Key Area 1 - Laboratory Techniques for Biologists Flashcards
Laboratory Techniques for Biologists
Health and Safety
What can present a hazard in a labatory?
Substances, organisms, and equipment in a
laboratory can present a hazard.
List hazards in the lab:
Hazards in the lab include toxic or corrosive
chemicals, heat or flammable substances,
pathogenic organisms, and mechanical
equipment.
Risk assessments control what in the lab?
Hazard, risk, and control of risk in the lab by risk assessment.
What is a risk and what do risk assessments involve?
Risk is the likelihood of harm arising from
exposure to a hazard.
Risk assessment involves identifying control measures to minimise the risk.
What do control measures include?
Control measures include using appropriate handling techniques, protective clothing and equipment, and aseptic technique.
Liquids and Solutions
Explain linear and log dilutions:
Dilutions in a linear dilution series differ by an equal interval, for example 0·1, 0·2, 0·3 and so on.
Dilutions in a log dilution series differ by a
constant proportion, for example
10-1, 10-2, 10-3 and so on.
Why is a standard curve produced?
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.
Why is a colorimeter used?
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.
Why are buffers used?
Buffers are used 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.
Separation Techniques
A centrifuge is used to…
Use of centrifuge to separate substances of
differing density.
More dense components settle in the pellet; less dense components remain in the supernatant.
What method is used to separate different substances?
Paper and thin layer chromatography can be 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.
What method is used to separate proteins?
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.
What is the principle of gel electrophoresis and its use?
Principle of gel electrophoresis and its use in separating proteins and nucleic acids.
Principle of gel electrophoresis and its use in separating proteins and nucleic acids.
How do native gels separate proteins?
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.
How do SDS–PAGE separate proteins?
SDS–PAGE separates proteins by size alone.
SDS–PAGE gives all the molecules an
equally negative charge and denatures them, separating proteins by size alone.
What mixture can proteins be separated from?
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
What techniques are used to detect and identify specific proteins?
Immunoassay techniques are used to detect and identify specific proteins.
These techniques use stocks of antibodies
with the same specificity, known as
monoclonal antibodies.
A chemical ‘label’ is linked to what?
An antibody specific to the protein antigen is linked to a chemical ‘label’.
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.
Microscopy
Explain 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.
Why is bright-field microscopy commonly used?
Bright-field microscopy is commonly used to observe whole organisms, parts of
organisms, thin sections of dissected tissue
or individual cells.
What does fluorescence microscopy use?
Fluorescence microscopy uses specific
fluorescent labels to bind to and visualise
certain molecules or structures within cells or tissues.
Aseptic Technique and Cell Culture
What does aseptic technique eliminate?
Aseptic technique eliminates unwanted
microbial contaminants when culturing microorganisms or cells.
What does aseptic technique involve?
Aseptic technique involves the sterilisation of equipment and culture media by heat or
chemical means and subsequent exclusion of microbial contaminants.
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.
Many culture media exist that promote the
growth of specific types of cells and
microbes.
Where are animal cells grown and what with?
Animal cells are grown in medium containing growth factors from serum.
What are growth factors?
Growth factors are proteins that promote cell growth and proliferation. Growth factors are essential for the culture of most animal cells.
What can primary cells do in culture?
In culture, primary cell lines can divide a
limited number of times, whereas tumour
cells lines can perform unlimited divisions.
What does plating out of a liquid microbial culture on solid media allow?
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.
Why is serial dilution often needed?
Serial dilution is often needed to achieve a
suitable colony count.
What is used to estimate cell numbers in a liquid culture?
Method and use of haemocytometer to
estimate cell numbers in a liquid culture.
Why is vital staining required?
Vital staining is required to identify and count viable cells.
