eChapters

True/False

Q.1.    The precision of an estimator increases as the sample size increases.
Q.2.    Statistical power is the probability that a statistical test will indicate a significant difference is true i.e. it is the probability of taking correct decision.

1. Introduction:- Overview of Good Laboratory Practices (GLP)
1.1 Definition and Scope of GLP
GLP refers to a set of standards that govern the organizational processes, personnel responsibilities, and conditions under which laboratory studies are conducted. It aims to regulate research environments and ensure the generation of high-quality, traceable, and reproducible results. The scope of GLP encompasses a wide range of scientific fields including pharmaceuticals, agrochemicals, veterinary products, and environmental safety assessments. Good Laboratory Practices (GLP) regulations were developed by the Organization for Economic Co-operation and Development (OECD) and well adopted by regulatory agencies world-wide. The primary objective of GLP is to promote high standards of accuracy and integrity in non-clinical research to protect human health and the environment.
1.2 Principles of GLP
The fundamental principles of GLP include:
• Study Planning and Protocol Development: Every study must have a clear plan and defined objectives, which should be outlined in a comprehensive protocol.
• Personnel Responsibilities: The roles of study directors, scientists, quality assurance personnel, and support staff are clearly defined.
• Test Systems and Facilities: Laboratories must maintain appropriate facilities and control systems for conducting studies. These include properly calibrated equipment, controlled environments, and standardized procedures.
• Data Integrity and Reporting: Data must be meticulously recorded, verified, and archived, ensuring it is traceable and reproducible.
• Study Documentation and Archiving: All records, raw data, and reports must be stored and accessible for a period defined by regulations, ensuring transparency and reproducibility.

I. CHALLENGES TO CYBERSPACE

The problems and prospects of copyright and information technology need to be deliberated in the context of present day jurisprudence. The subject is referable to the extensive use of information technology all over the world which has resulted in enhanced efficiency, effectiveness and optimum use of resources. The copyright law is concerned with the negative right of preventing copying of the physical material in the field of literature and arts. Trade and Merchandise Marks Act protects the patent, trade mark and design giving the registered proprietor an exclusive right to the registered material. The prohibition contained in rights in patent and copyright extend to the unauthorised manufacture or reproduction, so long as the exclusive right rests. Copyright has been held to be not the mere creature of the statute but a natural and civil right. The moral basis of the copyright is that the man is entitled to the fruits of its mental faculties as much as he is entitled to the fruits of his mechanical labour. The law rests on the English Commandment - thou shall not steal. It does not permit one man to make a profit and appropriate himself what has been produced by the labour, skill and capital of another. Blackstone in his commentaries Vol.II at page p.406 has stated:

There is still another aspect of property and this is the right which an author may be supposed to have in his own original literary compositions: so that no other person without his leave may publish or make profit of the copies. When a man by the exertion of his rational powers has produced an original work, he seems to have clearly a right to dispose of that identical work as he pleases and any attempt to vary the disposition he has made to it appears to be an invasion of that right.

Introduction
Cleavage is the process of cellular division without growth. It is also called segmentation division and it follows immediately after fertilization. By the end of this process, single cell zygote is transformed into multi-cellular stage called blastula. Daughter cells are called blastomeres. All divisions are mitotic and occur consequently. Ratio of nucleus to cytoplasm is very low at beginning but later they resemble somatic cell. Cleavage division always start at animal pole. Cleavage can be classified as:
1. Holoblastic/Total cleavage: Whole fertilized ovum is involved in production of blastomere. It is also called complete cleavage.
a) Equal: All blastomeres are of equal size. E.g. mammals
b) Unequal: Some blastomeres at vegetal pole are larger. E.g. amphibians
2. Meroblastic/Partial cleavage: When deutoplasm is abundant and only a part of fertilized ovum is involved in cleavage, it is called meroblastic cleavage.
a) Discoidal: Cleavage is confined to the germinal disc i.e. at animal pole. E.g. birds and reptiles.
b) Superficial: Cleavage is confined to the peripheral margin of cytoplasm. E.g. arthropods.
Dividing continuously, embryo attains a stage of 16-32 cells. 16-celled stage is called early/uncompact morula and 32-celled stage is called compact morula. This stage is called morula due to its close resemblance to mulberry fruit. Structure is surrounded by zona pellucida.
After morula stage, fluid accumulation begins and blastomeres are gradually pushed to the periphery. As fluid accumulation progresses further, it leads to formation of large cavity in central part of embryo known as blastocoels and morula stage is now transformed into blastula/blastocyst stage. Blastomeres now arrange themselves as single celled outer layer called trophoblast and small group of cells at animal pole called inner cell mass (ICM). Trophoblasts give rise to fetal membranes and will help embryo in its attachment to uterine wall (implantation). ICM gives rise to all organs of body. Development of blastula is called blastulation.

Introduction

Use of acid/base is common in laboratories for experiments like titration and proper safety in their handling laboratory is necessary. In this chapter we will briefly discuss about the safety measures necessary to be adopted while using acids/bases in laboratory for any experiment.

Handling of Strong Acid/Base

There are four major points that need to be taken care of while working with strong acids or bases that might be quite harmful if any accident happens.

1. Protect Yourself

Wear a lab-coat and acid-resistant gloves: Always use lab-coat/apron and make it sure that the sleeves properly cover your arms. More importantly, use gloves made of acid-resistant materials and also wear shoes to protect your feet while wearing long-pants/jeans. Female with long hair, must tie her hair properly (with rubber band) to avoid any disturbance caused by appearance in between.

Wear safety goggles: It is important to wear safety goggles to protect your eyes using larger and fitting goggles that cover front as well as sides of your eyes. Always use properly fitting and adjustable goggles because smaller/ loose goggles may not protect the areas around your eyes against accidental acid spills and it may fall down from your face, if it is too loose.

Locate emergency wash area: There must be an emergency washing area nearby the work place or laboratory containing lab spill management kits. All the workers in the laboratory must be aware of using the kits and emergency plans.

Neutralize acid: Don’t panic even if an acid or chemical spill happens. Stay calm and act as quickly as possible. In case an acid spills, neutralize it by using sodium bicarbonate so that they can be cleaned and disposed safely. If acid comes in contact of skin, wash it off under running tap water but do not use/ apply sodium bicarbonate on skin.

The cell is the unit of structure of all plants and animals. With increasing complexity of organization, masses of different cell types tend to become localized and to form recognizable patterns. An aggregation of cells forming a definite pattern is called tissue. A still higher level of organization, tissues aggregate to form organs. The animal cell is a highly organized structure. Plant cell is different from animal cell  by having a cellulose cell wall.  There are a number of well defined structures viz., nucleus, mitochondria, lysosomes, Golgi apparatus etc in the cell. Animal cells also have centrioles whereas plant cells contain plastids such as chloroplasts. The remainder of the cell is the cytoplasm.

Types of cells : Animal cells are usually defined by the tissue of their origin. On the basis of morphology, they can be divided into 5 types.

    1.    Epithelial cells : Epithelial tissues consist of a layer of cells which cover organs and line cavity. Example skin and the lining of alimentary canal. They grow well as single cell monolayer in culture.

    2.    Fibroblast cells : Connective tissue form the major structural component of animals. The tissue contains fibroblasts and most widely used cell in the laboratory. Fibroblasts are bound to the fibrous protein collagen in the connective tissue. They have excellent growth characteristics.

A finite growth capacity is a characteristic of all cells derived form the normal animal tissue. The cells pass through a series of age related changes before being incapable to divide further. The finite number of generations of growth is a characteristic of the cell type, age and species of origin and referred to as the “Hayflick limit”. The cells derived from embryonic tissue have a greater growth capacity than those derived from adult tissue.

Once cells are obtained from a culture collection or isolated from animal tissue,, cells are put in the flask with growth medium. The cells are inoculated at a density of 104-105 cells/ml to get a concentration of 106 cells/ml or 105 cells/cm2 in a solid surface in 2-3 days because of nutrient limitation, accumulation of toxic metabolite or lacking of growth surface.

The formation of the mammalian body plan is regulated by highly conserved gene regulatory networks. During this developmental stage, the notochord and primitive node play critical roles in establishing the cranial-caudal axis and left-right asymmetry. Ectoderm, the outer germ layer, differentiates into neuroectoderm and surface ectoderm. Neuroectodermal cells move from the developing neural tube’s lateral edges to a dorsolateral position. These neuroectodermal cells are known as neural crest cells. The central nervous system originates from the neural tube, whereas the peripheral nervous system originates from both the neural tube and the neural crest. Mesoderm cells proliferate and develops into three separate components: the paraxial, intermediate, and lateral plate of mesoderm. The paraxial mesoderm cells form whorl-like aggregations known as somitomeres on both sides of the neural plate. Paraxial mesoderm organises into separate blocks known as somites caudal to the seventh somitomere. Somatic cells make up the vast majority of the axial skeleton, accompanying muscle, and covering dermis. The embryonic gut tube, developing respiratory system, and accompanying glands all develop from endoderm, the inner germ layer. Endoderm is also responsible for the epithelial linings of the urinary bladder, middle ear, and auditory tube. Endoderm also gives rise to the parenchymal cells of the thyroid, parathyroid glands, pancreas, and liver.

Introduction
The nervous system is an intricate network that plays a crucial role in coordinating and regulating bodily functions, allowing organisms to interact with their environment. This complex system is fundamental for the existence, enabling to sense the world, process information, and respond to stimuli. Comprised of billions of specialized cells called neurons. The nervous system is responsible for various functions, from reflex actions to complex thought processes. The nervous system is a highly organized and complex network that serves as the primary means of communication within the body of all animals, including domesticated species. Understanding the anatomy and function of the nervous system is critical in veterinary medicine, as it governs essential processes such as locomotion, sensation, cognition, and homeostasis. Comprising the central nervous system (CNS) and peripheral nervous system (PNS), the nervous system enables the coordination of reflexes and voluntary movements, facilitating interactions with the environment. Structural Organization: the CNS is composed of the brain and spinal cord, which are protected by the bony encasement of the skull and vertebral column, respectively. The brain varies significantly across species, reflecting adaptations to specific ecological niches and behaviors. For instance, the canine brain shows notable development in areas associated with olfaction, whereas the equine brain has adaptations for vision and balance, crucial for flight responses. The spinal cord serves as a conduit for neural signals between the brain and the peripheral nerves. It is organized segmentally, with spinal nerves emerging from specific regions. This segmentation corresponds to the vertebral column and is vital for diagnosing spinal cord injuries and diseases in animals. Each spinal nerve innervates specific muscle groups and areas of the skin, forming a somatic map essential for clinical examination and treatment. Neurons and Neuroglia: Neurons are the functional units of the nervous system, specialized for the transmission of electrochemical signals. In veterinary anatomy, it is essential to understand the structure of neurons, which includes the cell body, dendrites, and axon. Neurons can be classified into various types—sensory (afferent), motor (efferent), and interneurons—each playing distinct roles in reflex arcs and neural circuits. Neuroglia, or glial cells, provide support, nourishment, and protection for neurons. In veterinary practice, the role of glial cells in pathology is increasingly recognized, particularly in conditions such as neuroinflammation and neurodegeneration. For example, astrocytes play a crucial

Cell growth and cell passage : Different types of cells have different growth requirements but in all cases the surrounding medium must supply all the essential nutrients. The temperature, pH, osmolarity and humidity must be kept within limits. Again, toxic or inhibitory substances must not be allowed to accumulate. Serum, a source of macromolecular growth factors, is essential to the growth of many cell types. Anchorage dependent cells require nontoxic or biologically inert glass or plastic surface for attachment.

A laboratory is essential for research work in science and development. Most of the research institutes have dedicated laboratories for researchers and students to conduct experiments, perform research work, as well as to provide hands-on learning/training to students. There are certain rules and regulations laid down by the laboratory in-charge which need to be followed strictly for safety purposes. Some of the laboratories keep ‘safety manual’ for researchers to read before entering into the laboratory to conduct experiments. Whenever safety is compromised in a laboratory, there is increase in risk of unwanted incidents in the laboratory. Storing food/beverages in the same refrigerator where laboratory chemicals are kept may lead to disastrous effects on health. This chapter aims to provide instructions in brief on the things to be performed (Do’s) before and after entering onto a laboratory as well as the things never to be done (Don’ts) in a laboratory for the safety reasons.

Good Laboratory Practices (Do’s)

• P Wear apron/labcoat while working in the laboratory.
• P Use gloves/respiratory mask/fumehood while handling toxic/volatile/inflammable chemicals/reagents.
• P Use pippet or pippetor for safety in transfer of solutions.
• P Protect eyes, face and naked parts of the body from UV radiations. Use safety goggles or face mask/apron.
• P Keep your eyes away from Liquid Nitrogen and use cryogloves while handling it.
• P Arrange all the necessary materials/solutions before starting the experiment.
• P Periodically fumigate/ozonize culture and inoculation rooms.
• P Autoclave all contaminated materials before discarding.
• P Pre-treat gel or solution containing Ethidium Bromide (EtBr) before discarding into laboratory waste-disposal bin, and use gloves while handling them.
• P Burn all the transgenic plant materials in an incinerator after taking necessary observations.
• P Make entry in the designated log-book and clean the instrument after using it properly.
• P Switch-off the instrument after use or when not in use.
• P Wash hands with soap and remove the labcoat before leaving/going out of the laboratory7.
• P Ensure that switches for fans/lights, and water taps are off while leaving the laboratory.
• P Follow the biosafety guidelines suggested for handling and disposal of the hazardous chemical.

Vero (monkey, African green, kidney) 
Morphology: Fibroblast
Species:, African green monkey 
Tissue: Kidney

Properties: Virology, virus titration, virus replication, plaque assays, bacterial cytotoxicity

2.1     Regulation of the Cell Cycle 
    
2.2     Mitosis
    
2.3     Meiosis

In many experimental situations, it becomes necessary to repeat an experiment over time, (for a number of seasons or years). This repetition (or replication) of the experiment broadens the scope of the experiment in the sense that our recommendations will be applicable for a number of seasons. In the case of agricultural experiments, for example, there may be present treatment x season interaction. So the results of a series of experiment, performed for different seasons with the same set of treatments, will have wider applicability.

We shall consider the simplest case of a repetition of experiments of identical structure for a number of seasons. Let us consider randomized block experiments with t treatments in r blocks and conducted in p seasons. The analysis of the experiment in a season is based on the linear model. Before attempting a combined analysis for the p experiments, it is necessary to perform the analysis for p experiments separately and interpret the results separately. It may be of interest to find out whether differences among the treatments are the same in the different seasons so that ‘ best ‘ treatment may be recommended for all seasons or whether different treatments are to be recommended for different seasons.

We next consider the analysis of the combined experiments considering the seasons as a random sample from the population of seasons. Analysis of Variance of series of experiments

Where Y is the average yield of a crop in a sequence over a period of years “n” and S is the standard deviation of yield over a period of years, Y max is the maximum yield of a crop in a certain year.

Example 1. Six crop sequences were evaluated in simple randomized block design replicated four times at Durgapura. The sequences tried are pearl millet-wheat, cluster bean-wheat, pearl milletmustard, cluster beanbarley, cowpea-mustard and pearl millet-Chickpea under irrigated conditions.

Small volume cultures are useful for experiments such as studying cell morphology, titration of viruses, plaque assay, pock assay, SNT and to compare the effect of agents on growth and metabolism. However, there are other application where there is a need of large number of cells such as extraction of DNA/RNA from cells, production of viruses for vaccine production and a number of products namely interferons, interleukins, hormones, enzymes, antibodies, glycoproteins etc. The large quantity of cells can be obtained by using a large number of flasks/bottles but it is tedius, labour intensive, expensive and there is a risk of contiamination. Although the unit processes are more cost effective and efficient, it needs some modification to overcome limiting factors such as oxygen depletion, shear damage and metabolic toxicity. There are a number of aberrations available to scale up cell culture described below.

A sample is just a part of a population selected according to some rule or plan. Following are some of the methods of sampling.

1. Simple Random Sampling

The simplest and the most commonly used type of probability sampling is simple random sampling. In this kind of sampling, each member of the population has the same probability of being included in the sample. Simple random sampling may be with or without replacement.