of their structure i.e. the kind of tissue material by which the bones are united, the other constituent parts that help in uniting the bones and the kind of movement performed at that joint. On this basis, the following three major types of joints exist in the skeleton of animal body.

In domestic animals, skeleton (Fig. 1-1) is a framework that is formed of bones, cartilages and ligaments binding together giving the specific shape, form and support to the body. In addition, the skeletal framework surrounds the major cavities of the body giving protection to other soft visceral organs of the body. There are three major parts of skeleton in the animal body (1) the axial skeleton (2) the appendicular skeleton and (3) the splanchnic skeleton.

The axial skeleton consists of the skull, vertebral column, ribs, and sternum. The appendicular skeleton consists of the bones of fore limb and hind limb

The splanchnic skeleton includes some of the bones embedded in the substance of visceral organs in the body such as os cordis in ox and os penis in dog.

Bones

The bones forming the skeleton of the body are of different shape, size, form and the constituting tissue material. The number of different types of bones of any species may differ in various stages of the growth of the animal. So also the numbers of various bones differ from species to species.

Mole

A mole is defined as the amount of chemical substance containing exactly 6.022 140 76 × 10₂₃ numbers of constitutive particles such as atoms, ions and molecules. This number is a fundamental constant named Avogadro’s number in honor of Italian scientist Amedeo Avogadro. This constant is properly reported with an explicit unit of “per mole,” and rounded version being 6.022 × 10₂₃/mol.

This definition has been adopted since November, 2018 revising the old definition based on the number of atoms in a sample of pure ₁₂C weighing exactly 12 g. Thus, as per the definition, 1 mole of any element contains the same number of atoms as 1 mole of any other element. However, the mass of 1 mole of a particular element is different from that of another element.

Molar mass

Molar mass of an element (or compound) is the mass in grams of 1 mole of that substance, a property expressed in units of grams per mole (g/mol).

Principle

Different ions migrate at different speed dictated by their size and by the amount of charges they carry. As a result, different ions can be separated from each other by electrophoresis. The magnitude of the electric force F exerted on the charged particle when subjected to electrophoresis is product of the charge q of the particle and the electric field E generated between the two electrodes.

Acid and alkaline phosphatase enzymes are among the extensively studied phosphatases and classified so because of their optimum activities in acid and alkaline pH ranges, respectively. Acid phosphatase is predominant in acid soil while alkaline phosphatase is dominant in alkaline soils. The colorimetric assay of phosphatase is based on release of p-nitrophenol from hydrolysis of p-nitrophenyl phosphate byaction of phosphatase enzyme in buffered soil solution (buffer pH 6.5 for acid and 11 for alkaline phosphatase assay).

Principle

β-Glucosidase (BG) plays a major role in the degradation of soil organic matter and plant residues. It catalyzes the hydrolysis of β-d-glucopyranosides, the final, rate-limiting step in the degradation of cellulose, the most abundant polysaccharide providing simple sugars for the soil microbial population. BG has been shown to be sensitive to changes in soil and residue management as well as an early indicator of changes in SOC before these changes are reflected in total or organic C analyses. The method is based on colorimetric determination of the p-nirtrophenol released by β-glucosidase when soil is incubated with buffered PNG solution and toluene.

Fluorescein diacetate (3’ 6’-diacetyl-fluorescein) is a fluorescein conjugated to two acetate radicals. This colourless compound is hydrolysed by both free (exoenzymes) and membrane bound enzymes, releasing a coloured end product, fluorescein. This end product absorbs strongly in the visible wavelength (490 nm) and can be measured by spectrophotometry. The enzymes responsible for FDA hydrolysis are plentiful in the soil environment. Non-specific esterases, proteases and lipases, have been shown to hydrolyse FDA. The hydrolysis of FDA by soil enzymes can be estimated by methods of Adam and Duncan (2001).

Biological activity or index of a soil is the function of number of organisms present in soil and their physiological efficiency. The rate of respiration can be used as an index of the biological activity of soil as it reflects the physiological efficiency of the organisms. Monitoring of dehydrogenase, which are respiratory enzymes and integral part of all soil organisms, will give a measure of biological activity of soil at a given time.

During respiration biological oxidation of reduced compounds occur which is catalyzed by dehydrogenase. In the process TTC gets reduced to a pink coloured compound Triphenyl formazan (TPF) which can be quantitatively extracted by methanol and measured colorimeterically. The soil dehydrogenase activity in the current experiment was measured as per the methods of Cassida, (1977).

Enumeration of microbial flora present in soil is done by dilution plating technique. The technique is based on the fact of diluting the source inoculums to the extent that after plating the known volume of inoculums on appropriate media, the colonies will appear in countable range and each colony will be assumed to have been derived from a single cell/colony forming unit. Three numerically dominant groups of soil microbes are bacteria, actinobacteria and fungi. The most commonly used culture media for their enumeration include

Nutrient Agar-for heterotrophic bacteria,

Actinomycetes isolation Agar or Kenknight and Munaier’s agar for actinobacteria

Martin’s Rose Bengal Agar for fungi.

Analytical methods for study of soil microbes

The analytical techniques for study of soil microbes are grouped under the following heads :

• Determination of form and arrangement of microorganisms in soil
• Isolation and characterization of soil microorganisms
• Detection of microbial activity in soil
• Determination of microbial biomass.

For the above, the different analytical methods used are described below.

This procedure uses Diethylene Tri-amine Penta Acetic acid (DTPA) as the chelating agent, which combines with free metal ions in soil solution and forms soluble complexes.

The hot water soluble method is the most common method to estimate available B content in soil

Reagents

1. Azomethine-H: Dissolve 0.45 g azomethine-H and 1.0 g L-ascorbic acid in about 100 ml de-ionized or redistilled water. If solution is not clear, it should be heated gently on water bath at about 30ºC till it dissolves. A fresh solution should be prepared every week and kept in a refrigerator.
2. Buffer solution: Dissolve 250 g ammonium acetate in 500 ml de-ionized or redistilled water. Adjust the pH to about 5.5 by slowly adding approximately 100 ml glacial acetic acid, with constant stirring.
3. EDTA reagent (0.025 M): Dissolve 9.3 g EDTA in de-ionized or redistilled water and makes the volume up to 1 litre.
4. Standard Boron solution: Dissolve 0.571 g boric acid (H₃BO₃) AR grade in a small volume of deionized water and make volume to 1000 ml to obtain a stock solution of 100 mg B/L.

In this method, plant available sulphur (S) is extracted with 0.15% CaCl₂ and the soluble sulphate is estimated on a colorimeter.

In soil system, potassium (K) exists as water soluble, exchangeable, non- exchangeable and lattice K. The plant available K refers to the sum of water soluble and exchangeable K. Estimation of available K is usually done using neutral normal ammonium acetate as the extractant.

Estimation of plant available phosphorus content in soil has been attempted using several extractants. However, Bray’s reagent and Olsen’s reagent are the two common extractants used in India. Bray’s reagent (0.03 N NH₄F and N HCl) is used for acid soils, where, the fluoride ion complexes with Fe and Al in soil and thus releases the bound P. The Olsen’s reagent (0.5 M NaHCO₃) is used for neutral-alkaline soils.

This is a distillation method proposed by Subbiah and Asija (1956), where the soil is distilled with alkaline KMnO solution. The ammonia liberated is trapped in boric acid and then determined by titration against a standard acid. This procedure estimates the easily mineralizable nitrogen in soil.

Soil respiration is a good estimator of overall biological activity and thus is often used for describing soil quality. Soil respiration is estimated as the flux of CO₂ emitted from the soil surface. It represents the sum of CO₂ produced by root respiration and by heterotrophic decomposition. This procedure is a laboratory method where soil is incubated for a given period of days and the released CO is trapped by sodiumhydroxide (NaoH). This is also known as alkali trap method.

Particulate soil organic matter (POM) is one of the important soil organic matter (SOM) pools and matches the characteristics of a slow, decomposable or stabilized SOM pool. The POM fraction is considered as one of the indicators to changes in management and thus has been used in soil quality assessment studies.

Organic carbon present in soil can be very labile, labile or non-labile. This procedure measures the extent of lability of soil organic carbon through relative degree of oxidation by K₂Cr₂O₇.

Labile fractions of soil carbon (C) refer to the fraction of soil carbon that are easily altered by microbial activities. These fractions serve as sensitive indicators to changes in management induced soil quality and are grouped under active soil C pool. The method described by Weil et al. (2003)estimates changes in biologically active soil C using a dilute KMnO . This is an improvement over the method used by Blair et al (1995), where a very high concentration (0.333 M) of KMnO₄ was used.

This is one of the commonly used method to estimate organic carbon content in soil. In this wet digestion method, soil organic matter is oxidised with a mixture of K₂Cr₂O₇ and concentrated H₂SO₄ utilizing the heat of dilution of H₂SO₄. The unused K₂Cr₂O₇ is back titrated with ferrous sulphate or ferrous ammonium sulphate.

In dry combustion technique, the combustion of soil sample (about 250 mg and mm sieved) is carried out at high temperature (about 1000 ºC) through feeding in an inert boat in the electric furnace. The CO₂ produced from the oxidation of carbon present in the soil sample at high temperature is carried towards non dispersive infrared detector (NDIR) by a carrier gas where it is quantified and total carbon content is determined.

Wet sieving procedure separates soil into different size aggregates. However, for comparing the aggregate size distribution among management treatments and soil types, it is convenient to use indices. Mean weight diameter (MWD) and water stable aggregate percentage are some of the common indices used in aggregate stability studies.

Mean Weight Diameter (MWD)

This index was proposed by Van Bavel (1949) and is being used widely. The entire soil sample is passed through an 8 mm sieve prior to analysis and is subjected to wet sieving procedure as described before. The parameter which Van Bavel (1949) called the MWD is equal to the sum of the product of (a) the mean diameter (d ) of each size fraction and (b) the proportion of the total sample weight (w ) occurring in the corresponding size fraction. The summation is carried out over all ‘n’ size fraction including the one that passes through the finest sieve.

Thisprocedure estimates aggregatestabilitytoimpact ofraindropsand water erosion. Wet sieving procedure was originally proposed by Yoder, 1936. Later, the procedure was modified byKemper and Rosenau, 1986.

Equipment

1. Yoder type sieve shaking electrically operated machine (3/4 inch stroke at 30 strokes per minute)
2. Concentric sieves of various sizes of pores to fit properly into the machine.
3. Weighing balance

Aggregation is one of the basic physical characteristics of soil. Under natural conditions, primary soil particles, particularly clays, tend to join together to form secondary units called aggregates. Size distribution of soil aggregates is important because the size of the aggregates determines their susceptibility to water and wind erosion. Also, their size is important in determining the pore size distribution and regulating pore space in cultivated soils.

There are a variety of procedures being in use to test the stability of aggregates and aggregate size distribution. However, choice of the method for analysis of aggregate size distribution is determined by the purpose of analysis. The developed measures are relative and purpose specific. This chapter discusses dry aggregate analysis.

Aggregation is a direct indicator of soil structure. Ahealthy soil is considered to have well aggregated structure. Though not routinely followed during soil testing and chemical analysis, aggregation is one of the major indicators of soil physical health. Wet sieve method (Yoder, 1936) is usually followed for soil aggregate analysis. This involves gently raising and lowering a nest of sieves in water to assess the stability of aggregates to rapid wetting. However, the Yoder’s wet sieving method has been modified to make it simpler and currently, the method of Kemper and Rosenau, 1986 is being commonly used in research laboratories. One of the pre-requisites in these methods is the availability of wet sieving apparatus and further, these methods are laborious and time consuming. On the other hand, the method described by Cambardella and Elliott (1993) is considered very ideal because of its simplicity and is being widely used for aggregate bound soil carbon fraction study. The soil sample is wet sieved with a series of three sieves (2000, 250 and 53 micron size).

Soil bulk density (BD) or apparent density is determined in -situ by core method. Stainless steel or metallic cores of cylindrical shape with known height and diameter are inserted into the soil till the rim of the core. The soil core is then excavated and the soil sample is oven dried to get the dry weight. The ratio of the oven dry weight to the total volume of the core gives the bulk density of the soil.

Soil texture refers to the relative distribution of sand, silt and clay on a sample of <2 mm size. It is an intrinsic soil property and basically refers to the particle size distribution in a soil. A number of physical characteristics and chemical properties of a soil are influenced by texture or particle size distribution. To estimate the content of sand, silt and clay particles, the soil samples are first brought into a dispersed state by mechanical and chemical treatments so that different binding agents are removed. In the dispersed soil samples, the soil particles settle down at a differential settling rate according to their size. In laboratory, the common methods for analysis for particle size distribution are : Buyoucos hydrometer method and International pipette method. In this chapter, the procedure of Buyoucos hydrometer method is described.

Principle

This method is based on the principle of variable rate of settling of particles as per their size and as described by Stokes’Law. Large particles shall settle fast. The measurement of density of the suspension is done at different times using a hydrometer (ASTM 152H-Type hydrometer is preferred). The measurement in this hydrometer is based on a standard temperature of 20 _oC and a particle density of 2.65 Mg m_-3 and the units are expressed in grams of soil per liter.The principal source of error in this procedure is the incomplete dispersion of soil clays as the clay particles are cemented by agents such as organic matter, CaCO and iron oxides. Incomplete dispersion results in low values for clay and high values for silt and sand. Thus, soils with higher organic matter (>1.0%) should be first treated with 6% hydrogen peroxide (H₂O₂) for removal of organic carbon. The rate of sedimentation is also affected by temperature and the density of the dispersing solution.

Exchanges sodium (Na+) is animportantparameter forsalt affected soils. This is estimated by extraction with ammonium acetate and then measuring the extract inflamephotometer.

Reagents

1. Neutral normal ammonium acetate solution (1 N CH₃COONH₄ of pH 7): Dissolve 77.09 g of ammonium acetate in distilled water and make up the volume to one litre. Adjust the pH of the solution to 7 with ammonia solution or acetic acid.
2. Standard solution of Na (500 mg/L Na): Dissolve 1.271 g of NaCl in distilled water and make up the volume to one litre.
3. Working standard solution of Na (100 mg/L): Dilute 200 ml of 500 mg/L stock solution to 1000 ml.

Ion exchange is a reversible process by which a cation or anion held on the solid phase is exchanged with another cation or anion in the liquid phase. Soil mineral and organic colloidal particles have negative valence charges that hold dissociable cations. The power to retain cations at the surface of soil colloids is referred to as the cation exchange capacity (CEC). Cation exchange is a reversible reaction in soil solution, dependent upon negative charges of soil components arising from permanently charged or pH-dependent sites on organic matter and mineral colloid surfaces. The CEC is the sum of cations held by a kilogram of soil and is expressed in Cmol (p₊)/kg soil. In most agricultural soils, the cation-exchange capacity (CEC) is generally higher than the anion exchange capacity (AEC).

Electrical conductivity (EC) is a useful indicator of soil salinity. Relationships have been developed between EC and salinity classes for a 1:1 soil: water suspension. With increase in salt content in a soil, the EC increases. Thus, for salt affected soils, measurement of EC is vital as accumulation of salts in the root zone is detrimental to plant growth. EC is a measure of the ability of a soil solution to conduct electrical current.

Equipment and Reagents

1. Digital Conductivity meter
2. 0.01 M potassium chloride (KCl) solution: Dissolve 0.7456 g dry KCl in distilled water and make up the volume to 1.0 litre. This solution has an electrical conductivity of 1.413 dS m_-1 at 25ºC.

Soil pH is the measure of hydronium ion (H₃O⁺) or more commonly hydrogen ion (H⁺) activity in the soil suspension. Soil pH can be measured in soil-water suspension, saturation extract or in 0.01 M CaCl₂ solution. Saturation extract is used to identify specific soil problems such as salinity or sodicity. Measurement of pH using 0.01 M CaCl₂ solution gives more reproducible results than measured through soil-water suspension. It also provides a good approximation of field conditions as the electrolytic composition of 0.01 M CaCl₂ is considered similar to that of soil solution. However, among the three methods, soil-water suspension is most commonly used in research laboratories as well as for fertilizer recommendation as it is simpler and more convenient.