Lab techniques for biologists

• Risk


  The likelihood of harm arising from exposure to a hazard
• Hazards (in the lab)


  Something that causes harm; toxic chemicals, corrosive chemicals, heat, flammable substances, pathogenic organisms and mechanical equipment.
• Risk assessment


  Involves identifying control measures to minimise the risk.
• Control measures


  Include using appropriate handling techniques, protective clothing, protective equipment and aseptic techniques.
• Linear dilution series


  Consists of a range of dilutions that differ by an equal interval
• log dilution series


  a log dilution series consists of a range of different dilutions that differ by a constant proportion.
• colorimeter


  used to measure the concentration of a pigment in a solution, the turbidity of the liquid or the density of cells in a culture.
• why use a suitable wavelength filter on a colorimeter?


  a suitable wavelength filter is used so that the concentration of a coloured solution can be determined.
• what does turbidity show on a colorimeter?


  For turbidity, a denser sample will show a longer degree of transmission.
• why is a colorimetric blank necessary?

  the machine is calibrated using a 'blank' cuvette containing solvent only, which acts as a baseline for the comparison.
• how is a standard curve made?


  A standard curve is made by plotting the absorbance readings for a series of known concentrations of a substance or culture.
• What is a standard curve used for?


  Can be used as a reference for any samples of unknown concentrations of the same substance or culture. Through interpolation, the concentration of the unknown can be estimated.
• Interpolation


  the prediction of data between two values and is a scientific technique that can allow confident predictions to be made.
• extrapolation


  prediction of a data point beyond the last known value, this means you'll have less confidence in the prediction.
• Buffers?


  buffers are aqueous solutions that show very little variation in their pH despite the addition of acids or alkalis.
• why use buffers?


  buffers can be selected so that the pH of the mixture of reactants can be kept constant.
• Centrifugation

  Centrifugation is a method for separating materials in suspension according to their differing density. The densest components form a pellet at the bottom of the tube and the least dense liquid fraction above is termed the supernatant.
• Paper / Thin-layer Chromatography


  Paper and thin-layer chromatography separate amino acids or sugars depending on their characteristic solubility. The different solubilities of the materials being tested result in different speeds of movement so the constituents travel different distances.
• Affinity chromatography


  An antibody or ligand specific for binding with the protein in question is immobilised in a gel or solid matrix inside a column. only the target protein with a high affinity to the antibody or ligand will bind. the other proteins will not bind and are washed out as they have a weaker affinity.
• Gel electrophoresis


  Gel electrophoresis uses current flowing through a buffer to separate either proteins or nucleic acids by size and charge. These molecules are in their native folded state.
• SDS-PAGE

  uses current flowing through a buffer to separate molecules by size alone. These molecules are in their non-native, unfolded state. To unfold the protein, it is denatured by heat in the presence of a detergent.
• Isoelectric point


  The isoelectric point of a protein is the pH at which it has no charge. proteins are insoluble at the isoelectric point and so will form a precipitate(solid) which can be separated from a solution of soluble proteins at this point.
• what is an immunoassay technique?

  immunoassay techniques are powerful tools in laboratory studies for the detection and identification of specific proteins.
• what are antibodies?

  antibodies are y-shaped globular proteins produced by B lymphocytes as part of the immune response in a vertebrate. B-lymphocytes are produced in the bone marrow and spleen.
• Polyclonal serum


  a serum containing many different antibodies.
• monoclonal antibodies


  a stock of identical antibodies which are used in immunoassay techniques
• reporter enzymes


  an enzyme that catalyses a colour-change reaction that is used to detect and quantify the presence of a specific antigen.
• ELISA Steps

  look for Human Chorionic Gonadotrophin(HCG) only produced by the placenta;

  
  

  1. Antibodies are attached to the bottom of a plastic well.
  2. A test sample is then added, if the sample contains the specific antigen, it would bind to the antibody.
  3. A second antibody is then added. This will bind only to the antigen. This antibody is enzyme-linked, which means a reporter enzyme is bound to the other side of it.
  4. A solution containing the reporter enzyme's substrate is added. This causes a colour-change and will confirm that the protein is in the mixture
• What does an ELISA show?


  Antibody presence.
• How can a false positive be prevented?


  thoroughly rinsed wells between each stage.
• Western Blotting


  Western blotting is a technique for identifying specific proteins that have been separated using SDS-PAGE gel electrophoresis. Once the proteins are separated they are blotted onto a solid medium. The medium is then flooded with fluorescent-labelled monoclonal antibodies or other reporters. If the reporter enzymes substrate is present a colour-change will occur.
• Histochemistry


  the study of tissues using stains and microscopy
• fluorescent immunohistochemical staining


  a technique used to visualise the distribution of specific cellular components in live cells
• monoclonal antibodies and disease


  monoclonal antibodies are increasingly used in the treatment of disease. This technology uses toxins attached to tumour-specific monoclonal antibodies. When the antibodies reach the tumour cells, they combine with their specific antigens, bringing the toxins into close contact with the target cell and killing them.
• fluorescence microscopy


  the use of specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.
• Bright-field microscopy


  Commonly used to observe whole organisms, parts of organisms, thin sections or dissected tissue or individual cells.
• Aseptic techniques


  Aseptic techniques are a set of precautions taken to eliminate unwanted microbial contamination. It involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contamination.
• How can a microbial culture be started?

  Can be started from an inoculum, where growing cells are added in a volume of liquid medium from a previous culture. Touching a broth or a culture plate with a sterile loop will gather enough microbes for an inoculum.
• What do most mammalian cells require for rapid cell division?


  The addition of a complex medium containing chemical growth factors from serum e.g. Foetal Bovine Serum which is a mixture containing growth factors, proteins, salts, vitamins and glucose.
• Why are antibiotics added to animal cell cultures?


  To minimise the chance of spoilage by micro-organisms.