eChapters

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

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

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

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.

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.

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.

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.

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.

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

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

The rhizosphere plays a pivotal role in the boundary where biological and chemical activities profoundly impact various landscape and global-scale processes. Understanding these processes is crucial for maintaining planetary health and sustaining the organisms inhabiting it (Morrissey et al., 2004). To enhance the yield potential of staple food crops, leveraging the root systems of plants becomes essential to meet the anticipated doubling of global food demand in the future (Gewvin, 2010; Zhang et al., 2010). Amidst the challenges posed by global climate change and the burgeoning global population, there’s an increasing need for greater productivity from cultivated food, animal feed, and fibers on less favorable and often infertile lands—conditions already prevalent in many developing nations (Tilman et al., 2002). Advancing our understanding and control of rhizosphere processes stands as a critical frontier in research for the next decade.

Introduction

Increasing fresh water demands for ever growing population and it has increased by 600% over the past 100 year (Wada, et. Al, 2016) the annual increment rate 1.8 % whereas the present growth rate is less, only 1 % but this figure may be optimistic. The global water demands will grow significantly over the next two decades in all three components as: Industry, domestic and agriculture (U.N, 2018) industry and domestic demand will grow faster than agriculture, but agriculture demands will remain largest (U.N, 2018). The global water demands for all purpose will increase 20% to 30% by 2050, up to 5,500 to 6,000 km3 per year (Burek et.al, 2016), whereas, agricultural demands will increase by 60% by 2025 (Alexandrators and Bruinsma, 2012).

Introduction

Agroforestry being old as an art and new in its scientific terms has been gaining importance in the modern era due to shift of world from technology to nature in search of resilient way of sustaining livelihood (Dutta et al., 2023, Sharma et al., 2023 Bishaw et al., 2022, Agnoletti et al., 2022). The simplicity of the agroforestry system is the flagship reason of its widespread applicability and reliability in agricultural domains. There has been change in the world perspective regarding the nature and natural resources in recent times. Although there has been forest over 31 per cent of the world’s total geographical area (FAO and UNEP, 2022) but the stabilization of ecosystem and sustainable development is difficult without subsequent interaction between the tree, crop and livestock component on farm lands, being collectively called as agroforestry.

Introduction

In recent decades, integrating trees with crops for food and wood production has received considerable attention in both tropical (Garrity et al., 2010) and temperate regions (Palma et al., 2007). The growing of trees in combination with the agricultural crop is a land management practice that farmers have been practicing since the ancient period. Seasonal agricultural/horticulture crops, woody perennial trees (multipurpose), and/or animals are major components of various agroforestry systems. According to Sanchez (1995), the science of agroforestry revolves around four aspects: competitiveness, complexity, sustainability, and profitability, and all of these factors must be balanced in order to produce beneficial outcomes. In fact, agroforestry is significantly assisting in meeting the United Nations Millennium Development Goals (MDGs) such as poverty and hunger eradication, improved health, nutrition, and education for people, gender equality, and environmental sustainability, particularly in developing countries (Garrity, 2004, 2006).

Introduction

`The living and nonliving components of ecosystems, as well as the conditions and processes that occur within them, create services thus connecting ecosystems and human cultures. The preservation of Earth’s biological diversity and the protection of the benefits, or “ecosystem services,” those functioning ecosystems bring to humanity are two primary goals of environmental conservation. Agroforestry is increasingly viewed as providing ecological services, environmental advantages, and economic gains as part of a multifunctional working landscape. The multifunctional importance of agroecosystems was emphasized in both the Millennium Ecosystem Assessment (MES 2005) and the International Assessment of Agricultural Science and Technology for Development (2008).

Introduction

More than a third is covered by drylands in the terrestrial land surface, which have a harsh and vulnerable natural condition (Maier et al., 2014). Due to insufficient water availability, dry areas have a scant population of vascular plants, as well as poor availability of the nutrient turnover with poor availability of the resources as a result of severe temperatures (Adeel, 2008). Biological soil crusts or Biocrusts commonly protects the soil surface in dry environments and it is considered “ecosystem engineers” of arid and semiarid areas, as well as signs of ecosystem health (Briggs and Morgan, 2011; Bowker et al., 2008). They are connected in dryland ecosystem restoration level or degradation (Assouline et al., 2015). When compared to chemical or physical crusts, biocrust is the largest word that explicitly implies that these are the crusts which are dependent on the living organisms’ activity.

Introduction

According to the Forest Survey of India (FSI) “all lands, more than one hectare in area, with a tree canopy density of more than 10% is defined as Forest” (FSI 2009). The status of forests and forest management systems contribute to the vulnerability of forests to climate change. Indian forests are extremely diverse and heterogeneous. Forests and Climate Change are intimately intertwined. Forests have regulated the climate, rain, groundwater, soil of the earth over millennia. Their transpiration act as a regulator of the balance of oxygen and carbon-di-oxide: the world’s forests and forest soils currently store more than one trillion tons of carbon – twice the amount found floating free in the atmosphere. The forest and soils are home of biological diversity.

Introduction

Future agricultural production potential is in danger due to the existing cropping system, characterized by irresponsible chemical utilization, extensive tillage, monoculture, and soil health degradation. Consequently, a significant challenge for modern agriculture lies in ensuring adequate nourishment for the entire world’s population while also safeguarding the environment. The intensification of agricultural practices negatively impacts vital natural resources like soil, water, air, and biodiversity throughout various developmental stages. While the conventional cropping system have enhanced crop productivity, its consequences including land degradation, resource depletion, reduced organic matter insoil, draught, soil salinity, erosion, and climate change pose significant global threats (Tomar et al. 2014; Yadav et al. 2014; Mandal et al. 2020; Rani et al. 2021).

Introduction

Soil is a vital, complex, and firm layer of the land surface that provides a source of minerals and organic matter interacting with air and water to harbor life forms. (Lobmann et al., 2022). Versatile micro and macro living organisms, i.e., bacteria, fungi, and actinomycetes, inhabit soil and contribute to its quality, health, and inevitable role in serving the environment (Elsakhawy et al., 2022). Soil roots for a variety of crucial functions which involve ecosystem services or socioeconomic activities, e.g., biomass production, climate gas fluxes of CO2 , N2 O, and CH4 , water cleaning, storage and supply, foundation of construction and provision of construction materials, or provision of habitats etc. The responsibility of soil in sustaining life on earth by providing and conserving resources is not negotiable.

Introduction

By enhancing microbial populations, enzyme activity, soil respiration, and microbial biomass, nano-black carbon is a novel method for the enhancement of soil biological characteristics. By reducing soil acidity and boosting soil fertility, nano-black carbon is believed to improve plant development. The application of nano-black carbon to agricultural soils can have a significant positive impact on plant productivity. In addition to potentially boosting soil resilience to climate change, nano-black carbon greatly boosted enzyme activity, microbial activity, and soil organic carbon retention.Due to a liming action, nano-black carbon has been proposed as a potential biotechnology to boost agricultural yields in acidic soils.