Advanced Higher Biology Unit 1 KA 1

What can present a hazard?

Substances, organisms and equipment in a laboratory

What are some examples of hazards in a lab?

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

What is risk?

The likelihood of harm occurring as a result of a hazard

What's does risk assessment involve?

identifying control measures to minimise risk

What are some examples of control measures in a lab?

Appropriate handling techniques, protective clothing and equipment, and aseptic technique

What can living organisms pose potential harm to?

The health and safety of people working in the laboratory and the environment

How are living organisms controlled?

By biological means

What may GM microorganisms have?

Genes which prevent their survival outside the lab

How do dilutions in linear dilutions differ?

By an equal interval, e.g 0.1, 0.2, 0.3

How do dilutions in log dilutions differ?

By a constant proportion, e.g 10^-1, 10^-2, 10^-3

What is a serial dilution?

Repeated dilutions from a stock solution

What does plotting measured values for known concentrations to produce a line or curve allow for?

The concentration of an unknown to be determined from the standard curve

What are buffers?

Aqueous solutions that show little variation in their pH despite the addition of an acid or alkali, allowing the pH to be constant

What can a colorimeter do?

Quantify concentration and turbidity of a pigmented compound

How is a colorimeter calibrated?

With an appropriate blank as a baseline

How is absorbance used in colorimetry?

To determine concentration of a coloured solution using suitable wavelength filters

How is percentage transmission used in colorimetry?

To determine turbidity such as cells in suspension

What are the 4 separation techniques?

1. centrifugation
2. chromatography
3. electrophoresis
4. isoelectric points

What is centrifugation used for?

It is used to separate substances of different density. More dense components settle in the pellet, less dense components settle in the supernatant

What is chromatography used for?

To separate different substances such as amino acids and sugars

What is the speed of solute dependent on?

Its differing solubility in the solvent used

How does thin layer chromatography work?

It uses a thin, uniform layer of silica gel onto glass metal or rigid plastic

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? And how does it work?

To separate proteins and nucleic acid and charged macromolecules move through an electronic field applied to a gel matrix

How does protein electrophoresis work? And what factors affect protein migration?

It uses current flowing through a buffer to separate proteins. Shape, size and charge are factor affecting protein migration in a gel

How do native gels work?

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

How does SDS-PAGE work?

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

What is isoelectric point (IEP)?

The pH at which a soluble protein has no net charge and will precipitate out of solution. If solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate

How can proteins 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

What are immunoassay techniques used for?

To detect and identify specific proteins

What are immunoassay techniques based on?

The ability of antibodies to bind to specific antigens

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 a chemical label?

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

In some cases, what does the assay use?

A specific antigen to detect the presence of antibodies

When is western blotting used?

After SDS-PAGE electrophoresis

How does western blotting work?

The separated proteins from the gel are transferred (blotted) onto a solid medium and 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 is fluorescence microscopy used for?

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

What does aseptic technique do?

Eliminates unwanted microbial contaminants when culturing micro-organisms or cells

What does aseptic technique 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?

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

What do many culture media promote?

The growth of specific types of cells and microbes

What are the 4 phases of microbial growth?

1. lag phase
2. log/exponential phase
3. stationary phase
4. death phase

What is cell culture?

A laboratory technique used to grow individual cells under laboratory conditions

How are animal cells grown?

In medium containing growth factors from serum

What are growth factors?

Proteins that promote cell growth and proliferation which are essential for the culture of most animal cells

In culture, what can primary cell lines do compared to tumour cells?

Divide a limited number of times, whereas tumour cell lines can preform unlimited divisions

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 serial dilution needed for?

To achieve a suitable colony count

What is a haemocytometer used for?

To estimate cell numbers in a liquid culture

What is vital staining required for?

To identify and count viable cells