The Ultrastructure of the Cell

1. The Ultrastructure of the Cell

2. Task 1 – Labelling cell diagrams Let’s check the homework.

3. Task 2 – Exam questions

4. Cell Fractionation and Ultracentrifugation Cells consist of a number of organelles. We can examine their structure and function if we can isolate them. Cell fractionation = breaking up a cell to release its organelles. Ultracentrifugation = a method for the separation of the organelles by density.

5. Extracting organelles from cells

6. Steps of Cell Fractionation & Ultra Centrifugation Cell Fractionation • Tissue to be studied is cut into small pieces and placed into an ICE COLD, ISOTONIC BUFFER solution. Why? ICE COLD to stop enzyme activity. ISOTONIC (same concentration/water potential as cytoplasm) to prevent osmosis which would damage organelles). BUFFER to keep pH constant so proteins are not denatured.

7. Steps of Cell Fractionation & Ultra Centrifugation Cell Fractionation • Ground into smaller pieces using a HOMOGENISER to release the organelles from the cell. • The HOMOGENATEis filtered to remove any complete cells and large debris i.e. cell walls/membranes.

8. Steps of Cell Fractionation & Ultra Centrifugation Ultracentrifugation • Homogenate is placed in a test tube and centrifuged. The faster the speed at which the tube is spun, the greater the force generated. • Larger, more dense fragments collect at the bottom of the tube to form a PELLET. Smaller ones remain near the top suspended in a liquid called the SUPERNATENT.

9. Steps of Cell Fractionation & Ultra Centrifugation Ultracentrifugation • Pellet is removed and the supernatant remaining is re-spun at a faster speed (more force). • Process is repeated at higher and higher speeds.

10. Important info! Organelle sizes are similar in all cells so we can predict when they will form a pellet. Since the whole process involves centrifuging at different speeds it is called DIFFERENTIAL CENTRIFUGATION.