eChapters

The terms “cultured dairy foods,” “cultured dairy products,” and “cultured milk products” are other terms for fermented dairy products. Lactobacillus, Lactococcus, Leuconostoc, and other lactic acid bacteria, along with yeasts in certain products, are used to ferment milk to make these dairy products. The product’s shelf life is extended through fermentation, which also improves the product’s taste and milk’s digestibility. There is a wide variety of fermented milk products available, each with unique flavors and attributes. The following categories apply to fermented milk products, based on the type of fermentation.

Changes in Nutrients Resulting from Fermentation

Fermentation lowers the lactose content in fermented milk products; however, if the starting milk is concentrated or fortified, the finished product’s lactose content may be higher than that of liquid milk.

The composition and content of protein and total amino acids are comparable to those of raw milk. Casein degradation is small compared to cheese, accounting for not more than 1% of the total protein. While some products of casein degradation are used by the starter microorganisms, others build up as free amino acids in the fermented kinds of milk. The amounts of free amino acids that build up differ depending on the species. Generally, proline is increased in large quantities, followed by serine, alanine, valine, leucine, and histidine. As a result of continuous starter culture activity, the level of free amino acids increases during storage. A limited degree of lipolysis occurs during the manufacture of fermented milk. The amount varies depending on the type of species, but the pattern and concentration of free fatty acids are both impacted. During fermentation and the breakdown of amino acids, trace amounts of volatile fatty acids are also produced.

Food adulteration is a global issue, and because there are insufficient regulations and oversight, developing nations are more vulnerable to it. But in many nations, food adulteration has gone unnoticed. Contrary to popular assumption, milk adulterants can, regrettably, present major health risks that result in fatal diseases. Modern milk adulteration involves increasingly complex techniques, necessitating state- of- the- art research to identify the adulterants. Because milk contains so many nutrients that are needed by both adults and infants, it is referred to as the ideal food. In terms of protein, fat, carbohydrates, vitamins, and minerals, it is among the best sources. Regretfully, milk adulteration is a very common problem worldwide. Potential causes could be the gap between supply and demand, the perishable nature of milk, the low purchasing power of the consumer, and the absence of appropriate detection tests. Because there is insufficient oversight and inadequate law enforcement, the situation is substantially worse in developing and underdeveloped nations. While quantitative detections of adulterants in milk are varied and complex, qualitative detections can be carried out simply using chemical reactions. The type of quantitative detection methods used depends on the milk’s adulterants.

Typical Adulterants in Milk

The addition of vegetable protein, milk from different species, whey addition, and watering are the main adulterants in milk that are known to be economically motivated. There is no serious health risk associated with these adulterations. Some adulterants, though, are too dangerous to ignore. Urea, formalin, detergents, ammonium sulfate, boric acid, caustic soda, benzoic acid, salicylic acid, hydrogen peroxide, sugars, and melamine are a few of the main adulterants in milk that have been shown to have serious negative health effects.

Electrophoresis

The mobility of ions in an electric field is the basis for the separation method known as electrophoresis. Ions that are negatively charged move toward a positive electrode, while those that are positively charged move toward a negative electrode. Usually, one electrode is biased either positively or negatively, and the other is at the ground for safety reasons. Ions can be distinguished from one another based on their size, shape, and overall charge, which determine their migration rates.

Poly Acrylamide Gel Electrophoresis (PAGE)

Introduction

Electrophoresis is an electrochemical process in which substances with a net electric charge migrate under the influence of an electric current. This migration occurs in an agar gel or a liquid- filled buffered matrix such as cellulose acetate. Positively charged substances travel toward the cathode (negative electrode), while negatively charged substances go toward the anode (positive electrode). Different substances move at different rates depending on their charge. This movement is called electrophoretic mobility.

Proteins are charged molecules. They have a positive as well as a negative charge. Among proteins, particularly caseins, they have different contents. The charge density also varies depending on the molecular weight. Proteins have a net negative charge at a pH above 4.6, and hence they move towards the anode (positive electrode) when subjected to an electric field under alkaline conditions. Due to differences in the charge of individual protein fractions, their migration rates also differ and are hence separated into distinct bands. These bands are stained with the dye amido- black, destained with 7% acetic acid, and observed for the electrophoretic pattern.

The primary constituents of milk are water, fat, protein, sugar (lactose), minerals, vitamins, and enzymes. Milk is a thin emulsion made up of two phases: A continuous aqueous colloidal phase and a dispersed phase of oil or fat. It is an opaque white liquid containing fat in an emulsion, protein, and a few minerals in a colloidal suspension, and lactose along with soluble proteins and a few minerals in a true solution. The physical characteristics of milk are comparable to those of water, but they are altered by the degree of dispersion of the colloidal and emulsified components as well as the presence of different solutes, such as salts, lactose, and protein, in the continuous phase.

Physical Appearance, Color and Optical Properties

Fat globules and casein micelles scatter light, giving the appearance of turbid and opaque milk. The way that molecules scatter light has an impact on optical characteristics. When the wavelength of light and the particle’s magnitude coincide, light scattering happens. Consequently, light with shorter wavelengths is scattered by smaller particles and vice versa. Because casein micelles scatter blue light more than red light at shorter wavelengths, skim milk appears slightly blue. Cow’s milk has a creamy color because of beta- carotene, a carotenoid precursor to vitamin A. The greenish tinge in whey is due to the presence of riboflavin. Turbid and opaque due to scattering of light mainly by protein micelles and fat globules.

Vitamins are naturally occurring, vital nutrients that are needed in small amounts. They are important for bone and tissue health, wound healing, growth and development, immune system function, and other biological processes. These vital organic substances serve a variety of biochemical purposes. Vitamins, like minerals, are not biosynthesized by the body. Consequently, in order to maintain our health, we must obtain them from the food we eat or, in rare circumstances, supplements.

Vitamins in Milk

Together with water- soluble vitamins like vitamin C and B complex and provitamins, milk also contains a variety of fat- soluble vitamins like A, D, E, and K. Some of these vitamins do, however, have very low concentrations.

Vitamin A (Retinol)

Plant foods contain carotenoids, which are the precursors of vitamin A, whereas animal foods provide preformed vitamin A in the form of retinyl esters.

The tissue/cell culture lab has the requirement of maintaining an aseptic environment. There are several aspects to the design of good tissue culture facilities. Ideally, cell culture work should be conducted in a single-use facility. Therefore, cell culture lab unit should be separated from the rest of the lab area. It can be either separate lab or a closed partition in a big lab. All new material should be handled as ‘quarantine’ material in a separate area, and materials known to be free of contaminants should only be allowed inside the culture room. In addition, the work surfaces should be thoroughly cleaned and disinfected between activities.

For most of the work involving non-infectious primary culture and cell lines, the laboratory should be designated to at least BSL2 facility. The location of the Biosafety cabinet is the most important point to consider. When operated correctly, the cabinet will provide a clean working environment, whilst protecting the operator from aerosols. Environmental monitoring with Tryptone Soy Broth agar settle plates inside the biosafety cabinet is a good indicator of the cleanliness of a cabinet. There should be no growth of bacteria and fungi after 3-5 days of incubation.

Things required for cell culture

1. Lab – having negative pressure (HEPA filter)

2. Safety cabinet (Hood/laminar flow)

a) Biosafety level 1 (BSL-1) – Basic level of protection. Agents handled under this cabinet are NOT infectious.

b) BSL-2 – Appropriate for moderate risk agents. Most of the non infectious cell cultures are done under this facility.

Working Principle: An autoclave is a sealed device that creates a different environment from the outside world under extraordinarily high pressure (Auto + clave = automatic locking). It uses moist heat or saturated steam under pressure to kill microorganisms and heat-resistant endospores. Its function is compared to that of a pressure cooker, i.e., steam is used to create a high pressure environment. However temperature of autoclave or steam sterilizer is generally maintained at 121°C for 15-20 minutes that is achieved by raising the pressure inside the device. This high pressure increases the boiling point of water and therefore raises the temperature that is too high for the microbes to survive, so used for sterilization.

Types of Autoclave cycle

1. Liquid cycle: Liquids take time to sterilize as they have high heat capacity. It is based on the principle of pressurized steam at 15 psi, 121°C temperature for 20 minutes. It has latent heat, which is 7× heat 100°C. Liquids and biological waste require slow exhaust to prevent the boiling over of super-heated liquids. This excess temperature is enough to hydrolyze the proteins of bacteria, viruses, or any other contaminant and therefore kill it. However, wet heat is seven times more intense than dry heat, so it requires less temperature than the dry heat cycle of the autoclave.

2. Dry Cycle: Here, steam is the sterilization medium, which penetrates inside the load and makes contact with all the surfaces, so a fast exhaust cycle is used. It has a high temperature of 160°C for 60 minutes.

In the realm of cell culture, the quality and composition of the growth media and reagents play a pivotal role in growth and sustaining cells in vitro. The preparation of these essential components requires precision, attention to detail, and adherence to the strict protocols to ensure optimal cellular growth, viability, and experimental reproducibility. This introduction serves as a foundational overview of the critical aspects involved in the preparation of media and reagents for cell culture experiments. Furthermore, the preparation of media and reagents for cell culture necessitates adherence to stringent quality control measures to ensure consistency and reproducibility across experiments. Validation of media batches, sterility testing, and periodic quality assessments are essential practic

What is primary cell culture?

In vitro culture of cells freshly obtained from the tissue is called primary cell culture. Unlike that of cell line, primary cell culture grows for a limited period of time. Examples, are primary mammary epithelial cells, primary hepatocyte culture.

Aim: To prepare the primary cell culture from chicken embryos.

Materials required for the primary cell culture

• 70% alcohol

• Cotton swabs

• Small beaker 20-50 ml

• Forceps

• Straight and required curved,

• 9 cm Petri dishes

• 11 day embryonated eggs

• Balanced Salt solution (BSS)

• 250 ml conical flask

• Magnetic centrifuge tubes

• Haemocytometer

• Growth medium with serum

• Culture flasks

What is keratinocyte?

The epidermal layer consists of keratinocytes, melanocytes, Langerhans cells, and Merkel cells. Their major function is to produce Keratinocytes which are the most abundant cells in the epidermis, keratin protein. Keratinocyte synthesizes major structural components of the epidermal barrier through a programmed process of differentiation. They are present in the innermost layer of the epidermis (Eckert et al?, 1989). The epidermis is composed of several layers, stratum corneum (horny layer), stratum lucidum (clear layer), stratum granulosum (granular layer), stratum spinosum (prickle cell layer), and stratum basale (basal layer), from the outermost to the deepest layer respectively. The process of maturation and movement of the keratinocytes from the stratum basale to the stratum corneum is what we call epidermal differentiation.

Procedures for isolation of canine karatinocytes

When studying skin cells, isolating keratinocytes from tissue samples, such as skin biopsies, is a critical initial step. Here is a general guide on how to isolate keratinocytes:

What is dermal fibroblast?

Skin fibroblasts are a type of cell found in the dermis, the middle layer of the skin. They are the most common cells in connective tissue and play a crucial role in maintaining the structural integrity of the skin. Fibroblasts are responsible for synthesizing and secreting extracellular matrix proteins such as collagen, elastin, and fibronectin, which provide support and elasticity to the skin. Additionally, fibroblasts play a role in wound healing by proliferating and migrating to the site of injury, where they aid in tissue repair and scar formation.

Aim: To prepare the primary cell culture of fibroblasts from canine skin.

Tools and instrument required for the procedure

1. Tissue collection box with ice cubes - To prevent contamination and maintain the stable temperature

2. Surgical Gloves - To prevent contamination

3. Tissue collection tube with 1X PBS - To collect sample

4. Laminar flow hood - To work in a sterile environment and prevent contamination

5. Cell culture dish - To digest the skin

6. Sterile 1000 μL and 200 μL pipette and tips-For accurate measurement and transfer of cell suspension and culture media.

7. Forceps - Fine-tipped forceps for handling and manipulating tissue.

8. Scalpel and blade holder - Scalpel for the dissection of skin tissue.

9. Surgical scissors - Chopping of the skin tissue

10. Falcon 70 μm cell strainer - To filter out undigested tissue debris and obtain a homogeneous cell suspension.

11. 15 ml and 50 ml Centrifuge tubes - To collect and centrifuge the digested cell suspension.

What is subculture?

In cell culture, a process called subculturing, or passaging, keeps cells alive and growing. It involves taking cells from an existing container, removing the used medium, and placing them in a new container with fresh nutrients. This allows the cells to continue multiplying and creates more cells for research.

Adherent subculture protocol Cells nearing 80% coverage need splitting!

1. Warm things up: Get your media, detachment solution, and incubator ready at 98.6°F (37°C).

2. Safety first! Disinfect your work area and supplies inside a biosafety cabinet.

3. Gently rinse: Remove the old media and wash the cells with a salt solution at room temperature. Be careful not to dislodge the cells.

4. Detach the cells: Add a pre-warmed enzyme solution to detach the cells from the flask surface. Keep an eye on them to make sure it works (usually 2 minutes). Note: Not all cells need this enzyme, and it can be harmful to some.

5. Stop the enzyme: Once all the cells are detached, add a culture medium with serum to neutralize the enzyme.

6. Collect the cells: Transfer the cell suspension to a tube, rinse the flask with fresh medium to get all the cells, and add it to the same tube.

7. Spin it down: Centrifuge the cell suspension for 5 minutes at room temperature (1000 rpm).

8. Get ready for counting: Remove the liquid, loosen the cell clump, and resuspend the cells in a fresh medium at a good volume for counting.

9. Count your cells: Use a hemocytometer to count the cells and determine their health.

Cell counting is the fundamental techniques in various fields of biology, medicine and biotechnology.

Importance of cell counting

1. Research: Cell counting is a critical component of biological and medical research. It helps scientists understand the behavior of cells in various contexts, such as studying cell proliferation, differentiation, and response to stimuli.

2. Disease diagnosis: Monitoring various medical conditions, including infections, autoimmune diseases, cancer and blood disorders. Complete blood counts (CBCs) are routinely used to assess the levels of different blood cell types and assess disease conditions.

3. Stem Cell Research: Cell counting plays a significant role in stem cell research, where precise quantification of stem cells is necessary for various applications, including regenerative medicine and tissue engineering.

4. Cell Culture: Cell counting is integral part to determine seeding density. Seeding of cells to available surface area for growth is important consideration for successful culture.

5. Cell-Based Assay: The cell-based assays are used for drug screening, toxicity testing, and evaluating the efficacy of new compounds. Accurate cell counts are essential to ensure the reliability of assay results.

Introduction

Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitization in antigen recall assays by scoring lymphocyte proliferation, c) immunophenotyping for surface markers as well as intracellular molecules in monocytes and lymphocytes etc. Activation of monocytes/macrophages by small molecules, cytokines and pathogen components can also be monitored. PBMCs can also be used for a variety of structural and functional studies for addressing issues in human immunology such as scoring for apoptosis and production of cytokines as well as other mediators in vitro.

Background information

Blood is a liquid mixture comprised of plasma, cells and proteins. The liquid portion, plasma, makes up half of the blood volume, and cells the other half. Isolation of lymphocytes is a common procedure in immunology and cell biology that involves separating and collecting lymphocytes.

Introduction

The protocol below describes the use of cell freezing methods involving an electric -80°C freezer for the cryopreservation of cell lines. ECACC routinely use a programmable rate-controlled freezer. This is the most reliable and reproducible way to freeze cells but as the cost of such equipment is beyond the majority of research laboratories the methods below are described in detail. If large numbers of cell cultures are regularly being frozen then a programmable rate-controlled freezer is recommended.

Materials

• Freeze medium (commonly 90% FBS, 10% DMSO or glycerol

• 70% (v/v) alcohol in sterile water

• PBS without Ca2+/Mg2+ • 0.05% trypsin/EDTA in HBSS, without Ca2+/Mg2+

• DMSO

Introduction

Structural changes in host cells by the viral infection is referred as cytopathic effect (CPE) and the responsible virus is called cytopathogenic virus. As a virus hijacks a cell to make copies of itself, the cell goes through distinct changes in its chemistry and structure. These effects are easiest to see in lab grown cell cultures. The severity of the damage caused by the virus depends on the specific virus itself, the type of cell it infects, how many viruses infect each cell (MOI), and other factors.

The early stages of this damage, called cytopathic effects (CPE), can be spotted directly under a microscope without any dyes or special treatments. While various types of CPE exist in living cells, some signs of viral infection, like inclusion bodies, require staining the cells to be visible. We can classify viruses based on how quickly they cause visible damage (cytopathic effect) in cell cultures. Viruses are typically considered slow if it takes 4-5 days to see this effect in cultures with a low starting dose of virus (MOI). On the other hand, viruses are considered fast if the damage shows up in just 1-2 days under the same conditions. It is important to note that at a high MOI all CPE can occur rapidly. So all decisions about rate of CPE appearance should be based on the lowest MOI that produces CPE

Principle

Isolating the virus in a laboratory is the gold standard for diagnosing many viral infections. Virus isolation is a highly specialised technique used to diagnose viral infections and produce a stock of viruses for research purposes. The isolation of the virus relies on the suitability of the sample, the ability of the cells to allow entry, the successful replication of the virus, and the release of viral particles from the cells. The choice of cell lines is dependent upon the suspected viral disease and the availability of such cell lines in the laboratory.

Materials required

Equipment

a) Biosafety cabinet level 2 (B2)

b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere)

c) Refrigerated centrifuge

d) Refrigerator

e) Inverted Microscope

f) Water bath

g) -86°C/-80°C deep freezer

h) Autoclave

i) Hot air oven

j) Filtration Assembly

Principle

The one-step growth experiment refers to a scientific methodology. A study aimed at observing the molecular events that take place during viral reproduction. This elucidates the fundamental features of the virus replication process and determines the optimal multiplicity of infection and timing for virus harvest. The initiation of modern bacteriophage research can be traced back to the development of the one-step growth curve by Max Delbrück and Emory Ellis in 1939, which utilised the Escherichia coli-T4 bacteriophage system. One step growth curve consists of three clearly defined phases 1. Latent period refers to the initial phase of a process where no significant changes or activities are observed. 2. Burst or Rise period is the subsequent phase characterised by a sudden increase virus particle. 3. Plateau period is the following phase where a stable and constant level virus replication.

Materials required

Principle

Unlike bacteria, which can be cultivated on an artificial nutrient medium, viruses rely on a living host cell for replication. Eukaryotic or prokaryotic host cells that have been infected with viruses can be used to produce viruses. Subsequently, the medium and cells can be collected and used as a virus source. The production of viruses usually consists of a two-step procedure: f irst, the growth of host cells, and then the production phase. Understanding the complex relationship between the host cell and virus is essential. Various crucial factors have been recognised, such as the concentration of host cells during infection and the multiplicity of infection (MOI).

Materials required

Equipment

a) Biosafety cabinet level 2 (B2)

b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere)

c) Refrigerated centrifuge

d) Refrigerator

e) Inverted Microscope

f) Micro pipettes(1-10 μl, 10-200 μl, 100-1000 μl)

g) -86°C/-80°C deep freezer

h) Autoclave

i) Hot air oven

j) Filtration Assembly

Principle

The quantification of infectious virus particles is commonly achieved through the utilisation of the Median Tissue Culture Infectious Dose (TCID50) assay. The assay functions through the introduction of a serial dilution of the virus sample into cells in a 96 well plate format. The chosen cell type is specifically intended to exhibit a cytopathic effect (CPE), which refers to observable changes in morphology or cell death resulting from viral infection. Following a period of incubation, the cells are examined for cytopathic effects or cell death, and each well is categorised as either infected or uninfected. The concentration at which 50% of the wells exhibit a cytopathic effect (CPE) is employed to determine the 50% tissue culture infectious dose (TCID50 ) of the viral sample. The end point is the dose or dilution that infects or kills or shows CPE of the cells. The calculation can be performed using various mathematical techniques, such as the Spearman-Karber method or the Reed Muench method. The virus titer is quantified as the number of tissue culture infectious dose 50 (TCID50 ) per millilitre (ml). The non-cytopathic virus can be quantified by employing the fluorescent antibody test, immune-peroxidase assay, or other appropriate methodologies.

Principle

Red blood cell sticking (hemadsorption) can reveal the presence of certain viruses. This happens because these viruses, like influenza or measles, sprout proteins (hemagglutinin) on their infected host cells. These proteins act like Velcro, attracting red blood cells and causing them to cling to the surface. A hemadsorption assay uses this natural process to check for viruses. It's faster than waiting for the cells to show damage (cytopathic effect), allowing for earlier detection. Haemadsorbing viruses can be detected several days prior to the appearance of a cytopathic effect.

Equipment

a) Biosafety cabinet level 2 (B2)

b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere)

c) Refrigerated centrifuge

d) Refrigerator

e) Inverted Microscope

f) Micro pipettes (1-10 μl, 10-200 μl, 100-1000 μl)

g) -86°C/-80°C deep freezer h) Autoclave

i) Hot air oven

j) Filtration Assembly

Principle

The plaque assay was developed in order to enumerate and quantify the infectivity of bacteriophages. This technique has been subsequently adapted for application in animal virology and has proven to be a dependable method for quantifying the concentration of various viruses that have been adapted for growth in cell cultures. This assay is designed to quantify the capacity of a solitary infectious virus to create a visible area of cell death on a layer of cultured cells. A plaque forms during the viral infection cycle, in which the host cell dies after viral replication. Some viruses cause cell lysis during replication, while others cause minimal damage to the cells. The plaque assay is a technique used to measure the quantity of viruses that cause the destruction of their host cells, resulting in cytopathic effects (CPE). During a plaque assay, the host cells and virus are briefly co-cultured to enhance the virus's attachment and entry into the host cell. Subsequently, the concoction is introduced into a partially solidified agar medium. The thick agar is poured onto a layer of "bottom agar" which supplies ample nutrients for the host cell. After one round of virus replication, an infected cell will undergo lysis, leading to the release of multiple new viruses. Within the gelatinous substance, these newly released viruses can only infect neighbouring cells. After a subsequent round of replication, these neighbouring cells will undergo lysis. The cells that manage to avoid infection will continue to proliferate. After a period of 24 to 48 hours, plates that were not contaminated with a virus will display a consistent layer of cells. The plates infected with a multitude of viruses exhibit plaques. Quantifying viral titer is essential for assessing viral infectivity. The determination can be made either by conducting a plaque assay or by calculating the infectious dose.

Principle

Neutralization refers to the process of reducing or nullifying the effect of a specific component. Neutralization is a process in which the effects of viral antigens or viruses reduce through Ag-Ab reactions. Neutralizing antibodies are a specific type of antibodies that play a role in neutralization. Not all antibodies generated within our body possess the capability to impede the effects of antigens. Only antibodies with the ability to neutralize can prevent the uptake of viral particles by cells and counteract their effects. The virus neutralization assay is a serological test utilized to identify the existence and extent of functional systemic antibodies that hinder the ability of a virus to cause infection.

Media and Materials

Equipment

a) Biosafety cabinet level 2 (B2)

b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere)

c) Refrigerated centrifuge

d) Refrigerator e) Inverted Microscope

f) Micro pipettes (1-10 μl, 10-200 μl, 100-1000 μl)

g) -86°C/-80°C deep freezer

h) Autoclave

i) Hot air oven

j) Filtration Assembly

Principle

The majority of cell lines are cultivated as monolayer adherent cells, meaning they only grow on the surfaces of culture vessels. However, there are particular cells that are non-adhesive and do not rely on external support for their growth, referred to as suspension cells. A suspension culture, is a cell culture where individual cells or small cell clusters grow and multiply in a stirred growth medium, forming a suspension. Subculturing, also known as passaging cells, involves transferring cells from a previous culture into fresh growth medium to allow the cell line to continue growing. Suspension cultures involve the suspension of cells in a liquid medium and agitation at a specific speed to ensure that the cells are uniformly exposed to nutrient media from every angle. This method prevents cell aggregation and enhances nutrient absorption, thereby promoting cell growth. The maintenance of these cultures is achieved by transferring the cells from the early stationary phase to a fresh medium through subculturing. During the incubation period, there is an increase in both cell division and cell enlargement, leading to rapid cellular growth. The viability of cells in the suspension decreases after the stationary phase due to the exhaustion of certain factors or the accumulation of toxic substances in the medium. This decrease in the viability of the cells further reduces the growth rate of the entire culture. Next, a portion of the cell suspension is added to the freshly prepared medium with the same composition.

Principle

Adenoviral, oncoretroviral, and lentiviral vectors are commonly utilised for gene delivery in mammalian cell culture and in vivo. Additional important examples of viral gene transfer involve baculovirus and vaccinia virus vectors. Stable cell lines produced with lentivirus technology provide consistent, long-term protein expression compared to the short-term expression achieved through transient transfection. This chapter focuses on cell transformation by lentiviruses.

Equipment

a) Biosafety cabinet level 2 (B2)

b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere)

c) Refrigerated centrifuge

d) Refrigerator

e) Inverted Microscope

f) Micro pipettes (1-10 μl, 10-200 μl, 100-1000 μl)

g) -86°C/-80°C deep freezer h) Autoclave

i) Hot air oven

j) Filtration Assembly

Principle

Cell viability refers to the quantity of healthy cells in a sample, while cell proliferation is a crucial indicator for comprehending the mechanisms and pathways involved in the survival of cells or death following exposure to toxic substances. The methods used to assess viability are typically the same as those used to detect cell proliferation. Cell cytotoxicity and proliferation assays are commonly employed in drug evaluations to determine if the test compounds impact cell proliferation or exhibit direct cytotoxic effects. Cell viability can be determined by calculating the ratio of total live cells to the total number of cells (live and dead). Staining also helps to observe the general cell structure. Cell proliferation assay by MTT. Metabolically active cells reduce the yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) through dehydrogenase enzymes to produce NADH and NADPH. The purple formazan produced inside the cell can be dissolved and measured using spectrophotometry. The MTT Cell Proliferation Assay quantifies cell proliferation rate and detects decreases in cell viability caused by metabolic events leading to apoptosis or necrosis. The MTT Reagent produces minimal absorbance values when no cells are present.

A key challenge for the years to come is feeding a rapidly growing human population while lowering the impact of food production on the environment. This is particularly true for low and middle-income countries where the demand for animal protein is likely to rise and further increase several folds in existing environmental changes (e.g., droughts, floods, extensive wildfires) have in recent years led to major food crises. To achieve these goals, food production not only needs to be increased, but most of all, good husbandry practices must be followed to reduce its negative impacts on the environment. Several studies have suggested that shifting the human diet towards increased consumption of f ish and seafood could be a solution to the need for protein that would sustain human and environmental health. In fact, fish and seafood consumption is forecast to increase by 27% on the horizon of 2030, mostly sustained by the aquaculture sector, which is expected to grow by 62% during the same period. The aquaculture industry contributes significantly to livelihood, food security, and poverty alleviation, with over 100 million people estimated to rely on aquaculture for their living. Fish is a cheap source of animal protein, and the current per capita consumption of fish in India is around 9 kg per annum, which is compared to the 11 kg recommended by the World Health Organisation (WHO). Presently, the contribution of the fisheries sector to the gross domestic product (GDP) and agriculture GDP has been estimated to be 1.2 and 4.2%, respectively.

Clinical Symptoms

• Gasping for air

• Discoloured gills with trapped materials

• Swollen gills with enhanced mucous secretion

• White to grey spots on the gills

• Diseased fish gather near the outlet of the pond

• Fish become lethargic and remain on the surface waters

Causative Agent: Flavobacterium branchiophilum, Cytophaga spp., Flexibacter spp.

Host (Common sp.)

• Goldfish • Koi carp

• Cichlids • Fighter fish

• Gourami fish

Clinical Symptoms • External haemorrhages

• Pale gills • Ascites

• Destruction of tissues in the kidney, spleen, and liver

• Excessive mucus secretion

• Skin hemorrhages with edema of the tissues

• Skin ulceration around the mouth and base of the fins

Causative Agent: Rhabdovirus

Host (Common sp.)

• Common carp

• Koi carp

• Crucian carp

• Silver carp

• Bighead carp

• Grass carp

• Goldfish

Clinical Symptoms

• Anchor worms can be seen with the naked eye

• Frequent rubbing or “flashing”

• Localised redness

• Inflammation on the body of the fish

• Tiny white-green or red worms in wounds

• Breathing difficulties

Clinical Symptoms

• White/brown cotton-like foci on the surface of the skin and/or gills

• Peritonitis

• Extensive haemorrhage • Necrosis

• Adhesions

Introduction and Scope of Biochemistry

Biochemistry is the field of study that focuses on studying chemical reactions occurring within living organisms. It is a specialized branch that combines the principles of both chemistry and biology. In order to comprehend the molecular mechanisms behind every biological event, this multidisciplinary f ield incorporates elements of biology, chemistry, and physiology. The scope of biochemistry extends from the study of small molecules and their interactions within cells to the analysis of complex metabolic pathways, enzyme kinetics, and the structure and function of biomolecules.

One of the main goals of biochemistry is to elucidate the chemical reactions that take place in living systems and understand how they regulate key biological processes. Biochemists investigate the structure, function, and properties of various biomolecules, such as proteins, carbohydrates, lipids, and nucleic acids. It helps us to study how these biomolecules interact and contribute to the overall functioning of cells, tissues, and organisms.

Biochemistry also plays a vital role in various areas of research and practical applications. For instance, in medical biochemistry, scientists study the molecular basis of diseases to develop diagnostic tests and therapeutic interventions. They investigate the biochemical mechanisms underlying genetic disorders, cancer, metabolic, and infectious diseases. Thus, understanding the biochemical basis of diseases can lead to the development of targeted and personalized treatments.

Carbohydrates are the macronutrients consisting of carbon (C), hydrogen (H), and oxygen (O) atoms. In simpler terms, carbohydrates can be defined as polyhydroxy aldehydes or ketones or substances, which yield them on hydrolysis. The general formula of carbohydrates is Cn (H2 O)n , where n is the number of carbon atoms. In nature, carbohydrates are the most abundant biological molecules. In the human diet, carbohydrates are one of the main nutrients that provide energy to the body. The green plants, through the process of photosynthesis, synthesize the carbohydrates.

Classification of carbohydrates

Carbohydrates are also known as saccharides, originating from the Greek word sakcharon, which means sugar. However, not all saccharides have a sweet taste. Carbohydrates can be classified into three distinct groups: monosaccharides, oligosaccharides, and polysaccharides. The number of sugar units determines this classification.

Monosaccharides

Monosaccharides, or simple sugars, are the most basic carbohydrates. They are the monomers that serve as the building blocks for forming polymers or complex carbohydrates. Monosaccharides are the most simple carbohydrates possessing carbonyl groups, i.e., aldehyde (CHO) and ketone (C=O) groups with two or more hydroxyl (OH) groups. Monosaccharide has the generic formula Cn (H2 O)n, where n is the number of carbon atoms. Monosaccharides are sugars that cannot be hydrolyzed further to form simple carbohydrates. These crystalline solids without color dissolve in water but remain insoluble in non-polar solvents.

Lipids are a class of organic compounds composed of hydrogen (H), carbon (C), and oxygen (O) atoms. These classes of biomolecules come together to create the fundamental building blocks that shape and govern the structure and functionality of living cells. The term lipid was used by the German biochemist Wilhem Bloor in 1943. Lipids are heterogeneous groups of substances that are insoluble in polar (water) and soluble in non-polar (ether, benzene, and chloroform) solvents. Lipids are the esters of fatty acids and their derivatives. Lipids play a crucial role in both the plant and animal domains. They are widely distributed in plants, but lipids are primarily stored in animal adipose tissues.

Classification of lipids

Lipids are classified into four different groups, as shown below:

1. Simple lipids

2. Complex lipids

3. Derived lipids and

4. Miscellaneous lipids

Amino acids are organic molecules made up of carbon (C), hydrogen (H), oxygen (O), and nitrogen (N). However, sulfur (S) and selenium (Se) are also found in some cases. Amino acids are essential organic compounds that come together to create proteins, thus making them the fundamental building blocks of proteins. These biomolecules play vital roles in various biological and chemical processes within the human body and are essential for the growth and development of an individual.

Amino acids consist of an acidic group (-COOH), a basic group (-NH2 ), and a side chain (R) or a hydrogen (H) atom, all bonded to the carbon atom. The features of amino acids are due to the side chain because they differ only in their side chain. All amino acids have the same basic structure except proline, which has a cyclic structure.

Various forms of amino acids like alpha (α), beta (β), gamma (γ) or delta (σ) form are found. The position of the amino group (-NH2 ) determines the form of the amino acid, i.e., if the NH2 group is attached to the α-carbon, it is called α-amino acid; if β-carbon, it is called β-amino acid, and so on. Mostly, α-Amino acids are found as the numbering of amino acids is done from the carboxylic group (-COOH).

Proteins are composed of amino acids and are large molecules that play a crucial role in various biological processes. They are considered the fundamental building blocks of life due to their abundance in the body, constituting approximately 60% of the dry weight of cells. These complex biomolecules are intricately folded and twisted strands of amino acids, actively participating in vital life processes, including metabolism, locomotion, immune response intercellular signaling, and molecular identification. As macronutrients proteins are present in all living organisms and directly contribute to numerous metabolic pathways. It is important to note that proteins are unique to each organism, exhibit species specificity, and vary within different organs of the same organism. With 22 different amino acids, the properties of a protein molecule are determined by the specific arrangement of these amino acids. While plants can synthesize all the necessary amino acids for protein production, animals rely on external sources for certain amino acids. Proteins consist of amino acids connected through peptide bonds, formed by the linkage between the amino (NH2 ) group of one amino acid and the carboxyl (COOH) group of another amino acid.

Nucleic acids are complex, chain-like polymers consisting of nucleotides as their monomers. Due to this characteristic, they are often referred to as polynucleotides. The largest molecules present in the biological system are nucleic acids. The two main types of nucleic acids are DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). The bulk of DNA is confined to the nucleus and some cell organelles like mitochondria and chloroplast. RNA is found in both the nucleus and cytoplasm. In 1869, Johannes Friedrich Miescher isolated a substance from the nuclei of the pus cell (leukocytes). The extracted material was named nuclein, which contained an unusually large amount of phosphorus; in addition to proteins, and devoid of sulfur. Richard Altman later named the nuclein as nucleic acid. These organic molecules are found in living cells and play a crucial role in transmitting genetic information from one generation to another.

Nucleotides

Nucleotides serve as the fundamental constituents of nucleic acids. Nucleotides have flat planar and heterocyclic origin. Nucleotides are small molecules composed of three components:

1. Nitrogenous base,

2. Pentose sugar and

3. Phosphate group.

Enzymes are biocatalysts that play a crucial role in accelerating chemical reactions without undergoing any changes themselves. Enzymes enhance the rate of a reaction by reducing its activation energy without affecting the equilibrium position of the reaction. These catalysts specifically target molecules called substrates and convert them into different molecules known as products. Most enzymes have a proteinous nature, and some have a ribonucleic acid nature (like ribozymes and peptidyl-transferase), showing high substrate and stereo-specificity. Enzyme catalysis is indispensable for the majority of metabolic processes in cells, as it ensures that these processes occur at a rate that is sufficient to sustain life. The progression of metabolic pathways heavily relies on the catalytic activity of enzymes to facilitate individual steps.

The proteinous enzymes are classified into simple and conjugated enzymes.

1. Simple enzymes: Enzymes are made up of only protein components.

2. Conjugated enzymes: Enzymes comprise both protein (apoenzyme) and non-protein components (cofactor).

Vitamins are natural and indispensable nutrients that are essential for our bodies in small quantities. Vitamins are indispensable for a multitude of biological functions, including growth, development, wound healing, maintaining healthy bones and tissues, supporting the immune system, and more. These vital organic compounds exhibit diverse biochemical functions and are essential for metabolic processes. The discovery of vitamins dates back to 1912 when Casimir Funk, a Polish American biochemist, initiated research on their sources, functions, and deficiency disorders. Because of his groundbreaking work, Funk is widely recognized as the pioneer of vitamins.

Types of vitamins

The classification of vitamins is determined by their solubility, resulting in two distinct groups: fat-soluble vitamins and water-soluble vitamins.

Fat-soluble vitamins

Fat-soluble vitamins, as their name implies, need fat for absorption and are stored in the fat cells. These vitamins include Vitamin A, D, E, and K.

Ultrasound is important tool for diagnosis and research that can be compared with other tools like radio-immunoassays. This has become an important tool applicable to farm and pet animals. Ultrasound uses high-frequency sound waves to produce images of soft tissues and internal organs. Electric current is applied to crystals in the transducer, producing vibrations characteristic of the crystals and resulting in sound waves. The sound beams that pass through the tissues are quite thin and a thin “slice” of tissue is sampled. The two dimensional image produced by ultrasound resemble to histological section. The sound waves are propagated or reflected by various tissues in different ways depending on their thickness and composition. The transducer receives those reflections and converts into electric signals and produces an echo on the ultrasound screen. Grey - scale is the term used for monochrome monitors on which a number of grey shades are distinguishable between black and white and constitute the image of reflected portion of tissue. Non-ecogenic (non echoic) tissues like fluids do not reflect sound waves and appear black on the screen. The echogenic tissues, having dense consistency, appear white as they reflect most portion of sound beam. Sometimes the artifacts create problem in the real image of the organ. These artifacts are created by certain tissues that cause bending or back and forth bouncing or blocking of sound beams. For imaging the reproductive tract of the domestic animals, B-mode (brightness modality), real time scanners are used. The amplitude of returning beams is reflected in the brightness of the dots on the screen. B-mode modulates brightness of the dots or pixels on the screen of echo displayer. The echo signals are presented as brightness of pixel corresponding to the amplitude of signal. The picture should be adjusted for clear display. In B-mode display the time-base brightness should be reduced just below the threshold of visibility so that even the smallest echoes could be visible.

Ultrasonography is the process of obtaining images of an area, organ or tissue with the help of ultrasound beam that can be displayed on a screen. Such images can be recorded on videocassettes or printed on a thermal paper. Ultrasonography in veterinary practice is a revolutionary step for diagnosis of various ailments and to assess normal physiological functions of the body organs of domestic animals. Ultrasonography is considered a wonderful tool particularly for the study of reproductive functions viz. follicular dynamics, pregnancy diagnosis and abnormalities of contents of tubular tract. In 1980, Palmer and Driancourt reported for the first time, real time ultrasonography for diagnosis of pregnancy in the mare. This report generated considerable interest among veterinarians and scientists about ultrasonography and this new discovery contributed significantly towards understanding of functioning of the uterus and ovaries of mare (Chevalier and Palmer, 1982; Ginther, 1983; Ginther and Pierson, 1984a & 1984b), cows (Pierson and Ginther, 1988, Reeves et al., 1984) and other species (Buckrell, 1988; Driancourt, 1991). The ultrasonography is fast replacing the age-old procedure of rectal palpation of genital tract for pregnancy diagnosis (Pieterse et al., 1990). This technique has been used and advocated for the use in the academics and field practice.

Most of ultrasound machines are designed for imaging with a particular resolution linear and convex transducers. These machines can be used for general purpose for abdominal, obstetrics, gynecological, small parts, urology and cardiac applications. Most of the machines have standard measurement software viz. distance, area, volume, circumference, and obstetrical gestational age measurement for BPD, HC, AC, FL, GS, CRL, AD and FW.

Assessment of probes: One should examine the probe whether it is linear array or curved (convex) array. The probe should also be checked for its frequency. These days multi-frequency probes are supplied with the ultrasound-machine. These multiple frequencies could be 2.0 to 5.0 MHz with centrally fixed frequency of 3.5 MHz. The frequencies could be 4.0 to 7.0 (5.0 MHz setting), 5.0 to 9.0 MHz (7.5 MHz setting). The area and depth covered by various frequencies should be known.

Before setting the ultrasound machine, one should make sure of all the auxiliary devices for the machines. These are Thermal video printer, Ultrasound gel, Biopsy needle, image saving devices (USB device or internal saving mechanism), wire, Uninterrupted Power Supply etc.

Identifying and knowing the main system: One should note down the name and model of ultrasound machine as these are quoted in all the documents published with the ultrascan images generated on that particular machine. It is written on one side of front panel. On the front panel, there are various knobs for proper setting. One should locate the auxiliary power switch of the machine to begin with. Generally, there is power indicator on the machine that shows light on when power button is pushed to on position. The auxiliary power switch may also have indications of its ON and OFF positions, may be marked with 0 and 1 or other symbols. As shown in the figure below, there are various operating buttons on the operating panel. These might vary from Machine to Machine.

In order to evaluate the correct status of the organs their correct disposition within the body becomes very essential. Although one cannot see the organs beyond the skin, but appropriate knowledge of the topography of the organs can act as a glass covered watch wherein one can see from outside the arms and the dial of the watch.

Horse

Heart: The heart is placed in the middle mediastinum of the thoracic cavity with its long axis directed caudo-ventrally. Three fifth of the heart lies on the left of median plane while two fifth is on the right of the median plane. The base is placed dorsally, with its highest point at the junction of the dorsal and middle thirds of dorso-ventral diameter of the thorax. As a whole it lies opposite to second intercostal space/third rib to sixth rib/sixth intercostal space. The apex of the heart lies in the center of the last segment of the sternum about half inch from the sternal part of the diaphragm. Heart is directly related to the lateral thoracic wall at the cardiac notches formed in the lungs. The left surface of the heart extends from third to sixth rib in the lower third of chest while the right surface extends from third to fourth intercostal space.

The affections of abdominal cavity incidence wise are on the top followed by thoracic cavity. Both these affections lead to high morbidity and heavy mortality losses. Most thoracic affections produce almost similar clinical signs but abdominal affections can show different clinical signs. Radiography and ultrasonography play an important role in differentiation and confirmation of various affections.

Radiography is an important diagnostic tool for both thoracic and abdominal diseases in small animals but for large animals it is more useful for thoracic affections as compared to abdominal affections. In large animals, the use of radiography is limited up to the ventral aspect of the abdomen only i.e. for diagnosis of diseases of reticulum, diaphragm and sternebrae. In thoracic cavity, there are numerous diseases that need to be differentiated. Pneumonia, lung cyst, lung abscesses, pleuritis and pleural effusions are some important diseases apart from presence of foreign bodies. In such a situation, radiography can play an important role as an aid in the diagnosis of various affections.

Similarly ultrasonography has also played a major role in the diagnosis of abdominal diseases in small animals such as dogs and newborn calves especially in the field of G I tract and urinary system. The ultrasonography is advantageous over radiography in the sense that internal anatomy of the soft organs can be better known and studied by ultrasonography as compared to radiography. It is also considered to be extremely safe for both the patient and the operator.

General Instructions

The monitor of ultrasound machine should be placed at the eye-level. The daylight should not be too bright. The setting of the machine should be done to obtain best picture on the screen. There are various knobs on the machine to set contrast and brightness. There are three other knobs that are used to set near gain, far gain and overall gain. There is also a brightness bar (help-bar) on the screen with different brightness areas. These knobs should be turned clockwise or anti-clockwise, so that all the areas on the help bar (brightness bar) are visible.

Transrectal Procedure

In cattle, trans-rectal ultrasonography is done in a way, similar to rectal examination done for palpation of genital organs. Following steps are suggested for proper procedure

1. Properly restrain the animal.

2. Evacuate the feces from the rectum.

3. Select a transducer of proper frequency (the linear array transducer of 5.0 MHz frequency is useful for general purpose in bovines, however for detailed examination of early conceptus and small follicles, a transducer of 7.5 MHz can also be used. Some transducers have range of these frequencies in single-equipment. Such transducers of switch-able frequency are found more useful.

Failure to accurately diagnose ovarian function is said to be one of the reason for poor reproductive efficiency in this species. The information regarding ovarian function can be gathered either by observing the reproductive behavior of the animal, status of reproductive hormones or palpation of ovaries per rectum for the presence of cyclic or pathological structures. However, due to poor expression of estrus signs behavioral observations poorly reflect the accurate information about ovarian function in buffaloes. Similarly, palpation per rectum does not provide reliable diagnosis since the CL is frequently embedded deep in the ovarian stroma of buffalo ovary and findings vary due to its subjective nature. Furthermore, measurement of hormones requires sophisticated laboratory procedures/ instruments for making a correct diagnosis. Therefore, real time ultrasonic examination is a better alternative to ascertain reproductive status of buffaloes and for devising suitable strategies to optimize fertility of this species (Sharma, 2003). However, it is important to mention that information gained from ultrasound must be used in conjunction with reproductive history of the animal, palpation of genital tract, and other diagnostic procedures rather than in isolation.

The recent availability and use of ultrasonography in veterinary science has revolutionized the study of bovine reproduction. The information- gathering capabilities of this technology are much higher than those of conventional rectal palpation. Ultrasonic imaging has provided new insights into the complex biology of various reproductive processes such as folliculogenesis and early fetal development. With the pressure on agricultural land, due to ever increasing human population, the grazing grounds for animals are fast squeezing and as a result more and more animals are being stall-fed. This practice has adversely affected the availability of essential nutrients to the animals and their reproductive efficiency. The problem is graverin small states, where pressure on land is more to accommodate the expanding developmental and commercial activities of the national capital situated next door. The routine applications of ultrasonography include evaluation of animals for pregnancy, twinning, cyclic structures on ovaries, uterine and ovarian pathologies etc.

Ultrasound is mainly used for reproductive purposes in bovine, now days it is also used for other applications. Ultrasound is an excellent tool for diagnostic imaging in large animals because it is portable, easy to use in the field, produces results at the time of the examination. It is totally non-invasive. Palpation of a gland can only detect certain levels of abnormality. Ultrasound can give an actual perception of mammary health, a picture of the inside anatomy and pathology. Tumors, scarring, stones, trauma, hematoma formation and level of damage due to mastitis can all be evaluated by this technique. Other diagnostic tools are still primary diagnostic tools, ultrasonogram gives yet another dimension to the hard to diagnose case. It will also allow a better estimate of treatment prognosis to aid in deciding whether to cull or treat a dairy animal.

Congenital anomalies at initial stages of the mammary gland development during gestation may result into presence of supernumerary teats or less number of external teats, in place of usual presence of four teats in buffaloes. Hence, cases with two / three / five or six teats are often encountered in the f ield.

Congenital anomaly of newborn buffalo calves is quite common in new born calves. Most common encountered are atresia ani, rectovaginal fistula etc. Sometimes, developmental defects interfere with physiological processes viz. in urination and defecation. Therefore, such cases, if not corrected well in time can cause death of the patient. In the present report, few newborn calves were brought to the veterinary clinic with the following abnormalities:

Agenesis of Urogenital System Without Anus

Five newborn buffalo calves were brought to the veterinary clinic with the complain of not passing urine and feces. There was no visible external genitalia and anal opening. At the site of vulva, there was only an impression of vulvar lips and below it, a cord like structure was hanging.

There was an impression of round structure below the skin; in some cases it was outside giving appearance of scrotum which was located in between the two thighs. On ventral side of abdomen, preputial sheath was not present, therefore, it was very difficult to decide about the sex of the calves and site of external preputial-orifice. There was an indication of both sexes, but organs were not developed, therefore it was difficult to decide about the sex of the calves. In some cases, impression of the anal sphincter was visible but there was no visible external anal opening. There was slight deviation of the tail from its base also. Intake of milk was normal. Abdomen was distended bilaterally. Respiratory rate was higher than the normal with slight discomfort .Heart rate was elevated. Blood picture revealed increased total blood cell count with neutrophilia. Radiography of the abdomen showed distended urinary bladder at the floor of pelvis indicating formation of the urogenital system without development of the external opening. Barium meal study revealed marking of gastrointestinal tract and stagnation of the barium up to the rectum.