eChapters

6.1 Introduction

Perennial crops are crucial for maintaining food security, despite making up only 4.2–4.7% of all agricultural acreage (Kreitzman et al., 2020). The value of fruit and nut production reached a value of USD 823 billion globally in 2020, with the top five fruit crops being apples (87 Mt), oranges (78 Mt), grapes (77 Mt), mangoes (55 Mt), and apricots (41 Mt) (Statista, 2020). The tree nut business is growing quickly, although making up a smaller share of the world’s agricultural output. Global tree nut output is expected to reach 4.2 Mt in 2019–2020, with almonds, cashews, walnuts, hazelnuts, and pistachios accounting for 31%, 17%, 21%, 12%, and 14% of the nut market share, respectively (Serna, 2020).

Tree crops require a longer investment period before producing a commercial yield; sweet cherry takes three years to attain a commercial yield, while pistachio takes ten (Rezaei et al., 2019). High-density monocultures of cultivars chosen for their potential yield, fruit quality, and disease resistance are part of optimal tree crop production techniques. These monocultures are maintained under intensive, high-input farming systems (Simon et al., 2017). However, inadequate pollination can seriously impair the quantity and quality of these agricultural products, prompting for meticulous dedication to pollination research and management (Bosch et al., 2021; Forbes et al., 2019).

Effective pollination, and on the contrary, situations in which pollination is restricted or deficient, depend on both internal and external factors (Figure 7.1, Table 7.1). The period of stigma receptivity, the lifespan of the ovule, and the quality and availability of compatible, suitable pollen are all considered as intrinsic factors (Howlett et al., 2015). Agronomic methods pertaining to plant nutrition, orchard layout, pest and disease control, pollinator activity, pollen quality, and weather patterns and their effects on flowering synchrony are examples of extrinsic factors(Toledo-Hernandez et al., 2017). Hatfield and Prueger assert that pollination is one of the phenological stages that is most sensitive to temperature, and as such, it has a significant impact on productivity (Hatfield and Prueger, 2015). Climate has been shown to be a key factor in determining pollination success and production in crops including peaches, apples, and almonds.

7.1 Introduction

Artificial intelligence (AI) is the imitation of human intellect in machines designed to emulate human activities and cognitive functions. These machines are designed to accomplish tasks that normally require human intellect, such as learning, problem solving, perception, and decision-making (Zhang et al., 2022). Horticultural crops, including fruits, vegetables, nuts, and ornamental plants, are of paramount importance in global agriculture, as they contribute significantly to food security, economic prosperity, and environmental sustainability. Nevertheless, the cultivation of these crops poses numerous challenges for growers, such as fluctuating market demand, insufficient resources, unpredictable climate conditions, and the constant threat of pests and diseases (Nowak & Grant, 2018). In recent years, the agricultural sector has experienced a significant shift owing to advancements in Artificial Intelligence (AI) technologies (Liakos et al., 2018). AI has emerged as a powerful tool for the cultivation of horticultural crops, providing solutions for boosting productivity, sustainability, and efficiency (Shrivastava & Kumar, 2021). By utilizing AI, horticulturists can overcome the traditional challenges associated with crop management, resource allocation, and environmental sustainability, paving the way for a more resilient and productive agricultural sector (Garg et al., 2019). The integration of AI into horticultural crop production encompasses a diverse range of applications, including precision agriculture, crop monitoring and management, predictive analytics, automated harvesting, and genetic improvement (Golhani et al., 2018). These applications leverage machine learning algorithms, remote sensing technologies, and data analytics to analyze vast amounts of agricultural data ranging from soil composition and weather patterns to crop health and market trends (Araus& Cairns, 2014). Through intelligent data-driven insights, AI enables farmers to make informed decisions in real-time, optimize resource utilization, minimize waste, and maximize yields (Kamilaris et al., 2017). This technological convergence offers promising prospects for food production, enabling stakeholders to explore new avenues for growth, resilience, and sustainability in horticultural crop cultivation (Mohanty et al., 2016).

Introduction

For proper and efficient experiment to be conducted, experimenters need so many chemicals to deal with. This includes different acids, base, salt, different kind of indicators and so on. Some of these chemicals are safe for health, but some of them are dangerous to health when come in close proximity to our body while some of the chemicals cause health issues on inhaling. These may cause improper functioning of some of our physiological activities. Acids and bases are very dangerous for our tissues when come in our contact. These may cause serious respiratory problem in addition to injuries on coming to our contact. Some chemicals are exploratory yet they may cause even death of the person working with them in laboratory. Therefore, mere use of the chemicals in laboratory is not only the objective of the experimenter. The person working in laboratory should also be concerned about the proper use and handing of the chemical while working with it. Thus, every researcher of an institution who has to work with chemicals in a laboratory should be made aware about the chemicals to be used in the laboratory and the consequences if any mishandling of a laboratory chemical occurs. Not only these, suitable precautionary measures should also be known to the researcher along with ensuring the availability of the required safety equipment/installations. Hence, a proper understanding of the ways of handling of chemicals in a laboratory is of prime importance for the personals like you who are working or have to work in laboratory.

Safe Handling of Chemicals in Laboratory

Safe use/handling of chemicals in laboratory include minimized exposure to chemicals, maintaining safety data sheets, proper labelling, storage, segregation, and transport of chemicals for personal hygiene.

Introduction

While experimenting with chemicals, sometimes we need to dry the chemical/ solution (solid, liquid or gas) using a process like evaporation or distillation, mainly to remove water or different solvents. Drying is the process of evaporation or distillation to remove water/solvent from a solution, solid or even from gas. Generally, a drying agent (usually an anhydrous inorganic salt) is used for this process, which reacts with the water (present in the chemical/ solvent to be dried) to form a hydrate. For example, anhydrous MgSO4 is one of the drying agents. The drying agents are recognized by their capacity (the rate at which they absorb water) and their intensity (the amount of water left behind in the solvent at equilibrium). No chemical reaction or change in phase occurs during the drying process.

Type of Solvents

Apart from water (a universal solvent), there are three families of organic solvents used in laboratories.

1) Oxygenated solvents: Alcohols, Ketones, Glycol ethers, Glycol esters, etc.

2) Hydrocarbon solvents: Aliphatic and Aromatic hydrocarbons.

3) Halogenated solvents: Chlorinated hydrocarbons.

Introduction

A variety of glassware, plasticware, and other apparatus are used for experimental purposes in a laboratory. They need to be washed, cleaned, dried, and sometimes sterilized before they can be used in the next set of experiments. Glassware needs special care of handling during washing, drying, sterilization, etc. because even a minor mistake in handling of glassware may result in its cracking/breakage and it may become unserviceable. All of these may result in a delay in experimentation if spare piece of the glassware is not available or it may affect serviceability of the associated apparatus.

A large number of glassware such as beaker, flask, measuring cylinder, burette, test tube, etc. are regularly used in a laboratory, which requires careful handling during the different stages of experiment as well as after completion of the experiment while preparing them for another set of experimentation.

Glass being a complex silicate, its properties depends on the type of silicate anions in the structure and the cations formed. Thermal properties of glass changes by the addition of boron oxide (B2 O3 ); hence, borosilicate glass is extensively used in the laboratories. With this brief background information, let us discuss about the necessity, procedures, and precautions during washing, cleaning, drying and sterilization of laboratory-wares. Figure 3.1 shows various advantages of using borosilicate glass in a laboratory.

Good laboratory practices require clean glassware, because an experiment may give erroneous result if dirty glassware is used at any step of the experiment. Hence, the glassware must be physically and chemically clean; and in many cases, it must be microbiologically clean or sterile. The commonly used glassware in a laboratory include test tube, measuring cylinder, pipettes, beakers, flasks, microscopic slides, etc.

A variety of glassware like burettes, pipettes, measuring cylinders, flasks, separating funnel etc. are used for experimental purposes in a laboratory. All of these need to be used and handled correctly, otherwise the chances of experimental error and damage to the glassware increase significantly. Being made of glass, they need special care while washing and using in experiments because even a minor mistake in handling of glassware may result in breakage of the apparatus. With this precautionary note, let us discuss about some of the specific glassware used in the laboratories.

Burette

Burette is a long graduated glass tube with a stopcock at one end for delivering a known/desired volume of a liquid, especially in titration experiments. It contains a stopcock at the lower end and a tapered capillary tube as the stopcock’s outlet. The flow rate of liquid from the tub stopcock’s outlet can be controlled with the help of stopcock valve. Burette is used for measuring the volume of a solution/chemical added in a quantitative (titrimetric) estimation experiment.

Procedure of using a Burette

The burette reading must be noted before and after delivering the chemical/ solution from the burette. The difference in these two readings is the volume of the chemical/solution released out of the burette. The solution should be released slowly/drop-by-drop to avoid any experimental error/mistake. If the solution/chemical is allowed to pour out too fast, the wall of the burette will not drain properly and some liquid may remain sticking to the surface of the walls. Moreover, excessive amount of chemical may be released/dropped during the experiment, particularly during the titration experiment wherein we need to know the exact amount of acid/base needed to get neutralization point. Thus, any minor mistake may lead to get a faulty reading. The measuring capacity of a burette usually used in a laboratory is 50 mL.

Different basic as well as advanced laboratory equipment need to be used in a laboratory while conducting experiments. Many of them like pH meter, laminar air-flow hood, viscometer, thermometer, magnetic stirrer, microwave ovens, incubators, sand-bath, water-bath, oil-bath, etc. are the basic and simple equipment which require to be used routinely while conducting experiments in a laboratory. However, basic knowledge of their operation, handling procedure, when to be used, how to be used, and more importantly what not to be done with it, etc. are essential for keeping them in serviceable condition as well as for the accuracy/precision in their operation. In this chapter, we will discuss about some of the routinely used laboratory equipment along, while some other sophisticated equipment will be discussed in other chapters.

pH Meter

A pH meter measures exactly the H+ concentration of a solution; thus indicates acidity or alkalinity of a solution with greater precision. Actually, it measures the difference in electrical potential between a pH electrode and a reference electrode, and display the results converted into the corresponding pH value. Electrode is the key part (rod-like structure, usually made of glass) of a pH meter, having a bulb at the bottom containing a sensor. The pH meters range from simple and inexpensive pen-like devices (portable/pocket pH meter) to complex and expensive laboratory instruments with computer interface (Figure 5.1).

Frequent calibration of a pH meter using the solutions of known pH (standard solution of pH 4.01, 7.00, 10.01, Figure 5.2), generally before each use, ensures the accuracy of the instrument. For very precise work the pH meter should be calibrated before each measurement. For normal use calibration should be performed at the beginning of each day. This is necessary because the glass electrode does not give a reproducible measurement over a longer period of time.

Since mouth pipetting of liquid reagents in a laboratory is not advisable, different tools are needed for safe, easier, and precise dispensing of liquid reagents. This chapter is devoted to make you aware of the tools like vacupette and micropipette which can be used for the handling of liquid reagents in a safe and precise way.

Vacupette

Glass pipettes have been traditionally used by sucking liquid solvent/solution directly using mouth. For safety reasons, mouth pipetting is no longer permitted in a laboratory. Hence, to fill a pipette vacupette is used by placing it on the top of glass or plastic pipette. It acts as a device to create vacuum for filling reagent in the pipette as well as for releasing the reagent with mechanical regulation on the flow out of the liquid. Vacupette can be of different types.

1. Rubber Bulb Vacupette

Rubber bulb vacupette is often used to transfer (pump up and discharge) liquid using a pipette when a larger quantity of chemicals is to be transferred. Small/tiny rubber bulb is attached to glass/plastic pipette for drawing little amount of reagents. Small vacupette is used to precisely discharge smaller volume/ drops.

Handling of Vacupette

• Insert the top of a pipette in the bottom of the rubber bulb vacupette (Figure 6.1).

• Remove air from the pipette by squeezing valve “A” at the top of the vacupette while simultaneously squeezing the rubber bulb. The amount of air released depends on the size of the rubber bulb.

Introduction

To accomplish experimentations in laboratories, sometimes we need microscope to test/validate our ideas/findings. A microscope is needed to examine objects which are very small which cannot be visualised by naked eyes. However, microscopic study requires some expertise in operating the microscope, particularly the sophisticated microscopes, as well as to prepare (peeling, sections, cutting, fixation, staining, dehydration, mounting, etc.) the sample, so that it can be appropriately examined for the anticipated details.

According to the scientific requirements different types of microscopes are used in the laboratories for research purpose. In this chapter, our aim is to acquaint you with the basics of using and handling of microscopes, particularly a compound microscope that is commonly used by the graduate and post graduate students for basic research.

Microscopy is often designated as the scientific investigation that is employed to review and analyse minute structures and objects using a microscope. The term “microscope” has been derived from ancient Greek words “micros” meaning “small” and “skopeîn” meaning “to look at or see,”.

Microscope

A microscope is a laboratory instrument used to examine objects (biological specimens) which are too small to be seen by the naked eye. The microscope must accomplish three tasks:

• Magnification- to provide a magnified image of the specimen.

• Resolution- to separate the details in the image.

• Contrast- to render the details clearly visible to the eyes.

Although we will tnroduce to you the different types of microscopes used for different speciments, our main focus is to discuss about compound microscope, which is commonly used in the laboratories. Compound microscope was developed around the beginning of 17th century.

Introduction

In a research laboratory, we frequently need weigh chemicals and prepare different solutions of varying strength, as per the requirements. Accuracy in weighing and preparation of solution, with respect to weight/volume and strength, determines the success of experiment. In turn, the accuracy depends on weighing of solid chemical and/or measuring the volume of liquid chemical/making up the volume. In this Chapter, weighing of chemicals using appropriate weighing balance, preparation of solutions of different strength, their dilution, and precautions while preparing will be discuss in brief.

Weighing of Chemical

The primary method to determine the amount of chemicals to be used is weighing the chemical by using a balance.

In general, there are two types of balances to weigh chemicals in a laboratory:

1. Standard laboratory balance

2. Analytical balance

Standard Laboratory Balance

Generally, a standard laboratory balance is used for weighing a chemical when an error of ±0.02 g is acceptable. It is also used for pre-weighing, where approximate mass needs to be determined. Precise mass of the chemical is then determined using an analytical balance. Following are the steps in using a weighing balance in a laboratory.

Introduction

For agricultural applications as well as for research work, often we need to prepare solutions of different agrochemicals of varying strength, as per the requirements. Agrochemicals are used to help crop plants grow, produce, and protect them from biotic/abiotic stresses. Many of the agrochemicals (insecticides, herbicides, fungicides, rodenticides, molluscicides, nematicides, etc.) are used to protect the crop from insect pests and diseases. However, some of them (fertilizers and soil conditioners) are used to augment crop yield. While in the previous chapter (Chapter 8) we discussed about preparation of laboratory solutions, in this chapter we will briefly discuss about the preparation of different agrochemicals.

Agrochemical/agrichemical is a generalized term used for the chemicals applied in agriculture. In general, it also includes the feed-supplements used in f isheries and animal husbandry.

Agrochemicals

Fertilizers

Fertilizers are used to augment growth of crop plants and to mitigate nutrient deficiency in the soil. Fertilisers can be organic (containing natural substances, prepared through natural processes) and inorganic fertilisers (synthetic fertilizers, manufactured artificially using chemical processes using natural deposits, by chemical alterations).

Soil Conditioners

Soil conditioners include manure, livestock and leaf compost, peat that help making the soil in good condition. They are laid on the top soil and then mixed properly. They not only increase nutrient contents in the soil but also improve aeration and water holding capacity.

Introduction

Measurement of pH of a solution and preparation of different types/strength of buffers are common practices in a laboratory, particularly in the Biochemistry and Molecular Biology laboratories. Therefore, in this chapter you will be acquainted with the procedures of measuring pH of a solution as well as preparation of some of the commonly used buffers in a laboratory.

Potential or Power of Hydrogen Ion (pH)

Measurement of pH of a solution is measuring the molar concentration of hydrogen ion [H+] in the solution, which is an indication of the acidity or basicity of the solution. The symbol pH stands for “power of hydrogen” and numerical value for pH is just the negative logarithm of the molar concentration of hydrogen ions [H+]. The ionic product of water (Kw ) is the basis for the pH scale. The total hydrogen ion concentration from all the sources and it can be experimentally measured.

The pH scale designates the concentration of H+ (and thus of OH−) in an aqueous solution, which ranges between 1.0 M H+ and 1.0 M OH−. The term pH is defined by the expression

Thus, the ionic product of water makes it possible to calculate the concentration of H+, with given concentration of OH−, and vice versa. The value of 7 for pH of a neutral solution is not an arbitrarily chosen figure; it is derived from the absolute value of the ionic product of water. A solution having pH greater than 7 is basic, and the concentration of OH− is greater than that of H+. A solution having a pH less than 7 is acidic.

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.

Introduction

Different types of culture media are used for culturing microbes and plant tissues. Preparing as well as sterilizing them properly following appropriate method with safety for the desired purpose are important parts of research work. In this chapter, we will discuss briefly about different types of media, their composition, preparation and sterilization in laboratory for microbial or plant tissue culture.

Culture Medium

Ingredients mixed to prepare solid, liquid or semi-solid substance to support the growth of microorganism or plant tissue in vitro (outside the body) is called culture medium. Frequently, it becomes important to grow microorganism or plant/animal tissue in vitro for any or all of the following purposes:

• To study characteristic features or properties of microorganisms.

• To identify the causal organism(s) of infection in a clinical sample, so that appropriate remedy can be determined.

• To culture/multiply microorganism or plant/animal tissues in vitro for research or commercial purposes.

• To prepare/produce biological molecules, vaccines, antigens, proteins, metabolites, etc. in vitro for research or industrial purposes.

Introduction

Tissue culture refers to the technique of growing cells, tissues, or organs under controlled, artificial conditions. This allows propagation of tissues from a small sample of cells/tissues under sterile conditions. Plant tissue culture is widely used for various purposes, including the production of disease-free plants, rapid multiplication of plants with desirable traits, genetic modification, conservation of rare or endangered species, etc. Tissue culture is a valuable technique because it enables consistent and large-scale production of plants, ensures genetic uniformity, and allows manipulation of plant biology at cellular level. In this chapter, we will discuss about the major types of tissue culture techniques and their advantages, disadvantages, as well as applications in brief.

Different Types of Tissue Culture

Callus culture involves induction and growing of unorganized, undifferentiated cell masses from an explant on a nutrient/culture medium. The part of plant used to induce callus is called explant which can be tissue, organ or part of a plant.

Organ culture is used to maintain or grow an entire organ or a part of organ in vitro. It preserves the structure and function of the organ for studies or transplantation.

Embryo culture involves isolating and growing embryos from seeds or ovules in a nutrient medium. It is used to bypass seed dormancy or rescue embryos from hybrid crosses that may not develop naturally.

Cell suspension culture involves growing dispersed cells or small cell clusters in a liquid medium under continuous shaking. This is used for large-scale production of cells, secondary metabolites, and studying cell biology..

Introduction

Seed is a living entity (mature ovule) consisting of a tiny plant in embryonic stage along with the stored food protected by an outer layer known as seed coat. Seed is an important biological input in agriculture, which is economically important for successful crop husbandry. Cotyledons serve as the source of nutrients (proteins, carbohydrates, and oils) that not only nurture the plant at the early developmental (seedling) stages of plant but also the vegetarian (heterotroph) organisms. Being one of the important inputs in agriculture, viability of seed needs to be assessed to know the germination percentage of seeds used to raise a crop. Since viability of seeds may deteriorate on storage depending on the storage conditions. Therefore, often it becomes important to test viability/germinability and vigour of seeds before being used for raising a crop or conducting an experiment. In this chapter, we briefly discuss about the different methods which can be used for testing the viability of seeds.

Seed Viability

Seed viability can be defined as the capability of the seed to germinate and produce a normal seedling within a specified period. In other words, seed viability indicates how many seeds in a lot are alive and could develop into plants under appropriate field conditions. It is very important that most of the stored seeds are capable of germinating when sown in a field. They must possess higher viability at the beginning of storage and maintain it over the storage. A seed with high initial viability survives longer in storage; however, the viability deteriorates slowly at first and then rapidly as the storage prolongs. It is important to know the duration of storage after which a considerable decline in viability occurs under given storage conditions so that to take necessary action to revive the seeds. Excessive deterioration in vianility of seeds may lead to loss of the seed/material/ germplasm.

Introduction

Test of pollen viability (extent of pollen viability) is important in pollination experiments to ensure fertility/seed setting. It is also important in monitoring state of pollens during storage in ecological/ taxonomic studies as well as during research on pollen biochemistry, genetics, stigma interactions, and incompatibility studies. Pollen viability refers to the ability of pollen to accomplish post-pollination events to achieve fertilization. Pollen viability test has gained importance particularly in plant breeding and hybrid seed production experiments/ practices. In this chapter, you will be acquainted with the basics of pollen viability test. However, for more professional knowledge on the topic readers are suggested to refer specialized books.

Pollen Viability Test

It is important to know the extent of viability of pollens used in pollination experiment, in finding fertility of a plant of interest, to monitor state of pollen on storage, in ecological/taxonomic studies, as well as in research on biochemistry, genetics, stigma interaction, incompatibility assay, etc. of pollen. Several pollen viability tests have been developed time-to-time. It is recommended to initially use different tests for a given pollen type to find out the most appropriate method that provides reliable result on pollen viability. It is important to note that every procedure requires standardization of protocol according to the sample used. Following different tests are used for assessing pollen viability.

1. In-vitro Pollen Germination Test

It is a simple, speedy, and quantitative test for assessing pollen viability. In this method, a collection/sample of pollen is germinated in vitro and observed under microscope. The percentage of germinating pollens (producing pollen tube) after appropriate treatment and a given period of time is determined. The percentage provides an index of viability of the pollen sample.

Introduction

Flowering plants (Angiosperms) are identified by their morphological characteristics like flower, fruit and seeds that help their taxonomic characterization into different species, genera, families and orders. The floral characters which are used for taxonomic characterization of plants include the type of inflorescences, perianth structure, floral symmetry, arrangement of floral leaves, type of androecium, stamens, gynoecium, carpel, ovule, etc. The type of fruit and seed also play important roles in taxonomic classification. In this chapter we will briefly discuss about various morphological features of flowering plants (in botanical terms) that are useful in taxonomy.

Nomenclature of Plant

Nomenclature of plant is the formal and scientific naming of plants. It is related to but distinct from taxonomy for grouping and classifying plants and provide botanical name.

The scientific/botanical nomenclature started with Species Plantarum by Carolus Linnaeus in 1753. Botanical nomenclature is governed by the International Code of Nomenclature for algae, fungi, and plants (ICN), which replaces the International Code of Botanical Nomenclature (ICBN).

ICBN and ICN

International Code of Botanical Nomenclature, (ICBN) was adopted by the VIIth International Botanical Congress held in Stockholm, July1950. While the International Code of Nomenclature for algae, fungi, and plants (ICN) was adopted by the XVIIIth International Botanical Congress held in Melbourne, Australia in July 2011.

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).

For a researcher, laboratory is another home where he/she spends much time every day. As we take care of cleanliness, safety, and design of our home, we must take care on these aspects of laboratory too. It is the responsibility of the laboratory personnel to ensure cleanliness/safety of the laboratory. Since every laboratory chemical and equipment have the potential to cause harms if not used with appropriate precautions. Hence, as a laboratory user, it is our responsibility to follow the basic safety/biosafety rules and regulations set up by the Principal Investigator of the laboratory, Institutional Biosafety Committee, as well as by other regulatory authorities, if any. We must be aware of the general/personal protective measures, cleanliness and laboratory waste disposal prior to joining a research laboratory. In this chapter we will briefly discuss about personal safety, chemical safety, cleanliness, waste disposal, etc.

A. Personal Safety

1. Personal Protective Equipment (PPE)

Personal protective equipment (PPE) is special arrangement to protect the worker from specific hazards because of using a hazardous substance. PPE does not reduce or eliminate the hazard, but protects the wearer from exposure to hazardous material. PPE should not be reused or brought outside the laboratory as these might increase the risk of spread hazardous material or contamination. Used PPE must be kept inside the laboratory in a designated waste storage area adjacent to the work area.

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.

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.

Cell culture refers to removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been establwashed.

Primary culture refers to the stage of culture after the cells are isolated from tissue and proliferated under appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth.

Pharmaceutical and chemical industries depend largely on toxicological evaluations to provide information regarding safety of their commercial products. Toxicologists so far have been relying on animal studies to investigate potentially beneficial or harmful effects of drugs and chemicals prior to testing them in humans. Traditionally, the safety assessment process during drug development has been based on screening indiscriminately a plethora of new chemical entities (NCEs) using large number of animals as testing models in order to identify new therapeutic agents with the expectation that one or a few of these NCEs have some competitive advantages over the existing therapeutic agents. That is why they are more efficacious, bioavailable, selective, and safer. This approach is costly, time-consuming, and requires large quantities of test material. Currently, it takes about 10-12 years and a huge expenditure for a drug to move from research laboratory to patent. Thus alternative cell culture models in the early phases of drug discovery process can save time and money both by eliminating least beneficial NCEs.

Environmental agents (xenobiotics) that are injurious to cells trigger a spectrum of responses that can range from cell death to adaptation, repair, and proliferation. Cell death, in turn, can take the form of apoptosis (a programmed form of cell death). Xenobiotics have been implicated in toxic cell injury, among them free radicals (nitrogen, oxygen, and carbon derivatives) are known to interact with cellular constituents including membrane lipids and chromagen materials.

Cytotoxicity

Cytotoxic: Toxic to cells, cell-toxic, cell-killing. Any agent or process that kills cells. The prefix cyto- denotes a cell. It comes from the Greek “kytos” meaning hollow, as a cell or container. Toxic is from the Greek “toxikon” = arrow poison.

In nutshell, cell cytotoxicity refers to the ability of certain chemicals or mediator cells to destroy living cells. By using a cytotoxic compound, healthy living cells can either be induced to undergo necrosis (accidental cell death) or apoptosis (programmed cell death).

Measuring cytotoxicity can prove to be very valuable tool in identifying compounds that might pose certain health risks in humans and animals. Cytotoxicity assays are widely used by pharmaceutical industry to screen for cytotoxicity in compound libraries from initial high-throughput drug screens for unwanted cytotoxicity. This can be of vital importance and prove to be quite indispensable during research phase of developing therapeutic new pharmaceutical products to ensure the safety of end-users. Additionally, understanding the mechanisms involved in cytotoxicity can likewise give researchers a more in-depth knowledge on the biological processes (both normal and abnormal) governing cell growth, cell proliferation and death. Compounds that have cytotoxic effects often compromise cell membrane integrity. Therefore, assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. There are three commonly used methods which can be employed to assess in vitro cytotoxicity (disruption of cell integrity) in cell cultures:

Safety is a prime concern for humans and animals. Without evaluating the safety of a chemical, no regulatory authority in the world allows the chemical to be used by humans. There are stipulated guidelines for toxicity evaluations. Among the toxicity assays genotoxicity assays are important and have occupied centre stage due to increase in understanding of the genome.

Genotoxicity describes the property of chemical agents to damage the genetic information within a cell to cause mutations, which may even lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic; however, not all genotoxic substances are mutagenic. Generally and depending on the agent, a cytotoxic agent may be genotoxic (causing DNA damage), mutagenic (causing gene mutation) or it may be harmful for cell organelles (e.g. cell membrane) or it may even be teratogenic. Therefore, every cytotoxic agent is not genotoxic.

Why we Need to Evaluate Genotoxicity?

Toxicity can be at any level according to the nature of chemicals and type of exposure. Duration of toxicity can also be short lived or long lived within a generation or continue to next generation. Genotoxicity effects are able to continue in next generation(s), in which the exposed population may not suffer seriously but the next generation may suffer without being exposed to the same chemicals. Therefore, genotoxicity evaluations need serious attention.

Apoptosis is triggered by extrinsic and intrinsic stimuli as radiation, oxidative stress and genotoxic chemicals. It is defined by characteristic changes in the nucleus morphology including chromatin condensation and fragmentation, overall cell shrinkage/rounding and formation of apoptotic cell bodies. Among the many markers of apoptosis, DNA fragmentation is considered a hallmark. The DNA ladder assay described here is a simple, sensitive, cost-effective and rapid method for estimating apoptosis in cells.

Isolation of Genomic DNA

Genomic DNA from primary cells culture of PMNC and splenocytes of rats is isolated by using standard phenol-chloroform DNA isolation protocol of Sambrook and Russel (2001) as described below:

1. Preparation of primary cells culture of different cells, namely-PBMNCs, thymocytes and splenocytes form Wistar rats and viability checked by trypan blue dye exclusion test.

2. 106 cells/ml of primary cell culture is used and seeded in 24 well culture plates containing along with treatment with xenobiotics at different µM concentration.

3. Cell culture plates are incubated in CO2 incubator at 37°C with 5% CO2 for 12 hrs.

4. The cells are collected in 1.5 ml of micro centrifuge tube and centrifuged at 3200 rpm for 10 min at 4°C; supernatant is discarded and pellet is washed with PBS (pH 7.2).

5. DNA extraction buffer (500 µl/tube) is added in tubes. Gently tap to disperse the cell pellet in extraction buffer and keep in water bath for 1.0 hr at 37 °C (it is to ensure that pellet is completely suspended so that cells are accessible to SDS and Proteinase K).

6. 10 % SDS is added (20 µl/ml) to cell suspension and the tubes are gently mixed by inverting the tube. The contents of tube become viscous which indicates lysis of cells. After that sample is handled gently from onward to avoid shearing of DNA.

7. Proteinase K (15 µl) of 20 mg Proteinase K/ml of buffer is added in tubes in two pulse i.e. half the requirement is added to tube in 1st pulse, mixed gently end to end and kept in water bath at 50°C. After 3-4 hrs, the second pulse of the remaining amount of Proteinase K is added and tubes are incubated at 50°C overnight.

8. Equal volume of equilibrated phenol (Tris saturated phenol pH> 7.8 ) is added in the tubes next day morning. Mix the contents of tube gently by inverting the tube for 15 min till a light coffee coloured uniform solution without any balls of phenol is formed and then centrifuge at 3400 rpm for 15 min.

The concept of single cell micro-gel electrophoresis (SCGE)/comet assay is f irst introduced by Ostling and Johanson (1984) as a method to measure DNA single-strand breaks under neutral conditions. Singh et al. (1988) modified this technique by using alkaline conditions (>13 pH) of electrophoresis, which enabled detection of not only frank strands breaks but also alkali labile sites, DNA cross- linking and incomplete excision repair sites. Now-a-days, this technique is extensively used to study the genotoxic effects of various chemicals. The advantages of SCGE technique include: (1) collection of data at the level of individual cell, allowing for more robust types of statistical analysis; (2) need for small number of cells per sample (<10,000); (3) its sensitivity for detecting DNA damage; and (4) that virtually any eukaryotic cell population is amenable to analysis. The idea is to combine DNA gel electrophoresis with fluorescence microscopy to visualize migration of DNA strands from individual agarose-embedded cells. If the negatively charged DNA contained breaks, DNA supercoils are relaxed and broken ends are able to migrate toward the anode during a brief electrophoresis. If the DNA is undamaged, the lack of free ends and large size of the fragments prevented migration. Determination of the relative amount of DNA that migrated provided a simple way to measure the number of DNA breaks in an individual cell.

Genotoxic effects of any xenobiotics in PMNC and splenocytes of Wistar rats can be studied according to the working protocol of Dhawan et al.

Genotoxicity potential of any environmental pollutants can be investigated using the micronucleus (MN) test. It is among the most sensitive DNA damage indicators and it has been applied to several organisms and tissues for evaluation of environmental contaminants (Lemos et al., 2011). Micronuclei (MN) assay is more rapid and simpler than chromosomal analysis (Gandhi et al., 2003). Compared to the chromosome aberrations, the final result is relatively easily scored and thus requires less time to make an assessment of the clastogenicity of a chemical (Garriott et al. 2002). An in vitro MN assay can detect the clastogens and aneugens as well as mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction (Corvi et al., 2008). Although, micronuclei constitute well-characterized biomarkers of chromosomal damage (Salazar et al. 2009) but in vitro MN test does not provide information on the origin of micronuclei (Corvi et al., 2008).

Experimental Procedure

Micronuclei assay is carried out in PMNC and splenocytes as per method of Hayashi et al. (1983).

Terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. The assay relies on the presence of nicks in DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. Here we perform the apoptotic assay test by TUNEL assay kit to identify the apoptotic property of any agent or chemical after treatment of primary cells culture at different concentration as per protocol provided with TUNEL Assay Kit and apoptotic cells are identified by using fluorescent microscope. Detailed procedure of TUNEL assay is as follows:

1. Preparation of primary cells culture as described in earlier methods.

2. Induction of apoptosis in primary cells culture is done by treatment of primary cells culture with different concentration of xenobiotics and incubation in CO2 incubator for 12 h as mentioned in micronuclei assay method.

3. The positive and negative control cells provided with the kit are already f ixed. However, negative control cells are prepared from control sample with absence of inducing agent.

4. Cell Fixation: Cell fixation is a required step in the TUNEL assay and it is done according to the procedure mentioned in the supplied kit by using paraformaldehyde solution. Suspend 1–2 × 106 cells (treated) in 0.5 ml of phosphate-buffered saline (PBS) and the cell suspension into 5 ml of 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes.

5. Centrifuge the cells for 5 minutes at 300 × g and discard the supernatant.

Spontaneous contractions of the uterus are controlled and coordinated for various reproductive functions. The non-pregnant uterus facilitates ova and sperm transport, provides adequate embryo placement for implantation, and contributes to the expulsion of menstrual debris through increased contractility, or periods of relative quiescence (Hutchings et al., 2009). Uterine dysmotility has been implicated in the pathogenesis of certain infertilities, implantation failure, endometriosis, dysmenorrhea, and abnormal menstrual events (Kunz and Leyendecker, 2002; Aguilar and Mitchell, 2010). Although these conditions may not contribute to mortality but can significantly affect patient’s reproductive health, and considerably alter their quality of life (Jones et al., 2004; Weissman et al., 2004; Chachamovich et al., 2010). Dysmenorrhoea refers to the occurrence of painful cramps in the lower abdominal region during menstruation and is one of the unresolved gynecological disorders in adolescent girls and women of reproductive age (Wang et al., 2004; Harel, 2008). This condition is mainly characterized by cramping pain in the lower abdomen immediately before or during menstruation, which does affect the quality of their life and work (Davis and Westhoff, 2001).

Introduction

Acute lung injury (ALI) is the manifestation of an inflammatory response of lung resulting from severe sepsis, bacterial or viral pneumonia, trauma, and burns in animals (Ali et al., 2020).It is distinguwashed by disruption to the alveolar capillary membranes, atelectasis of the pulmonary airspaces, and neutrophil and protein-rich fluid infiltration into the alveolar space (Li et al., 2019). Although advances in treatment, the disability and fatality rates of ALI remain high, that is around 30-40% (Ali et al., 2020). At the moment, new therapeutic alternatives are urgently required to treat ALI. As a result, this is a very active area of research to understand the molecular principles underlying pulmonary inflammation and ALI (Reiss et al., 2012). The use of animal models of inflammatory lung has allowed for the probing of pathways of inflammation responsible for injury. Specially, rodent models may serve a link between in vitro research to large animals and clinical studies by allowing for high quantities of samples under physiological circumstances at low expenses (Domscheit et al., 2020).LPS, found in the outer membrane of gram-negative bacteria, consists of a polar lipid head group (lipid A) and a chain of repeating disaccharides (Gao et al., 2023). LPS induced lung injury model, closely mimic sepsis and inflammatory lung diseases, is a well-establwashed model in this field of research (Reiss et al., 2012). To mimic acute lung injury, LPS can be administered either directly through intratracheal or intranasal route (direct pulmonary insult) or indirectly through intraperitoneally and intravenous routes (indirect extra pulmonary insult) (Perl et al., 2011). This chapter focuses mostly on rodent model of acute lung injury caused by lipopolysaccharide (LPS), both direct and indirect. Additionally, it also includes the characteristics and underlying molecular mechanism of LPS induced lung injury along with applications in drug development.

Mesenchymal Stem Cells

Recent advances in the field of regenerative medicine have opened up newer therapeutic modalities using cell-based and cell-free therapies, particularly stem cells, as potential candidates. As per the definition, stem cells are the undifferentiated progenitor cells that can regenerate and can also differentiate into various specialized cells (Hoang et al., 2022). They can be classified as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and postnatal adult stem cells (Locke et al., 2009). Among postnatal adult stem cells, mesenchymal stem cells (MSCs) have emerged as a popular choice of candidate for therapeutics due to the relatively easy availability, simple isolation techniques, low risk of teratogenicity, minimal ethical and legal concerns unlike embryonic stem cells, hence, are easy to apply in clinical research and practice (Voga et al., 2020). Among MSCs of different origin, bone marrow and particularly adipose tissue-derived stem cells (ADMSCs) received more attention and are one of the most common and promising stem cell types. These cells can be simply obtained from subcutaneous and omental fat or liposuction aspirates for the treatment of diverse clinical applications (Kim &Heo, 2014). These cells can also be expanded vigorously in vitro and differentiated into specific cell lineages such as adipocytes, osteoblasts, chondrocytes, and neurocytes and are even genetically stable in long-term cultures (Miana& González, 2018). Mesenchymal stem cells (MSCs) are multipotent progenitor cells possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages, according to the (ISCT) International Society for Cell and Gene Therapy (Dominici et al., 2006).

Injudicious and indiscriminate use of several agrochemicals viz. pesticides, herbicides, insecticides, dyes and medicaments in the field of agriculture and veterinary practices threatens the human and animal population for the toxicity of these environmental deterrents. These agrochemicals have been associated with several episodes of accidental poisoning and mortality among human and animal population. The episode of Mass ethion poisoning in Gujarat is one of the commonest example of inclusion of pesticides or agrochemicals in the environmental pollutants. There are modern tools to evaluate and analyze these chemicals in the environmental pollutants of human and animal use. The sensitivity of these modern highly sophisticated tools has now immensely changed the analytical environmental pollutants industry. Gas chromatography and Mass Spectrophotometry i.e. (GC-MS) technique is employed to detect the volatile residues from environmental pollutants. Whereas, High performance liquid chromatography i.e. HPLC is employed to analyse and detect non volatile drug residues. Gas Chromatography/Mass Spectrometry (GC/MS) instrument separates chemical mixtures (the GC component) and identifies the components at a molecular level (the MS component). It is one of the most accurate tools for analyzing environmental samples.

Flow Cytometry is a tool used to detect and measure chemicophysical characteristics of a population of cellular particles. Modern day biomedical research needs the molecular assembly of tools to proceed in the advent of biomedical research with real time diagnostic, prognostic and experimental detection of markers. For the same, a naïve tool is flow cytometry. In this technique, a sample having cells or particles is suspended into a fluid and injected into instrument. The ability to perform these measurements in a very rapid time span is one of the key advantages of the flow cytometry process. A single-cell suspension must first be prepared in order to analyse solid Tissues. A flow cytometer is look alike to microscope, except that, instead of producing an image of the cell, it generally offers a cell-by-cell basis high-throughput, automated quantification of specified optical parameters.

They can quantify up to three to six properties or components are quantitated in a single sample, cell by cell, for about 10,000 cells, in less than one minute. Sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. These cells are tagged with fluorescent markers in order to absorb the light and then emitted them in the form of band of wavelengths. Average ten thousand cells can be quickly examined and the data gathered are processed by a computer.

High-performance liquid chromatography or high-pressure liquid chromato graphy (HPLC) is a chromatographic method that is used to separate a mixture of compounds in analytical chemistry and biochemistry so as to identify, quantify or purify the individual components of the mixture. It involves the injection of a small volume of liquid sample into a tube packed with tiny particles (1.8- to 5-micron (μm) particle size called the stationary phase) where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump. These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”.

Aflatoxins are toxic and carcinogenic compounds produced as metabolites by many Aspergillus species. This fungus invades habitats such as soil, grain, nuts, or decaying vegetation when- ever suitable conditions such as high moisture and temperature are met. B1 is the most toxic of at least 14 different types of naturally occurring aflatoxins. Therefore, many countries and regulatory agencies impose strict limits on B1 as well as other common aflatoxins such as B2, G1, and G2 despite exhibiting lower toxicity. The U.S. FDA set the action level to20 μg/kg (ppb) for human food and to 300 μg/kg (ppb) for animal feed. Aflatoxin B1 is extremely widespread and is present in food and feed products such as cottonseed, corn, and peanuts. It is highly toxic and has been classified as a group 1 carcinogen by the WHO. The aflatoxins B2 , G1 , and G2 are usually found accompanying B1 , in lower concentrations in the contaminated samples. The order of toxicity is AFB1 > AFG1 > AFB2> AFG2.

Introduction

Minerals are present in all body tissues and fluids to carry out the physicochemical processes of life; both in animals and human. It is broadly classified into two categories: (1) macro and (ii) micro or trace elements. The macro elements include calcium, phosphorus, potassium, magnesium, sulfur, sodium and chloride, while elements such as iron, copper, cobalt, iodine, zinc, manganese, molybdenum, selenium and chromium come under micro elements. Animals need large amounts of calcium for construction and maintenance of bone and normal function of nerves and muscles. Phosphorus is a vital constituent of adenosine triphosphate (ATP) and nucleic acid and is also essential for acid-base balance, bone and tooth formation. Iron is an important component of the cytochromes for carrying out cellular respiration in addition to its vital role in red blood cells. Magnesium, copper, selenium, zinc, iron, manganese and molybdenum are important co-factors found in the structure of certain enzymes and are indispensable in numerous biochemical pathways. Animals need iodine to make thyroid hormones. Sodium, potassium and chlorine are important in the maintenance of osmotic balance between cells and the interstitial fluid. Excessive intake of some minerals can upset homeostatic balance and cause toxic side effects.

Isolated tissue preparations are widely used in physiological or pharmaco logical research to delineate the mechanism of action of drugs or any active biomolecules. Traditionally it is used as a tool for bioassay of drugs. The tissue used for such pharmacodynamics studies may be isolated from different animal models viz. frog, rodents, rabbits, sheep, goat, buffalo, chicken etc. Evaluation of drug-receptor interaction and underlying mechanism of action is then performed using isolated organ bath or myograph where the isolated tissues are mounted under optimum resting tension and changes in the tension following exposure of drug is recorded with the help of recording device. With the advancement of science and technology, the recording devices are also gradually upgraded from smoked drum to student’s physiograph to the most advanced Data Acquisition System (DAS) based polyphysiograph.

The isolated tissue preparations (uterine strips, arterial rings, tracheal chains etc.) need essential and appropriate nutrients and environment during experimental which is generally provided by using suitable salt solutions (Physiological Salt Solutions) which provide ionic balance, energy supply, maintain pH and tissue tonicity. The composition of physiological salt solutions is such that it provides an artificial media simulating with the inorganic composition of blood plasma together with a buffer mechanism to maintain an optimum pH of 7.0 to 7.4 and glucose to facilitate tissue metabolism. Commonly used PSS are Frog ringers, tyrode, Kreb’s, de Jalon, Ringer-Locke, modified Kreb’s Henseleit solution etc.

The cytological insult due to exposure of different environmental pollutant viz. pesticides, herbicides etc. impose great loss to the vitality and health of the individuals. The basic pathophysiological distrubances following the expo sure of these deterrents to the different visceral organs leads to dysfunction of these organs. Imbalance between body’s antioxidant defense and peroxidation of the lipid layer of cells’ causing free radical generation is defined as Oxidative stress. Free radicals are the molecules or entity that contains one or more than one unpaired electron. Pathophysiology of several diseases are associated with the generation of oxidative insult and many xenobiotics associated toxicity are also a result of oxidative damage and generation of reactive oxygen species and reactive nitrogen species. Alzheimer’s Disease is one of the fine example of oxidative damage associated neuronal degeneration.

Oxidative stress can be estimated using following parameters:

1. Lipid peroxidation (Paula et al., 2005)

2. Reduced glutathione (Sedlak and Lindsay, 1968)

3. Superoxide dismutase (Madesh and Balasubramanian, 1998)

4. Catalase (Aebi, 1983)

Tissue Collection

Rats are most commonly employed as test subjects to conduct the experiments related to drug metabolizing enzyme assays involving various enzymes i.e. microsomal and non microsomal enzymes. Rats are humanely sacrificed at the end of the exposure period of 28 days of any therapeutic agents or drug and the liver of all the animals has to excise and immediately wash with ice-cold normal saline, weigh and perfuse with ice-cold 1.15% KCl solution containing 0.05 mM EDTA. Livers are processed further for preparation of microsomes for determination of microsomal drug metabolizing enzymes.

Preparation of Microsomes

Livers tissues are homogenized in 3 volumes (1:3) of ice-cold (1-4o C) homogenization buffer containing 1.15% potassium chloride in 50 mM potassium phosphate buffer solution (pH 7.4). The homogenates are centrifuged at 15000 g for 30 min (Hitachi cs120GX II). The mitochondrial supernatant (8 ml) is transferred to ultracentrifuge tubes and spun at 105000 g for 1 hour in ultracentrifuge to sediment the microsomal pellet. Cytosolic fractions are collected from the middle separating the lipid layer on top in separate micro centrifuge tubes. The microsomal pellet is then washed by adding 8 ml of resuspension buffer (homogenization buffer containing 0.05 mM EDTA) and spun again 105000 g for 15 min. The microsomal pellet so obtained is resuspended in 4 ml of resuspension buffer and 1 ml aliquots are stored immediately at -80o C until assayed for drug metabolizing enzymes.

Acetylcholinesterase activity in brain is measured by the method described by Ellman’s et al. (1961).

Reagents

1. Phosphate buffer (0.1M) (pH 8.0) Solution A: 5.22 g of K2 HPO4 and 4.68 g of NaH2 PO4 are dissolved in 150 ml of distilled water. Solution B: 6.2 g of NaOH is dissolved in 150 ml of distilled water. Solution B is added to solution A to get desire pH 8.0 and then finally the volume is made up to 300 ml with distilled water.

2. DTNB reagent 39.6 mg of DTNB with 15 mg of NaHCO3 is dissolved in 10 ml of 0.1 M phosphate buffer.

3. Acetylthiocholine (ATC) 21.67 mg of Acetylthiocholine is dissolved in 1 ml of distilled water.

Procedure

The brain tissue is weighed and 10% homogenate is made in 0.1M Phosphate buffer (pH 8.0). Take 0.4 ml aliquot of homogenate in a cuvette containing 2.6 ml phosphate buffer (0.1M) and 100 µl DTNB. The contents of the cuvette are mixed thoroughly by bubbling air and absorbance is measured at 412 nm in a spectrophotometer. When absorbance reached a stable value, it is recorded as the basal reading .To this 20 µl of acetylthiocholine is added and change in absorbance is recorded for a period of 10 min at intervals of 2 min. Change in the absorbance per minute determined.

Pharmaceutical and chemical industries depend largely on toxicological evaluations to provide information regarding safety of their commercial products. Toxicologists so far have been relying on animal studies to investigate potentially beneficial or harmful effects of drugs and chemicals prior to testing them in humans. Traditionally, the safety assessment process during drug development has been based on screening indiscriminately a plethora of new chemical entities (NCEs) using a large number of animals as testing models in order to identify new therapeutic agents with the expectation that one or a few of these NCEs have some competitive advantages over the existing therapeutic agents. That is why they are more efficacious, bioavailable, selective, and safer. This approach is costly, time-consuming, and requires large quantities of test material. Currently, it takes about 10-12 years and a huge expenditure for a drug to move from research laboratory to patent. Thus alternative cell culture models in the early phases of the drug discovery process can save time and money both by eliminating least beneficial NCEs.

Preparation of Reagents

Culture media

For the culture of hepatocytes medium M199 (GibcoBRL, USA) is used. One pouch of the dry media is dissolved in 1 liter of filtered distil water. The pH of the media is adjusted by adding 0.4% sodium bicarbonate to be around 7.4. The media is than sterilized by filtration through seitz filter (0.22 mm) and stored in 50 ml sterilized conical flasks.

Isolation of Peripheral Blood Lymphocytes

Chemicals

1. Heparin

2. Sterile PBS (pH 7.2)

3. Histopaque-1077

4. RPMI-1640 Media

Protocol

Collect 5 ml of blood from the animal in 1 ml (20 IU/ml of blood) of diluted heparin and add 2 ml of PBS. Layer over the diluted blood on 5 ml of histopaque-1077 in 15 ml centrifuge tube and centrifuge at 1500 rpm for 40 min. Collect the buffy coat at the interface of plasma and histopaque in separate centrifuge tubes. Wash the cells thrice with PBS and suspended in 2 ml of RPMI-1640 media supplemented with 10% fetal calf serum. Determine the percentage cell viability by 0.1% trypan blue exclusion test, using hemocytometer. Finally dilute the cell suspension to 106 cells/ml in RPMI 1640 medium.

LD 50- It is defined as the concentration/dose that kills 50 % of the experimental population.

Acute toxic studies have been conducted in order to calculate LD50. There are several methods to calculate LD50, some are as follows:

Methods to Determine LD 50

• Karber’s method.

• Miller & Tainter method

• Lorke method

• Fixed dose method

• Acute Toxic Class method

• Up & down procedure

Karber’s Method

This method involves the administration of different doses of test substance to various groups, which has five animals each. The first group of animals is administered with the vehicle in which the test substance was dissolved or diluted

Introduction

The immense use of chemicals and pharmacological entities has put the public health at brisk of potential health hazards. These substances in modern days are presented in the form of common constituents of food, medicines, beverages and junk foods etc. However, long term consumption of these substances might be responsible for chronic toxicity, but the accidental ingestion of large quantities of these substances may result into adverse acute toxic reactions. Hence an experimenter should have the acumen to develop and perform the acute toxic tests in order to find out the lethal dose 50 i.e. LD50 of the substances.

Acute Toxicity Studies

Acute toxicity studies helps to establish the dose dependent adverse effect including mortalities. The LD50 has been used as a major parameter in screening of pharmacological agents for acute toxic potential. Apart from mortality, other biological effects and the time of onset, duration and degree of recovery on survived animals, are also important in evaluation of acute toxicity. Hence, acute toxicity study solely gives information about LD50, therapeutic index and the degree of safety of a pharmacological agent.

Acute toxicity is defined as adverse effects occur following oral or dermal administration of a single dose of a substance, or multiple doses given within 24 hours, or an inhalation exposure of 4 hours. Oral administration is the most common form of acute systemic toxicity testing.

Epididymal Total Sperm Count

The epididymal sperm is obtained as described above, incubated at 35°C, which is the optimum temperature of rat epididymal sperm. The epididymal f luid is then diluted to a volume of 5 mL of pre-warmed (32°C) the spermatozoa are counted by hemocytometer using the Improved Neubaur (Deep 1/10mm. LABART, Germany) chamber as described by Pant and Srivastava (2003).

Total viable cell/ml = (Average number of sperm per chamber) x 103 x (Dilution Factor)

Liveability and abnormal sperm

Per cent live spermatozoa is estimated by differential staining technique using Eosin-Nigrosin stain (NE) (Campbell et al., 1953). These slides are also used for estimating the per cent abnormal sperm morphologically on the basis of observable abnormalities of head, neck, mid-piece and trail region of the spermatozoa.

The percent live spermatozoa are determined adopting the differential staining technique using Eosin-Nigrosin stain.

• A drop of epididymal semen is taken on a clean, grease free pre warmed glass slide.

• 4-5 drops of Eosin-Nigrosine stain (1% Eosin and 5% Nigrosine in 3% sodium citrate dehydrate solution) is placed near the semen drop.

• Epididymal semen and stain are mixed gently using a blunt fine glass rod.

• After 30 seconds to 1 minute a thin smear is made on a clean, grease free glass slide.

• The smear is examined under oil immersion objective.

India has a unique social and cultural setting with diverse people, culture and religions. It is one of the oldest and greatest civilizations of the world. Geographically India extends snow clad Himalayan mountains in the north to the tropical rain forests of the south. It is the 7th largest country in the world and well recognised in Asia by its mountains, seas, deserts, and vast stretch of fertile plain land, which give the country a distinct geographical identity.

India has a very rich, diverse religious and cultural heritage. The four main religions of India are - Hinduism, Islam, Christianity and Sikhism. Hinduism is the largest religion in India, with approximately 80% of the population is Hindu while Islam (Muslim) is the second-largest religion having 14% (approx.) share.

Religion in India is characterised by the diversity of religious beliefs and practices. In Indian history, religion has been an important part of the country's culture. Indian subcontinent is the birthplace of four of the world's major religions, namely, Buddhism, Hinduism, Jainism and Sikhism, which are collectively known as native Indian religions and represent 83% of the total population of India approximately.

Hinduism

Hinduism is the main Indian religion. It is considered as a religious and universal order, a way of life which followed by the people who believed in Hinduism. The word ‘Hindu’ refers group of particular people who believes and follow Hinduism, the oldest religion in the world. It has also been described as ‘Sanatana dharma’.

The Ramayana written by sage Valmiki is one of the great epics of the Sanskrit language. It is most religiously regarded and popular book in Hinduism. The book is believed to be complied between 300 B.C.E. – 200 AD. However, the original story could be as old 500 B.C.E. (Goldman, 1984). There are many other versions of the epic in various regional languages found in India and South Asia (Raghunathan, 1981; Arya,1998; Dutt, 2004; Mukherjee, 2015; Sharma and Arya, 2022).

As per literature references, the name ‘Ramanaya’ is composed of two words, ‘Rama’ and ‘Aya?a’. Rama, the name of the main character of the Epic, has two contextual meanings. In the Atharvaveda, it means 'dark’, dark-coloured, ‘black' and is related to the word ‘ratri’ (night) which means 'darkness of night'. The other meaning, which can be found in the Mahabharata, is 'pleasing’, pleasant, charming, lovely, beautiful. The word ‘Ayana’ means travel or journey. Thus, Ramaya?a means ‘Rama's Journey’ from his kingdom to various places, forests during his life time (Anonymous, 1960; Tulsidas, 1966; Murthy, 2003; Kishore, 2007; Rajagopalachari, 2013).

Ramayana contains over 24,000 couplet verses, in its full form, which are divided into following seven ‘Ka??as’ (booklets) - Balaka??a, Ayodhyaka??a, Ara?yaka??a, Ki?kindhaka??a, Sundaraka??a, Yuddhaka??a, Uttaraka??a and about 500 sargas (chapters). It is regarded as one of the longest epic poems written in history (Keshavadas, 1988; Debroy, 2017; Gupta, 2021).

morally right man in the world? Narada replied that such a man is Rama, son of King Dasharatha of Ayodhya. Valmiki wanted to know more about Rama and proceeded to take bath when he saw two birds in playful mood and was happy. Suddenly, a hunter hiding in the bush shot one of the two birds. Valmiki became painful and chanted some dialogues in curse which ultimately created a new form of sloka. This was the beginning of the main narrative (Goldman, 1984; Sachithanantham, 2004).

The exile of Rama and his journey to the forests (vanvasa) for 14 years was started from Ayodhya (Uttar Pradesh, India) and ended to Lanka (Sri Lanka), through different forests, territorial regions, lands, hills, lakes of old India. The whole journey to those places was candidly portrayed in Ramayana with vivid descriptions, from narrations of the night sky, the position of different stars to descriptions of the flora, fauna and landscape. In this chapter, a retrospection of the places, forests, Rama passed through during his entire journey has been documented. The present status of those forests and places has been considered to evaluate a comparison over the thousands of years and the changes taken place besides significance they hold till date (Anonymous, 2024; Iyer, 2014).

Journey to the Forest and Route Map

Ayodhya, the capital city, kingdom of Kosala, was situated on the banks of holy river Saryu and was the birthplace of Rama. To fulfill the wish of his father, king Dasharatha, Rama left Ayodhya for 14 years of exile to the forest, along with Sita and Lakshmana. Their journey started from Ayodhya and passed through different places / forests.

In this chapter, a comprehensive list of all plants and trees mentioned in epic Ramayana has been furnished with salient details in order to establish their botanical identity and references of Sholka where it had been mentioned. Some of the plants and trees had been mentioned in multiple sholokas but the plant has been taken into account as a single reference to avoid repetition and to get correct count of the trees and plants in whole of the Epic (Amirthalingam, 2013, Arun Kumar, 2019; Balapure et al., 1987; Bast, 2022; Ghimire et.al, 2021; Mao and Dash, 2010; Prasad and Mishra, 1976).

The plant species mentioned in Ramayana were of different types having various usages. Considering their morphological characteristics, usages and other importance, they have been categorized broadly into the following heads.

1. Economically important plants (Timber value, product yielding etc. having commercial importance).

2. Edible (major and minor fruits etc.).

3. Culturally important plants (Plants used for plantation for shade, greening etc.).

4. Non-Edible Plants (Ornamental importance and other usages)

5. Medicinal Plants (having therapeutic uses).

6. Sacred Plants (having religious importance).

7. Others (having multipurpose uses).