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Tissue Culture
Unit oneCell and Molecular Biology
Advanced Higher Biology
Arrangements Differentiation of cells into tissues
and organs.
• Cells undergo differentiation to become specialised cells that are organised into tissues and organs.
• Cellular differentiation depends on changes in gene expression resulting in genes being switched on and off
Arrangements Cell and tissue culture
• The ability of stem cells to differentiate, unlike specialised cells.
• The lac operon in E.coli.– Repressor molecule, – regulator gene, – inducer, – operator– structural gene
Arrangements Mammalian cell culture.
• Requirement of aseptic conditions, solid surface, growth factors and nutrients in complex growth media.
• Use case studies to illustrate the applications of cell culture.
Arrangements Mammalian cell culture.
• The addition of animal serum such as fetal bovine serum (FBS) to promote cell proliferation and antibiotics to prevent bacterial growth. – Use of proteolytic enzymes to release
cells from source tissue. • Cells adhere to the surface, spread out
and divide until a monolayer is formed and the cells are confluent.
Arrangements Mammalian cell culture.
• Difficulty in maintaining cultures of mammalian cells due to cells dying after a finite number of divisions in culture.
• Cell lines prepared from cells which undergo a genetic change that makes them immortal or from cancer cells. A clone is the result of cell cloning in which a single cell is isolated and allowed to proliferate to form a large colony.
Arrangements Bacterial and fungal cultures • The advantages of the simpler
growth media requirements and culture conditions for bacteria and fungi compared to mammalian cells.
ArrangementsPlant tissue culture.
• Techniques used (including requirement for aseptic conditions and suitable growth medium).
• Growth of explants on suitable media to produce a callus.
• The use of growth regulators such as auxins and cytokinins to cause tissue differentiation.
ArrangementsPlant tissue culture.
• Production of pathogen-free plantlets and plants, generation of new varieties of plants and use in plant propagation.
• Use of terms protoplast and totipotent.
• Examine photographs of protoplasts at different stages of culture and as fusion products.
Differential gene
expression in developmentNeeded in similar depth to that
taught at Higher Biology
Revision Questions
• Name the process in which DNA is copied into RNA.
• Name the process in which RNA is read into protein
• What is a transcription factor?
Revision Questions
• Name the process in which DNA is copied into RNA.– transcription
• Name the process in which RNA is read into protein– translation
• What is a transcription factor?– A protein that interacts with RNA
polymerase, or other transcription factors, to regulate gene expression.
Control of gene expression
• Temporal• Spatial
• Drosophila melanogaster– Fruit fly– Used in studies
Development in Drosophila
Egg to organism
Mouse
Cell and Tissue Culture
Four major applications
• agriculture • pharmaceuticals • food production • biodegradation
Conditions for growth• a source of suitable cells; • the growth medium; • the type of growth container or
fermenter; • temperature; • pH; • gas exchange • aseptic conditions; • a method for monitoring cell growth; • safety measures
Data interpretation• Assume that bacterial cells have a
doubling time of 30 minutes, and that mammalian cells have a doubling time of 24 hours.– Calculate the number of cells that would
exist after one day of growth if you start with one cell in each culture.
– For the bacterial culture only, draw a graph to show the pattern of growth for the first 4.5 hours.
– Explain why the bacterial growth achieved after one day is unlikely to be achieved in practice.
Micro-organisms
• Categories of Micro-organisms– bacteria; – fungi; – protozoa; – algae.
• Microorganisms are relevant to many aspects of human existence– examples
Growth requirements
• nutrient medium • temperature• pH• gaseous environment• light
Bacterial Culture• Revision of bacterial growth
Questions• In which phase are the bacteria
dividing at a constant rate?• In which phase does the rate of cell
division equal the rate of cell death?• In which phase are the bacteria
metabolically active but not dividing?
• In which phase does bacterial cell death exceed cell division?
Mammalian Cell Culture
• More carefully controlled growth conditions
• Anchorage dependent–Monolayer– Confluence sub-culturing
• Non-anchorage dependent– E.g. blood– Grown in suspension
Growth medium
• Balanced salt solution with amino acids, glucose and vitamins
• Serum e.g. fetal bovine serum– Essential for animal cell proliferation– Usually 5-10% FBS
Primary cell culture
• Tissues treated with a proteolytic enzyme to separate cells from each other.
• Normal cells – finite division• Immortalised cell lines– E.g. cells derived from tumours– neoplastic
Uses of cell cloning
• Isolation of mutant cells• Investigating cell growth• Biotechnology– Vaccines– hormones
Plant Tissue Culture
• Nuclear totipotency– Plant cells have ability to regenerate
complete plants under appropriate conditions.
Plant tissue cultureexplant
Grown in media containing plant growth regulators
Cell proliferation
Callus
Sub-cultureChange growth regulators
Differentiation roots and shoots
Hybrid Plant cells
• Protoplast fusion– Revise• Formation of protoplasts• Growth of protoplasts
Essay• Discuss the requirements for the growth of microbial cells and describe, with the aid of a labelled diagram, the pattern of growth of a batch culture of microbial cells.