eChapters

Microscope is an instrument to magnify and enable to see the microorganisms by us.

Microscopes are of two types. Light and Electron microscope.

Basic Principles of light microscope

The light is the primary source on which magnification is performed. The magnification is obtained by a system of optical lenses using light waves. These light microscopes can be further classified into

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.

Mycotoxins are the secondary metabolites produced by moulds, which grow on food and feedstuffs causing pathological or physiological changes to humans and animals. They are toxic to vertebrates in low concentrations. Symptoms depend on the amount of toxin in the feed, the period for which the infested feed is ingested, the nutritional status of the feed and the susceptibility of the animal species. Toxic effects of chronic consumption of mycotoxins in human are carcinogenicity, hepatotoxicity, oestrogenicity, genotoxicity, nephrotoxicity, immunosuppression, reproductive disorders and dermal irritation (Anfossi et al., 2010). The most extensively studied mycotoxins viz., Aflatoxins, ochratoxins, deoxynivalenol (DON, vomitoxin), T-2 toxin, zearalenone, fumoninsins, mycophenolic acid, cyclopiazonic acid and ergot toxins are produced by the moulds Aspergillus, Fusarium, Pennicillium and Claviceps (Iqbal et al., 2015).

WHAT IS PLANT?

A. Definition - by most definitions, a plant:

is multi-cellular;

is non-motile

has eukaryotic cells

has cell walls comprised of cellulose

is autotrophic; and

exhibits alternation of generations - has a distinctive diploid (sporophyte) and haploid (gametophyte) phase.

Microbiology

Study of microorganisms, a large and diverse group of microscopic organisms, that exist as single cell or cluster. It also includes virus, which are microscopic, but not cellular.

Reproduction is one of the most serious biological problems that markedly influence the efficiency of livestock production. Immunology is a powerful tool for studying both normal fertility and infertility of farm animals. Reproductive immunology is in turn is an important tool for unlocking normal fertility problems and various forms of infertility or reproductive disorders. Although the immune and reproduction systems have been considered independent of each other, the cooperation of both the systems is crucial for initiation and maintenance of mammalian pregnancy. Therefore, reproductive immunology has now become a mature discipline and a major theme during advances in the field of reproductive immunology is to explain the successful gestation of the histcompatable mammalian embryos within the uterus of its immunologically competent mother.
 
Immune responses play an important role in various reproductive processes, including ovulation, menstruation and parturition. Clearly, during pregnancy, when the mother must accept a semi-allogeneic foetus, immune responses also play a very important role which  was first recognized by Medawar in 1953, when the concept of the foetal allograft was presented in order to explain the immunological relationship between mother and foetus. Since then, the immunology of pregnancy has been the leading subject within reproductive immunology research.

The term “nutraceutical” was coined from “nutrition” and “pharmaceutical” in 1989 by Stephen DeFelice, MD, founder and chairman of the Foundation for Innovation in Medicine (FIM), Cranford, NJ. According to DeFelice, nutraceutical can be defined as, “a food (or part of a food) that provides medical or health benefits, including the prevention and/or treatment of a disease”. In other words, nutraceuticals are chemical or natural feed ingredients which may enhance health by providing a physiological benefit by the provision of basic nutrients. Enzymes, prebiotics, probiotics, yeast and fungal extracts, predigested hydrolyzed carbohydrates and fats, phytogenic additives, acids etc are included in nutraceuticals. However, the term nutraceutical as commonly used in marketing has no regulatory definition.

Human beings have been concerned about the concept of food safety since time immorial. Around the globe there has been major improvement in food safety management in the last twenty years. Looking into the progress made on the ways to make food safe and how to make them free from pathogens, this book, will explain food hygiene, the journey of research been taken in food safety till date and the challenges that we are going to face in future to ensure food safety and its wholesomeness. It also includes the role and responsibilities of the various sectors of society, namely governments, food industry, consumers and academia.

In the beginning of the twentieth century many modern food safety policies have been established. In the packing of meat and in food processing industries a lot of work has been done (Sinclair 1906). Another second generation policy includes the outbreak in the United States in early 1990s’ of pathogen E. coli O157:H7 and the BSE scandal of the late 1990s in the UK Lopman et al., (2003) have discussed on viral outbreak in europe. Currently, the crisis has come due to the economic and technological transformations which have taken place in food and the food supply system. As these changes have many health risks the public is very concerned. Therefore, the control forms of safety regulation and food safety laws have come into the picture. These policies include continuous line inspection, visual product inspection and detailed specification of approved hygiene practices. There is a need of a public health-focused policy which works towards the preventive integrated management of foodborne hazards from farm to table. This includes the use of modern science based risk management tools which will increase the efficiency accurately towards public health. A lot needs to be worked out for better implications. Current efforts to modernize food safety policy around the world are being worked out by the use of risk analysis and cost-benefit analysis in public administration and also the quality management in industry. The role of public economics plays an important role in food safety policies. For the social benefits the policy makers need to take decision on the costs and strengthening of the risk assessment. Today there are globalized markets and this can influence the health globally. Emerging economies like China are industrializing and urbanizing rapidly like that of Europe and North America.

Everyone on this earth wishes to have a healthy life free from all ailments, stress and strain. But it is a common observation that some of us are more susceptible to diseases and fall victim of many infections while others have the capacity to resist many such infections. They are not so much susceptible to diseases. Is there anything which distinguishes them from others? An organism is made up of organs and organs systems. Like many other organ systems, in the body there is a system which is concerned with the protection of the body from infections.

IoT networks and smart sensors enable continuous livestock monitoring, overcoming the delay and subjectivity associated with manual observation. This chapter reviews sensor types, communication systems, cloud and edge computing, and their applications in animal-health and behaviour tracking. It proposes a framework integrating multimodal sensors with IoT networks and machine-learning models such as CNNs and LSTMs. The system can detect abnormalities, predict health conditions and provide real-time decision support. Practical benefits include earlier intervention, improved welfare, reduced labour and more efficient production. The chapter also addresses implementation problems, costs, connectivity, data security, technical reliability and ethical considerations associated with constant digital monitoring of animals.

Special Staining
A. Spore stain
Spores may be spherical or oval and may be central subterminal or terminal in position within the cell. Shape, size and position of the spore in the cell are useful for the identification of sporulated organisms. Spores are difficult to stain and when once stained, they are difficult to decolourize. With Gram’s stain, mature spores remain unstained bodies. Modified Ziehl-Neelsen’s method of staining will be used for the staining of spores.
Composition
a. Concentrated carbol fuchsin
b. Sulphuric acid 0.5 % solution.
c. Loeffler’s alkaline methylene blue
Materials
Old cultures of B. subtilis and Cl. Chauvoei and staining solutions.
Procedure
1. Make smears and fix over flame.
2. Stain with carbol fuchsin and steam for 3 to 4 minutes.
3. Wash with tap water.
4. Decolourize with 0.5% sulphuric acid.
5. Wash with water.
6. Counter stain with Loeffler’s methylene blue for 2 minutes.

The chief functions of Clinical Microbiological laboratory are to examine and culture specimens for microorganisms, to make accurate species identification of important isolates and to perform antibiotic susceptibility tests when indicated. These tasks will assist physicians in the diagnosis and treatment of infectious diseases.

Once the specimen is received in the laboratory, the specimens are examined visually depending on the physicians order and the nature of the specimen. Wet mounts and smears may be prepared and stained for microscopic examination. Observations may or may not be immediately reported to the physicians depending on the definitiveness of the results. Timely information may often be used to establish a presumptive diagnosis and institute a specific course of therapy. Specimens that require definitive identification of potentially pathogenic microbes are processed further. All agar plates are streaked for colony isolation; then plates and broths. All cultures are placed in an incubator with appropriate temperature and environmental conditions to maximize the growth and replication of microbes. Often a presumptive microbial identificationcan be made. A final report should be delayed while subcultures and additional test procedures are performed to identify the organisms definitively.

Preliminary Identification of Unknown Bacteria
A number of techniques may be used in the direct microscopic examination of clinical specimens to demonstrate the presence of microbes The examination of wet mounts of unstained materials by phase contrast or
dark field microscopy is useful for demonstrating motility, spirochaetes and endospores. Giemsa, Wright or acridine orange stains may be helpful in observing bacterial forms that stain poorly or that have little contrast from
back ground material.

Viruses are smaller than bacteria and require living cell for their growth. They can be detected through the bacteria to which they parasitise. Viruses are associated with both eukaryotes as well as prokaryotes and contain either DNA or RNA; only few have both DNA and RNA.

Viruses that infect bacteria are called bacteriophage. They consist of head or capsid and a tail. The head may be round, oval, polyhedral and is made up of proteins while tail is hollow and provides an exit for nucleic acid to enter in bacterial cell. The tail also helps in attachment to the cell wall surface of bacteria. After injection of the nucleic acid into bacteria they multiply and form large number of bacteriophage particles which cause lysis of the bacterial cell wall releasing many bacteriophages. Phages that cause lysis are known as lytic phages and that do not cause lysis are called temperate phage. The most common lytic phage is E.coli – a coliphage.

4.1 From Soil

Soil contains varied group of microorganisms such as fungi, bacteria, actinomycetes, algae and protozoa. The type of nutrients available, moisture, temperature and pH of the soil determine the nature of microbial population in soil. Different laboratory techniques for isolation of microorganisms have been detailed in chapter-III. These are used in particular for the isolation of specific type of microorganisms. All the techniques adopted are just to segregate the pathogens/ microorganisms from the mixture of microbial population present in nature.

(a) Isolation of Fungi

Soil contains large number of fungi and constitutes a major place of microbial population. They can be isolated in pure form by following the methods given below:

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:

Introduction
In order to study the cultural, morphological and physiological characteristics of an individual species, a pure culture of the microorganisms is required. Several different methods of separating the microorganisms from a mixed culture are available. The two most frequently used methods involve making a pour plate and/or streak plate. Isolated colonies are obtained from individual organisms after incubation.
Streak Plate Method
Plating is generally applied to the inoculation of a medium in petridish. Streak plate method is best for the separation of organisms from a mixed culture. In this method, agar medium is allowed to solidify as a thin layer in a petri dish and culture is streaked on the surface of agar medium and allowed to incubate to get isolated colonies.
Materials
1. Sterile petri plate
2. Wire loop
3. Nutrient agar medium
4. Bunsen burner
5. Mixed culture of E.coli and Micrococcus leutus

Whenever a person acquires desired qualifications, he needs a forum to express his talents and a place where he can fulfill his dreams and make important attainments. This may be achieved by two ways. One is, he becomes an entrepreneur and plans his own organization. As we are discussing biological research, there are good opportunities to start a Pharmaceutical firm to produce a drug, or a biotechnology firm to produce some biochemical molecule, enzymes, bio-fertilizers, vaccines against important human/animal/poultry diseases or a firm to supply diagnostic kits or immunological products etc. These are not hypothetical suggestions, difficult to achieve by a middle class person, but part of reality achieved by middle class family in India with laurels in their field of work. We may cite examples of firms like Dr Reddy Laboratory (Dr K.A Reddy), Biocon India (Ms Majumdar), Venky (poultry vaccines etc), Biorad (Bangalore based biochemical molecules supplier) who are working in India and leading their respective field. All this have been possible with tremendous change that has occurred in Indian scenario where there are many venture capitalists (or PE firms) who are willing to invest any amount of money provided they like your new idea of establishing a new enterprise. A somewhat more detailed information, in this regards, is provided elsewhere (see Research for money-chapter 13). Here I have not mentioned to pursue a Dairy or Poultry farm as these are not parts of research activities but related to animal production and any other person may also enter in such commercial activities.

Klebsiella pneumoniae (also known as Friedlander’s bacillus) is a Gramnegative, non-motile, encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterium found in the normal flora of the mouth, skin, and intestines.
Klebsiella spp. are Gram-negative, nonmotile, usually encapsulated rod-shaped bacteria, belonging to the family Enterobacteriaceae. These bacteria produce lysine decarboxylase but not ornithine decarboxylase and are generally positive in the Voges-Proskauer test. Members of the Enterobacteriaceae family are generally facultative anaerobic, and range from 0.3 to 1.0 μm in width and 0.6
to 6.0 μm in length. Klebsiella spp. often occurs in mucoid colonies. The genus consists of 77 capsular antigens (K antigens), leading to different serogroups. Morphology of Klebsiella pneumoniae (K. pneumoniae)
• Shape – Klebsiella pneumoniae is a short, plump, straight rod shape (bacillus) bacterium.
• Size – The size of Klebsiella pneumoniae is about 1–2 μm × 0.5–0.8 μm (micrometer).
• Arrangement Of Cells – K. pneumoniae is arranged singly, in pairs, or in short chains and sometimes in clusters.
• Motility – Klebsiella pneumoniae is a non-motile bacterium.
• Flagella – K. pneumoniae is a non-flagellated bacterium.
• Spores – The Klebsiella pneumoniae is a non–sporing bacterium.
• Capsule – Capsules are present in Klebsiella pneumoniae which can easily be demonstrated using India ink preparation, appear as a clear halo in a dark background.
• Gram Staining Reaction – Klebsiella pneumoniae is a Gram -ve (Negative) bacterium.

This chapter introduces the essential components of laboratory work that form the foundation for veterinary parasitology and molecular biology research. A well-equipped laboratory along with proper safety protocols and correctly prepared reagents, is the key to reliable experimental outcomes and a secure working environment.
The following sections describe the standard operating procedures and laboratory biosafety, standard equipment used in research work, and the fundamental principles of solution preparation. These basic laboratory skills are essential prerequisites for the specialised techniques covered in subsequent chapters on veterinary parasitology, molecular biology and bioinformatics. Understanding and applying these foundational concepts allows researchers to maintain experimental consistency, ensure reproducible results, and conduct investigations safely and efficiently. The methods and principles outlined here will be regularly applied throughout the various techniques detailed in this manual, serving as essential knowledge for all laboratory personnel as well short-term visitors.
1.1 Laboratory Standard Operating Procedures and Safety
Laboratory safety is fundamental to protecting personnel, maintaining sample integrity, and ensuring reliable experimental outcomes. This section outlines essential laboratory conduct, safety protocols for personnel and equipment, waste management, and emergency protocols for standard laboratories carrying out diagnostics as well as research.