Structure, function and growth of prokaryote and eukaryote cells

1. Structure, function and growth of prokaryote and eukaryote cells (iii) Differentiation of cells into tissues and organs. (iv) Cell and tissue culture Mammalian cell cultures Bacterial and Fungal Cultures (v) Plant tissue culture

2. Basic requirements for cell culture • Source of cell material • A suitable container for cell growth • Growth medium containing essential nutrients • Opportunity for gas exchange • Control of abiotic factors e.g. temperature and pH • Avoidance of contamination

3. Aseptic techniques • Methods employed to prevent contamination • E.g. sterilisation of equipment, media (substance in which the cells grow) and surfaces • Autoclaving = heating substance to a high temperature (usually 121oC) under high pressure to kill microbes and their spores. • Swabbing with disinfectant or alcohol • Running equipment through a flame before use. • Minimising exposure of equipment to the air

4. Aseptic Techniques cont…. • Biological Safety cabinets

5. Aseptic techniques cont…

6. Culturing Mammalian Cells • Source of Cell material • Tissue samples are treated with a proteolytic (protein digesting) enzyme e.g trypsin to separate cells from each other • Normal cells die after a finite number of divisions when grown in culture, making long-term culturing difficult. • For this reason cell lines are often prepared from cancer cells or cells that have been genetically modified to become immortal.

7. Culturing Mammalian Cells cont… • Suitable container • Mammalian cells require a solid surface for growth in culture

8. Culturing Mammalian Cells cont… • Growth medium • Is complex • Minimum growth medium contains balanced salt solution, amino acids, vitamins and glucose. • Additions are made to minimal growth media according to the requirements of the cells. • Antibiotics may be added to prevent contamination • Foetal Bovine Serum (FBS) (or other animal serum) is essential to promote cell proliferation (division)

9. Culturing Mammalian Cells cont… • Events in culturing mammalian cells • Growth medium is inoculated with cell source • Cells adhere to the surface of the container • Cells spread out and then divide to form a monolayer • When the entire surface is covered with cells just touching one another, they are said to be confluent or to have reached confluence.

10. Culturing Mammalian Cells cont… Cells spreading out and dividing Confluent cells Note: If a single cell was isolated and used to inoculate the culture, then all of these cells will be clones of one another.

11. Culturing Bacteria and Fungi • Source of Cell Material • Commercially bought • Isolated from environment • Suitable Container • Bacteria and liquid can be grown in solid or liquid medium and do not need a solid surface to adhere to.

12. Culturing Bacteria and Fungi cont.

13. Culturing Bacteria and Fungi cont. • Growth Medium • Is basic • Most microbes only require a source of carbon (usually a sugar for energy and to make basic cell components) • Some require special additives

14. Culturing Bacteria and Fungi cont. • Events in growing Bacteria and Fungi • After an initial lag phase microbes will undergo exponential growth. • Cultures then enter a stationary phase as nutrients become limited and wastes accumulate. • Death will eventually occur if not sub-cultured

15. Culturing Bacteria and Fungi cont.

16. Learning Activities • Read DART pg 20 – 22 • Scholar (Unit 1, 3.1 – 3.3) • Techniques in Microbial Culture • Cloning cells for an Interferon Bioassay