Unit 1 - Key Area 1: Laboratory Techniques Flashcards
Explain what is meant by a hazard in the laboratory
Anything that poses a potential threat to an individual or the environment
Give examples of hazards in the laboratory
Toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms and mechanical equipment
Explain what is meant by a risk in the laboratory
The likelihood of harm arising from exposure to a hazard
What control measures might be put in place to minimise risk?
Appropriate handling techniques, protective clothing and equipment, aseptic technique
Explain what is meant by a linear dilution series
Differ by an equal interval (eg 0.1, 0.2, 0.3)
Explain what is meant by a log dilution series
Differ by a constant proportion (10-1, 10-2)
Describe how a standard curve is produced
Plot measured values for known concentrations to produce a line or curve, allows the concentration of an unknown to be determined
Describe the use of a buffer to control pH
Buffer is not affected by acid or alkali, so is used to keep the pH of a reaction mixture to be kept constant
Describe the use of a colorimeter to quantify concentration
Absorbance is used to determine concentration of a coloured solution using suitable wavelength filters
Describe the use of a colorimeter to quantify turbidity
Percentage transmission is used to determine turbity such as cells in suspension
How is a centrifuge used to separate substances?
Centrifugation is used to separate by density. More dense components settle in the pellet, less dense remain in the supernatant
What type of substances can be separated by paper or thin layer chromatography?
Amino acids and sugars
What is the basic principle behind paper or thin layer chromatography?
The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used
Describe how affinity chromatography works
A solid gel column is created wuith specific molecules bound to the matrix or gel. Soluble, target proteins in a mixture have a high affinity to these molecules and become attached as the mixture passes through
Describe how gel electrophoresis works
Charged macromolecules move through an electric field applied to a gel matrix. Smaller pieces move faster and further than larger molecules
Explain the difference between native gels and SDS-PAGE
Native gels do not denature the molecule so that separation is by shape, size and charge. SDS-PAGE gives all the molecules an equally negative charge and denatures them, separating by size alone
Explain what is meant by the IEP of a protein
The pH at which a soluble protein has no net charge and will precipitate out of solution
How can proteins be separated by IEP?
Solution is buffered tia specific oH, only the protein(s) that have an IEP of that pH will precipitate
Describe the separation of proteins by IEP in electrophoresis
Soluble proteins can be separated using an electirc field and a pH gradient. A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.
What is meant by monoclonal antibodies?
Antibodies which have the same specificity
Describe the use of an immunoassay to detect and identify proteins
An antibody specific to the protein antigen is linked to a chemical “label” which reacts when it binds with the antibody
Give an example of the type of chemical label that might be used in an immunoassay
A reporter eznymbe producing a colour change, chemiluminescence or fluorescence
Describe the use of a Western blot
After SDS-PAGE electophoresis, separated proteins from the gel are transferred onto a solid medium. Proteins can be idenitfied using specific antibodies attached to reported enzymes
What is bright-field microscopy used for?
Observing whole organisms, parts of organisms, thin sections of dissected tissue or individual cells
What is fluorescence microscopy used for?
Uses fluorescent labels to bind to and visualise certain molecules or structures within cells and tissues
What is the purpose of aseptic technique?
To eliminate unwanted microbial contaminants when culturing micro-organisms or cells
What is involved in aseptic technique?
Sterilisation of equipment and culture media by heat or chemical means
What are the types of media used for growing a microbial culture?
A solid agar medium or in a broth with suitable nutrients
What do animal cells require in a growth medium?
Growth factors which are proteins that promote cell growth and proliferation
Describe the different growth of primary cell lines and tumour cell lines
Primary cell lines can divide a limited number of times wheras tumour cells can perform unlimited divisions
How can the density of cells in a liquid culture be estimated?
By plating out onto a solid meia allowing the number of colony-forming units to be counted
Describe the use of a haemocytometer
Counting the number of cells in a particular area of the grid, allows cell density to be calculated
What is meant by a total cell count?
the number of cells in a culture
What is meant by a viable cell count?
The number of living cells in a culture
