Unit 1 - Laboratory Techniques for Biologists Flashcards
What are some examples of hazards in a lab?
Toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms and mechanical equipment.
How are control measures to minimise risks and reduce hazards identified?
Carrying out a risk assessment.
What is risk?
The likelihood of harm arising from exposure to a hazard.
What are some examples of risk control measures?
Using appropriate handling techniques, protective clothing and equipment, and aseptic technique.
What are linear dilutions?
Dilutions that differ by an equal interval, eg 0.1, 0.2, 0.3 …
What are log (serial) dilutions?
Dilutions that differ by a constant proportion, eg 0.1, 0.01, 0.001 …
What is a standard curve used for?
To determine unknown values from a line or curve made with measured values.
What is a buffer solution?
A solution where adding acids or alkalis has very small effects on the pH. Allows pH of a reaction mixture to be kept constant.
What are colorimeters used to measure?
Concentration and turbidity (cloudiness).
What must be done before using a colorimeter?
The colorimeter must be calibrated with an appropriate blank sample to provide a baseline reading.
What can absorbance on a colorimeter be used to determine?
Concentration of a solution.
What can percentage transmission on a colorimeter be used to determine?
Turbidity such as cells in suspension.
What does a centrifuge separate substances according to?
Density.
What do more dense components form in a centrifuge?
Pellet.
What do less dense components form in a centrifuge?
Supernatant.
How does paper and thin layer chromatography separate substances?
By solubility in the solvent used - speed that each solute travels along the chromatogram depends on solubility in the solvent.
What is affinity chromatography used to separate?
Proteins from a mixture.
Explain the process of affinity chromatography.
A solid matrix or gel column is created with specific molecules (usually receptors) bound to it. Soluble, target proteins in a mixture, with a high affinity for these molecules, become attached to them as the mixture moves down the column. Other molecules with a weaker affinity are washed out.
What is gel electrophoresis used to separate?
Proteins and nucleic acids.
How does gel electrophoresis work?
Charged macromolecules move through an electric field applied to a gel matrix.
What do native gels in gel electrophoresis separate proteins by and how do they do this?
Shape, size and charge. They do not denature the molecule.
What does SDS-PAGE separate proteins by and how?
Size.
Gives all molecules an equally negative charge and denatures them.
What is the isoelectric point of a protein?
The pH at which a soluble protein has no net charge and will precipitate out of solution.
What is the name given to the techniques used to detect and identify specific proteins?
Immunoassay.
What are stocks of antibodies with the same specificity known as?
Monoclonal antibodies.
What is the chemical ‘label’ used in immunoassay?
It is often a reporter enzyme producing a colour change but chemiluminescence, fluorescence and other reporters can be used.
When is western blotting used?
After SDS-PAGE electrophoresis.
Explain the process of western blotting.
Once the proteins have been separated in the gel they are transferred or blotted onto a solid medium. The proteins can then be identified using specific antibodies with reporter enzymes attached (ELISA).
What 4 things is bright field microscopy commonly used to observe?
Whole organisms, parts of organisms, thin sections of dissected tissue or individual cells.
How does fluorescence microscopy work?
It uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.
What is aseptic technique?
Procedures used in laboratories to reduce contamination and unwanted growth or spread of microorganisms.
What does the aseptic technique involve?
Sterilisation of equipment and culture media by heat or chemical means (to remove microbial contaminants).
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 are animal cells grown in?
Medium containing growth factors from serum.
What cell lines can divide a limited number of times?
Primary.
What cell lines can perform unlimited divisions?
Tumour.
What are growth factors?
Proteins that promote cell growth and proliferation. They are essential for the culture of most animal cells.
What is used to estimate cell numbers in a liquid culture?
Haemocytometer.
What is required to identify and count viable (living, respiring) cells?
Vital staining.
What is the process in isoelectric point electrophoresis?
Two proteins with differing IEP’s loaded into a gel matrix
Proteins migrate towards the charges until they reach an area with the pH of their IEP
Proteins stop migrating as they have no charge and precipitate out
