Lab Techniques Flashcards
What can present a hazard?
Substances, organisms, and equipment in a laboratory can present a hazard.
What is an example of a hazard in a lab?
Hazards in the lab include toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms, and mechanical equipment.
What is a risk?
A risk is the likelihood of harm arising from exposure to a hazard.
What does risk assessment involve?
Risk assessment involves identifying control measures to minimise the risk.
What do control measures include?
Control measures include using appropriate handling techniques, protective clothing and equipment, and aseptic technique.
How do dilutions in a linear dilution series differ?
Dilutions in a linear dilution series differ by an equal interval, for example 0.1, 0.2, 0.3 and so on.
How do dilutions in a log dilution differ?
Dilutions in a log dilution series differ by a constant proportion, for example 10^-1, 10^-2, 10^-3 and so on.
How do you determine an unknown from a standard curve?
Plotting measured values for known concentrations to produce a line or curve allows the concentration of an unknown to be determined from the standard curve.
Use of buffers to control pH
Addition of acid or alkali has very small effects on the pH of a buffer, allowing the oH of a reaction mixture to be kept constant.
Method and uses of a colorimeter to quantify concentration and turbidity.
Calibration with appropriate blank as a baseline; use of absorbance ti determine concentration of a coloured solution using suitable wavelength filters; use of percentage transmission to detract turbidity, such as cells in suspension.
Use of centrifuge to separate substances of differing density.
More dense components settle in the pellet; less dense components remain in the supernatant.
What is paper and thin layer chromatography used for?
Paper and thin chromatography can be used for separating different substances such as amino acids and sugars.
What does the speed that each solute travels along the chromatogram depend on?
Its differing solubility in the solvent used.
Principle of affinity chromosome and its use in separating proteins.
A solid matrix or gel column is created with specific molecules bound to the matrix or gel. Soluble, target proteins inna mixture, with a high affinity for these molecules, become attached to them as the mixture passes down the column. Other non-target molecules with a weaker affinity are washed out.
Principle of gel electrophoresis and its use in separating proteins and nucleic acids.
Charged macromolecules move through an electrical field applied to a gel matrix.
Native gels
Native gels do not denature the molecule so that separation is done by shape, size, and charge.
SDS-PAGE
SDS-PAGE gives all molecules an equally negative charge and denatures them, separating proteins by size alone.
What does IEP stand for
Isoelectric points
What is IEP?
IEP is the pH at which a soluble protein has no net charge and will precipitate out of the solution.
If a solution is buffered to a special pH, only the protein(s) that have an IEP of the pH will precipitate.
How can proteins be separated using their IEPs in electrophoresis?
Soluble proteins can be separated using an electric 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 are immunoassay techniques used for?
They are used to detect and identify specific proteins.
What do immunoassay techniques use?
Stocks of antibodies with the same specificity, known as monoclonal antibodies.
An antibody specific to the protein antigen is linked to a chemical ‘label’. What often is this ‘label’ ?
The ‘label’ is often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used.
What is used after SDS-PAGE electrophoresis?
Western blotting
What is bright-field microscopy is commonly used for?
Observing whole organisms, parts of organisms, thin sections of dissected tissue or individual cells.
What does fluorescence microscopy use and what does it visualise?
Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.
Aseptic technique eliminates what?
Unwanted microbial contaminants when culturing micro-organisms or cells.
What does aseptic technique involve?
Aseptic technique involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of 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.
Many culture media exist that promote what?
The growth of specific types of cells and microbes.
What are animal cells grown in?
Animal cells are grown in medium containing growth factors from serum.
What are growth factors?
Proteins that promote cell growth and proliferation. Growth factors are essential for the culture of most animal cells.
What is the difference between primary cell lines and tumour cell lines?
In culture, primary cell lines can divide a limited number of times, whereas tumour cell lines can perform unlimited divisons
Plating out of a liquid microbial culture on solid media allows what?
The number of colony forming units to be counted and the density of cells in the culture estimated.
What is often needed to achieve a suitable colony count?
Serial dilution
What is required to identify and count viable cells in the use of a haemoctyometer.
Vital staining
