Laboratory Techniques Flashcards
Hazards in the lab include…
toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms and mechanical equipment
What is risk?
the likelihood of harm arising from exposure to a hazard
What do control measures include?
appropriate handling techniques, protective clothing and equipment and aseptic techniques
Dilutions in a linear dilution series differ by…
an equal interval
dilutions in a log dilution series differ by…
a constant proportion
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
Absorbance is used to determine..
the concentration of a COLOURED SOLUTION using suitable wavelength filters
Percentage transmission is used to…
determine turbidity
What can percentage transmission be used to estimate?
the number of cells in suspension
What do centrifuges do?
separate substances of differing density
What can paper and thin layer chromatography be used for?
separating different substances such as amino acids and sugars
More dense components settle in the…
pellet
less dense components remain in the…
supernatant
The speed that each solute travels along the chromatogram depends on…
its differing solubility in the solvent used
What happens in gel electrophoresis?
charged macromolecules move through an electric field applied to a gel matrix
Gel electrophoresis separates proteins based on their
shape, size and charge
SDS separates proteins by…
size alone
Why don’t native gels denature the molecule?
so that separation is by size, shape and charge
SDS PAGE gives all the molecules an equally…..
negative charge and denatures them
Proteins can be separated from a mixture using their…
isoelectric points (IEPs)
What is the IEP?
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 a technique called…
electrophoresis
soluble proteins can be separated using an electric field and a
pH gradient
A protein stops migrating through the gel at it’s IEP in the pH gradient because…
it has no net charge
What are immunoassay techniques used for?
Used to detect and identify specific proteins
What do immunassay techniques use?
monoclonal antibodies
What are monoclonal antibodies?
stocks of antibodies with the same specificity
What is western blotting?
A technique used after SDS-PAGE electrophoresis
Describe the process of western blotting
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
How can separated proteins be identified?
By using specific antibodies that have reporter enzymes attached
What is the ‘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
What is Bright-field microscopy commonly used to observe?
Whole organisms, parts of organisms, thin sections of dissected tissue or individual cells
What is fluorescence microscopy?
Uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues
What is the purpose of aseptic technique
eliminates unwanted contaminants when culturing micro-organisms or cells
How can a microbial culture be started?
Using an inoculum of microbial cell;s on an agar medium, or in a broth with suitable nutrients
How are animal cells grown?
in a medium containing growth factors from serum
In culture, primary cell lines can divide a __________ number of times
limited
tumour cell lines can perform ____________ divisions
unlimited
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 to be estimated
_______ ___________ is often needed to achieve a suitable colony count
serial dilution
What do haemocytometers do?
estimate cell numbers in a liquid culture
What is vital staining?
Vital staining is required to identify and count viable cells
What does asceptic technique involve?
The sterilisation of equipment and culture of media by heat or chemical means and subsequent exclusion of microbial contaminants
Many culture media exist that promote…
the growth of specific types of cells and microbes
What are growth factors?
Proteins that promote cell growth and proliferation
Growth factors are essential for the…
culture of most animal cells
