1.1 LAB TECHNIQUES FOR BIOLOGISTS Flashcards
What can chemicals and organisms be?
Intrinsically hazardous
What can chemicals and organisms pose risk to?
People, other organisms, environment
Give an example of a physical control measure.
Personal protective equipment (PPE)
Give an example of a biological control measure.
Using a more suitable strain of microorgansim
What is a linear dilution series?
A range of dilutions which differ by an equal interval
How do you perform a linear dilution series?
Add different volumes of stock solution to different volumes of solvent
What is a logarithmic dilution series?
A range of dilutions which differ by a constant proportion
How do you perform a logarithmic dilution series?
Add same volumes of stock to same volumes of solvent using each solution as stock for subsequent dilution
What is the result of an error being made while making stock?
Errors are compounded in any further dilutions made
What is used to determine the concentration of an unknown solution?
A standard curve for determination of an unkown
What is a ‘standard curve’?
A series of ‘standards’ of known concentrations are measured and depicted on a graph
What is a pH buffer?
A solution whose pH changes very little when either a small amount of acid or base is added
What is used to measure pH?
A meter or indicator
Name 5 tools used to measure liquid volumes.
Cylinders, pipettes, burettes, autopipettors, syringes
What is a colorimeter used for?
Quantifying the concentration of a pigmented compound
What can substances be separated by?
Solubility, size, shape, charge
What is centrifugation used to separate?
Pellet and supernatant
What is the pellet?
Largest and densest materials at bottom of tube
What is the supernatant?
The liquid remaining above the pellet
What separates pellets and supernatants?
Size and density
What is the purpose of chromatography?
To separate the components of a mixture - amino acids and proteins
Name the three types of chromatography.
Paper, thin layer, affinity
How does paper chromatography separate components?
Solvent is drawn up a piece of chromatography paper
How does thin layer chromatography separate components?
Using a TLC strip
How does affinity chromatography separate components?
Relies on binding interactions between protein of interest and a ligand
What does electrophoresis separate?
Proteins
How does electrophoresis separate proteins?
Uses a current flowing through a buffer
What factors affect protein migration in a gel?
Size and charge
How can proteins be separated by their pH?
They are separated at their iso-electric point
What happens at a protein’s iso-electric point?
Has overall neutral charge and precipitates out solution
What can antibody techniques be used for?
Detection and identification of specific proteins
What proteins are involved in immunoassay techniques?
Antibodies linked to reporter enzymes
What do antibodies linked to reporter enzymes in immunoassay techniques do?
Cause a colour change in presence of a specific antigen
What are antibodies also involved in?
Protein blotting and immunohistochemical staining of tissue
What is the first stage of protein blotting?
Proteins separated by electrophoresis and transferred to a membrane
What happens after the proteins are transferred to a membrane in protein blotting?
Membrane is probed for protein of interest using an antibody linked to a detectable label
How does the label indicate the presence of a target protein in protein blotting?
May be linked to a reporter enzyme which causes a detectable colour change in protein’s presence
What is immunohistochemical staining used for?
To visualise distribution of specific cellular components in live cells
How are monoclonal antibodies produced?
Using hybridomas
How are hybridomas produced?
Fusing a B lymphocyte with a myeloma cell
What is used to fuse a B lymphocyte with a myeloma cell?
Polyethylene glycol (PEG)
What is bright field microscopy used for?
Examination of whole/parts of organisms or thin sections of dissected tissue
How does bright field microscopy work?
Light transmitted through a specimen to an objective lens (for magnification) and then to an eyepiece
What does fluorescence microscopy enable?
Visualisation of particular protein structures
How does fluorescence microscopy work?
Fluorescent markers added to specific protein structures, cells placed on a slide, structures visualised through fluorescence microscope
What are aseptic techniques?
Practices and procedures used to prevent contamination from pathogens
Give two examples of aseptic techniques.
Sterilisation of containers, equipment and materials, disinfection of working area
What is inoculum?
Cells that are used to inoculate culture media
What are explants?
Small pieces of tissue
What are haemocytometers used for?
Estimation of total cell counts
What is vital staining used for?
Estimation of viable cell counts
What does culture media contain?
Requirements of the cells
What does complex culture media contain?
Factors from serum for animal cell lines
Compare the lifetime of primary cell lines vs cancer cell lines.
Primary cell lines have a limited lifetime whereas cancer cell lines grow and divide indefinitely
What are growth regulators used for in plant tissue culture?
Induction of embryogenesis to generate whole new embryonic plants
