Applied Veterinary Virology: The isolation and identification of viruses using cell cultures
Applied Veterinary Virology:
The isolation and identification of viruses using cell
cultures
Authors: Prof Estelle Venter
Licensed under a Creative Commons Attribution license.
TABLE OF CONTENTS
INTRODUCTION........................................................................................................................................... 3
Terminology ............................................................................................................................................. 3
SAFETY IN THE LABORATORY ..................................................................... Error! Bookmark not defined.
CELL CULTURE LABORATORY .................................................................... Error! Bookmark not defined.
Basic requirements .................................................................................. Error! Bookmark not defined.
Cleaning of tissue culture items ............................................................... Error! Bookmark not defined.
CELL CULTURE PROCEDURES .................................................................... Error! Bookmark not defined.
Cell culture media .................................................................................... Error! Bookmark not defined.
Basic salt media ....................................................................................... Error! Bookmark not defined.
Tissue culture technique .......................................................................... Error! Bookmark not defined.
Sub-culturing of cell lines ......................................................................... Error! Bookmark not defined.
Quantification of cells ............................................................................... Error! Bookmark not defined.
The use of different cell cultures .............................................................. Error! Bookmark not defined.
Contamination of cell cultures .................................................................. Error! Bookmark not defined.
Cryopreservation ...................................................................................... Error! Bookmark not defined.
Demonstration / confirmation of the presence of viruses in infected cell culturesError! Bookmark not defined.
Viral quantification .......................................................................................... Error! Bookmark not defined.
Infectivity assays for viruses .................................................................... Error! Bookmark not defined.
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Applied Veterinary Virology: The isolation and identification of viruses using cell cultures
Dose response curve and significance of ID50/TCID50 ............................. Error! Bookmark not defined.
Plaque assay............................................................................................ Error! Bookmark not defined.
STORAGE OF ANTIGEN ................................................................................. Error! Bookmark not defined.
Frequently Asked Questions ......................................................................... Error! Bookmark not defined.
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Applied Veterinary Virology: The isolation and identification of viruses using cell cultures
INTRODUCTION
Viruses cannot reproduce independently of living cells. For many years viruses were grown in natural
hosts, embryonated chicken eggs and laboratory animals. Currently many viruses are grown in
monolayers of animal cells. This technique was made possible by the development of growth media
for animal cells and the advent of antimicrobial drugs that can prevent bacterial and fungal
contamination.
Terminology
In vitro tissue culture: Tissue culture is commonly applied as a generic term that encompasses, and
is concerned with, the study of cells, tissues and organs maintained and grown in vitro.
Culture systems can be grouped into the following main categories based on their degree of
complexity and related in vivo topography:
Tissue culture: sometimes used as a specific term to denote the maintenance or growth of tissue
fragments in vitro. The original or characteristic architecture, and often the biochemical properties
associated with it, are not necessarily maintained.
Cell culture: denotes the maintenance and culture of cells in vitro including the culture of a single
type of cell. In cell cultures, the cells are no longer organized into tissues and are, therefore, usually
devoid of their histiotypic architecture and often of the biochemical properties associated with it. Cells
are cultured either as adherent mono- or multilayers on a solid substrate, or as a suspension in
culture medium. Some cells will grow in free suspension if mechanically agitated; otherwise they will
grow attached to the substrate.
Primary culture: cells derived directly from a particular organ or tissue. All such cultures are
considered primary until the first successful subculture, after which they become a serial culture.
Such cultures are usually heterogeneous and largely representative of the tissue from which they
were derived. Serial cultures, in which growth of a single histological cell type has been selected for,
are termed cell lines. These may be of continuous, established or finite nature.
Continuous cell lines are populations of cells within serial cultures which are apparently capable of
an unlimited number of population doublings and are often referred to as immortal cell lines. Such cell
lines may or may not express the characteristics of in vitro neoplastic or malignant transformation.
Established cell lines: A cell line in which the potential to be sub-cultured indefinitely in vitro has
been demonstrated.
Finite cell cultures or cell lines are capable of only a limited number of population doublings after
which the cells cease to proliferate. Both finite cell lines of sufficient doubling potential and continuous
cell lines can be propagated, expanded to produce large numbers of aliquots with characterized and
defined cell populations, and then preserved for future use by freezing.
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Applied Veterinary Virology: The isolation and identification of viruses using cell cultures
Explant: An excised fragment of a tissue or organ used to initiate an in vitro culture.
Monolayer: A single layer of cells growing on a surface (substrate).
Suspension culture: A culture arising from the passage of a primary culture.
Diploid cell line: A cell line in which, arbitrarily at least 75% of the cells have the same karyotype as
the normal cells of the species from which the cells were originally obtained.
Heteroploid cell line:
A cell line having less than 75% of cells with a diploid chromosome
constitution.
Pseudo-diploid cell line: A cell line in which the cells have the same number of chromosomes sets
as the normal cell of the species from which the cells were obtained, but which possess an altered
karyotype.
Cell strain: A cell strain can be derived either from a primary culture or cell line by the selection or
cloning of cells having specific properties or markers. The properties of markers must persist during
subsequent cultivation.
Sub-strain: A sub-strain can be derived from a strain by isolating a single cell or group of cells having
properties or markers not shared by all cells of the strain.
Clone: A population of cells derived from a single cell.
Cloned strain or line: A strain or line descended directly from a clone.
Passage: The transplantation of cells from one culture vessel to another.
Split: Synonymous with “passage”.
Subculture: Synonymous with “passage”.
Subculture (passage) number: The number of subcultures the line has undergone, from the primary
material to the culture in question. This term is loosely synonymous to generation number.
Fibroblasts: Cells of spindle or irregular shape which, as their name implies are responsible for fibre
formation.
Fibroblast-like cells: In cell cultures various types of cells acquire a similar morphology. Cells
acquiring an irregular or spindle shape are often referred to as “fibroblasts”. However, their derivation
or potentialities, such as production of fibers, are usually not known. Such cells are then called
“fibroblast-like”
Epithelial cells: Cells opposed to each other, forming mosaic-like sheets with very little intercellular
substance, as seen in vitro or in tissue or organ culture.
Epithelial-like cells: In cell cultures epithelial cells may assume various shapes to form sheets of
closely adherent polygonal cells. The degree of cohesion of the cells can vary.
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Applied Veterinary Virology: The isolation and identification of viruses using cell cultures
Transformation: Often used synonymously with “cell alteration”. It is recommended that this term is
used only for heritable changes, such as those brought about by the action of various viruses and
chemicals.
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