eChapters

Bielschowsky’s Method for Axis Cylinders and Dendrites
Tissues fix in 10% neutral buffered formalin and process for routine paraffin sections Protocol:
1) Deparaffinise the paraffin sections with two changes (10 minutes each) of xylene.
2) Descending grades of ethanol from absolute to 50% solution (2 minutes each).
3) Hydrate the sections with distilled water 5 minutes.
4) 2% Silver nitrate solution for 48 hours, in the dark.
5) Rinse quickly in double distilled water.
6) Ammoniacal silver solution 10-20 minutes or until sections turn a deep brown.
7) Rinse in distilled water.
8) Reduce in 20% formalin solution for 5 minutes. Sections appear a dark brownish black.
9) Rinse thoroughly in distilled water.
10) Tone in gold chloride solution for 1 hour. Sections will be a reddish violet colour.
11) Rinse thoroughly in distilled water.
12) Sodium thiosulfate solution for 1 minute.
13) Wash in tap water.
14) Dehydrate in ascending grades of 90% to absolute ethanol (one minute each).

Schultz Method for Cholesterol
Frozen sections; cold calcium-cadmium-formalin fixed sections
Protocol
1) Thoroughly wash the sections in distilled water for 24 hours, changing the water several times.
2) Incubate the sections in 2.5% ferric ammonium sulphate solution at 37oC for 7 hours.
3) Wash the sections three times in fresh acetate buffer, allowing 1 hour for each wash.
4) Rinse briefly in distilled water.
5) Immerse in 5% formalin for 10 minutes.
6) Mount the sections onto slides, blotting the edges to remove excess water. Do not let the sections dry out.
7) Place a drop of a 1:1 mixture of sulphuric and acetic acids on a coverslip. Invert the slide and lower it onto the coverslip. Then turn the slide rightside up and gently press the coverslip to flatten the section. Hold the coverslip by its corners and gently oscillate it a few times to ensure even spreading.

The Millon Reaction
Tissues fixation in 10% neutral buffered formalin solution
Protocol
1) Deparaffinise the paraffin sections with two changes (10 minutes each) of xylene.
2) Descending grades of alcohol from absolute to 50% solution (2 minutes each).
3) Hydrate the sections with distilled water for 5 minutes.
4) Transfer the sections to a small beaker with the reagent and gently boil.
5) Stop the heating process and let the solution cool to room temperature.
6) Remove the sections and rinse them three times in distilled water (2 minutes each).
7) Dehydrate in ascending grades of 90% to absolute alcohol (one minute each).
8) Clear in xylene, two changes of 30 minutes each.
9) Mount glass cover slip with DPX.

Lipids are naturally occurring fat like substances insoluble in water but are soluble in organic solvents like alcohol, ether, and chloroform etc. Some of the phospholipids are even soluble in water. All the lipids do not resemble fats.
Lipids are an important component of living cells. Together with carbohydrates and proteins, lipids are the main constituents of animal cells. Lipids include fatty acids, neutral fats, waxes and steroids (like cortisone). Compound lipids (lipids complexed with another type of chemical compound) comprise the lipoproteins, glycolipids and phospholipids.
Properties of lipids
Lipids are non-polar molecules a family of organic compounds, composed of fats and oils yield high energy and are responsible for different functions within the body. It is stored in the adipose tissue of the body. Lipids are a heterogeneous group of compounds, mainly composed of hydrocarbon chains. Lipids are energy-rich organic molecules, which provide energy for different life processes. Lipids are a class of compounds distinguished by their insolubility in water and solubility in nonpolar solvents. It is important constituent of cell membrane in biological systems, a mechanical barrier that divides a cell from the external environment.
Most of the lipids or fats are demonstrated by using fat soluble dyes. The lipid soluble dyes have greater affinity for fats, lipids, lipoproteins and triglycerides. The fat soluble dyes are:

A protein that is deposited in various tissues of body called amyloid. Amyloidosis is a disorder of protein folding, in which normally soluble proteins accumulate in the tissues as abnormal insoluble fibrils (or filaments), thus disrupting their function The deposited proteins, generically known as amyloid, damage the structure and function of the tissues and so cause serious disease which is often fatal when it affects major organs. There are several techniques to observe the amyloid which is as follows.
Highman’s Congo Red Technique
Tissues fix in 10% neutral buffered formalin and process for routine paraffin sections The formal saline is a better choice
Protocol
1) Sections to water, removing pigment where necessary.
2) Stain in Congo red solution, 5 minutes.
3) Differentiate with the alcoholic potassium hydroxide solution, 3-10 seconds.
4) Wash in water, stain nuclei in alum hematoxylin and differentiate blue.
5) Dehydrate, clear, and mount.
Observations
Red : Amyloid, elastic fibers, eosinophil granules: Blue : Nuclei
Chemical reagents
Immerse in alkaline sodium chloride solution for 20 minutes.

McManus’ Method for Glycogen (PAS)
Tissues fix in 10% neutral buffered formalin and process for routine paraffin sections
Protocol
1) Deparaffinise the paraffin sections with two changes (10 minutes each) of xylene.
2) Descending grades of ethanol from absolute to 50% solution (2 minutes each).
3) Hydrate the sections with distilled water 5 minutes.
4) 0.5% Periodic acid solution for 5-6 minutes.
5) Rinsing in distilled water.
6) Schiff’s’s reagent or Coleman’s Feulgen solution for 10-15 minutes.
7) Washing in running tap water for minimum 10 minutes until pink colour develops.
8) Counter stain with Harris’ hematoxylin for 2-3 minutes to stain nuclei.
9) Washing in running water for 2-3 minutes.
10) Quick dip in ammonia water to develop blue colour of the nuclei.
11) Washing in running tap water for 5 minutes.
12) Dehydrate in ascending grades of 90% to absolute ethanol (one minute each).
13) Clearing in xylene, two changes of 30 minutes each.

Van-Gieson Stain for Collagen Fibers
Tissues fix in 10% neutral buffered formalin and process for routine paraffin sections
Protocol
1) Deparaffinize sections and hydrate to water through descending grades of alcohol.
2) Remove mercury crystals if tissue fixed in Zenker’s or Helly’s fluid.
3) Stain in iron haemotoxylin solution for 2 to 3 minutes.
4) Wash in running water for 10-15 minutes.
5) Rinse in distilled water.
6) Counter stain in Van Gieson’ssolution for 2-3 minutes.
7) Two to three dips in 96% alcohol.
8) Dehydrate through absolute alcohol and clear in xylene.
9) Mount with D.P.X mountant.
Observations:
Red : Collagen fibers
Yellow : Muscles, RBC and cornified epithelium
Blue : Nuclei

Fixation is the first and utmost important step to take good histology of eye ball. The eye is a composite organ made up of layers of different consistencies that tend to separate during fixation and sectioning (Drury and Wallington, 1980).
Fixation (Davidson’s fluid)
Davidson’s fluid is an excellent fixative for fixation of whole eye ball but conventional 10% formalin causes artificial cellular shrinkage and poor cellular and nuclear resolution of the retina. Formaldehyde penetrates tissue well but takes more time because sclera stands as physical barriers that protects the retina and inhibit the penetration of fixative. Davidson’s fixative is acetic acid, alcohol- formalin based fixative in which alcohol denatured the protein by breaking hydrogen bonds and disturbing their tertiary structure and acetic acid increase the penetration. It has been advocated and widely used for the preservation of eye ball, maintaining retinal attachment during fixation and processing and providing better preservation of the retinal nuclear layer and sensory specialization of the rods and cones.

The hematoxylin and eosin stain are most widely used histological stain. Its popularity lies in its simple staining procedure and ability to demonstrate most tissue structures. Hematoxylin, also called natural black is a compound extracted from heartwood of the logwood tree (Haematoxylum campechianum) with a chemical formula of C16H14O6. This naturally derived dye has been used as a histologic stain, ink and as a dye in the textile and leather industry.
Hematoxylin, a natural dye was first used in about 1863. Hematoxylin itself is not a stain; it is its oxidation product, hematein. This process known as “ripening” takes several days or weeks unless it is hastened by the addition of an oxidizing agent. So the active coloring agent hematein can be produced from hematoxylin in two ways:
A. Natural oxidation by exposure to light and air.
B. Chemical oxidation: Sodium iodate is the most common oxidizing agent for this purpose, although there are others, such as potassium permanganate, iodine, bleach and mercuric oxide. It is now strongly recommended that mercuric oxide not be used for this oxidation as it eventually makes its way into the environment as a poison.
As the hematoxylin dye has little affinity for the tissue when used alone, it is usually used in conjunction with a mordant, therefore can be arbitrarily classified based on which mordant is used:
1. Alum hematoxylin
2. Iron hematoxylin
3. Tungsten hematoxylin

Types of bones
Bones forming the animal skeleton can be classified into two types such as compact bone (cortical) and spongy bone (trabecular / cancellous). The compact bone is found in diaphysis or shafts of all the long bones of forelimbs, hindlimbs and outer surfaces of the flat bones of skull and ribs. In contrast, the spongy or trabecular bone is found in epiphysis or extremities around the bone marrow cavities of long bones and centre of flat bones and vertebrae. As the name implies, the compact bone is solid and hard due to the arrangement of special collagen in the bone (Type I) forming the bands or lamellae parallel to each other surrounded by a cement of proteoglycan ground substance deposited with minerals. Whereas, the spongy bone is made up of meshwork of bone strands running in all directions arranged as trabeculae adapted for weight bearing ability in heads and condyles of long bones.
Structure of bone tissue
The bone consists of bone cells, extracellular organic matrix made of collagen fibres and minerals. The cells of bone are osteoblasts, osteocytes and osteoclasts. Osteoblasts are low columnar to squamous cells responsible for active synthesis and mineralization of bone matrix found mainly in the periosteum and endosteum. Osteocytes are responsible for structure of the bone and calcium homeostasis found in the mature bone. It is located in the lacunae with extensive cytoplasmic processes running through canaliculi with in the bone matrix to make communication between adjacent osteocytes. Osteoclasts are multinucleated (15-30 nuclei per cell) larger cells responsible for bone resorption and remodelling. The secretion of these cells composed of acid and lysosomal enzymes responsible for erosion of bone tissue thereby formation of Howship’s lacunae (Frappier and Eurell, 2006).

A useful method for quickly preparing tissue slides for microscopic analysis is the frozen section technique. There are numerous clinical and research contexts where the frozen section technique is employed. For quick intraoperative diagnosis in histopathology, frozen sections are frequently utilized, giving our surgical colleagues direction. To ascertain the extent of excision required to remove a skin tumour, the surgeon doing Mohs Micrographic Surgery depends solely on the frozen sections. The frozen section technique is used in many scientific applications to create microscopic slides using a variety of complex morphologic, immunohistochemical, and molecular techniques. There are several uses for frozen sections, including rapid diagnosis, immunohistochemistry, enzyme histochemistry, lipid detection, cancer margin research, and more. Making frozen section slides is a difficult technical procedure that calls for the mastery of advanced technical abilities in addition to knowledge of the pathology, microanatomy, and histology of the tissues under investigation. The outcomes will depend on our capacity to produce a high-quality preparation, whether it is utilized for research or intraoperative consulting. Dr. Louis B. Wilson’s 1905 description served as the basis for the frozen section technique used in medical labs today. At the request of surgeon Dr. William Mayo, one of the founders of the Mayo Clinic, Wilson devised the procedure based on previous studies. A frozen section was also used in earlier reports by Dr. Thomas S. Cullen of Johns Hopkins Hospital in Baltimore, but only after formalin fixation. Dr. William Welch, a pathologist at Hopkins, also tried Cullen’s technique, but there were no negative clinical effects. Wilson is therefore widely acknowledged as having really invented the procedure.

Sectioning of tissue is carried out by using an instrument called microtome and the procedure is known as microtomy. Most commonly used microtome is HYPERLINK http://paramedicsworld.com/microtome/ rotary microtome which allows the perfect sectioning of the paraffin-embedded tissue specimens. For histological purposes, section cutting is done at the thickness of 4-6 microns whereas for histochemical staining techniques, the thickness of sections may be increased.
Pre-steps involved in tissue section cutting
1. Ensure that microtome is placed on an even surface so that it will not be shaken during the procedure of section cutting
2. Ensure that Knob for the rotating handle of microtome is locked to avoid cutting the fingers of handler of microtome while fixing the blade in its holder
3. Ensure that paraffin shavings from the previous section cutting were removed from the various parts of microtome for smooth functioning of microtome
4. Thermostatically controlled water bath or tissue flotation bath should be kept ready with filled water to heat
5. Ice cubes should be kept ready in the refrigerator. Success of sectioning can be greatly improved by cooling the wax blocks before subjecting them to cut. To properly cool the blocks, placing them on a piece of aluminum foil on a levelled tray of ice for at least five minutes before sectioning is preferable. Chilled paraffin wax is harder and in general it allows thinner and higher quality sections .Cooling both the tissue and wax giving them a similar consistency, in addition to a smaller amount of water will be absorbed into the tissue and making sectioning easier.

Routine paraffin wax embedding technique can be conveniently discussed under the following five headings:
Stages of tissue processing
1. Fixation: Stabilizes and hardens tissue with minimal distortions of cells
2. Dehydration: Removal of water and fixative from the tissue.
3. Clearing: Removal of dehydrating solutions, making the tissue components receptive to the infiltration medium.
4. Infiltrating: Permeating the tissue with a support medium.
5. Embedding: Orienting the tissue sample in a support medium and allowing it to solidify.
Fixation-A
It is essential that tissues be fixed as soon as possible after death or removal from the body, and for this reason screw-capped specimen jars containing appropriate fixatives should be permanently kept wherever tissues for histological examination are taken regularly, for example in the operating theatre, the post- mortem room, or the animal house. The amount of fluid in the jars should be 15-20 times the bulk of the tissue to be fixed. Care should be taken to ensure early despatch of the specimens to the histology laboratory in order to avoid over-fixation in intolerant fixatives.
Tissues selected for sectioning should be sufficiently thin to be adequately fixed throughout in a reasonable time. The overall bulk of the tissue determines the volume of fixative required; the thickness will determine the speed of fixation.

Accurate estimation of embryonic/foetal age play vital role in the developmental anatomy research related to morphogenesis and histogenesis of an organ or system. The observations and recordings of normal developmental features of embryos/ foetuses at successive stages of gestationare very important for studying functional development of an organ in anatomical research. The documentation of normal embryonic and fetal growth can serve as a guide for understanding the consequences of harmful influences at various stages of gestation (Evans and Sack, 1973). Post-mortem studies have provided valuable information on fetal development, identifying stages such as implantation, appearance of forelimb bud, tail development and hind limb development (Alberto et al., 2013). The normal development of external features of animals has been described by Evans and sack (1973), however, subjective assessment of foetal age from the external features alone is not much reliable, although it may be useful adjunct to more precise measurements (Richardson et al., 1990). For accurate estimation of developmental age, fetal measurements are needed which show a consistently rapid rate of growth and which are least variable at a given age (Richardson et al., 1990), however, these parameters are affected by individual variation and by breed differences within a species (Greenwood et al., 2005).
A number of methods have been discussed by many workers for estimation of age of foetus in animals. The method mainly focus on foetal biometry recorded either inside uterus during normal gestation process or after taking out foetus during post-mortem examination. In-utero foetal biometry allows estimating gestational age and time of parturition by ultrasonographic methods (Manning

It is an inherent, distinctive and implicit human nature to preserve and care for objects that seem unique, rare and adorable. This instinctive desire of the human mind must have invented the concept of museum. The word museum derives from the Greek word “Mousein” which mean “Seat of the Muses”. The muses are the divine patron of arts in Greek Mythology.
A museum by definition is “An institution that houses and cares for a collection of artifacts and other objects of scientific, artistic or historical importance and makes them available for public viewing through exhibitions that may be permanent or temporary (Alexander and Alexander 2008; Kamath et al., 2014). The anatomy museum comes under the category of science museum. Anatomy museum define as a place where bones, models, charts wet specimen of permanent value are kept and displayed. It has to be designed to function as an institute where active leaning can take place (Kamath et al., 2016).
The museums now witnessed a transition from an institute housing a collection of models and artistic sketches to one housing formalin preserved specimens, corrosion casts and plastinates. The contemporary museums have sophisticated interiors, display, lightings and use computerized catalogues and audio-visual aids to teach anatomy.
History
The concept of anatomy museum was first conceived by the Edinburg Surgeons in 1763 A.D. A cabinet with anatomical picture books and few specimens was first prepared and named as cabinet of curiosities. Early museums housing a simple collection of wood, clay, wax or ivory models of anatomical specimens.
The discovery of formalin in 1859 by Alexander Mikhailovich Butlerov was a great milestone in anatomical history and changed the, manner of anatomical specimen presentation. Later on discovery of different methods significantly make transition in the nature of collection exhibited in anatomy museum.

1. Fix the organ or the animal in 5-10 % formalin preferably neutral buffered formalin. If the organ is an isolated thoracic limb, for example, then cannulate the axillary artery and perfuse the formalin. I find sometimes it is advantageous to pump some buffered saline solution to flush out any blood before pumping in the formalin. Organs, such as the heart, could be nicely flushed with water through major blood vessels, and immersed in formalin to fix. The same can be done with liver, kidney, etc. It takes a week for fixing a large structure (e.g., 10 kg) by immersing in formalin.
2. Dissect the specimen as needed. Remove all fat and clean as much as possible so the specimen looks nice.
3. If the specimen is dark brown, it can be lightened by leaving 1-2 days in 5% hydrogen peroxide. Check the specimen 1-2 times during the day to make sure desired light tone is achieved. Leaving it too long in the hydrogen peroxide may result in tissue degradation.
4. Drain the specimen well, and dehydrate in acetone. The dehydration should be thorough. To achieve this, use 100% acetone, at least 2-3 changes. The acetone can be recycled by repeated distillation. Acetone tends to evaporate, so use a container that has a tight lid. Acetone is also flammable, so use required precautions when handling acetone. If concentration of acetone remains up to 99% even after leaving organ for 4-5 days it is sure that dehydration is complete. If you are using any thin sheets of organ or Brain for dehydration keep slice between glass plates and then dehydrate.
5. Immerse the specimen in glycerin. Buy industrial glycerin, which may be cheaper. It may take 2-3 weeks for the glycerin to permeate into the tissues.

Veterinary anatomy is a backbone as well as basic discipline in the veterinary sciences at large. The new entrants of the first professional year of B.V.Sc. & A.H. get more exposure in gross anatomy, histo- architect models and developmental anatomy of their current curriculum in our country.
Laboratory demonstration plays a vital role module in virtual teaching and learning program of their better understanding of the subject. The visualization and memorization is the basic requirement for thorough understanding of the anatomy of the individual in medical sciences. The various method of teaching have been debated because it has own limitations. To improve the anatomical understanding the traditional way of using cadavers but with current scenario of restrictive laws which implemented by Veterinary Council of India (VCI) and other organizations viz, SPCA, PFA, CPCSEA/IAEC etc. The scarcity of specimens which are invited the other alternative way of keeping and preserving the specimens for laboratory teaching. Under the unusual circumstances, the normal conventional method of cadaver/specimen/organ preservation should be replaced by the advanced technologies for easy and interesting to learn. In the traditional curriculum method of anatomical specimens taught and learned by formalin fixed cadavers/specimens/tissues. Since, the conventional method creates health problem due to extensive use of formalin as preservative chemical for cadaver preservation. This method of preservation causes wet slippery tissues, respiratory irritation, topical allergic reactions, and students should wear gloves to protect themselves from the fixative like formaldehyde (act as a potential carcinogenic agent). However, the anatomists have been challenged for an alternative laboratory teaching aids in near future. In recent past, the alternative method has developed teaching materials by the innovative novel techniques like Plastination, corrosion cast, pneumatic air dried specimen etc.

Taxidermy
The word ‘Taxidermy’ is derived from the Greek taxis meaning “fixing” or arrangement and derma meaning ‘skin’. It is possible to perform taxidermy of mammals, birds, fishes, reptiles and amphibians and procedure to perform taxidermy is also variable accordingly. In this case we will see the procedure applied to prepare a big cat (Tiger, lion, leopard etc.).
Skinning
To skin an animal make an inclusion from the chin along the belly to tip of tail, Then from paw to paw across the chest, and deal similarly with the hind legs. If they are done haphazard, The appearance of the skin will be completely wrecked. Place the carcass on its back and make an incision through the chin and carry it along the centre of the throat to the chest. There is a whorl of hair which runs from the chest along the belly to the vent, then carry the incision along from the vent to tip of tail. Fore- legs, after cutting through the ball of the pad the incision must be made exactly through the center of the white till it reaches the incision which is made from chin to tip of tail, the other foreleg is treated in the same way, both these incision should meet on the chest, and not end one above the other, Hind-legs after cutting through the ball of the pad. The incision runs behind the leg to end of the calcaneum, then turn inside the thigh. The hind leg, of course must be done in the same way. It will be better to mark with charcoal on proposed incision lines before the skinning carried out. After the pads have been skinned and turned back, also inside of the thigh and belly, there is not much need for the knife as the skin ought to be removed by what butchers term ‘ fisting ‘. Get someone to hold the leg to keep the carcass rigid, when the skinners pull the skin taut with his left hand and, with his right first clenched, process vigorously on the skin close to the

The whole brain or its slices could be stained for differentiation of the areas of gray and white matter for better understanding of macroscopic neuroanatomy. Various researchers have developed the methods of staining of either the whole brain or its slices. The review of all these methods is being detailed below. These would definitely help the teachers and students to teach and grasp the macroscopic details of central nervous system viz. nuclei, laminae, fascicule, capsule and other structures with little or no magnification.
Staining of Whole Brain and Sections from its Slices (Hewitt, 1959)
Fixation of whole brain in either 70% alcohol or 10% formaldehyde without removal of membranes and blood vessels.
Protocol
1. The collected whole brain is immersed in ten volumes of staining fluid.
2. The staining fluid can be resaturated, when ever it becomes paler and clearer in colour, then it can be used in smaller volume in proportion to the volume of brain for the next staining.
3. Complete staining is assessed by uniform red colouration of the brain. A cats’ brain requires at least 1 month and the human brain not less than 6 months. The time required is governed by size of the brain. The brain stem is cut of about 5 mm thickness to examine the completion of the staining.
4. The stained brain is immersed in ten volumes of 70% alcohol for differentiation.

Biometry is the measurement of various grossly visible dimensions of organs and their application in statistical analysis to generate biological data.
Various biometrical parameters used for different organs
The biometry of different organs is conducted with the help of non-stretchable thread, Vernier callipers, measuring scale, protector, glass beaker of various sizes, calibrated measuring cylinders, etc. While conducting biometry, it should be kept in mind that the way or the procedure for conducting biometry of different organs varies due to variation in shapes of the same and their positions. Some of the biometrical parameters of different organs and bones are summarized below.
1. Length: May be used to measure the distance between the highest points along the length of any bone or organ. Usually expressed in cm. Non-stretchable thread, Vernier callipers and measuring scale are used for this purpose.
2. Breadth: May be used to measure the distance between the highest points along the breadth of any bone or organ. Usually expressed in cm. Non-stretchable thread, Vernier callipers and measuring scale are used for this purpose.
3. Thickness: May be used to measure the distance between the highest points in regard to the thickness of any bone or organ. Usually expressed in cm. Non-stretchable thread, Vernier callipers and measuring scale are used for this purpose.
4. Volume: It is measures by water displacement of any organ. Usually smaller organs are used. The fresh organ is dipped into a calibrated

Animals experience the effects of force in everyday life. For example, the animal body exerts a force on the body and to the objects during various agricultural operations. Furthermore, the gravitational force exerts continuously, for instance while sitting, walking, etc. All parts of the body in one way or the other are loaded by forces. The bones provide rigidity to the body and can sustain high loads. The skin is resistant to force. The cardiovascular system is continuously loaded dynamically owing to the pulsating blood pressure. The bladder is loaded and stretched when it fills up. The intervertebral discs serve as flexible force- transmitting media that give the spine its flexibility. Beside force, animals are using levers all the time in their daily life to increase the ‘force’ that is applied to some object.
Animal soft tissues are complex materials that can exhibit nonlinear, time dependent, inhomogeneous, and anisotropic behaviors. The mechanical responses are reflected in all levels of organism (molecules, organelle and body levels). Several biological activities are under the action of forces, which even govern the gene expression. The study dealing with the forces and their effects on living systems is the Biomechanics.
Biomechanics applies the laws and the techniques of mechanics in the study of biological systems and phenomena. Indeed, mechanics provides the tools for:

The study of Veterinary Anatomy is an integral part of Veterinary Science as it evolved as a keystone of veterinary education through ages. Osteology (study of bones) is the essential part of the anatomy teaching curriculum. The bones are unsurpassed in the ability to provide the information on the three dimensional form and architecture of the body. It also helps in understanding the disposition and relations of soft tissues along with the course of neurovascular structures in that region. For osteological study, the bones are collected from the carcasses and are duly processed. Bone preparation involves soft tissue removal, cleaning and bleaching of bones and joints, articulation and labeling. The skin, visceral organs and the maximum possible amount of flesh adhering to the bones are removed by knife. Such bones are called wet bones. The whole skeleton is disarticulated; however, the pedal bones are left with the hoof. In case of skull, the mandible is dismembered and the mouth parts are removed. The tongue is carefully sliced to locate the hyoid bone. It is separated at the hyoid process of the petrous temporal bone. The brain is lacerated and goosed out of cranial cavity with a stick through the foramen magnum. The eyeball is carefully extirpated without any injury to the orbit. The nasal cavity is left untouched with the turbinates and cartilages intact. Following steps are involved in processing of bones and preparation of skeleton.
A. Maceration
B. Degreasing
C. Bleaching
D. Varnish
E. Categorization of bones
F. Preparation of articulated skeleton

Teaching and research in anatomy is mainly based on cadaver dissections. A sound knowledge of anatomy is essential from the beginning of veterinary education and the knowledge obtained through dissection of animal body is an indispensable part of the veterinary science.
History of embalming
Embalming is the art and science of preserving the animal by treating with chemicals to forestall decomposition. Embalming destroys the colloid nature of the proteins and neutralizes the active centres of the molecules forming many cross linkages of adjacent protein molecules. Proteins are thus converted to high molecular inert solid materials that can no longer serve as food for bacteria or as a substrate for enzyme action. Embalming preserves the body from decay. The embalmed bodies are stored in tanks containing a fluid composed of formalin, phenol, thymol and rock salt in water in varying proportions.
Claudius Galen (130-200 A.D) did dissections of animals such as the pig or monkey. Leonardo Da Vinci (1452-1519) a pioneer anatomist introduced anatomical drawings. During 15th-16th century the motive was to advance the techniques for the preservation of the dead and to study detailed anatomy by dissections. Andreas Vesalius (1514-1564) believed that dissection was necessary to find out how bodies worked. His lectures were based purely on his observations. He carried out dissection on human cadavers as well as animals to understand Anatomy. As an Anatomist he represents the end of the middle ages obscurantism and the beginning of modern Anatomy and scientific thinking. He is considered the father of modern Anatomy. In 17th century Antonie Van Leeuwenhoek (1632-1723) opened up new horizons

Climate change is one of the most pressing issues of our time, with far-reaching consequences for the environment, human health, and the economy. One of the most significant impacts of climate change is on the world’s water resources. Rising temperatures, changing precipitation patterns, and increased frequency of extreme weather events are all taking a toll on the availability, quality, and management of water resources. Water is essential for human survival, and its availability and quality are crucial for human health, food security, and economic development. However, climate change is altering the hydrological cycle, leading to changes in precipitation patterns, increased evaporation, and altered water flows. These changes have significant implications on demand for water resources, including reduced water availability, decreased water quality, and increased risk of waterrelated disasters which will ultimately impact agricultural production and food security.
15.1. Changes in Precipitation Patterns
One of the most significant impacts of climate change on water resources is the alteration of precipitation patterns and projected per cent changes in average annual precipitation over 1850-1900 period at 2.00C increase in temperature are depicted here (Fig.15.1).

Hydrometeorology forecasting is limited to forecasting of occurrence of rainfall to a greater extent. Although several countries all over the world have developed and established hydrometorological forecasting with focus on occurrence of floods and droughts, still there is very limited success. Floods are associated with occurrence of continuous heavy rainfall due to movement of low-pressure systems like cyclones and depression overflow into rivers, reservoirs and breaching of bunds. In countries like India, the floods occur on the average in 70 lakh hectors of area and the economic loss due to floods and expenditure on relief operations mounting year after year besides loss of life. Foods occur at all of a sudden leaving little time for planning mitigation or rescue operation. Flood forecasting in based on impact assessment of heavy rainfall assessments to identify the exact area that is likely to be affected. It requires more intensive research. Forecasting of droughts is still an area of serious concern. There is need to strengthen regional capabilities to develop forecasting techniques for occurrence of flood and droughts through proper analysis of historical data in conjunction with meteorological forecasts and remote sensing data. Though flood alerts are issued, the identification of exact area and the extent to which flood impacts are not very clear most of the times heavy rainfall warnings are not just sufficient until and unless the vulnerable areas can be clearly specified. The medium range forecasting will be extremely useful for issuing flood alerts.
14.1. Forecasting Droughts
Long range forecasting of seasonal rainfall can indicate the possibility of receiving excess or deficit rainfall over larger regions. In India, there is considerable progress in development of long range forecast of Southwest monsoon rainfall. This forecast also indicating the regions which may get surplus or deficit rainfall and for indifferent parts of a given region as well. However, there is serious dearth of local expertise to further analyse and interpret the long-range forecast for a given area on the basis of local climate data. The advantages of reliable long-range forecast are still unexpected. Almost one fifth of the area in India gets affected by draught most of the years. The drought affects are more severe in arid and semi arid regions. Prediction of occurrence of drought and its intensity, period of occurrence and the areas to be affected by drought is very important. During recent years, there is considerable increase in the occurrence of droughts due to climate change which is attributed mainly to atmospheric pollution. The physics of the pollution is consistent and not the policies support in handling hydrometerological disasters. There are instances where in flood and droughts occurred in the same

A watershed is an area that provides pathway to the surface or sub surface water flow to a given drainage system. The watershed is a hydrological unit. The area of watershed may vary from few hectares to thousands of square kilometres. The watershed development involves effective management of land and vegetation in order to conserve and improve the quantity of water. The watershed development is a comprehensive and sustainable solution to water conservation (Fig.13.1).
The watershed development has several advantages. It helps in
• Conservation, upgradation and utilization of natural resources particularly land, water and vegetation
• Minimise the inequalities between rainfed and irrigated areas
• Promoting cost effective adoptable simple technologies
• Supply water for domestic ,agricultural and industrial use
• Poverty alleviation and employment generation
• Reducing chemical pollution
• Adopting flood control measures by constructing water storage or impounding structures

A disaster is sudden event that causes significant damage to the people, environment and the economy. Disasters are serious disruptions to the functioning of a community that exceed its capacity to cope up using its own resources. The disasters that result either due to excess water or severe water deficit are floods and droughts.
12.1. Floods
The flood is an overflowing of water onto land that is normally dry. Floods may occur due to
• Heavy rainfall
• Ocean waves
• Snow melting
The economic losses due to floods will be very high and nearly 68 per cent of the economic losses are attributed to floods only. During the year 2018, India suffered a loss of nearly 90,000 crores due to floods alone. There are several causes for occurrence of floods in India. The major causes are
• Severe cyclonic storms in coastal areas which produce continuous heavy rainfall for 3 to 4 days in succession
• Encroachment of flood plains with residential structures
• Inadequate strength of river banks when there is high flows of water in the rivers
• Unplanned growth of urban areas
• Silting of water storage structures due to increased soil erosion thereby decreasing the storage capacity of reservoirs
• Simultaneous flooding of rivers and tributaries due to excessive water flows

Agriculture sector alone uses nearly seventy per cent of fresh water in the world. Therefore, water use economy in the agriculture sector is becoming a necessity. Increasing flood production is possible reducing water used for irrigation if we can adopt precision irrigation (Fig.11.1).
The precision irrigation has to be practiced by providing water to the crop plants in its root zone to meet the water requirements. The precise irrigation can result in-
• Water saving
• Avoid weed control
• Preventing loss of water due to infiltration
• Preventing loss of water due to surface evaporation from the soil
• Minimise salinity problems
• Improving water use efficiency of crops
Traditionally farmers were practicing flood irrigation. It is a method of watering crops delivering to a field and allowing it to flow over the ground among the crops. It is a simple and inexpensive technique followed in less developed areas. Flood irrigation is not very efficient way as nearly 50 per cent of the water applied in the field will be lost through run-off, soil evaporation and other factors. Flood irrigation is only suitable in wet climates and not effective in sandy soils. It leads to water logging in clay soils. There are several types of flood irrigation such as basin, border, furrow and contour irrigation. Although flood irrigation is not very efficient, the water wastage can be avoided to some extent using some techniques

The purpose of irrigation scheduling is to decide upon the amount of water to be applied for irrigation and timing of irrigation in order to avoid wastage of precious water. The irrigation scheduling is also used to determine the number of irrigations required for a crop during its growth cycle and judicious use of water depending upon the weather forecasts issued from time to time. The irrigation scheduling has to be planned for either full irrigation or supplementary irrigation depending upon the availability of water resources and the season during which crops are grown. Crops grown during the dry season requires full irrigation and crops grown during the rainy season require supplementary irrigation. The third category of irrigation is protective irrigation which is practiced in dryland areas. The protective irrigation is given when the crops are experiencing severe moisture stress due to prolonged dry spells wherever the farmers has access to small ponds and tanks developed within his own field as water harvesting structures.
10.1. Full Irrigation
Under full irrigation, the crops are provided with irrigation right from land preparation till the crop is getting ready for harvest maturity. The full irrigation will be usually planned for maximum yields of the crop (Fig.10.1).

Remote sensing involves measurement of the physical properties of a body without actual contact with it. It is based on the reflectance when the electromagnetic radiation is incident upon a body or surface. The spectral characteristics of the reflected radiation vary depending upon the nature and condition of the surface. In case of vegetation, it absorbs light and can reflect light in the visible range. The leaf pigments dominate the reflectance properties (Fig.9.1). The radiation bands within which the reflectance of electromagnetic radiation takes place are classified as follows-
Spectrum Wave Length (nm)
Visible 400-700
Near Infrared 701-1300
Middle infrared 1301-2500
Far infrared >3000

9.1. Vegetation Indices
The vegetation index is a mathematical expression that uses spectral imaging data to characterize the health and growth of the vegetation indices are calculated using data collected by remote sensors like Satellites. The vegetation indices have wide range of applications in agriculture and hydrology including
• Classification of vegetation
• Apex health of vegetation
• Changes in land cover
• Drought monitoring
• Estimation of soil moisture
• Evaluate crop growth, vigour, biomass and chlorophyll content
• Irrigation scheduling

Stress is force per unit area. When matter is subjected to stress; it may undergo changes in size, shape and displacement. Plants are grown in open environment and are likely to experience stress due to environmental conditions or when subjected to pests and diseases. The stress experienced by plants due to environmental conditions is called abiotic stresses. These include stresses caused by weather factors and climatic conditions and include moisture stress and thermal stress. The plants may also experience stress due to strong winds, dryness and as well as humid conditions (Fig.8.1).
The plants are generally classified into three types depending upon the ability to cope up with moisture stress as exhibited by-
• Drought resistance
• Drought tolerance
• Drought adaptations
The moisture stress may be to shortage of Water Compared to its requirements for evapotranspiration and metabolic activities. The plants may also suffer moisture

It is estimated that the world requires nearly 70 per cent increase in agriculture production to meet the demand for food by the year 2050 based on current trends in population growth it can be achieved either by increasing land under cultivation (which is not possible), increasing the availability of more water for agriculture and efficient utilisation of available land and water resources. The best available option is to increase in productivity of unit of land for unit water. However, the availability of water for agriculture production cannot be presumed to be same year after year as the major source of water is precipitation more known for its uncertainty due to change in weather patterns from season to season and year to year all over the world. The element of uncertainty is increasing since the beginning of the present century due to climate change under the present circumstance, the crop production is practiced -
• In Irrigated Areas
• In Rainfed Areas
• Under Conserved Moisture Condition Without Any Source Of Irrigation
For increasing crop production under irrigated condition, it is necessary to identify crops and varieties that can produce more yield per unit of water even when there is some decrease in availability of water through irrigation. Therefore, the concept of water production function is used for evaluation of water use efficiency of the crops.

The movement of water from soil to the crop canopy and leaves to provide water for transpiration and the effect of weather factors on transpiration is most important area of study. When the crop is just sown, there will be an only evaporation of water from the soil. After germination of seeds there will be emergence of seedlings. After the emergence of seedlings there will be formation of leaves. The small leaves start intercepting the incoming solar radiation and absorb some portion of solar energy, get heated and there will be evaporation turn off water from the stomata of the leaf. When water vapour escapes into the atmosphere through the stomata of leaf, the plant absorbs carbon dioxide which results in photosynthetic activity. During night time, there will not be any photosynthesis due to absence of sunlight. The plants use parts of the photosynthetic accumulate for its metabolic activity and the process is called maintenance respiration. The balance of the photosynthetic accumulate contributes to the expansion of leaves and increased dry matter accumulation in crop plans. The process of dry matter accumulation in plants is called crop growth. When the seedlings start growing, there will be formation of tillers or branches. When the leaves grow it reaches a stage when the crop will be completely shading the ground and the soil evaporation will be minimum and maximum amount of water is lost only through evapotranspiration (Fig.6.1).

The plants draw water from the soil for growth and development. The soil acts a reservoir for soil moisture (Fig.5.1). The basic aim of a soil water balance model is to quantify the manner in which the water supplied to the soil either through precipitation and irrigation is disposed off. In the process, the soil water balance models were found to be extremely useful to determine the amount of water utilized by the crop for evapotranspiration.
The concept of water balance was originally developed by Thornthwaite, an American geographer and it formed the basis for development of more scientifi c advances due to its wide range of applicability including
• Choice of crops/ cropping systems based on water availability
• Irrigation scheduling
• Optimisation of limited water resources for higher productivity
• Characterization of droughts and agricultural droughts
• Assessment of crop yield and pre harvest forecasting of crop yields
• Designing irrigation projects
• Development of water storage structures
There are quite a large number of water balance models available in literature nowadays. Several of these models may be applicable for regions where there are developed. There were considerable efforts to develop water balance models can be used by the crop on daily basis in case of different crops as well. The choice of a water balance model to a large extent depends on the availability of data required for running the model. It may not be possible to cover all models available in

The soil consists of many solid particles of different sizes and is capable of absorbing and retaining water in the soil within certain limits when water is received through either from precipitation or by irrigation. The water that is held in the soil is called soil water (Fig.4.1). It can exist also in the form of water vapour as well. The crop production depends upon the soil water. All the water that is present in the soil may not be available for plants.
The water availability to the plants from the soil depends upon some physical properties of the soil as well as on the physical processes that govern the existence of water in the soil. Therefore, it is necessary to understand about the relationships that govern the water availability in the soil as explained in the following text-
4.1. Energy Concept of Soil Water
Soil water energy concept is all about potential energy, gravitational potential, osmatic potential, pressure potential energies.
4.1.1. Soil Water Energy: The total energy state of soil water is defined by its
equivalent potential energy as determined by various forces acting on the water per unit quantity. Different energies are involved including kinetic and potential energy.

The major source of water supply on the earth is precipitation. The water received at the earth’s surface from the atmosphere either in solid or liquid form is called precipitation. The water received in solid form may be snow or hail. The precipitation is seasonal in character in many parts of the world. The precipitation may occur due to –
• Conventional physical process
• Orographic effect and
• Convergence of air masses
3.1. Conventional Physical Process
It occurs when the air on the surface of the earth and few metres above the surface of the earth is heated by absorbing long wave radiation emitted by the earth’s surface. As the air is heated, it becomes lighter. The water vapour formed due to evaporation from the soil surface or water bodies will mix with the air and the air will be moist. When the moist air gets heated it becomes lighter and rises vertically upwards. After reaching certain height, the air gets cooled enough to get saturated with water vapour. The moisture in the saturated air will condense on small solid particles to form tiny drops. These solid particles may be dust or smoke or solid particles suspended in the atmosphere and are called aerosols.

Water is the most important natural resource that governs and exercises control on living organisms on earth besides supporting all human activities. The demand for water is almost increasing at exponential rate due to increase in population and industrialization. All sectors of agriculture including crop production, horticulture, animal husbandry, poultry and fi sheries primarily depend upon water. Water scarcity can cause all kinds of miseries. The water exists in all 3 different forms on earth. It exists as snow or ice in glaciers and ice caps, liquid water in rivers, lakes, streams, tanks, ponds and as groundwater storage. The water exists in all three different forms in the atmosphere. The clouds may contain fi ne ice particles, tiny water droplets and as well as water vapour. The air in atmosphere also contains water vapour. It is estimated that 97% of water is salt water and remaining 3% of water is pure and fresh water. Approximately two-thirds of the fresh water is trapped in glaciers and ice caps. Most of the fresh water (30%) exists as groundwater. About 0.7% of fresh water exists as soil water and 0.3% a fresh water accounts for surface water resources and water present in the atmosphere as water vapour, water drops and ice particles. The circulation of water between oceans, earth and atmosphere forms hydrologic cycle.
2.1. Hydrological Cycle
The hydrological cycle explains the continuous recirculation and transport of water between the atmosphere, oceans and earth. It is a very complex system that has many sub cycles associated with it. For example, the water from earth surface, oceans and water bodies get evaporated due to heat energy received from the sun in the form of incident solar radiation (Fig.2.1).

The entire world is now facing a crisis situation. The problems encountered to address the crisis are interactive with each other and there is no easy way to find solution. The world’s population is expected to increase by 70 per cent by the end of year 2050 and there is need to step up agricultural production for a hunger free world. Nearly 70 per cent of the fresh water is used in agriculture sector. The demand for water in agriculture sector can be judged by the fact that several thousands of litres of water is required to produce one kilogram of food grains. Whatever maybe the source from which water is drawn for agriculture, all the sources depend upon the precipitation for replenishment on continuous basis. The precipitation patterns are not consistent and are known for greater variability from season to season, year to year and from region to region. These inconsistencies are further increasing due to recent threat of climate change triggered by the atmospheric pollution. The atmospheric pollution due to increase of greenhouse gases in the atmosphere is responsible for rise in temperatures. The rise in temperature induced changes in global pressure and wind systems. The consequence effect of it is changes in rainfall patterns, frequent occurrence of extreme weather events like floods, droughts, heat waves, intensified cyclonic activity. Rise in temperatures is increasing evaporation of water and water requirement of crops as well. There is considerable increase of the chemicals in the agriculture sector in the form of fertilizers, pesticides, fungicides and weedicides how to increase crop production and protection of crops from biotic and abiotic stresses. Consequently, there is increased pollution of water resources which may adversely affect the availability of fresh water, which is essential and critical input in agriculture for increasing agricultural production. It calls for efficient utilization of water resources in agriculture. More concerted efforts are required immediately to:
• Improve water use efficiency in agriculture
• Reduce pollution of water resources
• Conservation of fresh water resources
• Reduce emissions in agriculture sector and
• Prevent dwindling of fresh water resources

Laboratory pipettes are classified by their method of liquid transfer. There are two types of pipette one is plastic which is made up of polystyrene and are essentially disposable and second one is glass pipettes are very resistant to chemicals. Glass pipette can be used frequently by washing, sterilising and dry heat sterilisation is also available. Some of the common types of pipettes are using in laboratory such as serological, volumetric, micropipettes, multichannel, and Pasteur pipettes. Serological and graduated pipettes are used for transferring various volumes, while micropipettes and volumetric types of pipettes are only considered for precise measurement and transfer. Multichannel pipettes are used for dispensing into multiple wells, and Pasteur pipettes are simple for transferring small amounts.
1. Serological Pipettes
• Also known as graduated pipettes, made of glass or transparent polystyrene they are calibrated with markings to measure and dispense varying volumes.
• Appropriate for cell culture applications, transferring liquids and mixing.
2. Multichannel Pipettes
• It is design to transfer multiple samples at once, often used for 96 well plates or pcr tubes.
• It can be manual or automated, with multiple heads for faster dispensing.
3. Volumetric Pipettes
• It is designed to supply for single and particular volume of liquid.
• Marked by single graduation for an exact volume, such as 1, 5, 10, 25, and 50 mL.
• Used in chemical reactions and analysis where accuracy is crucial.

The total plate count (TPC) is also called the Bacterial plate count (BPC), aerobic plate count, standard plate count (SPC), total viable count (TVC) or mesophilic count represents the number of colony forming units (CFU) per g (or per mL) of growing microorganisms such as bacteria, yeast and mold on a nonspecific solid bacteriological growth medium under the specified conditions. Temperature, incubating time, medium type, and any other conditions used in the enumeration varies upon reference standards and laboratory objectives. Laboratories participating in the program choose their own routine conditions.
Principle
Usually direct count of too many bacteria in a sample is difficult but the bacterial colonies may be used as a measure of the bacterial cells in the known serially dilution and plated out on an agar surface in such a way that single isolated bacteria form visible isolated colonies. However, colony-forming unit might actually consist of a chain of bacteria rather than a single bacterium if the organism often develops various cell configurations, such chains. Furthermore, a portion of the bacteria might be grouped together. Thus, typically refer to the process of using the plate count technique as a means of estimating the number of colony forming units (CFUs) in that known solution. Then further this figure use to extrapolate the total amount of CFUs present in the original sample.

Sulfate can have harmful effects on aquatic life in freshwaters, sulfate cause osmotic stress or specific ion toxicity in aquatic organisms, especially in soft waters where Ca2+ and Mg2+ concentrations are low. Egg fertilization and early embryonic development were the most sensitive developmental stages of whitefish to sulfate (Karjalainen et al., 2021).
Sulphate (SO4 2-) may be estimated by either turbidimetric or gravimetric methods (APHA, 1995)
1. Turbidimetric method is more applicable in the range of 1- 40 mg Sulphate (SO4 2-)/ L
2. Gravimetric method is suitable if the concentration of Sulphate (SO4 2-) is above 10 mg/ L
Turbidimetric Method (Range 1 - 40 mg/L of sulphate) Principle
Sulphate ion (SO4 2-) precipitated with barium chloride (BaCl2) in an acetic acid medium in order to form crystals of barium sulphate (BaSO4).
BaCl2 + SO4 2-+ BaSO4 (precipitate)
The formation of these crystals is increased in the presence of an acetic acid solution containing sodium acetate, potassium nitrate, acetic acid and magnesium chloride. The suspended barium sulphate (BaSO4) is measured by spectrophotometer and concentration of sulphate (SO4 2-) is determined by comparison of the reading with a standard curve. Minimum detectable concentration is approximately 1 mg (SO4 2-) / L.

Phosphorus is nearly exclusive found as phosphates in waters and wastewater and it categorised in to: a) orthophosphate, (b) organically bound phosphate and (c) condensed phosphates. Theses all three forms of phosphorus occur in solution, in the body of aquatic organisms through food chain. All the forms of phosphorous, whether dissolved or particulate are converted to inorganic forms (phosphate) after digestion of sample. Various methods use for the digestion of the sample including use of perchloric acid, H2SO4- K2SO4, H2SO4- HNO3 etc. The H2SO4- HNO3 digestion technique is discussed here.
Reagents
1. Phenolphthalein indicator
2. Conc. sulphuric acid (H2SO4)
3. Conc. nitric acid (HNO3)
4. Sodium hydroxide (NaOH), 1 N: Dissolve take 40 g of NaOH in 1000 ml of distilled water
Procedure
Digestion
1. Take 50 ml of sample in a Kjeldahl flask
2. Add 1 ml H2SO4 and 5 ml HNO3
3. Once the HNO completely removed, digest the sample on a hot plate until the volume approaches 5 ml. Then, keep heating the solution until it loses colour (you may also heat it in a beaker covered with a watch glass to prevent excessive evaporation).

The most oxidizable form of nitrogen is nitrate nitrogen (NO3 -N), which is found in minimal amounts in surface waters but can reach considerable concentrations in some aquifers. An essential plant nutrient, nitrate also contributes to eutrophication in receiving water bodies.
Principle
Nitrate reacts with phenol disulphonic acid and produces a nitro-derivative which in alkaline solution develops yellow colour due to rearrangement of its structure. The yellow colour follows Beer’s law and is proportional to the concentration of nitrate present in the sample. Ultraviolet (UV) technique measures the absorbance of nitrate at 220 nm, which is suitable for screening uncontaminated water (low in organic matter). A second measurement made at 275 nm, may be used to correct the nitrate value (because 275 nm is not absorbed by nitrate, but absorbed by other matter). The nitrate calibration curve follows Beer’s law up to 11mg NO3 -N /L.
Reagents
1. Stock nitrate solution (1ml=100 µg NO3 -N): Dry potassium nitrate (KNO3) in an oven at 105 oC for 24 h. Take 360.9 mg potassium nitrate and dissolve in distilled water and dilute to 500 ml. Preserve it with adding 1 ml of chloroform in per 500 ml stock nitrate solution. This solution will stable for at least 6 months.
2. Intermediate nitrate solution (1 ml = 10µg NO3 -N): Take 100 ml of stock nitrate solution and dilute it to 1000 ml of distilled water. This solution can be preserve by adding 2 ml of chloroform per 1000 ml. This Intermediate nitrate solution will stable for at least 6 months.
3. Hydrochloric acid (HCL) solution, 1N: Take 83 ml of hydrochloric acid and dilute it to 1000 ml of distilled water

Nitrite (NO2-) is formed in waters by oxidation of ammonia compounds (by aerobic nitrifying bacteria, e.g. Nitrosomonas) or by reduction of nitrate (by facultative anaerobic denitrifying bacteria, e.g. Pseudomonas). Such oxidation and reduction may occur in wastewater treatment plants, water distribution system, and natural waters. As an intermediate stage in nitrogen cycle, it is unstable. Very high nitrite levels are usually associated with waters having unsatisfactory microbiological activity.
Principle (Colorimetric method)
When nitrite (NO2 -) as nitrous acid (HNO2) mixes with sulfanilamide in an acidic environment (pH 2-2.5), a diazonium salt is created. This salt then combines with N-(1-naphthyl)-ethylenediamine dihydrochloride (NED dihydrochloride) to make a vivid pinkish red azo-dye. The amount of nitrite in the sample directly correlates with the colour that is created. At 543 nm, the colour follows Beer’s law up to 180 µg/L with a 1 cm light path. By diluting a sample, a higher nitrite concentration can be determined. Sulfanilamide + HNO2 + HCl ? Diazonium salt + H2O.
Diazonium salt + NED-dihydrochloride ? Red colouredazo dye

Fish excrete Ammonia, is a byproduct from protein metabolism and bacteria break down organic waste such as discarded food, faeces, dead planktons, sewage, and so on. When ammonia is unionized form (NH3) is extremely dangerous; yet when ammonia ionized form (NH4 +) it is harmless; both forms are known as total ammonia. Fish suffering from ammonia toxicity often”become lethargic and frequently emerge near the surface gasping for oxygen. common effects of ammonia in the range >0.1mg/l include Gill damage, breakdown of mucous-producing membranes, “sub-lethal” effects like reduced growth, poor (FC) feed conversion, and reduced disease resistance at concentrations lower than lethal concentrations, osmoregulatory imbalance, and kidney failure.
Principle
When Ammonia react with alkaline Nessler reagent (K2HgI4 or 2KI + HgI2) it produces a yellowish-brown coloured compound. Pretreatment with ZnSO4 and NaOH precipitates Ca, Mg, Fe and sulphide, which form turbidity and apparent colour. Addition of ethylene diamine tetraacetic acid, EDTA (before nessler’s reagent) or Rochelle salt solution prevents precipitation of residual Ca and Mg in presence of the alkaline Nesslers’s reagent.

Principle
Chloride ion is determined by Mohr’s method, titration with standard silver nitrate solution in which silver chloride is precipitated first. The end of titration is indicated by formation of red silver chromate from excess AgNO3 and potassium chromate used as an indicator in neutral to slightly alkaline solution.
AgNO3 +Cl- ? AgCl+NO3
2AgNO3+ K2CrO4 ? Ag2CrO4+2KNO3 (Reddish Brown)
Reagents
1. Standard Silver nitrate 0.0141N : Take 1.189 g and dissolved it in 500 ml distilled water.
2. Sodium chloride 0.014N: Dissolve 0.28 g sodium chloride in 500 ml of distilled water.
3. Potassium Chromate indicator
Procedure
1. Take 50 ml sample in a 100 ml conical flask
2. Adjust its pH in between 7.0 and 8.0 either with alkali or acid solution. Otherwise, AgOH is formed at high pH level or CrO4 -2 at low pH level.
3. Add 1 ml of potassium chromate (K2CrO4) colour turn to light yellow.
4. Titrate with standard silver nitrate (AgNO3) solution till colour change from light yellow to brick red colour.
5. Note the volume of AgNO3 added (A)
6. For better accuracy, titrate distilled water in the same manner
7. Note the volume of silver nitrate added for distilled water (B)

Hardness is the total soluble Ca and Mg Salts (in same cases Fe salts). It includes sulphates and chlorides along with carbonate, bicarbonate and hydroxide salts. Soft water: 0-100 mg/L, Hard water: 100 - 300 mg/L, Very hard water: > 300 mg/L CaCO3. Total hardness should be 50- 300 mg/L. Very hard water causes osmoregulatory stress to fish. Hardness below 50 mg/L reduces growth of plankton.
Reagents
1. 10.01M EDTA solution: Dissolve 3.72 g disodium salt of EDTA in distilled water and make the volume 1L by shaking.
2. Eriochrome Black T (EBT) Indicator: Add 0.2 g dry Eriochrome powder to 50 ml of triethyl amine. Add 5 ml absolute alcohol and shake it.
3. Ammonium chloride-Ammonium hydroxide buffer solution: Dissolve 17.5g ammonium chloride (NH4Cl) in 142 ml of ammonia solution (NH4OH). Add distilled water to make the volume 250 ml.
Procedure
1. Take 50ml water sample in a 250 ml conical flask.
2. Add 2 ml buffer solution and 5 drops of EBT indicator.
3. Wine-red colour will appear.
4. Titrate against 0.01M EDTA till the colour changes into bright greenish blue.

Common elements of natural water that significantly contribute to its hardness include calcium and magnesium ions of these elements leached from rocks. Water hardness reduces the utility of water for domestic use. Calcium hardness sometimes may be helpful because it provides a coating in the pipes which protects them against corrosion.
Principle
When calcium and magnesium are present in the water, EDTA reacts with calcium first. EDTA can be used to directly measure calcium when the pH is raised to a point where magnesium hydroxide is mostly precipitated and an indicator that only combines with calcium is employed. At a pH of 12 to 13, several indicators change colour to indicate that all of the Ca has been incorporated by the EDTA. The variations between an aliquot titrated at pH 10 and one titrated at pH 12 or 13 are what define the amount of magnesium.

Principle
Alkalinity of a water sample is can be determined by titrating with standard sulphuric acid. Titration to pH 8.3 or de-colourization of phenolphthalein indicator will indicate complete neutralization of OH and ½ of CO3 while to pH 4.5 or sharp change from yellow to pink of methyl orange indicator, that will indicate total alkalinity (complete neutralization of OH, CO3, HCO3)
Chemicals
1. Phenoplthalein indicator
2. Methyl orange indicator
3. Sodium carbonate, Na2CO3
4. Sodium hydroxide, NaOH
5. Sulphuric acid, H2SO4
Reagents
1. Phenolphthalein indicator: Dissolve 0.5 g in 500 ml 95% ethyl alcohol.
2. Methyl orange indicator: Dissolve 0.5 g of methyl orange in 100 ml of water and dilute to 1000 ml in deionized or distilled water (CO2 free deionized or distilled water should be use).
3. Standard Sulphuric acid, H2SO4 (0.02N): to prepare 0.1N H2SO4 take 3 ml conc. H2SO4 and dilute it in to 1000 ml of deionized or distilled water. Standardise it against standard Na2CO3.
4. Sodium carbonate, Na2CO3 (0.05 N): Take 7g standard Sodium carbonate (Na2CO3) dry at 250 oC for 4 hr and cool in desicator then weigh 5.3 g and transfer it a 1000 ml volumetric flask after that fill to mark with distilled water. Solution should be use within 1 week.

Chemical oxygen demand (COD) is measurement of oxygen demand of waste in terms of the total quantity of oxygen required for oxidation of the waste to carbon dioxide and water. This test is based on the fact that all the organic compounds, with few exceptions, can be oxidized The test is based on the fact that all organic compounds, with a few exceptions, can be oxidized by the action of strong oxidizing agents under acid conditions. Organic matter + Oxidizing agent = CO2 + H2O. The major advantage of COD test is the short time required for evaluation. The determination can be made in about 3 hours rather than the 5-days required for the measurement of BOD
Principle
The oxidizing agents like potassium permanganate and potassium dichromate is usually used to determine COD. Potassium permanganate is selective in the reaction like it reacts with carbonaceous on the other hand it shows nonreactive with nitrogenous matter. Estimating of COD with any method, oxidizing agent must be present to ensure that organic matter is oxidized completely. The organic matter and oxidisable inorganic substances present in water or wastewater get oxidised completely by standard potassium dichromate (K2Cr2O7) in the presence of H2SO4 to produce CO2 + H2O.The excess K2Cr2O7 remaining after the reaction is titrated with ferrous ammonium sulphate [Fe(NH4)2 (SO4)2]. The dichromate consumed gives the 02 required for oxidation of the organic matter.