1.1 Lab techniques for biologists

• Hazards in the lab include toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms, and mechanical equipment.
• 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.
• 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.
• 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.
• 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.
• Colorimeters can be used to quantify concentration and turbidity(the cloudiness).
• Before use the colorimeters need to be calibrated with an appropriate blank sample to provide a baseline reading. 
• Absorbance can be used to determine concentration of a coloured solution using suitable wavelength filters
• Percentage transmission can be used to determine turbidity, such as cells in suspension.
• In the centrifuge, more dense components settle to form the pellet​

  whilst 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 technique used to separate proteins based on their affinity for a specific binding site.
• (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 can be used to separate proteins and nucleic acids
• Native gels will separate proteins by their shape , size and charge . They do this by ensuring they do not denature the molecule.
• SDS-PAGE will separate proteins by  size alone. They do this by giving all the molecules an equally  negative  charge and denatures them. 
• Isoelectric point can be used to separate proteins from a mixture
• The IEP is the specific pH at which a soluble protein has no net  charge and will precipitate out of a solution.​

  If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate.​

  This can be used along with electrophoresis to separate out proteins.
• Immunoassay techniques are 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.
• ELISA (enzyme-linked immunosorbent assay) is an analytical technique which uses antibodies to detect the presence of an antibody within a solution. There are three forms of ELISA: direct,  indirect and sandwich.
• Western Blotting is a technique which is used after SDS-PAGE  electrophoresis. 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).
• 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
• Aseptic technique eliminates unwanted microbial contaminants when culturing microorganisms 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
• 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 can divide a limited number of times, whereas tumour cells lines can perform unlimited divisions
• Plating out of a liquid microbial culture on solid media allows the number of colonyforming units to be counted and the density of cells in the culture estimated
• Serial dilution is often needed to achieve a suitable colony count
• Haemocytometers are specialised microscope cells which are used to estimate cell numbers in a liquid culture.
• In order to identify and count living cells, vital  staining is required. Any living cells will absorb the stain whilst non-living cells do not.