eChapters

Introduction

Until the 20thcentury, bacterial infections were the number one cause of human death. In the 1940s, antibiotics became the lifelines for treating serious infections that saved millions of lives. Despite the advantages derived from the use of antibiotics, resistance to these drugs emerged. Unfortunately, resistance to most antibiotics emerged in time-course even for the most powerful antibiotics developed.Resistance to antibiotics is a challenge to the scientific community in keeping up the pace of antibiotic discovery.World Economic Forum’s Global Risks Reporthas listed antibiotic resistance as one of the greatest threats (WEF, 2020).United Nations (UN) has warned that drug-resistant diseases could account for 10 million deaths each year by 2050. By 2030, antimicrobial resistance (AMR) could likelylead up to 24 million people into extreme poverty(IACG,2019). Currently, at least 7,00,000 people die each year due to drug-resistant diseases (IACG,2019). The emergence and widespread of antibiotic-resistant organisms is a global challenge and needs a multi-dimensional approach to address the fundamental issues to minimize drug-resistance.The Antibiotic Resistance Genes Database (ARDB) contains a repository of around 23,000 antibiotic resistance gene or protein sequences conferring resistance against over 240 antibiotics (ARDB). Computational Biology (Bioinformatics) has become one of the important tools in studying antimicrobial resistance. It employs a range of computation techniques to make predictions about the biological phenomenon.

Introduction

Resistance to antibiotics in the bacterial world is a genetically determined phenomenon that was first observed within a few years after the introduction of antibiotics for therapeutic purposes. In Nature, ARGs are believed to have originated about three billion years ago as an intrinsic defense mechanism in antibiotic-producing bacteria (Perry et al., 2016; Iskandar et al., 2022), and subsequently, evolved by complex and quantum jump trajectories to mobilize, diversify and interconnect all ecosystems in the micro-biosphere. Bacterial genomes exhibit plasticity and continue to evolve under the selection pressure of antibiotics and other stressors (Patel, 2016). The ARGs in circulation at present are thus a mixture of ancient and modern counterparts. Recently, the interplay between bacterial metabolism and antibiotic resistance has also been revealed (Zampieri, 2021).

The use of antimicrobials in health management, livestock, and food industry is driving the emergence of resistant microbes at a rapid pace (Wall et al., 2016; Adegoke et al., 2016; Hu et al., 2017). As a result, AR pathogens have spread globally to cause loss of lives, production, productivity, and negatively impact economic growth. Recently, AR pathogens in a large study have been found directly responsible for millions of deaths, and the figures are likely to increase in the coming decades (Murray et al., 2022). The pace of the emergence of AR can however be slowed down by improving human interventions. The global and local AR combat plans implemented during the past few years have enhanced the mass awareness of the pandemic problem (WHO, 2015; FAO, 2016; NAP, GOI, 2017; WHO, 2022; WOAH, 2022). These Action Plans on AR containment encourage avoiding of indiscriminate use of antibiotics for the treatment of sick humans and animals, recommend the ban on the use of antibiotics as growth promoters in food animals, promote AR surveillance and monitoring, and support the ‘One-Health’ strategy and application of the biosecurity principles for containment of the resistant microbes.

Introduction

Antibiotic-resistant bacteria are a major cause of health care-associated infections around the world, and resistance has also emerged in infections in the wider community. Infections caused by multi resistant organisms significantly increase morbidity, mortality, and health care costs. Molecular analyses have revealed that widespread multi resistance has commonly been achieved by the acquisition of preexisting determinants followed by amplification in response to selection. The capture, accumulation, and dissemination of resistance genes are largely due to the actions of mobile genetic elements (MGE), a term used to refer to elements that promote intracellular DNA mobility (e.g., from the chromosome to a plasmid or between plasmids) as well as those that enable intercellular DNA mobility.

Insertion sequences (IS) and transposons (Tn) are discrete DNA segments that areable to move themselves (and associated resistance genes) almost randomly to newlocations in the same or different DNA molecules within a single cell. Other elements, such as integrons (In), use site-specific recombination to move resistance genes between defined sites. As these types of MGE are often present in multiple copies indifferent locations in a genome, they can also facilitate homologous recombination(exchange of sequences between identical or related segments). Intercellular mechanisms of genetic exchange include conjugation/mobilization (mediated by plasmidsand integrative conjugative elements [ICE]), transduction (mediated by bacteriophages), and transformation (uptake of extracellular DNA). Interactions between thevarious types of MGE underpin the rapid evolution of diverse multi resistant pathogens in the face of antimicrobial chemotherapy. Primarily the most important and/ or topical elements in Gram-negative and Gram-positive bacterial species of particular concern clinically, namely, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (the so-called ESKAPE group) (Rice LB., 2008) as well as Escherichia coli (giving the ESKAPEE group) (Llaca-Díaz J.M. et al.,2012); nonetheless, many of the MGE types described are of course broadly relevant to many other bacterial taxa.

Antibiotics, often referred to as “magic bullets” in the battle against bacteria, stand out as one of the most remarkable medical breakthroughs of the 20th century. Their advent revolutionized the therapeutic landscape, consistently saving millions of lives from bacterial infections (WHO, 2020). Undoubtedly, antibiotics are a blessing to humanity, not merely for their medicinal applications but also for their diverse uses. They have long been employed in various contexts, including animal husbandry and production, serving as preventive measures in numerous underdeveloped and developing nations for decades (Majumder et al., 2020). A range of antimicrobial agents, including antibiotics, antifungals, antivirals, disinfectants, and food preservatives, function to either inhibit microbial growth or eradicate them altogether. Among these, antibiotics, specifically tailored to combat bacterial infections, are the most widely used class of antimicrobials. With their increasing and often improper use, microorganisms have developed antimicrobial resistance (AMR) (Murray et al., 2022).

Antimicrobial resistance is an inevitable evolutionary process observed in all microorganisms, and is driven by genetic mutations aimed at resisting the lethal pressures of selection. Bacteria, adapt to environmental selection pressures by developing resistance to antibacterial drugs, thereby diminishing the efficacy of these treatments (Murray et al., 2022). The widespread use of antibiotics, particularly in developing nations, further flares up the process, leading to significant repercussions such as increased rates of morbidity and mortality (WHO, 2020). The incidence and prevalence of antimicrobial resistant bacterial infections have reached alarming levels in the 21st century, posing a substantial threat to global public health akin to a silent pandemic. Antibiotic resistance knows no borders and can impact individuals of any age or gender, regardless of their location. In its present state, antimicrobial resistance (AMR) stands as one of the foremost threats, not only to global health but also to food security (Murray et al., 2022).

The response of any living cell to a xenobiotic (including antibacterial agent) is significantly affected by the prevailing local environmental conditions. In case of bacterial infection, it may include the presence of other microbes, no matter commensal or pathogenic, or biochemical alterations at that particular site of bacterial infection in host. All these host and bacterial factors play a critical role in the evolutionary responses of bacteria but usually go unconsidered while determining the microbial sensitivity to a particular antibiotic in a clinical setting. These also include the direct and indirect interspecies interactions that can affect the responses of individual bacterial species and surviving microbial communities to a particular antibiotic therapy. These interactions may significantly alter the qualitative and quantitative responses, potential phenotypic changes and the final trajectory of evolution of antimicrobial resistance. Due acknowledgement and plausible interpretation to these interactions may help to reduce the upsurge in AMR and its associated morbidity and fatality and pave path to more proficient clinical success.

The factors participating in drug- microbe interaction can be classified into two broad groups: host factors and microbial factors. The environmental milieu of the interaction is chiefly regulated by the gut microbiota with stressors causing deflections. The gastrointestinal bacteria amend host mental and physiological cadence via microbial metabolites such as butyrate, polyphenolic antioxidants, vitamins, and amines, affecting the overall ready perception and active rebuke to a stimulus. Lifestyle stressors such as fluctuating sleep and erratic eating patterns (western diet) disturb the host circadian system along with the gut microbiome. The overall impact is impaired detoxification (decreased conjugation of bile acids or increased production of hydrogen sulphide), substrate oxidation and energy regulation (reduced butyrate production) in the host affecting the overall health and productivity.

Globally, antimicrobial resistance (AMR) is an important public health and development threat (WHO, 2020). Being one of the top 10 global public health threats, the ever-increasing rates in the occurrence of drug-resistant bacterial infections have become a grave public health concern for the governments (Laxminarayan et al., 2013). Moreover, such infections would be difficult to treat and therefore can lead to worse clinical outcomes (Laxminarayan et al., 2013). Worldwide, each year about 700,000 people die from antimicrobial resistant infections and the mortality figures are projected to reach 10 million/ year by 2050 (O’Neill, 2016). The crude infectious disease mortality rate in India is more than 400 per 100,000 persons and at least 23,000 deaths in adults and 58,000 neonatal deaths per year are caused by AMR bacteria (Laxminarayan and Chaudhury, 2016). A 2017 estimate puts the cost of developing an antibiotic at around US$1.5 billion (Towse, et al. 2017). Meanwhile, industry analysts estimate that the average revenue generated from an antibiotic’s sale is roughly $46 million per year. “That’s tiny and nowhere near the.

Introduction

Antimicrobials, viz. antibiotics, antivirals, antifungals and anthelmintics are substances widely used to prevent and treat infections in humans, fish & prawn, livestock animals, and crops. In a situation where microorganisms such as bacteria, viruses, parasites or fungi become resistant to antimicrobial treatments to which they were previously susceptible, it is apprehended that antimicrobial resistance in the microbes has evolved. In fact, antibiotic resistant bacteria evade the effects of antibiotics and multiply in their own way leading to severe health consequences in human and animals. The menace is such that antimicrobial resistance (AMR) hinders treatment modalities targeted for infectious diseases. The hazards of antimicrobial resistance is frequently observed in various pathogenic and non-pathogenic organisms, viz. Salmonella spp., Campylobacter spp., Yersinia sp., E. coli serotype O157:H7, enterococci, Pasteurella sp., Actinobacillus sp. with varying severity (McEwen and Fedorka-Cray, 2002). Microorganisms that develop resistance to commonly used antimicrobials are referred to as superbugs. Globally it is a matter of great concern that it spreads rapidly and threatening the ability to treat common infectious diseases, resulting in prolonged illness, disability and death. Without any effective antimicrobial agent, treatment of infectious diseases, many medical procedures like cancer therapy, diabetes management, organ transplantation, and major surgery will not be possible. The microbes showing resistance to multiple antimicrobials are commonly known as multidrug resistance (MDR) organisms (Magiorakos et al., 2012).

Introduction

Today, antimicrobial resistance (AMR) has become a global issue, contributing to rise in mortality and cost of treatment. Antimicrobial resistance (AMR) is the resistance of a microorganism against an antimicrobial agent to which it was originally sensitive (WHO, 2013). Dispersion of antibiotics is done at large scale, in India even sometimes without any proper prescription. Animal Husbandry is not untouched and antibiotics are used to a great extent, which needs enforcement of strict government rules and regulations for their use in f ield conditions (PHFI, 2011).

Extended-spectrum β-lactamases (ESBLs) are enzymes that hydrolyse most of the β-lactam antibiotics and thus mediate resistance to penicillins, 3rd and 4th generation cephalosporins (Saravanan et al., 2018). Beta-lactamase inhibitors, inhibit these ESBLs such as clavulanic acid, sulbactam and tazobactam (Trott, 2013). The genes encoding for these enzymes are commonly found both on the chromosomes and plasmids among Enterobacteriaceae family (Kotsoana et al., 2019).

Among Enterobacteriacae, E. coli and Klebsiella species are main environmental pathogens associated with various illnesses such as acute bovine mastitis (Koovapra, 2015), pathogenic E. coli with diarrhoea and extra intestinal infections in humans and animals (Levine et al., 1987), K. pneumoniae with respiratory and urinary tract as well as bloodstream infections (Daehre et al., 2018). Resistance to extended-spectrum β-lactams occurs mainly in members of Enterobacteriaceae family through plasmids encoded ESBL genes. (Sharma et al., 2010).

Introduction

The antibiotics are low molecular weight natural, semi-synthetic, or synthetic substance that kills or slows the growth of germs while causing little or no harm to the host is considered an antimicrobial (Walsh, 2000). Antibiotics are the type of medicine produced by some microorganisms that kill or slow the growth of other microorganisms. It includes any of a large group of chemical substances, as penicillin or streptomycin, produced by various microorganisms and fungi, having the capacity in dilute solutions to inhibit the growth of or to destroy bacteria and other microorganisms, used chiefly in the treatment of infectious diseases (De Vetten et al., 2003). However today, a large number of bacteria are resistant to various antibiotics. Antibiotic resistance occurs when a bacterium becomes able to survive and multiply despite the presence of an antibiotic that would normally control or kill it. In other words, the bacteria become “resistant” and continue to grow even at therapeutic levels of the antibiotic. While antibiotic resistance is a natural phenomenon, it is accelerated by the selective pressure exerted when an antibiotic is used. Bacteria that are naturally resistant to the antibiotic have a better chance of survival compared to those that are susceptible. As the susceptible bacteria are killed or inhibited, resistant strains are favored and continue to thrive (Holme et al., 2006). Some resistance arises without human influence, as bacteria can naturally produce antibiotics against other microorganisms, resulting in a low-level, natural selection for antibiotic resistance. However, the current surge in antibiotic resistant bacteria is largely attributed to the overuse and misuse of antibiotics. One of the main issues facing public health is antimicrobial resistance, particularly in developing nations where access to and use of medications are easier than in developed nations, leading to a disproportionately higher rate of inappropriate antibiotic use and higher resistance levels (Ganguly et al., 2011). Antimicrobial resistance will make it more difficult to control infections in the community and make health care services less effective (Bhatia et al., 2010). It is well known that India has one of the highest rates of infectious disease burdens in the world, and that the country’s high rates of malnutrition and inadequate sanitation make these circumstances worse (http://www. resistancestrategies.org/wpcontent/uploads/2011/03/Indiareport).

Antimicrobial Resistance abbreviated as AMR is defined as the process by which microorganisms like bacteria, fungi, viruses, and parasites change over a period and become resistant to a drug to which they were earlier susceptible to. This makes the medicines ineffective for treatment overtime, thus making infections difficult to treat thereby increasing the risk of spread of diseases caused by them and severe illness which may lead to death. This in turn necessitates the need for development of newer antibiotics. However, there has been a sharp decline in the development and manufacture of new antibiotics in the recent years resulting in development of only few antibiotics in the past two decades. The increase in emergence of antibiotic resistance concurrent with decline in development of newer antibiotics poses a serious threat to health of both humans and animals. Every year about 700,000 deaths have been reported to occur due to resistant infections and this rate is likely to increase further with estimated infections due to resistant pathogens expected to reach over 50 million globally by 2050 (O’Neill, 2016). The worrisome trends of increasing antimicrobial resistance across the globe poses a serious threat to both animal and human health as AMR not only increases the morbidity and mortality rates but also increases the economic burden on health care (Zhu et al., 2022). This presents an extremely grave situation as the supply of new antibiotics is inadequate to cope with the increase of AMR pathogens. The unnecessary use of antibiotics globally especially in the livestock sector as growth promoter further helps in selective enrichment of AMR pathogens which further increases health risks. It is well known that resistant pathogenic and commensal bacteria can persist and spread within and between premises despite zero antimicrobial drug use. This may be explained due to maintenance, replication, and further dissemination of resistance genes by horizontal transfer of genes via natural ecosystem and can occur either by conjugation, transformation and transduction; and these mechanisms can rapidly spread AMR genes among microbial community in natural environment and human microbiome, where these resistant bacterial species may be selected due to presence of antimicrobial drugs (Zhu et al., 2022). The physical transfer of bacteria via movement of animals, workers, and equipment and ineffective cleaning and disinfection may also be responsible for increasing the AMR pathogens in the natural environments.

AMR has been prevalent in almost all European countries from past decades. A 2021-2022 report published by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) on AMR in zoonotic and indicator bacteria from humans, animals and food origin reported vast differences in antimicrobials resistance levels among the European countries, with comparatively higher AMR percentages in the southern and eastern parts of Europe (ECDC, 2022, 2023). The resistance levels ranged from very high for tetracyclines, sulfonamides and ampicillin, moderate for ciprofloxacin to low levels for third-generation cephalosporins (cefotaxime and ceftazidime) in human cases infected with Salmonella spp. in 2022 (ECDC, 2023). The trends are dynamic and over a decade of 2013 2022, the human isolates from 12 and 15 countries reported a statistically significant reduction in tetracycline and ampicillin resistance, particularly for S. typhimurium (ECDC, 2023). The ciprofloxacin and cefotaxime combination, a highest priority critically important antimicrobials (CIA), had very low and rare resistance levels for Salmonella isolates of humans and animal and their derived meat.

Livestock species wise variation was also observed in the resistance pattern in imported meat, being higher in poultry meat isolates compared to pig or cattle meat for Escherichia coli (ECDC, 2023). High to extremely high levels of f luoroquinolone resistance from Campylobacter jejuni and C. coli of both human and animal origin isolates exist, due to which the fluoroquinolones can no more be used as a treatment option for human Campylobacter infections (ECDC 2023). The combined occurrence of erythromycin resistance and ciprofloxacin in Campylobacter spp is considered of high public health importance, and the report mentioned that the combined resistance for these antimicrobials was higher in C. coli compared to C. jejuni from food-producing animals and humans. However, while the ciprofloxacin resistance showed an increase in over 13 study countries, seven countries reported reduced erythromycin resistance for C. jejuni. Overall, resistance to conventional antimicrobials was frequent in Campylobacter and Salmonella from humans and animals. Increasing trend in resistance to one of two CIA for treatment in humans has been observed in poultry-originated Salmonella serovars and Campylobacter while combined resistance was is low in few Salmonella serovars and C. coli isolates from humans and animals in some countries (ECDC, 2023).

Introduction to Antibiotic Discovery

Antibiotics are the drugs that are used to treat illnesses caused by bacteria in humans and animals. These act by killing or inhibiting the growth and multiplication of bacteria. Antibiotics were considered as “magic bullets” that selectively kills microbes responsible for disease causation, but at the same time would not affect the host (Zaman et al., 2017).

Louis Pasteur in 1877, first discovered the properties of antibacterial compounds with the inhibition of anthrax by saprophytic bacteria. In 1928, Alexander Fleming observed that the growth of Staphylococcus aureus in petri dishes was inhibited by substances produced by the fungus Penicillium chrysogenum, which led to the discovery of the first antibiotic, penicillin (Aminov, 2017). Selman Waksman, discovered multiple antibiotics from filamentous actinomycetes living in the soil and defined an antibiotic as “a compound made by a microbe to destroy other microbes”. Following the discovery of Penicillin, various antimicrobial compounds such as sulphonamides, aminoglycosides, tetracyclines, lipopeptides, oxazolidinones, glycopeptides, streptogramins, and quinolones were discovered in the “Golden era” of antibiotics during the period from the 1940s to the 1960s and most of the antibiotics still in current use were discovered during that period (Salam et al., 2023). Millions of metric tons of newer classes of antibiotics have been produced in last 60 years since its inception.

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.

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.

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

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).

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

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

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

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.