This document discusses protoplast isolation and cell culture. It begins by defining a protoplast as a plant cell without a cell wall that contains normal cell organelles. Protoplasts can be isolated from actively growing tissues using mechanical and enzymatic methods. The mechanical method involves plasmolysis and cutting of tissues, while the enzymatic method uses lytic enzymes to remove the cell wall. Cell culture methods are also discussed, including primary cell cultures derived directly from tissues that have a finite lifespan, and continuous cell lines derived from tumors that can divide indefinitely. Different cell types, culture media, and tissue culture techniques are described.

1. VIROLOGY

2. Definition :
 Protoplast is a cell without a cell wall are called protoplast.
 They contain all the normal cell organelles plus the nucleus.
 The cell wall of a plant cell can be decomposed and removed by the treatment of the lytic
enzymes like cellulose and pectinase
Protoplasts can be isolated from all types of actively growing young and healthy tissues.
METHODS OF ISOLATION :
1.Mechanical method
2. Enzymatic method
3. Combination of above methods
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3. During the process of isolation, the cells are first separated by mechanical method and
subsequently protoplasts are isolated by enzymatic method
Mechanical method
 The cells are first placed in a suitable plasmolyticum .This treatment makes the
protoplasms of these plasmolysed cells shrink away from their cell walls( this makes
the removal of cell wall easy)
 Further ,they are cut with a knife.
 Then the protoplasts are released from the cells through the cell wall , and then the
tissue is again deplasmolysed.
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4. Thomas Huckle Weller Frederick Chapman Robbins
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5. Cytopathic effect
Vesicular stomatitis
Indiana virus was
modified to
attack HIV-infected
T-cells. The modified
virus was called a
"trojan horse" virus
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8. • The culture medium is the Combination of ingredients will support the cell growth by providing
all the essential nutrients , growth factors, and hormones for cell growth, as well as
regulating the pH and the osmolarity of culture.
• The three basic classes of media are: (differ in their requirement for supplementation with
serum. )
Basal
media
Serum-
free
media
Reduced-
serum
media
The choice of culture media is
dependent on the requirements of cells
being cultured.
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9. Basal (Basic) Media :
• Basal Medium is a defined medium that contains essential and nonessential amino acids, vitamins, inorganic salts,
organic compounds, and trace elements, but does not contain the Growth Supplements necessary for cell growth.
• Balanced salt solutions (BSS) e.g. phosphate-buffered saline (PBS)
• DMEM and RPMI 1640 (with or without glutamine)
Reduced-Serum Media :
• Reduced-serum media are basal media formulations enriched with nutrients and animal-derived factors, which
reduce the amount of serum that is needed.
Serum-Free Media :
• Serum-free media (SFM) circumvents issues with using animal sera by replacing the serum with appropriate
nutritional and hormonal formulations.
• Serum-free media formulations exist for many primary cultures and cell lines, including Chinese Hamster Ovary
(CHO), hybridoma cell lines, VERO, MDCK, MDBK cell lines etc.
• One of the major advantages of using serum-free media is the ability to make the medium selective for specific
cell types by choosing the appropriate combination of growth factors
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10. Types of tissue culture
Primary Continuous
Finite Indefinite
Normal cells cultured
without any change in
their division rate
Single cell type roughly
thirty times of division,
enhanced by growth
factors
It is nearly the same as
finite but the cells here
can divide indefinitely
by transformation
into tumor cells,
They are called cell line
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11. Primary cultures
Cells when surgically or enzymatically removed from an organism and placed in suitable culture
environment will attach and grow and are called as primary culture
Primary cells have a finite life span
Primary culture contains a very heterogeneous population of cells
Sub culturing of primary cells leads to the generation of cell lines
Cell lines have limited life span, they passage several times before they become senescent
Cells such as macrophages and neurons do not divide in -vitro so can be used as primary cultures
Continuous cell lines
Most cell lines grow for a limited number of generations after which they cease
Cell lines which either occur spontaneously or induced virally are chemically transformed into continous
cell lines
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12. Cell types
On the basis of morphology (shape & appearance) or on their functional characteristics. They are divided into three.
Epithelial like- Attached to a substrate and appears flattened and polygonal in shape
Lymphoblast like- Cells do not attach, remain in suspension with a spherical shape
Fibroblast like- Cells attached to a substrate, appear elongated and bipolar Dr. Farzin Asghari Sana

13. Cell Cultures/ Dispersion Cultures
Primary cell cultures
a cell line derived directly from the parent tissue. Cells in primary culture have the same karyotype and
chromosome number as those in the original tissue.
Can be subculture only one or twice e.g-primary monkey, baboon kidney
• Diploid cell strains.
Which are derived from human fetal tissue and can be subcultured 20-50 times e.g-human diploid
fibroblast such as MRC-5(human fetal lung fibroblast establish from normal lung tissue of 14 week old
male foetus)
Some primary cells can be passed through secondary and several subsequent subcultures while retaining
their original morphological characteristics and karyotype. Subcultures will have fewer cell types than
primary cultures. After 20 to 50 passages in vitro, these diploid cell strains usually undergo a crisis in
which their growth rate slows and they eventually die out. Diploid strains of fibroblasts derived from
human fetal tissue are widely used in diagnostic virology and vaccine production.
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14. Continuous cell lines. Derived from tumors of human or animals e.g- Vero, Hep2
Certain cultured cells, notably mouse fetal fibroblasts, kidney cells from various mammalian
species, and human carcinoma cells, are able to survive the growth crisis and undergo indefinite
propagation in vitro.
After several passages, the growth rate of the culture slows down; then isolated colonies of cells
begin to grow more rapidly than diploid cells, their karyotype becomes abnormal (aneuploid),
their morphology changes, and other poorly understood changes take place that make the cells
immortal. The cells are now "dedifferentiated," having lost the specialized morphology and
biochemical abilities they possessed as differentiated cells in vivo. Continuous cell lines such as
KB and HeLa, both derived from human carcinomas, support the growth of a number of
viruses. These lines and others derived from monkey kidneys (e.g., Vero), mouse fetuses (L929),
and hamster kidneys (BHK) are widely used in diagnostic and experimental virology. Continuous
cell lines have been established from many types of vertebrate and invertebrate animal tissues and
are available from the American Type Culture Collection (ATCC).
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15. Continuous Cell Line
A HeLa cell, also Hela or hela cell, is a cell type in an immortal cell line used in scientific
research. It is the oldest and most commonly used human cell line. The line was derived
from cervical cancer cells taken on February 8, 1951from Henrietta Lacks, a patient who died of
her cancer on October 4, 1951. The cell line was found to be remarkably durable and prolific
which warrants its extensive use in scientific research.
Henrietta LacksDr. Farzin Asghari Sana

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17. 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.
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18. • 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.
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21. Immunofluorescent staining with GTX36623_RSV-nucleoprotein
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22. EMBRYONATED EGGS
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.
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.

23. Francis Peyton Rous
Rous sarcoma virus
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24. SITES SUITABLE FOR CULTIVATION OF VIRUSES
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29. DRILL THE HOLE
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30. INJECT THE SUSPENSION WITH SYRINGE
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31. HOLE SEALED WITH PARAFFIN WAX
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32. EGGS INCUBATE AT 36C FOR 2-3 DAYS
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33. 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.
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34. POCK LESIONS ON CAM
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35. ADVANTAGES
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36. 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.
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37. 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
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38. 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
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39. 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
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40. • 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.

41. PLAQUE ASSAY RESULTS
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42. 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
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43. 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.
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44. 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
101 102 103 104 105 106
TCID₅₀
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45. Formation of multinucleated cells syncytium = CPE
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46. Syncytium formation induced by Murine leukemia virus
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48. VIROLOGY