eChapters

Under natural habitats microorganisms usually grow in mixed populations containing several species. This is really a problem for microbiologists because a single type of microorganism can not be studied adequately in a mixed culture. In laboratories these populations can be separated into pure cultures. The pure cultures contain only one type of organism and are thus suitable for studying the cultural, morphological and biochemical characters of an individual species. In the present days there are several ways of developing pure cultures in laboratories. Some of which are as follows:

Molecular biology is a branch of science that deals with understanding the biological activities at the molecular level. Molecular biologists are involved in understanding the concepts behind various intra and intercellular interaction of molecules, contributing to the knowledge about interrelationship of DNA, RNA and protein synthesis, i.e., how genes are transcribed into RNAs and how RNA is translated into protein, the phenomenon which is commonly known as “Central Dogma”stated by Francis Crick in 1956.

“The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid”-Francis Crick.

Prebiotic foods have been consumed for centuries, either as natural components of food, or as fermented food. However, interest in dietary use of prebiotics blossomed in the later 1800s and early 1900. Preliminary researches revealed that lactose had a profound effect on the pH of the caecal contents and intestine due to lactic acid fermentation resulting in enhanced calcium and phosphorous absorption. Further, it was also noted by several workers that inclusion of lactose in poultry diets resulted in better growth and reduced mortality.

A prebiotic can be defined as “a non digestible food ingredient that beneficially affects the host by selectivity stimulating the growth and/or activity of one or limited number of bacteria in the colon, and thus improves host health” (Gibson and Roberfroid, 1995). Based on this definition, Russell (1998) formulated the criteria according to which a substance can be a prebiotic. First, prebiotics are always feed ingredients that are not digested by the host, not or little used and/or metabolised as they pass through the upper portion of the intestinal tract, so they can reach the f lora of the large intestine. Secondly, they have to be able to serve as a substrate for one or more bacterial species with a potentially beneficial effect on the host. Finally they have to be able to cause a shift in the microf lora that improves the health of the host. In principle only non-digestible, fermentable feed components are prebiotics.

Molecular biology involves the study of DNA, RNA and proteins in cells. It emphasizes the biomolecules that carry out biological processes. One should understand the biomolecules participating in the process to understand the cellular function. In diagnostic microbiology, biomolecules are the analytes for testing and characterization. The analytes for molecular testing are the genomes (DNA), transcriptomes (RNA) and proteomes (Proteins) of the microorganism. Traditional methods of microorganism characterization depend on the culturing of the organisms, which is time-consuming and sometimes challenging to perform. Molecular techniques involve the characterization of DNA, RNA and protein to identify microorganisms. Nucleic acid sequence details are used to classify bacterial species, and mass spectrometry is used to identify microorganisms based on peptide profiles.

DNA (Deoxy ribonucleic acid)
DNA contains the genetic information required for the development and functioning of the living organism. It is the central informational molecule comprising a sequence of A, T, G and C nucelotides linked together by a phosphodiester bond. DNA is double helical in structure, composed of two complementary strands of nucleotides held together by hydrogen bonds between nitrogenous bases.

Theileriosis is a tick-borne protozoan disease that affects a wide range of domestic and wild animals, particularly cattle. While multiple species of Theileria exist, Theileria parva and Theileria annulata are the most important pathogens in terms of their impact on domestic cattle. This disease is transmitted by ixodid ticks and has a significant impact on cattle populations in tropical and subtropical regions. Theileriosis poses a substantial threat to livestock productivity, affecting economic stability in many countries, especially those that rely heavily on agriculture and cattle farming.
The disease has two primary forms: East Coast fever (ECF), caused by T. parva, which predominantly affects cattle in eastern, central, and southern Africa; and tropical theileriosis, caused by T. annulata, which affects cattle across northern Africa, the Middle East, and parts of Asia. Both diseases are highly pathogenic and, if left untreated, can lead to high morbidity and mortality rates, significantly impacting livestock production.

Transplantation refers to the implantation of a cell, tissue or organ from one site to another site in an individual or from one individual to another. It involves implanting of organs or grafting of tissues to repair defects and to stimulate regeneration.

A tumor or neoplasm is an abnormal growth of the cells to form a lump or mass of transformed cells. This growth occurs due to loss of regulatory control of cell division. The majority of cells and organs have a balance between cell renewal and cell death. 

Animal Biotechnology

Biotechnology is defined as “the application of biological organisms, systems or processes to manufacturing and service industries” (Coombs, 1984).

It is also defined as “the application of scientific and engineering principles to the processing of materials by biological agents to produce goods and services” (Coleman, 1986).

Animalbiotechnologyis the application of scientific and engineeringprinciples to the processing or production of materials by animals to provide goods and services to human kind.

The very basics in one’s life is whether he has judged his interests or decided goals of his life or has taken life casually. The first is to know what you wish to achieve in your life or in the words of our former president and Bharat Ratna APJ Abdul Kalam (14) what are your dreams ? If you have dreams than only you can achieve them hence it’s essential to know what is your passion ?, passion is the key to success. In pursuing your passion, all things become secondary ; in fact, desired changes occur within you, unnoticed and automatic to achieve your goals. It may not be out of contest to mention that basically I belonged to a business family and being eldest son in the family, my father asked me to join family business, after doing my intermediate, with the liberty to join part time BA classes while carrying business activities. But business was not my passion hence I struggled to join to the academics and carried all my Veterinary education with distinctions - because of my interests. I am not sure what would have happened to my life have I joined least interested business line- perhaps a failed business man.

History
Pseudomonas is comprised of two Greek words-Pseudo meaning false and Monas meaning unit, so literally the term means “false unit”. However Pseudomonas is a real bacteria so we really do not know the basis of the nomenclature by Walter Migula. A scientist by the name of Migula gave the Genus name of Pseudomonas in 1894 to these bacteria. Classification
The classification of Pseudomonas is given below:
• Class : Gamma Proteobacteria
• Order : Pseudomonadales
• Family : Pseudomonadaceae
• Genus : Pseudomonas
• Eight groups : P. aeruginosa ; P. chlororaphis; P. fluorescens;
P.pertucinogena; P.putida; P. stutzeri; P. syringae; P.
incertae sedis.
The family has 191 valid species. These include bacteria which are saprophytic, free living, and human, animal and plant pathogens. The type species is Pseudomonas aeruginosa.
Usual Habitat
Pseudomonas species are environmental organisms which occur worldwide in water and soil, and on plants. Pseudomonas aeruginasa is also found on the skin, on mucous membranes and in faeces. Burkholderia pseudomallei, which is found in soils, occasionally infectsanimals and man. Wild rodents can act as reservoirs of this organism. It is widely distributed in some tropical and subtropical regions of Southeast Asia and Australia.Although B.mallei can survive in the environment for upto 6 weeks its reservoir is infected Equidae.

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 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.

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

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

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.

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.

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).

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.