Aseptic Techniques Flashcards
What is aseptic technique and why is it required
Set of precautions taken to eliminate unwanted microbial contamination
To study a cell system you need the cell in pure culture so all competition must be removed
How do you achieve aseptic conditions
Autoclave, disinfectant, Lab coat, flaming
How to start cell cultures
Single celled organisms can be cultured from a single cell
Tissue samples from multicellular organisms are used to start cultures
These are used to inoculate a culture or medium
Why are mammalian cells difficult to grow
They have anchorage dependence They will grow to confluent growth then stop(contact inhibition) They require complex growth medium They grow slowly There is low productivity They are fragile cells Low hay flick number
How to get around low growth and medium requirements
By adding media that give a large number of growth factors you can get the cells to increase in number faster
Most effective- fetal bovine serum
Some particular cell types may require more specific growth factors e.g. experimental growth factor or platelet derived growth factor
How to get around anchorage dependence
Protease digestion
If you need single cells you need to break the things that hold them together
Links between cells are created by protein filaments
Proteolytic enzymes are used to break cells into single cells
Which cells will not be affected by contact inhibition/ density dependent inhibition or hayflick number or have strong anchorage dependence
Cancer cells
Immortalised cells
What must you do in order to contain optimal conditions
Cut down competition Correct temperature Correct pH Correct oxygen level Food and nutrient source In solution keep stirring
How to grow plant cell cultures
Separate cells from the linked cells in the tissue
Involves using a pectinase
Will only grow in a culture medium if there is a certain level of cells = minimum inoculation density
How to work out if you have enough cells (plants )
Dye exclusion- treating the cell sample with a dye that will only be taken into the cells if there is an inactive membrane
Fluorescein diacetate conversion- provides the cells with a substance which I’m living cells will be metabolised into fluorecein which glows in UV
Cell counting - flow cytometry how it works and what can it be combined with
Uses a machine to produce a single cell suspension In a flow of liquid
Droplets are formed that contain single cells and then each drop containing сell is counted using a laser
Can be combined 2irh antibody technology to produce fluorescence- activated cell counter
