Viruses are small, acellular particles that can replicate only in a host cell. They are obligatory intracellular parasites.They
consist of a nucleic acid genome enclosed in a protective protein shell or capsidBacteriophage is the virus that infect bacteria.Bacteriophages were discovered by Frederick Twort(1915)and Felix d'Herelle(1917).
Viruses are small, acellular particles that can replicate only in a host cell. They are obligatory intracellular parasites.They
consist of a nucleic acid genome enclosed in a protective protein shell or capsidBacteriophage is the virus that infect bacteria.Bacteriophages were discovered by Frederick Twort(1915)and Felix d'Herelle(1917).
INTRODUCTION:
The first plant virus shown to have a DNA genome and the first shown to replicate by reverse transcription.
Worldwide but only causes significantly losses locally.
It is transmitted by aphids .
Type member of the Caulimovirus genus, contains 11 species and 6 possible members.
significantly impact on plant virology and plant molecular biology.
The virus is an important source of gene regulatory elements, used exclusively in the genetic manipulation of plants.
STRUCTURE:Icosachedral with a diameter of 52Â nm built from 420 capsid protein subunits.
It contains a circular double-stranded DNA molecule of about 8.0 kB .
Dna is interrupted by sitespecific discontinuties resulting from its replication by reverse transcription.
After entering the host, the single stranded nicks in the viral DNA are repaired, forming a supercoiled molecule that binds to histones.
DNA is transcriped into a full length .
Replication
Risk Factors:The Cauliflower mosaic virus promoter (CaMV 35S) is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant. It is inserted into transgenic plants in a form which is different from that found when it is present in its natural Brassica plant hosts. This enables it to operate in a wide range of host-organism environments which would otherwise not be possible.
A bacteriophage (informally, phage) is a virus that infects and replicates within a bacterium. The term is derived from "bacteria" and the Greek (phagein), "to devour". Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and may have relatively simple or elaborate structures. Their genomes may encode as few as four genes, and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm. Bacteriophages are among the most common and diverse entities in the biosphere.
Phages are widely distributed in locations populated by bacterial hosts, such as soil or the intestines of animals. One of the densest natural sources for phages and other viruses is sea water, where up to 9×108 virions per milliliter have been found in microbial mats at the surface,] and up to 70% of marine bacteria may be infected by phages. They have been used for over 90 years as an alternative to antibiotics in the former Soviet Union and Central Europe, as well as in France. They are seen as a possible therapy against multi-drug-resistant strains of many bacteria (see phage therapy). Nevertheless, phages of Inoviridae have been shown to complicate biofilms involved in pneumonia and cystic fibrosis, shelter the bacteria from drugs meant to eradicate disease and promote persistent infection
Poxviruses are brick or oval-shaped viruses with large double-stranded DNA genomes. Poxviruses exist throughout the world and cause disease in humans and many other types of animals. Poxvirus infections typically result in the formation of lesions, skin nodules, or disseminated rash.
Largest viruses that infect vertebrates
Can be seen under light microscope
Poxvirus diseases are characterized by skin lesions – localized or generalized
Important diseases caused by poxviruses are-
Smallpox
Monkeypox
Cowpox
Tanapox
Molluscum contagiosum
Direct methods of measurement of microbial growth includes various methods of enumeration of both viable and non viable cell also includes growth curve. Helpful for UG and PG programs of microbiology
Mechanism of pathogenicity-Exotoxin and endotoxinaiswarya thomas
Brief description on mechanisms of pathogenicity, actions of toxins produced by various bacteria and notable endotoxins and exotoxins. Mechanism of action of some of the commonest endotoxins and exotoxins are explained.
INTRODUCTION:
The first plant virus shown to have a DNA genome and the first shown to replicate by reverse transcription.
Worldwide but only causes significantly losses locally.
It is transmitted by aphids .
Type member of the Caulimovirus genus, contains 11 species and 6 possible members.
significantly impact on plant virology and plant molecular biology.
The virus is an important source of gene regulatory elements, used exclusively in the genetic manipulation of plants.
STRUCTURE:Icosachedral with a diameter of 52Â nm built from 420 capsid protein subunits.
It contains a circular double-stranded DNA molecule of about 8.0 kB .
Dna is interrupted by sitespecific discontinuties resulting from its replication by reverse transcription.
After entering the host, the single stranded nicks in the viral DNA are repaired, forming a supercoiled molecule that binds to histones.
DNA is transcriped into a full length .
Replication
Risk Factors:The Cauliflower mosaic virus promoter (CaMV 35S) is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant. It is inserted into transgenic plants in a form which is different from that found when it is present in its natural Brassica plant hosts. This enables it to operate in a wide range of host-organism environments which would otherwise not be possible.
A bacteriophage (informally, phage) is a virus that infects and replicates within a bacterium. The term is derived from "bacteria" and the Greek (phagein), "to devour". Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and may have relatively simple or elaborate structures. Their genomes may encode as few as four genes, and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm. Bacteriophages are among the most common and diverse entities in the biosphere.
Phages are widely distributed in locations populated by bacterial hosts, such as soil or the intestines of animals. One of the densest natural sources for phages and other viruses is sea water, where up to 9×108 virions per milliliter have been found in microbial mats at the surface,] and up to 70% of marine bacteria may be infected by phages. They have been used for over 90 years as an alternative to antibiotics in the former Soviet Union and Central Europe, as well as in France. They are seen as a possible therapy against multi-drug-resistant strains of many bacteria (see phage therapy). Nevertheless, phages of Inoviridae have been shown to complicate biofilms involved in pneumonia and cystic fibrosis, shelter the bacteria from drugs meant to eradicate disease and promote persistent infection
Poxviruses are brick or oval-shaped viruses with large double-stranded DNA genomes. Poxviruses exist throughout the world and cause disease in humans and many other types of animals. Poxvirus infections typically result in the formation of lesions, skin nodules, or disseminated rash.
Largest viruses that infect vertebrates
Can be seen under light microscope
Poxvirus diseases are characterized by skin lesions – localized or generalized
Important diseases caused by poxviruses are-
Smallpox
Monkeypox
Cowpox
Tanapox
Molluscum contagiosum
Direct methods of measurement of microbial growth includes various methods of enumeration of both viable and non viable cell also includes growth curve. Helpful for UG and PG programs of microbiology
Mechanism of pathogenicity-Exotoxin and endotoxinaiswarya thomas
Brief description on mechanisms of pathogenicity, actions of toxins produced by various bacteria and notable endotoxins and exotoxins. Mechanism of action of some of the commonest endotoxins and exotoxins are explained.
LD 50 is a certain dose is expressed in milligrams of test material weight per kilogram of body weight (BW) test animals that produce 50% mortality in the population response of test animals within a certain time frame.
LC 50 is the median lethal concentration or concentration of a substance in a water that can cause death is estimated at 50% of the population of a particular test organism. LC 50 also illustrates the relationship between several concentrations of test solution with the response (mortality) amounted to 50% of the population in the test biota in the specified time period.
CT or chronic toxicity is the toxicity test that includes observation of stimuli that can obstruct or interfere with the test organism's life is continuously in a relatively long period of time. Chronic toxicity tests should consider things related to life events such as the test organism growth, reproduction etc
Viruses are obligate intracellular parasites so they depend on host for their survival. They cannot be grown in non-living culture media or on agar plates alone, they must require living cells to support their replication.Cultivation of viruses can be discussed under following headings:
Animal Inoculation
Inoculation into embryonated egg
Cell Culture
Viruses are obligate intracellular parasites which means they can only grow or reproduce inside a host cell.
The primary purpose of virus cultivation:
To isolate and identify viruses in clinical samples.
To do research on the viral structure, replication, genetics, and effects on the host cell.
To prepare viruses for vaccine production.
Isolation of the virus is always considered a gold standard for establishing the viral origin of the disease
topics covered
CULTIVATION OF VIRUSES
Animal inoculation
Embryonated eggs
CAM
Allantoic cavity
Amniotic cavity
Yolk sac
Tissue culture
Organ culture
Explant culture
Cell culture
Primary cell culture
diploid cell culture
Continues cell lines
viruses are intracellular obligate parasites. They are either DNA or RNA viruses. In order to grow in labs, tissue culture is used. Some general characteristics of viruses are discussed here.
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
263778731218 Abortion Clinic /Pills In Harare ,sisternakatoto
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Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
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Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
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NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
3. INTRODUCTION
Viruses are obligate intracellular organisms
Must be grown in living cells.
They can't be grown in culture media or on agar plates alone, they
must have living cells to support their replication.
The easiest viruses to grow are bacteriophages…..WHY?
Animal viruses – difficult, due to the properties of the animal host
Under natural conditions, many viruses are relatively host-specific.
Moreover, they may show a marked predilection for certain tissues of
the host such as nervous tissue, epithelial tissue etc.
The majority can be adapted to foreign hosts by passage
4. VIRUS CULTIVATION
Also known as viral propagation or growth.
Necessary to supply the virus with appropriate cells in
which it can replicate.
Phages are supplied with bacterial cultures.
Plant viruses may be supplied with specially cultivated plants
or with cultures of protoplasts (plant cells from which
the cell wall has been removed),
Animal viruses may be supplied with whole organisms, such as mice,
eggs containing chick embryos, insect larvae or animal cells.
Viruses can be isolated from different specimens.
5. SPECIMENS USED TO ISOLATE VIRUSES
-Blood
specimens
EDTA
Heparin
Serum
-Stool
-Throat swabs
-Naso-
-Stools, rectal swabs
-Urine
-Saliva
-Cerebro-spinal fluid
-Biopsy
Skin (filoviridae)
Organs (fixation
with formaldehyde
10%)
6. PURPOSE OF VIRUS CULTIVATION
The primary purposes of viral cultivation are:
1. To isolate and identify viruses in clinical specimens
2. To prepare viruses for vaccines
3. To do detailed research on viral structure, multiplication
cycles, genetics, and effects on host cells.
7. VIRUS CULTIVATION SYSTEMS
Tissue culture system
Embryonated eggs system
Whole animal systems
a) Natural host
b) Experimental animals
c) Transgenic animals
8. TISSUE CULTURE SYSTEM
HISTORY OF CELL CULTURE
Cultured cells could only survive for a few days.
In 1951, cells taken from Henrietta Lacks (cervical cancer
patient)
Cell line was found to be remarkably durable and prolific.
George Gey was able to isolate one specific cell, multiply it,
and start a cell line.
Named the sample HeLa. First human cells grown in the lab
that were immortal.
The use of the antibiotics, chemically defined medium and
use of trypsin greatly enhanced the cell culture technique.
9. HeLa cells cont….
1.polio vaccine
2. Gardisil developed by studying HeLa
cells
3.MMR vaccine.
4. Understanding of TB, HIV, HPV-HeLa
cells have been used to understand how
these diseases impact cells
5. Human chromosome number and
mitosis-scientists used HeLa cells to
determine the exact number of
chromosomes in
10. TISSUE CULTURE SYSTEM cont….
Use isolated cell from animal that are cultured invitro.
It is the preferred type of growth medium for viruses.
• Three discoveries greatly enhanced the usefulness of cell cultures for
virologists and scientists
1. The discovery and use of antibiotics made it possible to prevent bacterial and
fungal contamination
2.The discovery of proteolytic enzymes (e.g. trypsin) can free animal cells from
surrounding tissues without injuring freed cells
3.This technique has also become possible by the development of growth media
for animal cells.
11. STEPS IN TISSUE CULTURE TECHNIQUE
Cultivating animal viruses using tissue culture technique
involves following three main steps:
1. Monolayer preparation
2. Clonal cell line preparation
3. Infection with virus
The first two steps are summarised with the notes on cell
culture in the next slides.
12. 1.Monolayer and clonal cell line preparation
CELL CULTURE
Cell culture refers to the removal of cells from an animal or plant
and their subsequent growth in a favourable artificial
environment.
The cells may be removed from the tissue directly and
disaggregated by enzymatic or mechanical means before
cultivation, or they may be derived from a cell line or cell strain
that has already been already established.
Can be classified under the following cell lines.
i) Primary culture
ii) Diploid cell lines
iii) Continuous cell lines
13. PRIMARY CULTURE
Primary culture refers to the stage of the culture after the cells are
isolated from the tissue and proliferated under the appropriate
conditions until they occupy all of the available substrate (i.e., reach
confluence) (e.g. Primary monkey kidney, mice fibroblasts)
-Heterogeneous – many cell types
-Technical hassle
-5 to 20 cell divisions
-Normal chromosome number
-Contact inhibition
-need constant source
-Closest to animal
15. DIPLOID CELL LINES
After the first subculture, the primary culture becomes known
as a Diploid cell line or subclone. E.g(human fetal lung)
-Lines-up to 100 cell divisions
-Homogeneous population of a single
type.
-Typically derived from tumors.
Remain diploid
-Further from animal
-Technically less hassle
16. CONTINUOUS CELL LINES
When a finite cell line undergoes transformation and acquires the ability to
divide indefinitely, it becomes a continuous cell line.
Become immortal through a process called transformation.
Can occur spontaneously or can be chemically or virally induced.
-Immortal
-Most homogeneous
-Genetically weird – furthest from animal
-Hassle free
-Suspension or monolayer
-Aneuploid- abnormal in chromosome
morphology and number, Grow rapidly.
(e.g.various types of cancer cells - HeLa
cells, Hep 2 cells, or human amnion cells,
continual monkey kidney cell line, dog kidney
cell line, etc.).
18. 3.Infection with virus
The clonal cell lines suspended in suitable media
are infected with any desired virus which
replicates inside the multiplying cells. If the virus
is virulent, they cause lysis of cells and virus
particles are released in the surrounding medium.
These newly produced virus particles (virions)
infect the adjacent cells. As a result localized
areas of cellular destruction and lysis (called
plaques) often are formed
19. CULTURE CONDITIONS
Culture conditions vary widely for each cell type.
The artificial media invariably consist of a substrate or medium that
supplies the essential nutrients (amino acids, carbohydrates,
vitamins,minerals), growth factors, hormones, and gases (O2, CO2).
It also regulates the physicochemicalenvironment (pH, osmotic
pressure, temperature).
Most cells are anchoragedependent and must be cultured while
attached to a solid or semi-solid substrate(adherent or monolayer
culture),
Others can be grown floating in the culture (suspension culture)
21. Differences between Adherent and
Suspension Cultures
ADHERENT CULTURES SUSPENSION CULTURES
Most cells can be cultured this way Cells which are adapted to suspension
cultures or non-adhesive cultures
Passaging required at certain intervals Passaging is much easier, can dilute
culture to stimulate growth
Allows easy visualisation of cells Harder to view cells
Cells dissociated enzymatically or
mechanically
Not require enzymatic or mechanical
dissociation
Surface area limits growth Cell concentration in medium limits growth
Used for cytology, harvesting products Used for bulk protein production, batch
continuously and also research harvesting and also research applications
applications
23. Viral detection:
Detection of a growth of a virus is observed by the changes in the
cell culture monolayer - cytopathic effect (CPE).
CPE are Changes of morphology of cells e.g:
1. Lysis of the cells 2. Vacuolation,
3. Formation of syncytia 4. Presence of inclusion bodies
Uninfected Cell Culture Infected Cell Culture with CPE
29. Cont…
As some viruses do not cause CPE in cell lines,
they can be detected by other techniques.
Hemadsorption of erythrocytes onto cells
infected with viruses which do not form CPE and
contain hemagglutinin can be used in myxovirus
and paramyxovirus detection.
Influenza viruses can be released into the culture
medium and then detected by hemagglutination.
30. DISADVANTAGES OF CELL CULTURES
Long period (up to 4 weeks) required for result.
Often very poor sensitivity, sensitivity depends on a large extent on
the condition of the specimen.
Susceptible to bacterial contamination.
Susceptible to toxic substances which may be present in the
specimen.
Many viruses will not grow in cell culture e.g. Hepatitis B,
Diarrhoeal viruses, parvovirus, papillomavirus.
32. EMBRYONATED EGGS
INTRODUCTION
The Embryonated hen’s egg was first used for cultivation of viruses by
Good Pasteur and Burnet (1931).
Cultivation of viruses in organized tissues like chick embryo necessitates
a different type of approach..
For all practical purposes they all themselves behave as tissue cultures.
The process of cultivation of viruses in embryonated eggs depend on the
type of egg which is used.
The egg used for cultivation must be sterile and the shell should be
intact and healthy.
33. Cont…
Use embryonated chicken, duck or turkey for
inoculation of viral suspension
Are used especially for the influenza viruses
isolation.
7 - 10 days old embryonated eggs are used.
The egg must be cleaned, the shell
decontaminated with a disinfectant and checked
in ovoscope if it is alive
Ovoscope is the equipment used for candling.
43. DETECTION OF VIRAL GROWTH
The signs of viral growth include:
i) Death of the embryo,
ii) Defects in embryonic development, and
iii) Localized areas of damage in the membranes, resulting in
discrete, opaque spots called pocks (a variant of pox).
iv)The embryonic fluid and tissue can be prepared for
examination with an electron microscope.
v) Some can also be detected by their ability to agglutinate
red blood cells or by their reaction with an antibody of
known specificity that will affix to its corresponding virus, if
it is present.
46. ADVANTAGES
Isolation and cultivation of many avian and few mammalian viruses
Ideal receptacle for virus to grow
Sterile & wide range of tissues and fluids
Cost- much less
Maintenance-easier
Less labour
Readily available
48. WHOLE ANIMALS
- using live animal eg.mice, rats, rabbits, guinea pigs, hamster, chickens, and monkey.
- the animal is exposed to the virus by injection of a viral preparation or specimen into the brain, blood,
muscle, body cavity, skin, or footpads.
- use in example research to study the immune system’s response to viral infections.
- HIV: immunodeficient mice grafted to produce human T cells and human gamma globulin.
- Only system for studying pathogenesis & immune responses
- Used if it’s the only method through which the virus can be isolated.
49. ANIMAL MODEL
usually a purpose-bred animal
Mouse model
Advantages
• in-breed strains reduce genetic variability
• genetics are well understood
• Introduce, mutate or inactivation specific genes thought to control the immune response.
Disadvantages
• Sometimes not infected-therefore virus has to be adapted or use a closely related surrogate virus
• Does not always cause same disease state
• Mice are not humans
51. DETECTION OF VIRAL GROWTH
The signs of viral growth include
i)death of the animal
Ii) defects in animal development.
The infected animal tissue can be prepared for examination with an electron
microscope
52. VIRAL QUANTIFICATION
Virus quantification involves counting the number of viruses in a specific
volume to determine the virus concentration.
It is utilized in both (R&D) in commercial and academic laboratories as well as
production situations where the quantity of virus at various steps is an
important variable
The methods used include but not limited to:
i) Hemagglutination assay
ii) Plaque assay
iii)TCID₅₀
53. HEMAGGLUTINATION ASSAY
A direct method to titre virus.
Based on the ability of some viruses to agglutinate RBCs
Virus is tittered by making serial two fold dilutions of the virus and
determining the highest dilution of virus that causes agglutination of RBCs.
54. PLAQUE ASSAY
When cells grow as monolayers, they can be used to
quantify the number of viruses using plaque assay.
- The virus is serially diluted in a liquid medium.
- For each dilution a set amount is added to separate
plate containing monolayer of tissue culture cells
and the viruses in that solution are allowed to
attach to the tissue culture cells.
- After attachment has been allowed to occur, a semi
solid medium is added to restrict the movement of
new viruses produced so that only adjacent cells
will be infected.
55. CONT…
Where virus has infected the tissue culture cells, the infected
cells will die causing the formation of a clear zone amongst
the otherwise intact monolayer of cells
This clear zone is called a plaque and it theoretically
represents an area where one virus has infected a single
tissue culture cell, has multiplied and been released, and has
gone on to infect adjacent cells.
The number of plaque forming units (pfu)/ml can be
calculated based on the dilution of the original viral solution.
The term pfu/ml is used rather than the number of
viruses/ml because it is possible that occasionally more than
one virus infects a single cell.
Often the cells or plaques are stained to help in visualization
of the plaques.
59. CALCULATION OF PFU/Ml
Plaques are
enumerated
Plaque Counts are
averaged over wells
The average is then
divided by the
dilution times the
volume
(43+40+38)/3
(10-4 x 0.1)
= 3,730,000 pfu/ml
43 4 1 0
40 3 0 0
38 6 2 0
Plaques formed per well
60. TCID₅₀
TCID50 is the measure of infectious virus titer.
This endpoint dilution assay quantifies the amount of virus required to kill
50% of infected hosts or to produce a cytopathic effect in 50% of inoculated
tissue culture cells
This assay may be more common in clinical research applications where the
lethal dose of virus must be determined or if the virus does not form plaques.
When used in the context of tissue culture, host cells are plated and serial
dilutions of the virus are added. After incubation, the percentage of cell
death (i.e. infected cells) is manually observed and recorded for each virus
dilution, and results are used to mathematically calculate a TCID50 result.
61. TCID₅₀ calculation
The outcome of a TCID50 determination can be used
to estimate a virus titre in pfu, or vice versa, using the formula
100
75
50
1 TCID₅₀ = 0.7 pfu
101 102 103 104 105 106
TCID₅₀
62. Other methods for TCID50 calculation
Two methods commonly used to calculate TCID50 are:
i) Spearman-Karber
ii) Reed-Muench method