(a) Health and safety       

  • Substances, organisms, and equipment in a laboratory can present a hazard
  • Hazards in the lab include toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms, and mechanical equipment.

Hazard, risk, and control of risk in the lab by risk assessment

  • Risk is the likelihood of harm arising from exposure to a hazard.
  • Risk assessment involves identifying control measures to minimise the risk.
  • Control measures include using appropriate handling techniques, protective clothing and equipment, and aseptic technique.

 (b) Liquids and solutions

Method and uses of linear and log dilution

  • Dilutions in a linear dilution series differ by an equal interval, for example 0·1, 0·2, 0·3 and so on.
  • Dilutions in a log dilution series differ by a constant proportion, for example 10-1, 10-2, 10-3 and so on.

Production of a standard curve to determine an unknown

  • 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 pH 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 to determine concentration of a coloured solution using suitable wavelength filters;
  • use of percentage transmission to determine turbidity, such as cells in suspension.

 (c) Separation techniques 

Centrifuge

  • Used to separate substances of differing density.
  • More dense components settle in the pellet; less dense components remain in the supernatant.

Paper and thin layer chromatography

  • Can be used for separating different substances such as amino acids and sugars
  • The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used.

Affinity chromatography

  • Used in separating proteins
  • A solid matrix or gel column is created with specific molecules bound to the matrix or gel.
  • Soluble, target proteins in a 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.

Gel electrophoresis

  • Used in separating proteins and nucleic acids
  • Charged macromolecules move though an electric field applied to a gel matrix.
  • Native gels separate proteins by their shape, size and charge
  • Native gels do not denature the molecule so that separation is by shape, size and charge.
  • SDS–PAGE separates proteins by size alone
  • SDS–PAGE gives all the molecules an equally negative charge and denatures them, separating proteins by size alone.

Isoelectric Points (IEPs)

  • Proteins can be separated from a mixture using their isoelectric points (IEPs)
  • IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
  • If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate
  • Proteins can also 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.

(d) Detecting proteins using antibodies   

Immunoassay techniques

  • Used to detect and identify specific proteins
  • These 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’
  • The ‘label’ is often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used.
  • In some cases the assay uses a specific antigen to detect the presence of antibodies.

Western blotting

  • A technique, used after SDS–PAGE electrophoresis
  • The separated proteins from the gel are transferred (blotted) onto a solid medium
  • The proteins can be identified using specific antibodies that have reporter enzymes attached

(e) Microscopy       

  • Bright-field microscopy is commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells
  • Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues

(f) Aseptic technique and cell culture     

  • Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells
  • Aseptic technique involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants.
  • A microbial culture can 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 the growth of specific types of cells and microbes.
  • Animal cells are grown in medium containing growth factors from serum
  • Growth factors are proteins that promote cell growth and proliferation. Growth factors are essential for the culture of most animal cells.
  • In culture, primary cell lines (sourced from animal tissue) can divide a limited number of times, whereas tumour cells lines (sourced from tumours) can perform unlimited divisions
  • Plating out of a liquid microbial culture on solid media allows the number of colony-forming units to be counted and the density of cells in the culture estimated
  • Serial dilution is often needed to achieve a suitable colony count
  • A haemocytometer is used to estimate cell numbers in a liquid culture

Vital staining is required to identify and count viable cells.

  • A viable cell count identifies the number of actively growing/dividing cells in a sample.
  • A stain/dye is added to a cell culture, which is taken up by dead cells but not by living cells.
  • A live cell count can then be performed where only unstained cells are counted.

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