1.1 Lab Techniques

Lab hazards

Lab hazards

Toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms, and mechanical equipment.

Risk

The likelihood of harm arising from exposure to a hazard.

Risk assessment

Identifying control measures to minimise the risk.

Control measures

Using appropriate handling techniques, protective clothing and equipment, and aseptic technique.

Linear dilutions

Dilutions differ by an equal interval, for example 0·1, 0·2, 0·3

Log dilutions

Dilutions differ by a constant proportion, for example 0.1, 0.01, 0.001

Standard Curve

Plotting measured values for known concentrations to produce a line or curve allowing the concentration of an unknown to be determined

Buffers

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.

Use of a colorimeter to measure absorbance

Can determine concentration of a coloured solution using suitable wavelength filters

Use of a colorimeter to measure % transmission

Can determine turbidity, such as cells in suspension.

Centrifugation

Separation by 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

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

Charged macromolecules move through an electric field applied to a gel matrix. Used to separate proteins and nucleic acids.

Native gels

These do not denature the molecule so that separation is by shape, size and charge.

SDS-PAGE

Gives all the molecules an equally negative charge and denatures them, separating proteins by size alone.

Isoelectric point

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. Soluble proteins can be separated using an electric field and a pH gradient.

Immunoassay techniques

Used to detect and identify specific proteins

Monoclonal antibodies

Stocks of antibodies with the same specificity used in immunoassay techniques

Chemical labels

Often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and specific antigens 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

Bright field microscopy

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

Aseptic technique

Eliminates unwanted microbial contaminants when culturing microorganisms or cells. It involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants.

Starting a microbial culture

By using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients

Animal cell cultures

Grown in medium containing growth factors from serum. In culture, primary cell lines can divide a limited number of times, whereas tumour cells lines can perform unlimited divisions

Growth factors

Proteins that promote cell growth and proliferation.

Haemocytometers

Used to elimate a cell count in a liquid culture

Vital staining

Used to determine a viable cell count by staining.