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Bacteria can be defined as a group of diverse and ubiquitous prokaryotic single cell microorganisms. The size of bacteria varies with genera/species. In cocci the diameter varies between 0.5-1 µm. The measurement of bacillus / rod shaped bacteria is 4-8 µm ? 1-1.25 µm. The surface area and volume ratio of bacteria is very high. The nutrients are absorbed through a large surface area and it is rapidly distributed into a small volume. So the growth rate and metabolism of bacteria is very high. The bacterial cell mass is packed near the surface and no circulatory system is required to distribute the nutrients. The growth and metabolism rate of bacilli are more than cocci because surface area and volume ratio of a sphere (cocci) is lowest among all the geometrical forms.

Shape of bacteria

The shape of bacteria is mostly regulated by the rigid cell wall covering the bacteria. Different shapes of bacteria are observed.

Introduction
Microbiology techniques are used for various objectives like:
• To observe and isolate the micro-flora
• To test the microbiological quality of various food and dairy products
• To find the presence of any particular pathogenic microorganism
• To maintain a pure or mixed culture of specific microorganisms
1. Serial Dilution Techniques
• Bacteria are often present in such huge numbers that they can be difficult to count. Yet accurate counts are necessary for a variety of reasons like, for instance, assessing the quality of water or the safety of food. There are a number of counting techniques but most rely on the dilution of the sample to reduce the bacterial numbers down to a quantity that can be counted accurately.
• All bacterial plate count methods require the sample to be serially diluted until an estimated count of 30-300 colony forming units (CFU) on the plate is obtained. Plates with more than 300 CFU are very difficult to count. Plates with less than 30 CFU are not statistically reliable.
• If turbidity is observed in a broth culture, it might have millions more bacteria than you need. If this turbid broth is used directly for plating, all the CFU would grow together into a confluent mass and we would not be able to distinguish one colony from another.

Introduction
A) Cultivation of Fungi
A fungus is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. These organisms are classified as a kingdom, Fungi, which is separate from the other eukaryotic life kingdoms of plants and animals. A characteristic that places fungi in a different kingdom from plants, bacteria, and some protests is chitin in their cell walls. Similar to animals, fungi are heterotrophy; they acquire their food by absorbing dissolved molecules, typically by secreting digestive enzymes into their environment. Fungi do not photosynthesize. Growth is their means of mobility, except for spores (a few of which are flagellated), which may travel through the air or water. Fungi are the principal decomposers in ecological systems. These and other differences place fungi in a single group of related organisms, named the Eumycota (true fungi or Eumycetes), which share a common ancestor (form a monophyleticgroup), an interpretation that is also strongly supported by molecular phylogenetics. This fungal group is distinct from the structurally similar myxomycetes (slime molds) and oomycetes (water molds). The discipline of biology devoted to the study of fungi is known as mycology. In the past, mycology was regarded as a branch of botany, although now known fungi are genetically more closely related to animals than to plants.

Our exposure to wide variety of chemicals has increased markedly in the recent past. Most of these compounds are mutagenic as well as potential carcinogens. Emphasis is being laid to develop method for detection of environmental carcinogens so that their use can be avoided.

Despite the fact that mammalian cell structure and human enzymatic pathways differ from those in bacteria, the chemical nature of DNA is common to all organisms; this permits the use of bacterial test systems for rapid detection of possible mutagens.

The Ames test which was developed by Bruce Ames in 1970, at the University of California, U.S.A. is a mutational reversion assay employing several special strains of Salmonella typhimurium, each of which has a different mutation in the amino acid histidine biosynthesis operon.

S. typhimurium, the indicator organism, used in this test is the mutant strain that cannot synthesize histidine (His^-), lack the enzyme to repair DNA so that mutation shows up readily and has leaky cell wall that permit the rapid entry of chemicals. In addition, this S. typhimurium strain carries mutation enhanced plasmid, the combined effect of these characters increase the sensitivity of the organism to mutagenic treatments.

(A) Effect of Temperature
Introduction
Temperature is one of the most important physical factors affecting microorganisms. Bacteria are different from higher plants and animals in the lack of homeostatic mechanism and cannot regulate heat generated by metabolism and are, therefore, directly and readily affected by temperature. Over a limited temperature range, there is a two-fold increase in the rate of enzyme catalyzed reaction for every 10°C rise in temperature.
Bacteria may be divided into three major groups with respect to their temperature requirements: (i) Psychrophiles, those with optimum temperature between 0°C and 20°C; (ii) Mesophiles, with optimum temperature between 20ºC and 40°C and (iii) Thermophiles, with optimum temperature between 40°C and 80°C. Thermophiles are of two types: (a) Facultative thermophiles with an optimum temperature of growth between 45°C and 60°C and (b) Obligate thermophiles with optimum temperature above 60°C. Temperatures in the range between 50°C and 100°C are normally in the lethal range for bacterial cells and spores. The range of temperature preferred by bacteria is genetically determined, resulting in enzymes with different temperature requirements.
Each organism grows within a particular temperature range (i.e. cardinal temperature points). The minimum growth temperature is the lowest temperature at which growth of a species will occur. The highest temperature at which a species can grow is its maximum growth temperature, and a species grows fastest at its optimum growth temperature.  Time of exposure is a vital factor in assessing the

If you have prepared yourself to make research as your career, it is most advantageous to read research papers/reviews/chapters/books published by the eminent scientists. It is not necessary to confine reading to your subject but a voracious reading will enable you to develop an idea about general problems related to science and our society; how scientists managed to solve them and with development of new knowledge, how you may tackle fresh or existing problems. A deep analysis may be done of those works which are so accurate that even after lapse of so many years, they still hold true. In our opinion, it is better to launch websites/blogs highlighting important works of eminent Parasitologists for the benefit of our research scholars; a beginning, in India in Parasitology, has been made in this direction with launch of a website on first time Rafi Ahmed Kidwai award winner (in Animal Sciences) late Dr SC Dutt (www.drscdutt.com). 

Introduction
Antimicrobial resistance (AMR) is rapidly becoming one of the most critical challenges in global health, with profound implications for both human and veterinary medicine. The increasing prevalence of drug-resistant bacterial infections has eroded the effectiveness of many frontline antibiotics, making even routine treatments more complex and less predictable. Surveillance data from the World Health Organization’s GLASS initiative highlights the scale of the problem: resistance to third-generation cephalosporins in Escherichia coli and methicillin in Staphylococcus aureus has reached alarming levels in dozens of countries, complicating the management of common infections and raising the stakes for therapeutic innovation. In veterinary settings, the crisis is equally urgent. Resistance has emerged across nearly all major antibiotic classes used in livestock, poultry, and companion animals. Contributing factors include widespread antibiotic use in agriculture, limited diagnostic stewardship, and the ease with which resistance genes spread within and between microbial communities. These trends not only threaten animal health and productivity but also increase the risk of zoonotic transmission, blurring the boundaries between veterinary and human public health. Global health authorities have long warned of a “post-antibiotic era” - a time when conventional antibiotics may no longer offer reliable protection against bacterial pathogens. Recent reports suggest that this era is no longer hypothetical. If current trajectories continue, drug-resistant infections could account for up to 10 million deaths annually by 2050, with staggering economic consequences. To address this escalating threat, researchers are exploring alternative therapeutic strategies that go beyond traditional antibiotics. Among the most promising are antimicrobial peptides (AMPs) and bacteriophage therapy. AMPs are naturally occurring molecules found in animals and other organisms, known for their broad-spectrum activity and rapid bactericidal effects. Their ability to disrupt bacterial membranes through electrostatic and hydrophobic interactions makes them less susceptible to resistance mechanisms that target conventional antibiotics. Bacteriophages - viruses that specifically infect and lyse bacteria - offer a complementary approach. Phage therapy is highly targeted, capable of eliminating specific bacterial strains while sparing beneficial microbiota. Phage’s can also evolve alongside bacterial populations, potentially outpacing resistance development. In veterinary medicine, phage-based treatments have shown promise in managing infections in poultry, swine, cattle, and companion animals, particularly where antibiotic options are limited or ineffective. The combined use of AMPs and bacteriophages represents a novel and potentially synergistic strategy for tackling AMR. By leveraging distinct mechanisms of action and delivery profiles, these agents may enhance therapeutic efficacy, reduce resistance selection pressure, and improve outcomes across a range of veterinary applications. This chapter explores the pharmacological and pharmacokinetic challenges associated with delivering such combination therapies, with a focus on optimizing their use in the fight against antimicrobial resistance.

A. Answer the following.

Q.1.     What are the key cytokines produced by Th1 cells ?
Ans.     IL-2 and IFN-g. 

Q.2.     What is class III MHC genes ?
Ans.    The class III MHC genes encode various secreted protein that have immune functions including component of C’ and molecules involved in inflammation.

B. Write true of false about the followings.

Q.1.     Macrophages, dendritic cells and B cells can acts as antigen presenting cells.
Ans.    True.

All blood cells arise from a basic cell called the hematopoietic stem cell. Stem cells are undifferentiated embryonic cells.They are self renewing and their population by cell division. Stem cells have the ability to develop into any type of blood cell. The formation of blood cells begins in the embryonic yolk sac during the first week of development. Every mature cell is derived from the same type of stem cell. In contrast to an unipotent cell which differentiates into a single cell type, a hematopoietic stem cell is multipotent or pluripotent. In the mammalian embryo, the blood corpuscles are normally formed in yolk sac, liver, spleen and thymus gland.

Introduction
Some species of rod shaped bacteria e.g. E.coli, Ps. fluorescence, Bacillus subtilis are known to be motile. Bacterial motility is generally associated with the presence of hair-like appendages called “Flagella”. There are several ways for determining the motility of a culture such as the wet mount method, hanging drop method and a culture method (on semisolid medium). Bacterial cells in suspension also exhibit a vibratory or oscillatory movement without changing their relative position. This is termed ’Brownian Movement’ and it should not be confused for true motility of bacteria. In motility the cells change their relative position and move across the microscopic field in different directions.
1. The Wet Mount Technique
This is the simplest and quickest way to determine the motility of bacteria.
Materials
1. Clean glass slide
2. Broth culture of bacteria (E.coli, Ps.fluroscenes)
3. Cover glass
Procedure
1. Place a drop of organisms on clean glass slide and cover with a cover glass.
2. Examine the slide with phase contrast microscope or bright field microscope with reduced light under oil-immersion.

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.

With increasing industrialization, water sources available for consumption as potable water are being adulterated with industrial, animal and human wastes. As a result water has become a formidable factor in disease transmission. Polluted water usually contains large amount of organic matter which forms nutritional base for growth and multiplication of microorganisms. Usually the water with non-pathogenic organisms, like autotrophs and saprophytic heterotrophs, are not of major concern, but presence of intestinal contaminants of fecal origin are of great concern. These pathogens are responsible for intestinal infection such as bacillary dysentery, typhoid fever, cholera and paratyphoid fever.

The detection of all the intestinal pathogens on a routine basis would be a tedious and difficult task, however, it would be easy to demonstrate the presence of some of the nonpathogenic intestinal organisms like Escherichia coli and Streptococcus faecalis. These are always found in intestine but their detection confirms the contamination of water with fecal material. Such organisms are also called pollution indicator.

Introduction

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

Seed Viability

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

Introduction
Antimicrobial resistance (AMR) has become a major worldwide health issue, requiring the search for natural, safe, and efficient substitutes for synthetic antibiotics. The ancient medical system known as Ayurveda lists a variety of polyherbal compositions having jwaraghna (antipyretic) and krimighna (antimicrobial) qualities. A traditional decoction preparation that is frequently prescribed for microbial infestations (krimi), wound infections, fevers, and skin conditions called Nimbadi Kwatha.Strong herbs that have been separately shown to have antibacterial and immunomodulatory properties, including Nimba (Azadirachta indica), Guduchi (Tinospora cordifolia), Triphala (Haritaki, Bibhitaki, Amalaki), Vasa (Adhatoda vasica), and Katuki (Picrorhiza kurroa), make up Nimbadi Kwatha. Triphala has antimicrobial and antioxidant qualities, Guduchi boosts host immunity and has antibacterial qualities, and Neem provides broad-spectrum antibacterial, antifungal, and antiviral activity. According to experimental studies, giloy extracts are effective against a variety of pathogens, such as bacteria, viruses, and fungi. It has a distinct antimicrobial activity that involves two mechanisms: it enhances phagocytosis, changes cytokine production, and activates macrophages. In light of antimicrobial resistance (AMR), T. cordifolia is a prospective option for the development of plant-based antimicrobials and integrated therapies. Its use is backed by both modern pharmacological research and ethnomedicinal history for infectious disorders and immune health. Nimbadi Kwatha and its derivatives, including Nimbadi Ubatan and soap formulations, have been shown to have antibacterial properties by modern pharmacological research, which also showed a notable inhibition of bacterial and fungal growth in vitro. These results demonstrate the value of Ayurveda in treating infectious diseases and lend credence to its traditional claims. To close the gap between traditional knowledge and contemporary evidence, a thorough analysis of Nimbadi Kwatha’s antibacterial impact is essential. It also identifies the plant’s potential as a natural remedy for drug-resistant infections.

Introduction 
The discovery of antibiotics—one of the greatest medical breakthroughs of the 20th century—began when Sir Alexander Fleming noticed that the fungus Penicillium notatum inhibited the growth of Staphylococcus spp. This finding sparked a search for similar properties in other fungi and bacteria. From 1940 to 1960, known as the ‘Golden Era of Antibiotic Discovery’, most modern antibiotics such as streptomycin, tetracycline, rifampicin, and nalidixic acid were identified through natural product screening (Clardy et al., 2006; Lewis, 2013). However, the declaration that “it is time to close the book on infectious diseases and declare the war against pestilence won” proved to be premature (Davies & Davies, 2010). It was not a final victory but a fragile truce. In the ongoing war between humans and microbes—nature’s own creations—our successes are brief, and the microbial battleground keeps shifting. Bacteria’s genetic adaptability, coupled with human complacency, led to antimicrobial resistance (AMR), where microbes gradually evolved resistance to almost all known antibiotics (Ventola, 2015). Resistance appeared soon after antibiotics entered clinical use, but the emergence of methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Mycobacterium tuberculosis, and extended-spectrum ß-lactamase (ESBL)-producing bacteria marked major setbacks (Prestinaci et al., 2015; WHO, 2023). AMR gained global attention with the rise of pan-drug-resistant strains—resistant to all antibiotic classes—seen in Klebsiella (India), Acinetobacter, and Pseudomonas (China, Italy, Greece), and extensively drug-resistant tuberculosis (XDR-TB). The infamous case of

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.

One prime fact in research is not to take the matter for granted and do not believe in status quo. This is most critical mindset for creating something new. Even change may be made in existing circumstances with minimum requirements. Here, I may cite my experience while starting research in 1976 for my doctorate program on immunity against schistosomiasis. The experimental model was albino mouse where schistosomes, by their nature, develop into blood vascular system of the host hence require a special technique called “Perfusion technique” for retrieval of mature and immature schistosomes. This ‘perfusion technique’ requires use of a “Peristaltic pump” and was followed in some western countries (2). However, No body in India followed this perfusion technique hence recovered schistosomes from mouse or from any other host species by teasing the blood vessels or/and cutting and soaking liver,spleen, kidney, lung pieces in normal saline.

Immunity is defined as the resistance to infection. This is carried out by the process of identification and removal of non-self material. In contrast to this, our body has to maintain immune tolerance to our own cells and tissues and develop the ability to distinguish these self-antigensfrom non-self (foreign) antigens. Tolerance means unresponsiveness of the immune system to an antigen. Immune tolerance is the process by which the immune system does not destroy an antigen. Tolerance can be either natural or induced.

Trans-boundary Animal Diseases (TADs) are highly contagious diseases of livestock in the world and transmissible diseases which have the potential for very serious and rapid spread, irrespective of national borders, which has serious socio-economic or public health consequence and they are importance in the international trade of animals and animal products. With rapidly increasing globalization, an associated risk of movement of trans-boundary diseases is emerging. Trans-boundary animal diseases represent a serious threat. They reduce production and productivity, disrupt local and national economies and also threaten human health (zoonotic). Trans-boundary animal disease is a concern globally; cumulative effort is needed at international level to minimize the spread of infectious diseases across the borders.
Considering that livestock rearing constitutes a significant share in the national economy of a developing country, it is imperative to take up disease control initiatives. Measures are required to safeguard the livestock industry from epidemics of infectious diseases and to uphold safe international trade of  livestock and their products. In this regard, it is essential to develop scientific and risk-based standards that facilitate the international trade in animal commodities.
Introduction
Trans- boundary Animal Diseases (TADs) are highly contagious diseases of livestock in the world. Moreover, their economic importance is a major constraint in international trade. Their implication on human Health and National food security cannot be over emphasized. Zoonotic diseases among TAD’s include diseases like West Nile Virus (WNV), Rift Valley Fever (RVF), Mad Cow disease (BSE), Bovine Tuberculosis and Highly Pathogenic Avian Influenza (HPAI). Other important TADs are Foot and Mouth Disease (FMD), Contagious Bovine Pleuropneumonia (CBPP), Lumpy Skin Disease, African
Swine Fever (ASF) and Newcastle Disease (ND). They have the potential for very rapid spread, irrespective of national borders and these diseases can cause serious socio-economic and possibly public health consequences.