advanced higher biology unit 1 ka 1

what is and can present a hazard

Substances, organisms, and equipment in a laboratory can present a hazard

Risk is the likelihood of harm arising from exposure to a hazard.

like toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms

risk assessment

Risk assessment involves identifying control measures to minimise the risk.

Hazard, risk, and control of risk in the lab by risk assessment

Method and uses of linear and log dilution

Dilutions in a linear dilution series differ by an equal interval, for example 0·1, 0·2, 0·3 and so on.

you would add different volumes of stock solution to different volumes of solvent

Dilutions in a log dilution series differ by a constant proportion, for example 10-1 , 10-2 , 10-3 and so on.

each dilution solution acts as a stock for the subsequent dilute

Production of a standard curve to determine an unknown

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.

made by plotting absorbance readings for series of standards of known concentrations of substance

Use of buffers to control pH

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.

Method and uses of a colorimeter to quantify concentration and turbidity

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.

density of cells in culture

Use of centrifuge to separate substances of differing density

More dense components settle in the pellet

less dense components remain in the supernatant.

ensure tubes are balanced

Paper and thin layer chromatography can be used for separating different substances such as

and rf calc

amino acids and sugars

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

amino acid have varying affinity to mobile phase high affinity travel faster

Rf= distance travelled by component/ distance travelled by solvent

Principle of affinity chromatography and its use in separating proteins

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.

Separation of one specific target protein

Principle of gel electrophoresis and its use in separating proteins and nucleic acids

proteins and nucleic acid separated upon charge size or shape

current flows through gel immersed in buffer which acts as sieve

Charged macromolecules move through an electric field applied to a gel matrix.

Native gels separate proteins by their shape, size and charge

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

SDS–PAGE ( form of gel electrophoresis ) separates proteins by size alone

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

then migrate towards positive electrode

small proteins travel further through gel than large proteins

brings folded proteins down to linear molecules

Proteins can be separated from a mixture using their isoelectric points (IEPs)

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

Proteins can also 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.

Detecting proteins using antibodies Immunoassay techniques are used to detect and identify specific proteins

These techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies

generation of monoclonal antibodies- a supply of antibodies that are identical and will bind to exactly same feature of antigen

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.

in some cases the assay uses a specific antigen to detect the presence of antibodies.

western blotting

Western blotting is 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

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

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.

how can microbial culture be started

A microbial culture can be started using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients

Many culture media exist that promote the growth of specific types of cells and microbes.

growth factors

Growth factors are proteins that promote cell growth and proliferation. Growth factors are essential for the culture of most animal cells.

Animal cells are grown in medium containing growth factors from serum

cell culture-

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

Serial dilution is often needed to achieve a suitable colony count

Method and use of haemocytometer

allows estimation of cell numbers within a liquid cell culture to be made

Vital staining is required to identify and count ( dead ) viable cells and needed to identify and count viable cells

vital staining

Technique used to differentiate between viable and non-viable cells

sample treated with dye

living cells actively pump out dye

non-viable cells cant pump the dye out

non viable cells remain stained

estimate number of living cells only unstained cells are counted as they are viable cells