Lec 3: Methods Flashcards
methods in virology are used also in
cell & molecular biology
most cells taken from body…
don’t grow well in culture
cell line =
If cells from a primary culture can be subcultured they are growing as a cell line
continuous cell line =
cells can be subcultured only a finite number of times unless they are immortalized, which can be subcultured indefinitely as a continuous cell line
cancer cells are already…
&…
immortalized,
…& continuous cell lines may be established from these without further treatment
Normal Cells from Animal –> _____ –> —-(___)—-> ____ —-(______)—-> _____
Normal Cells from Animal –> 1* Cell Culture –> —-(subculture)—-> Cell Line —-(immortalization)—-> Continuous Cell Line
Cancer Cells from Animal –> _____
Cancer Cells from Animal –> Continuous Cell Line
primary culture cells have
finite life span
Primary culture contains
a very heterogeneous population of cells
Subculturing 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 not be used as primary cultures
cell strain
Lineage of cells origination from the primary culture is called a cell strain
Continuous Cell Lines –>
HeLa cells
senescence =
Normal cells usually divide only a limited number of times before losing their ability to proliferate, which is a genetically determined event known as senescence; these cell lines are known as finite.
Some cell lines become…
through a process called…
which can…
…become immortal,
…through transformation,
…can occur spontaneously or can be chemically or virally induced
continuous cell lines form when
a finite cell line undergoes transformation and acquires the ability to divide indefinitely
continuous cell lines have undergone significant…
this can…
…mutations to become immortal,
…can alter the biology of the cell and must be taken into consideration in any analysis.
Why do you need agar in the media? (2)
- To keep virus particles in place, otherwise they would disperse everywhere
- Allows you to control movement of the virus
(after centrifugation)
top band =
bottom band =
each band =
lowest density
highest density
pure sample of virus
viral structure studies uses
microscopy:
- light = not suitable due to limited resolution
- electron = negative staining (stain w/ heavy metal; stained portion will reflect the electron beam) (heavy metal = white dot ) (no metal/structure = black dot)
x-ray beam array =
viral protein analysis
allows for diffraction pattern determination and relative positions of molecules to be determined
(important for drug design)
Detection of: Virions –>
Electron Microscopy
Detection of: Infectivity of cell culture –>
Examine change in characteristics of the cells (plaque arrays?)
Detection of: Virus Antigens –>
ELISA
Detection of: Virus Antibodies –>
ELISA
Detection of: Virus Nucleic Acids –>
Hybridization (southern, northern) & PCR
SDS =
this will make…
“Sodium Dodecyl Sulfate”
breaks bonds in proteins, making them all simple 1* chains
…this will make proteins move through electrophoresis gel at a rate dependent only on MW
PAGE =
Polyacrylamide gel electrophoresis
use PAGE for
proteins
PAGE makes proteins move from
cathode (-) —> anode (+)
Principles of tests to detect virus antigens:
Direct Test = anti-virus antibody labelled
Indirect Test = second antibody labelled
Blotting and probing for detection of:
- DNA =
- RNA =
- Proteins =
= southern blot/ hybridization
= northern blot/hybridization)
= western blot
PCR aka
Polymerase chain reaction
(infectivity assays)
eclipse period =
period during which no intracellular infectious virus can be recovered;
- infectious nucleic acid might
be recoverable in some cases
(infectivity assays)
bust size
average yield of infectious virus per cell
