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 .
0.3
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
4
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
to ensure that the cells are not overexposed to trypsin, as this may result in extensive damage to the cell surface, eventually resulting in cell death. It is therefore important that the ____ reaction is quickly terminated by the addition of ___ ___ containing serum to inactivate the ___
proteolysis
complete medium
trypsin
As with all tissue culture procedures, aseptic techniques should be adopted an in the case of a trypsin stock, which temp should it be stored?
-20, and when needed just water bath (37) right before thawing
While the working solution should be kept at 4, once made and can be stored for up to 3 months.
Regarding subculture of cells in suspension,if the medium is spent and appears acidic. What should one do?
the cells must be collected by centrifugation at 1000 rpm for 10 min, resuspended in
fresh medium and transferred into a new flask.
Similarly, cells that grow in clumps
are collected by centrifugation and resuspended in fresh medium as single cells using
a glass Pasteur or fi ne-bore pipette.
if cells are seeded
at a lower seeding density they may take ___ to reach confluency
longer
What is the most common way of cell quantification (density)?
Name one other alternative
use of haemocytometer
electronic Coulter counter .
After quantification of subcultured cells, cells should be …
seeded at a density that promotes optimal cell growth
As a guide,
a) cells which are under __% confluency may be
cultured in approximately 1 cm3 of medium
per 5 cm2
b) those between __% and ___% confluency should
be supplemented with 1.5 cm3 and 2 cm3 culture
medium per 5 cm2 , respectively.
25
25
40–45
When changing the medium,
it is advisable to pipette the latter onto the ___ or the opposite surface of the flask to where the cells are attached. This is to avoid making direct contact with
the ____, as this will damage or dislodge the cells.
sides
monolayers
Regarding growth kinetics for animal cells in cultures having optimum conditions follow a characteristic growth pattern, which 4 phases exist?
lag
log
stationary
decline
Compared to animal cells, pure bacterial cultures exhibit ___ wider degree of diversity in terms of both their nutritional and environmental requirements, conditions for their cultivation are diverse and the precise requirements highly dependent on the species being cultivated.
much
Does bacteria require much more complex condition¨than animal cells?
no
Due to the wider and diverse growth conditions for bacteria, two categories are present, which two?
autotrophs
heterotrophs –> chemoautotrophs & photoautotrophs
Culture Media for Bacterial Cell Culture:
2 categories:
____ – consist of natural susbtances
____ – made of known compounds, thus simpler –> selective cultivation
complex
defined
Culture Procedures for Bacterial Cells:
the volume of medium added to the flasks should not exceed more than __% of the total volume of the flask
20
Once cultures for beacterial cells are isolated, the cells can be cultivated in two ways!
batch
a) for routine liquid growth
b) Such systems are referred to as closed
–> cellular composition and physiological status of
the cells will vary throughout the growth cycle
due to limited nutrient source
continuous cultures
a) the medium is refreshed regularly to replace that
spent by the cells.
b) The objective of this system is to maintain
the cells in the exponential growth phase
c) give more defined physiological status due to growth
over steady-state-conditions giving little variation in
cellular composition –> coupling between
cell division & biosynthesis
Determination of Growth of Bacterial Cultures:
1. directly counting cells using a haemocytometer, only
suitable for cell in ___
2. When cells are grown on solid agar plates, ___ ___
can be used instead to estimate growth, assuming
that each colony is derived from a single cell
(subculturing technique)
3. When cells are grown in suspension, changes in the
turbidity (Sec. 13.2.2 ) of the growth medium could be
determined using a ______ and the apparent
absorbance value (–> cell number) using a
standard curve of optical ___ vs. __ ___
suspension colony counting spectrophotometer density (see Section 13.2.2 ) cell number ------- Such calibration curves should be constructed for each cell type by taking the readings of a series of known numbers of cells in suspension.
