eChapters

MENDEL’S RULES OF GENETICS DO NOT ALWAYS WORK

Mendel’s rules:

Segregation of alleles

Independent assortment of genes for different characters

Hold for characters:

Which have dominant & recessive traits

Determined by single genes

Genes are on separate chromosomes

Different types of chromosomes, or

Different homologues of same type of chromosome

These conditions are not always met, so there are many complications

BACTERIA HAVE SMALL, COMPACT GENOMES

We now know the complete DNA sequences for about a dozen bacteria

Bacteria seem to need about 500 to 5000 genes to conduct their lives

Because they are small, bacterial genomes are highly organized

Most of the time there are only a few bases between the end of one gene and the beginning of the next

Few introns are found in bacteria

Bacteria contain accessory “chromosomes” called plasmids; each has only a few genes

Viruses are much simpler than cells

Protein capsid

Genetic information stored on DNA or RNA:

Types of genome:

Double strand DNA: sometimes circular

Single strand DNA: sometimes circular

Double strand RNA

Single strand RNA

Some can be crystallized

Introduction

The study of infectious diseases, alongside other clinical and natural strengths, is going through fast change introduced on by the approach of reasonable next generation sequencing (NGS) advancements (Koser et al., 2012).These technologies become adopted as routine methods easily, and laboratory techniques are transformed throughout the process. The amount of genomic bacterial data generated is huge. As of this writing, for example, over 200,000 Salmonella genomes alone are in the public domain with hundreds being added weekly. A whole genomic DNA sequence characterizes the maximum practicable level of structural detail on the individuating traits of an microbial organism or population. It can therefore be used to provide more accurate microbial genome recognition, evolutionary relations and a detailed catalogue of epidemiologically important characteristics. This has a significant role to play for outbreak research, diagnosis and treatment of infectious diseases, microbiology and epidemiology (Allard et al., 2019). Besides, DNA sequences are also a universal dataset which can theoretically be used to determine any biological function. This means that antimicrobial resistant (AMR) can be detected and to track the development and spread of AMR bacteria in a hospital or the community.

Place of Microorganisms in Living World

Haeckel’s kingdom Protista

E.H. Haeckel, 1866 proposed this concept. Apart from plant and animal kingdom, the third kingdom “Protista” was developed by him. The protista includes unicellular organisms that are typically neither plant nor animal. These organisms, the protists, include bacteria, algae, fungi and protozoa. Bacteria are referred as lower protists and others are called as higher protists.

Whittaker’s five kingdom concept

R.H.Whittaker (1969) proposed the concept of five kingdom. He divided the living things in to 5 kingdoms.

Growth of bacteria can be defined as change of total population mass, not individual increase in size or mass. Macroscopically visible growth of bacteria on the solid medium is known as ‘colony’. It is estimated that if Escherichia coli are allowed to grow, a single bacterial cell can produce a daughter population with the mass of the whole earth within 2 days! In the environment the bacteria are not actively growing all the time. The mechanisms behind the growth-arrested states of environmental bacteria are not well understood.

Bacterial reproduction

Bacterial reproduction is an asexual process which occurs by binary fission, budding, fragmentation and condiospore or sporangiospore formation. There is no possibility of genetic exchange (reassortment) as it is originated from a single parent cell. Bacterial gene transfer occurs by transformation, transduction and conjugation methods (discussed later in details). These are one-directional DNA transfer methods and are not associated with reproduction. The donor bacteria can give the genetic material to the recipient and the vice-versa does not occur.

Binary fission: In binary fission, bacterial DNA is replicated first and then the parent cell separates. Each daughter cell receives a complete genome, identical to the parent cell. Bacterial DNA replication begins when the cells reach a specific mass. If the cell diameter is constant, longer cells will begin DNA replication earlier than the shorter cells. The shorter cells need time to reach a critical mass required for the initiation of DNA replication. The ugtP proteins were identified to be associated with shorter cells and delayed DNA replication in Gram-positive bacteria (Bacillus subtilis). It was confirmed later that the ugtP act as inhibitor for Z-ring formation.

Principle
Some viruses like Orthomyxo, Paramyxoviruses etc. have the property to clump the erythrocytes of certain species of birds or animals. This is not an antigen-antibody reaction but a network of erythrocytes formed by the bridging virions. These virions on their envelop have peplomers that binds with the receptors on the erythrocytes. If large quantities of virus are used for HA and allowed to act on red blood cells
for a long period of time, the agglutinated red cells shed the virus particles which then float freely in the suspending fluid and neither attach to the same red cells nor agglutinate them. This process of dissociation of virus from the red cells is known as elution, and is due to the destruction of receptors on red cells by the virion associated receptor destroying enzyme - neuraminidase. The red cells freed from the virus particles are known as stabilized red cells. The elution will occur most rapidly if the temperature is raised to 37°C as the enzyme neuraminidase will be more active at this temperature.

Practical Application of Haemagglutination
a) Spot test: For rapid detection of the presence of a haemagglutinating virus. This can be done by mixing two or three drops of suspected sample (infected chick embryo fluids or cell culture fluids) with equal quantity of 5% chicken red cells in normal saline. The appearance of the clumps of agglutinated red cells, indicate the presence of a haemagglutinating
virus in the sample.
b) Titration of the virus using the haemagglutinating property.

Introduction
The genus Haemophilus contains small, nonmotile, nonsporing, oxidase positive, pleomorphic, gram negative bacilli that are parasitic on human beings or animals. Haemophilus means blood loving organisms.

Haemophilus species are small (less than 1 pm x 1 to 3 μm), Gram-negative rods, which often appear coccobacillary and may occasionally form short filaments. These motile organisms, which are facultative anaerobes with variable reactions in catalase and oxidase tests, do not grow on MacConkey agar. They are fastidious bacteria requiring one or both of the growth factors X (haemin) and V (nicotinamide adenine dinucleotide, NAD). Optimal growth occurs in an atmosphere of 5-10% CO2 on chocolate agar which supplies both X and V factors. Small, transparent, dewdrop-like colonies are formed by most Haemophilus species after incubation for 48 hours.

Colonies of H.somnus have a yellowish hue and some isolates are haemolytic on sheep blood agar. The main pathogens in the genus are H.somnus in cattle and sheep, H.parasuis in pigs and H.paragallinarum which is responsible for infectious coryza of chickens.

The haemophili are motile, facultative anaerobes, produce acid from glucose, reduce nitrates and are variable in the oxidase and catalase tests. They are nutritionally fastidious, will not grow on MacConkey agar and grow best on chocolate agar (supplying the X and V factors) under 5- 10 per cent CO2 at 37°C.

The growth of many of the Haemaphilus species is enhanced by 10 per cent CO2, As this is not inhibitory for any of them, CO2 should be used for routine isolation.The inoculated chocolate agar plates are incubated under 10 per cent CO2 at 35-37°C for 3-4 days, although some growth may be seen after 24 hours.

Introduction

Use of acid/base is common in laboratories for experiments like titration and proper safety in their handling laboratory is necessary. In this chapter we will briefly discuss about the safety measures necessary to be adopted while using acids/bases in laboratory for any experiment.

Handling of Strong Acid/Base

There are four major points that need to be taken care of while working with strong acids or bases that might be quite harmful if any accident happens.

1. Protect Yourself

Wear a lab-coat and acid-resistant gloves: Always use lab-coat/apron and make it sure that the sleeves properly cover your arms. More importantly, use gloves made of acid-resistant materials and also wear shoes to protect your feet while wearing long-pants/jeans. Female with long hair, must tie her hair properly (with rubber band) to avoid any disturbance caused by appearance in between.

Wear safety goggles: It is important to wear safety goggles to protect your eyes using larger and fitting goggles that cover front as well as sides of your eyes. Always use properly fitting and adjustable goggles because smaller/ loose goggles may not protect the areas around your eyes against accidental acid spills and it may fall down from your face, if it is too loose.

Locate emergency wash area: There must be an emergency washing area nearby the work place or laboratory containing lab spill management kits. All the workers in the laboratory must be aware of using the kits and emergency plans.

Neutralize acid: Don’t panic even if an acid or chemical spill happens. Stay calm and act as quickly as possible. In case an acid spills, neutralize it by using sodium bicarbonate so that they can be cleaned and disposed safely. If acid comes in contact of skin, wash it off under running tap water but do not use/ apply sodium bicarbonate on skin.

Introduction

For proper and efficient experiment to be conducted, experimenters need so many chemicals to deal with. This includes different acids, base, salt, different kind of indicators and so on. Some of these chemicals are safe for health, but some of them are dangerous to health when come in close proximity to our body while some of the chemicals cause health issues on inhaling. These may cause improper functioning of some of our physiological activities. Acids and bases are very dangerous for our tissues when come in our contact. These may cause serious respiratory problem in addition to injuries on coming to our contact. Some chemicals are exploratory yet they may cause even death of the person working with them in laboratory. Therefore, mere use of the chemicals in laboratory is not only the objective of the experimenter. The person working in laboratory should also be concerned about the proper use and handing of the chemical while working with it. Thus, every researcher of an institution who has to work with chemicals in a laboratory should be made aware about the chemicals to be used in the laboratory and the consequences if any mishandling of a laboratory chemical occurs. Not only these, suitable precautionary measures should also be known to the researcher along with ensuring the availability of the required safety equipment/installations. Hence, a proper understanding of the ways of handling of chemicals in a laboratory is of prime importance for the personals like you who are working or have to work in laboratory.

Safe Handling of Chemicals in Laboratory

Safe use/handling of chemicals in laboratory include minimized exposure to chemicals, maintaining safety data sheets, proper labelling, storage, segregation, and transport of chemicals for personal hygiene.

Introduction

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
Antimicrobial resistance (AMR) constitutes a profound global health crisis, jeopardizing the efficacy of antibiotics and posing a severe threat to the foundation of modern medical practice. The rise of multidrug-resistant (MDR) pathogens, which exhibit resistance to multiple classes of antibiotics, has significantly escalated the global burden of infectious diseases, complicating treatment protocols and increasing morbidity and mortality rates. According to the World Health Organization (WHO), AMR could lead to 10 million deaths annually by 2050 if current trends continue, accompanied by economic losses projected to reach trillions of dollars due to prolonged hospitalizations, increased healthcare costs, and reduced productivity. The traditional antibiotic development pipeline has stagnated, hampered by scientific complexities, high financial costs, and stringent regulatory frameworks, resulting in a critical shortage of novel antimicrobial agents. This necessitates the exploration of innovative strategies to combat AMR and preserve the utility of existing antibiotics. Among these strategies, Crispr-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and Crispr-associated proteins) systems have emerged as a groundbreaking gene-editing platform with transformative potential for addressing AMR. Initially identified as a bacterial adaptive immune mechanism, Crispr-Cas has been repurposed to enable precise targeting of genetic elements, offering novel therapeutic, diagnostic, and environmental solutions. This chapter provides a comprehensive analysis of Crispr-Cas systems in the context of AMR, exploring their molecular mechanisms, diverse applications, current limitations, and future prospects across clinical, agricultural, and environmental domains. By leveraging the precision and programmability of Crispr-Cas, researchers aim to develop targeted interventions that mitigate the spread of resistance while minimizing collateral damage to beneficial microbial communities.

Research is the basics which have changed human life and surrounding environment what existed on earth million years ago into present form. As soon as man realized himself as a separate entity from other animals, he started thinking about himself, creatures co-inhabiting with him and the universe. In that sense, biological science is the oldest subject which fascinated man, though it was called natural sciences in ancient times. Perhaps, the first curiosity was about himself, his birth, death and alike matters. As soon as he realized mortality of life or faced death of his dear ones, he attempted to become immortal or ‘amar’. This is reflected by worshiping of ‘Asur ’(Hirnakashyap, Bhasmasur, Ravan etc) to ‘Brahma’ or ‘Shiva’ for receiving the blessings of becoming immortal . However, even God refrained to bless these personalities becoming ‘amar’ confirming the realization by all that life is strongly linked with death ; as such they were blessed with certain conditional ties which may lead their death. Eventually, they met with their death under specific circumstances (3). When God or His little part (Ansh) took birth on earth as incarnation, He also followed all the rules and regulations of cosmic laws. But He never tried to become immortal and left His body on the earth, after finishing His duties.

This chapter introduces the historical development and fundamental concepts of immunology. It highlights the contributions of Edward Jenner, Louis Pasteur, Elie Metchnikoff, Paul Ehrlich, Emil von Behring and other pioneering scientists. Important discoveries related to vaccination, phagocytosis, antibody formation, cellular immunity, ELISA, radioimmunoassay and hybridoma technology are addressed. The chapter also explains antigens, immunogens, epitopes, haptens, adjuvants and the desirable characteristics of an effective immunogen. Questions concerning smallpox and rinderpest eradication, vaccine development and primary and secondary immune responses help readers understand the scientific milestones that established immunology as an essential discipline within veterinary and biomedical sciences.

Obviously human have had to deal with microbes even before the recorded history. The first record of human using microorganisms comes from ancient tablets from mid east.

Babylonians were using yeast to make beer over 8000 years ago and acetic acid bacteria to make vinegar over 6000 years ago. About 5000 years ago, Persia (Now Iran) region recorded the wine making.

The Romans had God for specific microorganisms. The roman God of mold and mildew were “Robigus” and “Robigo” which mean crop rust. (Rust is one of the plant disease caused by fungus). God Robigus was very much feared because of crop lost. About 2000 years ago, Romans proposed that diseases were caused by tiny animals. But, fundamentalist religions had a strong hold over the progress. The real microbiology history starts from 1600s, when people began to make crude lenses and microscopes.

The following are the scientists and their role in the development of microbiology:

It will be unjust not to describe history of Parasitology (31) and a little bit about Parasitology (chapter 8) so that our students may know the earlier works done in this field and how research has advanced by diligent observations of the eminent scientists when they were not having such sophisticated facilities.

As helminth is visible with naked eyes, it was this parasite which man encountered first in its journey of life sciences or natural sciences,as was known in ancient times. Some of these were known even before the era of biblical Palestine and ancient Egyptian civilization. Hippocrates (460-357BC), ‘Father of Medicine’ knew about pinworms (Oxyuris equi) of horses. In his “History of Animalium”, Aristotle (384-322 BC)(32) has mentioned three kinds of helminthes-flatworms, cylindrical worms (Ascaris lumbricoides) and thinworms (Enterobius vermicularis). The treatises of India had the information on large roundworms of intestine (Ascaris), pinworms, leeches, bed bugs and mosquitoes.

According to Futter (2001) it is very important the way we cook our food. If the cooking is inadequate it causes food poisoning. While cooking there can be cross contamination starting from raw foods. It includes hands or utensils which can cause food poisoning. Most foods, especially meat, poultry, fish and eggs should be cooked thoroughly to kill most types of food poisoning bacteria. Generally the food should be cooked to a temperature of 75 °C or hotter. When food is cooked, it should be eaten promptly, kept hotter than 60 °C or cooled, covered and stored in the fridge or freezer. Some people are more at risk from food poisoning than others. In order to buy safe food in the market like buying pre-cut produce as a half a watermelon or cut veggies from a salad bar, it is important to choose items that are refrigerated or surrounded by ice. They must be kept in separate bags. FDA (2019) has studied the contamination in lettuce. Shopping at a farmer’s market is a great way to get locally-grown, fresh fruit, vegetables, and other foods. However, safety of these produce needs to be checked. Many markets have their own food safety rules and vendors must comply with them along with the government regulations. Fruits and vegetables must be washed thoroughly under running water just before eating, cutting or cooking. Fruits and vegetables must never be washed with soap or detergent or using commercial produce washes. Even if it has to be peeled before eating, it is still important to wash it first. Any bacteria present on the outside of items like melons can be transferred to the inside when it is cut or peeled. They should be refrigerated within two hours after preparation. The juice or cider must be treated (pasteurized) to kill harmful bacteria. Pregnant women, children, older adults and people with weak immune systems should drink only pasteurized or treated juice. Raw milk can harbor dangerous microorganisms like Salmonella, E. coli, and Listeria. World India (2017) has looked into the food regulations. These can pose serious health risks. Pregnant women, older adults and people with weakened immune systems are at higher risk for illness caused by Listeria. One source for these bacteria is soft cheese made from unpasteurized milk. It is vital to check the label to make sure that it is made from pasteurized or treated milk. Make sure that eggs are properly chilled at the market. FDA requires that untreated shell eggs must be stored and displayed at 45°F. While buying eggs it needs to be ensured that the eggs are clean and the shells are not cracked. Meat should be properly chilled even in the market and kept in closed coolers with adequate amounts of ice to maintain cool temperatures. If it is brought in an insulated bag or cooler during transit it is very beneficial to keep the quality. It should be always kept separately from your other purchases, so that the juices from raw meat (which may contain harmful bacteria) do not come in contact with produce and other foods. Essays (2018) have written a very comprehensive overview of food safety.