Tissue Culture Unit one Cell 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.

Arrangements Plant 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.

Arrangements Plant 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 development Needed 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 culture explant Grown in media containing plant growth regulators Cell proliferation Callus Sub-culture Change 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.