1.1 Laboratory techniques for biologists

What do hazards in the lab include

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

How does the addition of acid or alkali effect the pH of a buffer

Very small effect, allowing the pH of a reaction mixture to be kept constant

What is the use of a colorimeter

To quantify concentration and turbidity

What does a centrifuge do

Separate substances of different densities

What can be used for separating different substances such as amino acids and sugars

Paper and thin layer chromatography

How does a centrifuge work

More dense components settle in the pellet; less dense components remain in the supernatant

The speed hat each solute travels along the chromatogram depends on its differing solubility in the solvent used

What is affinity chromatography used for

Separating proteins

How does affinity chromatography work

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

What is gel electrophoresis used for

Separating proteins and nucleic acids

How does gel electrophoresis work

charged macromolecules move through an electric field applied to a gel matrix

What is native gels used for

Separating proteins by their shape, size and charge

How do native gels work

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

What is SDS-PAGE used for

Seperating proteins by size alone

How does SDS-PAGE work

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

How can proteins be separated from a mixture

Using their isoelectronic points (IEPs)

what is an IEP

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 what protein will precipitate

Only the protein(s) that have an IEP of that pH will precipitate

How can a soluble protein be separated

Using an electric field and a pH gradient

A protein stops migrating through the gel at it's IEP in the pH gradient because it has no net charge

What is used to detect and identify specific proteins

Immunoassay techniques

What do immunoassay techniques use

Stocks of antibodies with the same specificity, known as monoclonal antibodies

What is an antibody specific to the protein antigen linked to

a chemical 'label'

What is the 'label'

Often a reporter enzyme producing a colour change, but chimiluminescence, fluorescence and other reporters can be used

Why in some cases do the assay use a specific antigen

To detect the presence of antibodies

What technique is used after SDS-PAGE electrophoresis

Western blotting

What is western blotting

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

What is bright field microscopy used for

to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells

What does fluorescence microscopy use

specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues

What do aseptic techniques do

Eliminates unwanted microbial contaminants when culturing microorganisms or cells

How can a microbial culture be started

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

What does aseptic techniques involve

the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants

What do many existing culture media promote

The growth of specific types of cells and microbes

How are animal cells grown

In medium containing growth factors from serum

In culture how many times can primary cell lines divide

a limited number of times

In culture how many times do tumour cell lines divide

Unlimited number of times

What are growth factors

Proteins that promote cell growth and proliferation

Why are growth factors essential

for the culture of most animal cells

What does plating out of a liquid microbial culture on solid media allow?

the number of colony-forming units to be counted and the density of cells in the culture estimated

What is needed to achieve a suitable colony count

Serial dilution

How can you estimate cell numbers in a liquid culture

Using a haemocytometer

What is required to identify and count viable cells

Vital staining