AH Biology U1: KA1 Laboratory Techniques for Biologists

Examples of hazards

• toxic or corrosive chemicals

• heat or flammable substances

• pathogenic organisms

• mechanical equipment

A risk assessment involves…

Identifying control measures to minimise the risk

Control measures include the use of:

• appropriate handling techniques

• protective clothing and equipment

• aseptic techniques

Linear dilution

Dilutions differ by an equal interval, e.g. 0.1, 0.2, 0.3, etc

Log dilution

Dilutions differ by a constant proportion, e.g. 10-1, 10-2, 10-3, etc

Formula for dilutions

V1C1 = V2C2

Standard curve

Where measured values of known concentration are plotted to produce a line or curve to allow the concentration of an unknown to be determined from the standard curve

Buffer

Solution where the addition of acid or alkali has very small effects on the pH, allowing the pH of a reaction mixture to be kept constant

Why are buffers useful?

Buffers are extremely useful for biological reactions as pH can affect proteins and thus the overall reaction

Colorimeters are used to quantify what?

Concentration and turbidity (cloudiness)

Describe how to use a colorimeter

• colorimeter is calibrated before use with an appropriate blank sample to provide a baseline reading

• the absorbance of a coloured solution is used to determine its concentration using suitable wavelength filters

• percentage transmission can be used to determine turbidity, such as cells in suspension

Risk

The likelihood of harm arising from exposure to a hazard

Centrifuge

Separation technique where samples are spun at incredibly high speeds to separate substances according to density. More dense components settle in the pellet whilst less dense components remain in the supernatant.

Paper and thin layer chromatography

• 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, i.e. more soluble solutes travel further

What is affinity chromatography used to separate?

Proteins

Describe the process of affinity chromatography

• A solid matrix or gel column is created with specific molecules bound to the matrix or gel (LOADING)

• Soluble, target proteins in a mixture, with a high affinity for these molecules, become attached to them as the mixture passes down the column (SEPARATION)

• Other non-target molecules with a weaker affinity are washed out (ELUTION)

Define what gel electrophoresis is and explain how this technique works

• Gel electrophoresis is a technique used to separate proteins and nucleic acids

• Charged macromolecules move through an electric field applied to a gel matrix towards the opposing charge

• Smaller molecules will travel faster than larger molecules so will travel further

Native gel electrophoresis

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

SDS-PAGE

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

Isoelectric Point

The IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution

Explain how IEP can be used alongside electrophoresis to separate proteins

• 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

Immunoassay techniques are used to…

Detect and identify specific proteins

Chemical label in immunoassay

• Reporter enzyme linked to the antibody, which indicates the presence (positive result) or absence (negative result) of the protein of interest

• Reporter enzymes often produce a colour change, but chemiluminescence, fluorescence and other reporters can be used

Western blotting

• Technique used after SDS–PAGE electrophoresis where separated proteins from the gel are transferred (blotted) onto a solid medium

• The proteins can then be identified using specific antibodies that have reporter enzymes attached

Bright-field microscopy is commonly used to observe…

• whole organisms

• parts of organisms

• thin sections of dissected tissue

• or individual cells

Fluorescence microscopy

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

Define what is meant by aseptic technique and describe what it involves

• Aseptic technique describes procedures used in laboratories to reduce contamination as well as the unwanted growth or spread of microorganisms

• 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

Microbial culture

• Method of multiplying microorganisms by letting them reproduce in a culture medium under controlled laboratory conditions

• 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

What are animal cells grown in?

• 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

Describe the difference between primary cell lines and tumour cell lines in culture

Primary cell lines can divide a limited number of times, whereas tumour cells lines can perform unlimited divisions

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

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

When plating out of a liquid microbial culture on solid media, what is often needed to achieve a suitable colony count?

Serial dilution

Haemocytometer

Specialised microscope cells which are used to estimate cell numbers in a liquid culture

Calculating cell density

1. Calculate the volume of each square

2. Count the number of cells in the grid including those that are touching the TOP and LEFT sides

3. Cell density = average cells in a square / volume of the square

State what is required to identify and count viable cells using a haecytometer and explain why

Vital staining, as living cells will not absorb stain whilst non-living cells will

Explain how isoelectric point is used to separate proteins

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

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

Requirements for immunoassay techniques

Immunoassay techniques use:

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

• Chemical ‘labels’ - The antibody specific to the protein antigen is linked to a chemical ‘label’

In some cases a specific antigen is used to detect the presence of antibodies, this is known as ELISA (enzyme-linked immunosorbant assay)

Enzyme-linked immunosorbant assay (ELISA)

Type of immunoassay which uses a specific antibody to detect the presence of antigens

Monoclonal antibodies

Stocks of antibodies with the same specificity