1.1 Lab techniques for biologists Flashcards
What presents a hazard in a lab?
Substances, organisms, and equipment in a laboratory can present a hazard.
Give four examples of hazards in a lab
Hazards in the lab include:
- Toxic or corrosive chemicals
- Heat or flammable substances
- Pathogenic organisms
- Mechanical equipment.
What is risk?
Risk is the likelihood of harm arising from exposure to a hazard.
What is risk assessment?
Risk assessment involves identifying control measures to minimise the risk.
Give three examples of control measures used to minimise risk
Control measures include:
- Using appropriate handling techniques
- Protective clothing and equipment
- Aseptic technique.
Linear dilutions
Dilutions in a linear dilution series differ by an equal interval, for example 0·1, 0·2, 0·3 and so on.
Log dilutions
Dilutions in a log dilution series differ by a constant proportion, for example 10-1, 10-2, 10-3 and so on.
How does a standard curve work?
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.
How do pH buffers 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.
What are colorimeters used for?
Colorimeters can be used to quantify concentration and turbidity (cloudiness).
Before use, colorimeters must be…
Calibrated with an appropriate blank sample as a base line.
In a colorimeter, what is absorbance used to determine?
Absorbance can be used to determine concentration of a coloured solution using suitable wavelength filters.
In a colorimeter, what is percentage transmission used to determine?
Percentage transmission can be used to determine turbidity, such as cells in suspension.
What is a centrifuge used for?
A centrifuge is used to separate substances of differing density.
How does a centrifuge work?
Samples are spun so that more dense components settle in the pellet (solid) and less dense components remain in the supernatant (liquid).
What substances do paper and thin layer chromatography separate?
Paper and thin layer chromatography can be used for separating different substances such as amino acids and sugars.
How do paper and thin layer chromatography work?
The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used.
What is affinity chromatography used for?
Affinity chromatography is used to separate proteins.
How does affinity chromatography work?
A solid matrix or gel column is created with specific molecules (usually receptors) bound to it.
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?
Gel electrophoresis is used to separate proteins and nucleic acids.
How does gel electrophoresis work?
Charged macromolecules move through an electric field applied to a gel matrix.
Native gels
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, separating proteins by size alone.
What is isoelectric point?
IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
What is IEP used for?
Proteins can be separated from a mixture using their isoelectric points.
Proteins can also be separated using their IEPs in electrophoresis.
pH buffers and IEP
If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate.
How does separation using IEPs work?
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?
Immunoassay techniques are used to detect and identify specific proteins.
What do immunoassay techniques use?
Immunoassay techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies
Chemical ‘labels’
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.
What is sometimes used with immunoassay techniques?
In some cases the assay uses a specific antigen to detect the presence of antibodies to test for diseases.
When is Western blotting used?
Western blotting is a technique used after SDS–PAGE electrophoresis.
How does Western blotting work?
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
Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues
What is aseptic technique?
Aseptic technique involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants.
What does aseptic technique do?
Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells.
How can a 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.
Growth of specific cells or microbes
Many culture media exist that promote the growth of specific types of cells and microbes.
Culture of animal cells
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.
Primary cell lines
In culture, primary cell lines can divide a limited number of times.
Tumour cell lines
Tumour cells lines can perform unlimited divisions.
Colony counts and estimating the density of cells
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
What are haemocytometers used for?
Haemocytometers are used to estimate cell numbers in a liquid culture.
How do you use a haemocytometer?
- Calculate the volume of the ‘large’ squares in the grid.
- Count the number of cells in each ‘large’ square and calculate an average.
- Cell density = average no. of cells per square / volume of the square.
What is vital staining needed for?
Vital staining is required to identify and count viable cells.
