1.6 Aseptic Technique and Cell Culture Flashcards
What is aseptic technique?
Aseptic technique involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants.
Examples of aseptic technique
work surfaces are disinfected before and after working with 70% ethanol or virkon.
All equipment and culture media sterilised to exclude microbial contaminants – e.g. use of heat (dry in an oven), flame inoculating loops or steam (using an autoclave) or chemicals (such as bleach).
Purpose of aseptic technique
Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells
How can a microbial culture be started?
Using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients
What is microbial culture?
A method of multiplying microorganisms by letting them reproduce in a culture medium under controlled laboratory conditions.
What does cell culture require?
Cell culture requires environmental factors such as temperature and pH to be controlled and the growing cells must have the opportunity for gas exchange.
What does the medium that animal cells are grown in contain?
Growth factors
What are growth factors
Growth factors are proteins that promote cell growth and proliferation. Growth factors are essential for the culture of most animal cells.
Where do growth factors come from?
Serum
How many times can primary cell lines divide in culture
A limited number of times
How many times can tumour cell lines divide in culture?
Tumour cells lines can perform unlimited divisions
What does a typical culture media contain?
Water, salts, amino acids, vitamins and glucose.
What does the plating out of a liquid microbial culture on solid media allow?
The number of colony-forming units to be counted and the density of cells in the culture estimated
What type of dilution may be needed to achieve a suitable colony count?
Serial dilution
Equation used to calculate the number of bacteria in a diluted cell culture
number of colonies x dilution of sample = number of bacteria/ml
What is a haemocytometer?
Haemocytometers are specialised slides that have a counting chamber with a known volume of liquid.
What are haemocytometers used for?
They are used to estimate cell numbers in a liquid culture
How would you calculate the number of cells per cm^3 of culture if 15 cells were counted in a haemocytometer?
15 cells counted = 15 cells per 0.1 μl (or 1mm^3)
15 × 10 = 150 cells per μl
150 × 1000 = 150000 (1⋅5 × 10^5) cells per cm^3 of culture
How are cells on a haemocytometer made visible?
They are stained
How can living cells prevent the stain entering through the cell membrane?
Closing pores in the membrane.
Actively transporting the stain out the cell.
Breaking down the stains.
What kind of cells can the stain be taken up by?
Non-viable cells (dead cells) take up the stain and can be visualised as the stain colour (usually blue).
What is a viable cell count?
Number of live actively growing/dividing cells in a sample.
What is a total cell count?
The number of dead and live cells
What state are the cells that take up Trypan Blue dye?
Dead
Equation for calculating percentage viability of cells
Live cell count/ total cell count = percentage viability
