Laboratory techniques for Biologists Flashcards
What can present a hazard
Substances, organisms and equipment in a laboratory
What are examples of hazards
- A toxic or corrosive chemical
- Heat or flammable substances
- Pathogenic organisms
- Mechanical equipment
What is a risk
The likelihood of harm arising from exposure to a hazard
How can Biologists identify and control measures to minimise risks and reduce hazards
Creating a risk assessment
What are examples of control measures
- Using appropriate handling techniques
- Protective clothing and equipment
- Aseptic techniques
What are linear dilutions
When dilutions differ by an equal amount e.g. 0.1, 0.2, 0.3
What are log/serial dilutions
Dilutions that differ by a constant proportion e.g. 0.1, 0.01, 0.001
What is a standard curve
Plotting known measurements onto a graph to determine unknown measuremeants
How does a buffer control pH
A buffer is a solution where adding acids or alkalis has very little effect on the pH. This allows pH in a reaction mixture to be kept constant
What can a colorimeter be used for
A colorimeter can be used to quantify absorbance and turbidity (the cloudiness)
How do colorimeters work
Before use the colorimeters need to be calibrated with an appropriate blank sample to provide a baseline reading. Light is split into its component colours and filtered so there is one wavelength of light. This is then passed through the sample solution where a detector picks up how much light has been absorbed by the sample or transmitted (passed through)
What can absorbance be used for
To determine the concentration of a coloured solution using a suitable wavelength filter
What can percentage transmission be used for
Percentage transmission can be used to determine turbidity, such as cells in suspension
How does centrifuge separate substances
Samples are spun at incredibly fast speeds. More dense components settle to form the pellet whilst less dense components remain in the supernatant
How does paper and thin layer chromatography separate different amino acids and sugars
The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used
How does affinity chromatography separate proteins
A solid matrix or gel 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
How does Gel electrophoresis separate proteins and nucleic acids
Samples are loaded into wells in a gel which will have an electric current running through it. As they are usually charged molecules they will move towards the opposing charge. Smaller molecules will travel faster than larger molecules so they will travel further
How do native gels separate proteins
They separate proteins by their shape, size, and charge, They do this by ensuring they do not denature the molecule
How do SDS-PAGEs separate proteins
They separate by size alone. They do this by giving all molecules an equally negative charge and denature them
How do IEPs separate proteins from a mixture
Proteins with different IEPs are loaded into a gel matrix. The proteins migrate towards the charges until they reach an area with the pH or their IEP. Proteins stop migrating as they have no net charge and precipitate out
What happens if the solution is buffered (IEP)
Only the proteins that have an IEP of that pH will precipitate out
Explain the relationship between electrophoresis and IEPs
Proteins can be separated using their IEPs in electrophoresis
What is immunoassay
Its a technique which is used to detect and identify specific proteins
What do immunoassay techniques use
Stocks of antibodies with the same specificity, known as monoclonal antibodies
What is linked to a chemical label
An antigen that is specific to an antibody
What is a chemical label
Often a reporter enzyme producing a colour change,.
What is immunoassay used for
Using a specific antigen to detect the presence of antibodies to test for diseases
What is western blotting
A technique used after SDS-PAGE electrophoresis
How does western blotting work
Once the proteins have been separated in the gel they are transferred or blotted onto a solid medium. The proteins can then be identified using specific antibodies with reporter enzymes attached
What is bright field microscopy
Form of microscopy commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells
What is Fluorescence Microscopy
Form of microscopy which uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues
What does an aseptic technique do
Eliminates unwanted microbial contaminants when culturing micro-organisms or cells
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 are animal cells grown in
A medium containing growth factors from serum. The growth factors promote growth and proliferation
What is the difference between primary and tumour cells
Primary cell lines can divide a limited number of times, whereas tumour cell lines can perform unlimited divisions
How can the number of colony-forming units be counted and the density of the cells in the culture be estimated
Plating out of a liquid microbial culture on solid media
Why is a serial dilution often used
To achieve a suitable colony count
What is a haemocytometer and what does it do
Specialised microscope cells which are used to estimate cell numbers in a liquid culture
What is needed in order to identify and count living cells
Vital staining is required, any living cells will absorb the stain whilst non-living cells do not
