Cultivation of Viruses - Microbiology

1. Cultivation of Viruses
MICROBIOLOGY
Islam Ghassan Sarakbi : 39

2. A virus is a biological agent that reproduces inside
the cells of living hosts.
When infected by a virus, a host cell is forced to
produce many thousands of identical copies of the
original virus, at an extraordinary rate. Unlike most
living things, viruses do not have cells that divide;
new viruses are assembled in the infected host
cell.

3. Viruses 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 (because the easiest cells
to grow in the lab are bacteria).

4. On the other hand, Viruses can be cultivated
within suitable hosts, such as a living cell. To
study bacteriophages, for example, bacteria are
grown in a suitable growth medium, then
bacteriophages are added.
The bacteriophages multiply within the bacteria
and increase their numbers substantially.

5. 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.

6. Virus Cultivation Systems
 Tissue culture system.
 Embryonated eggs system.
 Whole animal systems.

7. Tissue culture system For Cultivation
Of Viruses
Animal and plant viruses are cultivated in cell
cultures. A cell culture is prepared by
encouraging cell growth outside the animal or
plant source. The cells are kept alive in a
suspension of growth factors within a Petri
dish. A thin layer of cells, or monolayer, is
then inoculated with viruses, and replication
takes place.

8. Tissue culture system For Cultivation Of
Viruses
(Fertilized eggs and living animals can also be
used to cultivate viruses.)
For research study, viruses can be cultivated
in large volumes by inoculations to tissue
culture systems. After a time, the cells are
degenerated, and viruses are harvested.

9. Growing Bacteriophages in the
Laboratory:
Bacteriophages can be grown either in suspension of
bacteria in liquid media or in bacterial cultures on solid
media.
Solid media makes possible the plaque method for
detecting and counting viruses.
A sample of bacteriophage is mixed with host bacteria
and melted agar.
The agar containing the bacteriophages and host
bacteria is then poured into a Petri plate containing a
hardened layer of agar growth medium.

10. Growing Bacteriophages In The
Laboratory (cont.) :
The viral particles are concentrated by
precipitation methods and purified by
repeated centrifugations.
Highly purified viruses can be obtained by
crystallization and concentration under
established conditions.

11. Flasks containing tissue
culture growth medium
which provides
nourishment to
growing cells.

12. The Cell Cultures
• In modern usage, tissue culture generally refers
to the growth of cells from a tissue from
a multicellular organism in vitro.
• These cells may be cells isolated from a donor
organism, primary cells, or an immortalised cell
line.
• The cells are bathed in a culture medium, which
contains essential nutrients and energy sources
necessary for the cells' survival.

13. The Cell Cultures
 The term tissue culture is often used
interchangeably with Cell Culture.
 Cell cultures have replaced embryonated
eggs as the preferred type of growth
medium for many viruses.
 The literal meaning of tissue culture refers to
the culturing of tissue pieces.

14. The Cell Cultures
Tissue culture is an important tool for the
study of the biology of cells from
multicellular organisms.
It provides an in vitro model of the tissue in a
well defined environment which can be easily
manipulated and analysed.

15. The Cell Cultures
 Contain cell lines, called diploid cell lines, developed
from human embryos can be maintained for about
100 generations and are widely used for culturing
viruses that require a human host.
 When viruses are routinely grown in the laboratory,
continuous cell lines are used. These are
transformed (cancerous) cells that can be
maintained through an indefinite number of
generations, and they are sometimes called mortal
cell lines.

16. The Cell Cultures
 This cell deterioration is called cytopathic effect
(CPE), can be detected and counted in much the
same way as plaques caused by bacteriophages on
a lawn of bacteria.
 Primary cell lines, derived from tissue slices, tend to
die out after only a few generations.

17. Cell cultures

18. 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).

19. Suspension Adherent

20. 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

21. 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.

22. Steps of cultivating animal viruses using
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.

23. 1,2) Monolayer & 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.

24. i) 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 and mice
fibroblasts).

25. ii) Diploid Cel Lines:
• After the first subculture,
the primary culture
becomes known as a
Diploid cell line or
subclone. (e.g. human
fetal lung).

26. iii) 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.

27. 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.

28. Growing Bacteriophages In Living
Animals:
Animal inoculation may be used as a
diagnostic procedure for identifying and
isolating a virus from a clinical specimen.
After the animal is inoculated with the
specimen, the animal is observed for signs of
disease, or is killed so that infected tissues
can be examined for the virus.

29. Growing Bacteriophages In
Embryonated Eggs:
Growing viruses in an embryonated egg can
be a fairly convenient and inexpensive form
of host for many animal viruses.
A hole is drilled in the shell of embryonated
egg, and a viral suspension or suspected
virus-containing tissue is injected into the
fluid of the egg.

30. Growing Bacteriophages In
Embryonated Eggs (cont.):
There are several membranes in an egg, and the
virus is injected near the one most appropriate for
its growth.
Viral growth is signaled by the death of the
embryo, by embryo cell damage, or by the
formation of typical pocks or lesions on the egg
membranes.
This method was once the most widely used
method of viral isolation and growth, and it is still
used to grow viruses for some vaccines.

32. Detection Of Viral Growth In
Embryonated Eggs:
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.

33. HARVESTED
EMBRYOS

34. Viral Measurements
Viruses are generally too small to be seen
under the light microscope, and an electron
microscope is usually necessary to make
them visible.
Once viruses have replicated and been
harvested the concentration of viral particles
(virions) in the viral stock solution must be
determined.

35. Viral Measurements
The most important method and the most
commonly used to determine the
concentration of a stock solution of
bacteriophages is the Plaque Assay (plaque
method).

36. The Plaque Essay (plaque method) :
The plaque assay is performed by cultivating
viruses on a “lawn” of host cells and noting
the presence of clear areas where viruses
have replicated and destroyed the cells (the
virus lyses the bacteria). Each plaque is
assumed to come from a single viral particle.

37. The concentration of
the stock solution of
the virus is given in
plaque forming units.

38. Viral Measurements
Another way of determining virus infectious
units is by cultivating viruses in living animals
and determining which dilution of virus is
lethal to the animals. The end-point
dilution can be calculated by this method.