eChapters

A cell culture laboratory should allow the sterile handling of cultured cells with no or minimum level of contamination. The growth of bacteria and fungi is very high and no level of contamination can be allowed in animal cell culture. However, it is impractical to design a laboratory free from potentially contaminated microorganism. Further, it is possible to establish a laboratory with minimal risk of contamination. Several points should be taken into consideration while designing a new cell culture laboratory.

Introduction

We come across a variety of chemicals every day in a laboratory. While some of them are relatively safer, others are extremely hazardous. Thus, it is very important to know the potential hazards that can be caused by the chemicals present in the laboratory and the safety measures needed to be followed to avoid the hazards. Additionally, we should also be aware of the steps necessary to be taken on such hazardous situations. According to IUPAC, a hazardous chemical mean the chemical which has the potential to cause damage in various ways. In a laboratory, there are chemicals that might be irritant, carcinogen, respiratory fibrogen, systemic poison, asphyxiant, flammable, etc. which have the potential to cause hazard to the workers or environment. Therefore, it is important to use appropriate procedure to reduce the risk and protect the health/safety of laboratory personnel, the public and the environment.

Types of Hazards Associated with Chemicals

Chemical hazards: The hazards caused by chemicals can be broadly classified into three categories.

1. Health hazard: This is also known as toxic effects of chemical. The risk associated with a chemical in laboratory depends on the extent of exposure and the inherent toxicity of the chemical. Many chemical reactions form the product that might be much more toxic than the reacting chemicals. For example, inadvertent mixing of formaldehyde (a common tissue fixative) and hydrogen chloride results in the generation of bis(chloromethyl) ether, a potent human carcinogen. For most of the chemicals, the route of exposure (through skin, eyes, gastrointestinal tract, or respiratory tract) is more important (Figure 17.1) that must be considered during risk assessment.

Introduction

Proper electrical wiring and earthing are crucial for laboratory safety and functionality. A laboratory houses several installations/equipment that demand stable/continuous power supply. Inappropriate or faulty wiring and earthing may lead to significant risks, including electrical shocks, damage to equipment, and even incidence of fire. Therefore, understanding the requirements of electrical wiring and earthing is essential for designing, construction, and maintenance of laboratory. This chapter aims to provide a basic knowledge of electrical wiring and earthing necessary for a laboratory. Importance of earthing in mitigating electrical hazards and ensuring integrity of electrical installations are also described.

Basics of Electrical Wiring

Basic understanding of electrical wiring is necessary for laboratory safety and efficient electrical functioning. The potential difference between two electrical points (negative and positive) is known as Voltage, which drives the flow of electric current. This is measured as volt (V). The rate of flow of electricity in a circuit is known as Current (I), which is measured as ampere (A). Current can be either direct (DC) or alternating (AC). Opposition/resistance experienced in the flow of current in circuit is known as Resistance (R) and it is measured in ohms (Ω). Higher the resistance means lesser the current flows at a given voltage. The rate at which electrical energy is transferred/utilized in an electric circuit is known as Power (P), which is measured in watt (W). The Power can be calculated as the product of voltage and current (P = V × I).

Duck farming in India is characterized by nomadic, extensive, seasonal, and is still held in the hands of small and marginal farmers. Traditionally Assam, West Bengal and Kerala are the major consumer states for duck egg and meat and one of the reasons is that duck egg and meat highly suits and impart taste for their fish based culinary preparations.

Different housing system for Duck rearing

In general, ducks do not require elaborate houses. The house should be well ventilated, dry, and rat proof. In semi-intensive system of rearing, the house should have easy access to outside run as the ducks prefer to come out during the day time, winter and rainy time. 

Introduction

Several hazardous chemicals, microbes, and techniques like recombinant DNA technology are used for research and development work in research Institutes and universities. Therefore, the researchers, technical, helping and supporting staffs as well as the laboratory outputs must be kept free from contaminations. Thus, biosafety is necessary to prevent unintentional exposure/ release of hazardous chemicals, microbes/pathogens, and toxins to the laboratory staff and environment. Following the biosafety guidelines and ethical considerations are essential to maintain safety standards for the researchers as well as the environment. This chapter deals with various biosafety measures and ethical issues to be strictly followed while working in a research laboratory. The required safety level needs to be determined as per the chemical/biological materials are used in the laboratory to keep the researcher/environment safe for sustainable research and developmental activities.

Biosafety

The term biosafety is used to describe the procedure and policies needed to ensure environmental and personal safety. Biosafety refers to the containment principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens, toxins, or their accidental release into the environment. A fundamental objective of any biosafety program is the containment of potentially harmful chemical/biological agents as precautionary safety measures.

Why Biosafety?

With the increasing number of countries adopting molecular biology tools and techniques in life science research and development activities, especially in the areas of agriculture and medicine, the biosafety issues are gaining importance to ensure biological safety for the public and the environment. Conducting research in safe and sustainable manner is not only a personal requirement but essential collective efforts to ensure biological safety for a clean and safe environment. This requires rules, regulations, monitoring bodies, and awareness among the workers as well as the public. Therefore, biosafety per se is an integral part of laboratory research and requires awareness among the researchers so that biological safety can be well taken care from the grassroot level.

Introduction
The advent of antibiotics, beginning with penicillin in 1941, transformed medicine by rendering bacterial infections treatable [1]. However, antimicrobial resistance (AMR) has emerged as a profound threat, enabling bacteria to survive in the presence of drugs designed to inhibit or kill them and these are rooted in ancient microbial ecosystems evidenced by resistome genes in 30,000-year-old permafrost [2]. AMR has been exacerbated by human practices: approximately 50% of antibiotic prescriptions are inappropriate, 70% of global antibiotics fuel agriculture, and environmental pollution creates resistance hotspots [1]. By 2025, AMR directly causes 1.3 million deaths annually, with economic losses projected at $100 trillion by 2050 [1]. Priority pathogens, including carbapenem-resistant Enterobacteriaceae (CRE), methicillin-resistant Staphylococcus aureus (MRSA), and colistin-resistant strains via mcr-1, challenge last-resort therapies, particularly in low-resource settings [3]. This chapter delivers a technically extraordinary exploration of AMR, structured into genetic, biochemical, and evolutionary mechanisms, each elaborated with molecular precision and step-by-step clarity for learners. Genetic alterations provide the DNA-level foundation, biochemical pathways execute molecular defenses, and evolutionary forces drive adaptation and dissemination. It ensures scientific rigor and broad appeal for students, researchers, clinicians, and policymakers.
Historical Context and Global Burden
AMR emerged shortly after antibiotics’ introduction, with penicillin-resistant S. aureus identified by 1942 due to ß-lactamase production [1]. The 1960s

Introduction

The study of infectious diseases, alongside other clinical and natural strengths, is going through fast change introduced on by the approach of reasonable next generation sequencing (NGS) advancements (Koser et al., 2012).These technologies become adopted as routine methods easily, and laboratory techniques are transformed throughout the process. The amount of genomic bacterial data generated is huge. As of this writing, for example, over 200,000 Salmonella genomes alone are in the public domain with hundreds being added weekly. A whole genomic DNA sequence characterizes the maximum practicable level of structural detail on the individuating traits of an microbial organism or population. It can therefore be used to provide more accurate microbial genome recognition, evolutionary relations and a detailed catalogue of epidemiologically important characteristics. This has a significant role to play for outbreak research, diagnosis and treatment of infectious diseases, microbiology and epidemiology (Allard et al., 2019). Besides, DNA sequences are also a universal dataset which can theoretically be used to determine any biological function. This means that antimicrobial resistant (AMR) can be detected and to track the development and spread of AMR bacteria in a hospital or the community.

Carbohydrates (glucose) are the primary energy source for a body; when digested, glucose is the end product in human beings and simple stomach animals, but it yields volatile fatty acids in ruminants. Precise regulation of levels of blood sugars/glucose in the body is the net result of various processes like digestion, absorption, and metabolism operating under the interaction of various a) enzymes and b) hormones secreted by the different organs of the body. An abnormal secretion of any of the enzymes or hormones associated with the metabolism of carbohydrates disrupts the precise regulation of blood sugar, leading to various disorders.
14.1 Enzyme Deficiency and Disorders in Carbohydrate Metabolism
Usually, various enzymes are involved in the breakdown of carbohydrates, and if their secretion is insufficient, missing, or does not work correctly, it leads to different disorders.
I. Glycogen storage diseases (GSDs, glycogenosis, and dextrinosis)
Glucose is needed by the cells for the generation of energy and other cellular activities. If glucose happens to be in excess, it is stored as glycogen, mainly in the liver and muscles, for future needs. The stored glycogen can be converted back to glucose when the need arises. Many enzymes are related to this interconversion of glucose and glycogen. Deficiencies of one or more enzymes may lead to abnormal glycogen metabolism, leading to glycogen accumulation of normal or abnormal chemical structure in tissue, collectively called glycogen storage diseases (GSDs). Roman numerals identify different types of glycogen storage conditions. They are also classified under various categories like inherited metabolic diseases, autosomal recessive type, and X-linked type (VIII/IX type).

Protozoa are single celled eukaryotes which are either parasitic or free living belonged to family apicomplexa. These protozoan affects the different birds like chickens, turkey, ducks, pigeons, game birds and also various pet birds. The infection caused by protozoan diseases may affect one system or more than one system of the body depending on the causative agents involved. The common protozoan diseases like leukocytozonosis, chochlosomiasis, cryptosporidiosis, coccidiosis, histoplasmosis causes infection in the ducks. The protozoan diseases cause severe economic loss to the poultry industry due to death of birds, expenditure on the treatment and prevention of disease and loss of egg and meat production.

Introduction

Urinalysis, the examination of normal and abnormal components of urine, is one of the most important aspect of clinical pathology that aids in accurate and timely diagnosis in veterinary practice. Urine is considered as one of the most accessible bio-fluid that can be collected using non- invasive techniques in adequate quantities. It includes the evaluation of colour, odour, turbidity, volume, pH, specific gravity, protein and glucose content, ketone bodies, blood, erythrocytes, leukocytes, epithelial cells, casts, crystal, micro-organisms, etc. Urinalysis gives us valuable information regarding the several important attributes of renal function. For example, specific gravity of urine gives valuable insights over the potential of the Henle’s loop to dilute urine and the distal convoluted tubules to concentrate urine after ruling out the extra-renal factors affecting the urine specific gravity. It is highly useful to evaluate the different types of urogenital diseases and it also provides significant information regarding several systemic disorders. It is comparatively quicker to perform and is relatively a low-cost test that could be easily done in most clinical setups.

Introduction
The emergence and spread of antimicrobial resistance has changed the landscape of infectious disease treatment, creating an urgent need for new diagnostic and therapeutic approaches4’7. Traditional phenotypic antimicrobial susceptibility testing, while remaining the gold standard, requires 24-72 hours for results and provides limited insights into resistance mechanisms. Limited turnaround time is often a constraint that requires empirical use of antibiotics, potentially driving the propagation of other resistance emergence as well as suboptimal patient outcomes. Thanks to mass spectrometry, clinical microbiology has greatly benefitted through the rapid detection of pathogens. However, the application is far broader than species identification. The integration of multiple omics fields like proteomics, metabolomics, genomics, and transcriptomics through mass spectrometry provides unprecedented means to unravel, predict, and combat antimicrobial resistance (AMR) on a molecular level. The use of a multiomics approach is demonstrated to show that AMR is a complex phenotype that arises from the intersection between genetic mutations, variations in gene expression, metabolic differences, and changes in protein structures that all act in unison to affect bacterial survival in the presence of antibiotics. The conversion of raw mass spectrometry outputs into clinical insights amenable to decision-making is dependent upon bioinformatics. Through the use of sophisticated computational methodologies, such as machine learning and network analysis, bioinformatics provides the means to incorporate heterogeneous omics datasets. Through this approach, resistance-related biomarkers can be identified, predictions for therapeutic efficacy can be made, and new targets for intervention can be identified. The intersection between high-resolution mass spectrometry, broad-based omic profiling, and strictbioinformatics provides a powerful platform for addressing the intractable challenge of antimicrobial resistance.

11.1 Total DNA Extraction by cTAB Method
    
11.2 Extraction, Purification Estimation of DNA
    
11.3 Potassium Acetate Procedure
    
11.4 DNA  Estimation
    
11.5 Restriction Enzymes

Acetic acid            
    

Molecular weight    60.05        
    Melting point (^ºC)    17        
    Boiling point (^ºC)    118        
    Flash point (^ºC)    40        
    Auto-ignition point (^ºC)    426        
    Specific gravity at 20^ºC    1.05        
    Molarity of the concentrated acid (M)    17.4    
    1 M solution    57.5 ml concentrated acid per litre

Transport systems are crucial to survival. Unicellular organisms rely on simple diffusion for transport of nutrients and removal of waste. Multicellular organisms have developed more complex circulatory systems. The circulatory system is responsible for the transport of the various essential compounds and other factors around the body, as well as the removal of the metabolic wastes that accumulate in the tissues from body activities to places of elimination.

Introduction Antimicrobial resistance (AMR) has emerged as one of the gravest threats to modern medicine, undermining decades of progress in infectious disease control. It is cited by the World Health Organization (WHO) as one of the top 10 threats to global health. In 2019 alone, AMR was associated with approximately 4.95 million deaths, including 1.27 million directly attributable to resistant bacterial infections. This makes AMR a leading cause of death worldwide, surpassing HIV/AIDS (0.86 million deaths) and malaria (0.64 million deaths) in the same year. Projections suggest that if the trend continues unchecked, AMR could cause 10 million deaths annually by 2050, representing a profound human-cost escalation. Livestock and Animal burden While the quantitative burden of AMR in animals is not yet globally mapped, its effects in livestock are already both profound and alarming. Over 70% of all antimicrobials globally are used in farm animalsfrequently for growth promotion and disease prevention—not just treatment. This widespread use contributes to emerging resistance in key pathogens—Salmonella, E. coli, Campylobacter in poultry, and severe swine dysentery in pigs. In some outbreaks, swine dysentery, resistant to conventional treatment, has caused up to 90% morbidity and 30% mortality in weaned pigs. Animal-to-Human Transmission and the One Health Paradigm Antimicrobial resistance in livestock doesn’t stay on the farm—it seeps into the environment and crosses over to humans via food chains, direct contact, and environmental pathways. For example, substandard veterinary medicines and antibiotic misuse has been traced in zoonotic infections and veterinary-tohuman AMR transmission.Surveillance gaps remain: the World Organisation for Animal Health (WOAH) acknowledges that while human-related AMR mortality is well-documented, the animal health burden is not yet quantified globally—though numerous projects are underway to address this gap. The failure of conventional antibiotics to treat resistant infections highlights the urgent need for alternative therapeutic strategies. Among the most promising options is bacteriophage therapy—the use of viruses that specifically target and kill bacteria. Bacteriophages Bacteriophages, often referred to simply as phages, are viruses that infect and replicate within the bacteria. They are considered as the most abundant biological entities on Earth, with an estimated amount 10³¹ phage particles present in the biosphere—outnumbering bacteria by approximately tenfold. Phages are naturally found in diverse ecosystems including soil, ocean, sewage, and even within the microbiomes of humans and animals.

Domestic animals –including chicken and mammals, belong to the category of homeotherms, as opposed to poikilotherms. Homeotherms or warm blooded animals are those which are able to maintain constant body temperature in the face of varying environmental temperature. The poikilotherms or the cold blooded animals change their body temperature according to the temperature of the surroundings.

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.

Characterization of Germplasm / Hybrids, Varietal identification, mutants etc.

Accurate identification of plants is desired for patent protection of plant varieties. Accurate characterization is also necessary to trace out the parents of new varieties. Use of present day molecular markers in addition to the classical methods provides more positive identification of new cultivars. RAPD and other molecular techniques have been used in a variety of plant materials for such studies (Sneath and Sokal 1973, Saitou and Nei 1987, Demeke et al., 1992, Selvam and Rajasekaran  1993, 1994, Dubouzet, et al., 1997, 1998, Hallden, et al., 1994, Hu and Quiros 1991, Jain et al., 1994, Matsumoto and Fukui 1996, Williams et al., 1990, Wolff et al., 1995). RAPD technique has been used in the genus Bougainvillea. It is often difficult to distinguish both mutants and hybrids and sports having a common progenitor, or progenitors themselves. Therefore, the industry needs a DNA-based fingerprinting technique to distinguish mutants or cultivars reliably. Reports are available on investigation about the molecular systematics and genetic differences for better understanding of varietal relationships in bougainvillea (Chatterjee et al., 2007, Kumar et al., 2006, Kumar et al., 2014, Hammad 2009, Pavan Kumar et al., 2015). 

Veterinary epidemiology is the study of disease within a livestock population, dealing with the investigation (including identification, quantification, and examination), of the direct and indirect determinants of disease distribution in animal populations. Any epidemiological investigation starts with whether there is an animal health problem in a population, which requires data collection on animal, management, economic, and environmental factors. These data are then analyzed descriptively to assess the frequency of disease occurrence. The results are then used to decide whether investigations of causal relationships between disease occurrence and potential risk factors are necessary for the development of control or prevention policies. Once the relationships are understood, necessary interventions for the disease are then planned and their impacts assessed. Hence, the goal of epidemiological investigations is the control and prevention of animal disease problems by integrating the available pieces of evidence into the decision-making process for disease control policy development.

For crop improvement and more specifically to develop new varieties a number of plant breeding methods are available. CSIR-NBRI from very beginning selected all available techniques and standardized their practical application for all ornamental crops. All crop specific basic information was collected/generated for successful application of techniques for development of new varieties by creating genetic variability. Ornamental plant species fall into two main categories. In the first group the plants that are capable of sexual reproduction, but are commercially propagated vegetatively. The second group of plant species are the apomicts. In obligate apomicts hybridization fails to generate any variability. For development of new variety creation of genetic variability is pre-requisite. Breeder should have all up-to-date knowledge about all the available techniques and their merits and demerits for improvement and development of new varieties.