Unit 1: KA1 - Lab Techniques Flashcards
Define the term Hazard.
A source of potential harm
Define the term Risk.
The likelihood of a hazard causing significant harm.
What 3 things must a risk assessment include?
- Identification of risk
- Likelihood it occurs
- control measures to minimise risk
Name 3 appropriate measures to minimise risk
- appropriate handling techniques
- PPE
- Aseptic techniques to culture cells
Describe linear dilution and what it’s used for.
- Equal intervals
- Used for plotting standard curves
Describe log dilution and what it’s used for.
- Constant proportion (eg. 1x10^-1, 1x10^-2)
- Used for counting cell colonies
What is the formula for calculating dilutions?
V₁C₁ = V₂C₂
What is the purpose of adding a buffer to a reaction?
To allow the pH to remain constant within the reaction mixture.
What is the use of a colorimeter?
To quantify colour variation or turbidity of solutions.
What does absorbance measure?
Concentration of pigment?
What does Transmission determine?
Turbidity (cloudiness)
How does a Centrifuge separate components?
It spins at a high speed to separate components of differing densities into a Pellet (solid) and a supernatant (liquid).
How does paper and thin layer chromatography separate components and what is an example?
Distance travelled is dependent on a substance’s solubility in the solvent.
Amino acids and Sugars
Describe Affinity Chromatography.
- A solid matrix or Gel column is created with specific molecules.
- Soluble target proteins in a mixture with high affinity for these molecules become attached when passed through.
- Other non-target molecules are washed out.
Describe gel electrophoresis
Charged macromolecules move through an electric field applied to the gel matrix.
Size and charge are factors that affect rate of which any macromolecules migrate through the gel.
How do native gels separate proteins?
By shape size and charge.
Fully folded proteins
How does SDS-Page electrophoresis separate proteins?
By size alone.
Denatured proteins
Describe Western blotting.
- Used after SDS Page electrophoresis.
- Separated proteins are transferred to a solid medium.
- The proteins can be identified using specific antibodies with reporter enzymes.
Define the term isoelectric point
The pH at which a particular molecule carries no net electrical charge.
Give two ways proteins can be separated by isoelectric point.
1- Buffer to specific pH- only proteins of that IEP will precipitate out
2- (electrophoresis) soluble proteins can be separated using an electric field and a pH gradient.
A protein stops moving through the gel at its IEP in the pH gradient because it has no net charge.
What are the two types of microscopy?
Bright field
Fluorescence
What 4 things is bright field microscopy used to observe?
- Whole organisms
- parts of organisms
- thin sections of dissected tissue
- individual cells
How does Fluorescent microscopy work?
Uses specific fluorescent labels to bind to and visualise:
- certain molecules
- structures within cells or tissues
What is the purpose of immunoassay techniques?
to identify and detect specific proteins
What are monoclonal antibodies?
Identical antibodies with the same specificity.
Describe immunoassay.
- Monoclonal antibodies are bound to plate
- substance being tested for specific protein is washed through and binds to antibodies
- plate is washed and any unbound proteins are washed away.
- enzyme substrate for protein is added creating detectable change if specific protein is present.
What is the advantage of aseptic technique?
Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells
What does aseptic technique include?
- sterilisation of equipment using heat or chemicals
- exclusion of microbial contaminants.
Define the term inoculum.
An original stock of 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 serum?
It is added to animal cell culturing media as it contains growth factors.
What is the difference between primary cell lines and tumour cell lines?
Primary cell lines can divide a limited number of times, whereas tumour cell lines can divide an unlimited number of times.
Describe plating out.
- Liquid microbial culture is plated onto a solid media, allowing a number of colonies to form
- The number of these colonies can be counted, giving an indication of cell density.
What is serial dilution’s use?
to allow a countable number of colonies after plating out.
What is a haemocytometer?
A special slide used to estimate total and viable cell counts in a sample
What are the rules in counting viable cell count in a haemocytometer?
- Include cells touching middle top and left line
- Exclude cells touching middle bottom and right line
What does vital staining achieve?
Vital staining is required to identify and count viable cells.
How does vital staining work?
Dye is only taken up by non-viable cells, so viable cells are easily counted.
