Unit 1.1 - Laboratory Techniques for Biologists Flashcards
Name hazards in a lab
Toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms and mechanical equipment
Define risk and what could be done to minimise it
Risk is the likelihood of harm arising from a hazard.
To minimise risk a risk assessment is used to identify control measures
What is the difference between linear and log dilutions?
Linear dilution - series differ by an equal interval
Log dilution - series differ by a constant proportion
What is a standard curve?
Measured values for known concentrations are plotted to produce a line or curve allowing the concentration of an unknown to be determined
During colorimetry when is absorbance and percentage transmission used?
Absorbance - to determine the concentration of a coloured solution using suitable wavelength filters
Percentage transmission - determine turbidity
Define turbidity
The cloudiness of a fluid caused by a large number of particles
When using a centrifuge, what components go in the pellet and supernatant?
Pellet - More dense components
Supernatant - Less dense components remain here
How can paper and thin layer chromatography be used to separate different substances such as amino acids and sugars?
The speed each solute travels along the chromatogram depends on its differing solubility in the solvent used
How does affinity chromatography work?
A solid matrix or gel column is created with specific molecules bound to the matrix of gel. Soluble target proteins have a high affinity for these molecules and become attached to them as the mixture passes down the column . Other non-target molecules are washed out
How does gel electrophoresis work?
Charged macromolecules move through an electric field applied to a gel matrix
How do native gels separate proteins?
Don’t denature molecules so the separation is based on shape, size and charge
How does SDS-PAGE separate proteins?
Gives all molecules an equally negative charge and denatures them, separating the proteins based on size alone
What is an isoelectric point (IEP)?
The pH at which a soluble protein has no net charge and will precipitate out of solution
How can IEPs be used in electrophoresis?
Soluble proteins can be separated using an electric field and a pH gradient. A protein will stop moving through the gel at its IEP in the pH gradient as it will have no net charge
What are immunoassay techniques used for?
To detect and identify specific proteins
How do immunoassay techniques work?
Uses stocks of antibodies with the same specificity (monoclonal antibodies). An antibody specific to the protein antigen is linked to a chemical label. The label is often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used
What is western blotting?
A technique used after SDS-PAGE electrophoresis where the separated proteins are transferred onto a solid medium. The proteins can then be identified using specific antibodies that have reporter enzymes attached
Define bright-field microscopy
Commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells
Define fluorescence microscopy
Uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues
What is the purpose of aseptic technique?
Eliminates unwanted microbial contaminants when culturing microorganisms or cells
How is a microbial culture started?
Using an inoculum of microbial cells on an agar medium or broth with suitable nutrients to promote the growth of specific types of cells and microbes
What is in the medium animal cells are grown in?
Serum which contains growth factors which are proteins that promote cell growth and proliferation
Differences between primary cell lines and tumour cell line
Primary - can divide a limited number of times
Tumour - can perform unlimited divisions
How can the number of colony forming units be counted and the density of cells in a culture counted?
Plating out of a liquid microbial culture on to solid media. A serial dilution is often needed to achieve a suitable colony count
What is vital staining used for?
Required to identify and count viable cells
