Chapter 3: cell cultures Flashcards
In animal cell cultures, bacterial and fungal infections are relatively easy to identify and isolate. The other most common contamination originates from mycoplasma.
These are the smallest (approximately ___mm in diameter) self-replicating prokaryotes in existence.
They lack a rigid cell wall and generally infect the cytoplasm of mammalian cells. There are at least five species known to contaminate cells in culture:
- Mycoplasma hyorhinis
- Mycoplasma arginini
- Mycoplasma orale
- Mycoplasma fermentans
- Acholeplasma laidlawii .
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Both bacterial and fungal contaminations are easily identified as the infective agents are readily visible to the naked eye, even in the early stages. This is usually made noticeable by the increase in ___ and the change in ___ of the culture medium owing to the
change in __ caused by the infection
turbidity
colour
pH
In addition, bacteria can be easily identified under
___ examination as motile round bodies.
Fungi, on the other hand, are distinctive by their long ___ growth and by the ___ colonies they form in the medium.
In most cases, the simplest solution to these infections is to?
microscopic hyphal fuzzy remove and dispose of the contaminated cultures
Testing of bacterial or fungal infections
involves culturing a suspension of cells or products in an appropriate medium such as:
1. ____ for bacterial detection
2. ____fungal detection
The mixture is incubated for up to ___ days, but
examined daily for turbidity, which is used as an indication of microbial growth. It is essential that both positive and negative controls are set up in parallel with the sample to be tested
tryptone soya broth (TSB) thioglycollate medium (TGM) 14
How does mycoplasma infections manifest
and which 3 methods can be used for detection analysis?
What is the rapid alternative to DNA staining dye method?
a slowdown in growth and in changes in cellular metabolism and functions
ELISA, PCR & DNA staining using the fluorescent dye Hoechst 33258
DNA binding method
A new generation of bactericidal antibiotic preparation referred to as ____ is effective against mycoplasma even at relatively low, non-cytotoxic concentrations
Apart from antibiotics inhibiting growth, other products have been introduced to eradicate mycoplasma effi ciently and quickly without causing any adverse effects
to eukaryotic cells or viruses. One such product is ___
The antibiotics contained in this product are actively transported into cells, thus facilitating killing of intracellular mycoplasma without any adverse effects on actual cellular metabolism.
Mynox ®
Describe primary cell cultures
Disadvantage?
Primary cultures are cells derived directly from tissues following enzymatic dissociation or from tissue fragments referred to as explants. These are usually the cells
of preference, since it is argued that primary cultures retain their characteristics and reflect the true activity of the cell type in vivo
their isolation can be labour-intensive and may produce a
heterogeneous population of cells and primary cultures have a relatively limited lifespan and can be used over only a limited period of time in culture.
Describe continuos cell lines?
Cell lines consist of a single cell type that has gained the ability for infinite growth due to loosing of growth regulation, usually occuring after cell transformation –> loose original in vivo characteristics
In addition, they require less serum for growth, have a shorter doubling time and can grow without necessarily
needing to attach or adhere to the surface of the fl ask.
Cell Culture Media and Growth Requirements for Animal Cells
The cell culture medium used for animal cell growth is a complex mixture of:
1. nutrients (amino acids, a carbohydrate such as glucose, and vitamins)
2. inorganic salts (e.g. containing magnesium, sodium, potassium, calcium, phosphate, chloride, sulfate and
bicarbonate ions)
3. broad-spectrum antibiotics.
a) Penicillin - inhibits the last step in bacterial cell wall
synthesis In certain situations
b) Streptomycin - block protein synthesis
c) it may be essential to include a fungicide such as
amphotericin B, although this may not always be
necessary.
4. Serum (10-20%) - provided as a buffer, that enchances cell attachment and hormone-like-growth factors
A cell Culture Media for animal growth, once prepare is called complete growth medium, which should be kept at __°C until used
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What is subculturing?
the process by which cells are harvested, diluted in fresh growth
medium and replaced in a new culture fl ask to promote further growth
What are cells that are considered confluent?
Which state do they go in?
that adherent cells grow in a continuous layer that eventually
occupies the whole surface of the culture dish
Resting state, where they will stop growing
Which two ways can adherent cell be harvested?
For which way, does require test run on cell viability and growth?
mechanically, using a rubber spatula (also
referred to as a ‘rubber policeman’) or enzymatically using proteolytic enzymes
-
Cells
in suspension are simply diluted in fresh medium by taking a given volume of cell
suspension and adding an equal volume of medium.
mechanically
Regarding enzymatic harvesting of adherent cells, name on enzyme
What is the end result of these enzymes
Trypsin
Release single cells that can grow on their own. thus enhancing the propagation of the cultures
Regarding which chemical is typically used in combination?
And why?
EDTA
enhances the action of the enzyme by chelating Ca2+ thus negating the cell adhersion sequestering chelated divalent cations that inhibit trypsin activity
