Laboratory techniques for biologists

Examples of hazards in the lab

• toxic or corrosive chemicals,

• heat or flammable substances,

• pathogenic organisms

• mechanical equipment

What is a risk?

The likelihood of harm arising from exposure to a hazard

What does a risk assessment involve?

identifying control measures to minimise the risk

Examples of control measures

• using appropriate handling techniques, protective clothing and equipment

• aseptic technique

Dilutions in a linear dilution series differ by what?

an equal interval, for example 0·1, 0·2, 0·3 and

so on

Dilutions in a log dilution series differ by what?

a constant proportion, for example 10-1, 10-2

,10-3 and so on

The addition of acid or alkali has what effect on the pH of a buffer and allows what?

has very small effects on the pH of a buffer, allowing the pH of a reaction mixture to be kept constant

What is the use of colorimetry

To quantify concentration and turbidity

What are the uses of a colorimeter?

• 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.

What is the use of a centrifuge?

To separate substances of differing density

What happens to substances of differing density’s in a centrifuge?

More dense components settle in the pellet and

less dense components remain in the

supernatant.

What is the use of paper and thin layer chromatography?

To separate different substances

The speed that each solute travels along the

chromatogram depends on what?

its differing solubility in the solvent used.

What is the use of affinity chromatography?

To separate proteins

Principle of 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.

What is the use gel electrophoresis?

To separate proteins and nucleic acids

Principle of gel electrophoresis

Charged macromolecules move through an

electric field applied to a gel matrix

Native gals separate what?

proteins by their shape,size and charge

Native gels do not what?

Denature the molecule

What is the use of SDS-PAGE?

Separates proteins by size alone

How does SDS-PAGE separate proteins?

By giving all the molecules an

equally negative charge and denatures them,

How can proteins be separated from a mixture?

By using their isoelectric points (IEPs)

What is an IEP?

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 what will happen?

only the protein(s) that have an IEP of that

pH will precipitate

What other separation technique can IEPs be used in?

Electrophoresis

How can a soluble protein be separated?and how?

by 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.

Immunoassay techniques are used to do what?

Detect and identify specific proteins

Immunoassay techniques use what?

Stocks of antibodies with the same specificity (monoclonal antibodies)

An antibody specific to the protein antigen is linked to what?

A chemical ‘label’

What is a ‘label’

often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used

What can happen in some cases of immunoassay?

the assay uses a specific antigen to detect the presence of antibodies

What is western blotting? And how does it work?

• 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

What is bright-field microscopy commonly used for?

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

Function of fluorescence microscopy?

uses specific fluorescent labels to bind to and visualise

certain molecules or structures within cells or tissues

What do areptic techniques do?

eliminates unwanted microbial contaminants when culturing micro-organism or cells

What do aseptic techniques involve?

the sterilisation of equipment and culture media by heat or

chemical means and subsequent exclusion of microbial contaminants

How can a microbial culture be started?

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

Many culture media exist that promote the growth of what?

Specific types of cells and microbes

What type of medium are animal cells grown in?

A medium containing growth factors from serums

What are growth factors?

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

In culture, tumour cell lines can do what?

Perform unlimited divisions

Plating out of a liquid microbial culture on

solid media allows what?

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

What is often needed to achieve a suitable colony count?

Serial dilution

What is the use of a haemocytometer?

To estimate cell numbers in a liquid culture

What is required to identify and count viable cells?

Vital staining