Objectives • Learn preparing and staining procedure to identify the stages of mitosis in onion root tip. • To differentiate between the different stages of mitosis. • Calculate the mitotic index.
Types of Cell Division • Mitosis • Produces two new daughter cells with the same number and kind of chromosomes as the parent cell • Meiosis • Reduction division produces progeny cells with one-half the genetic content and number of chromosomes as parent cell • Produces gametes/spores
Mitosis • Purpose: • Mitosis occurs in order for organisms to grow and develop. • In order to replenish dead or dying cells such as skin cells, and cells in the digestive tract. • Karyokinesis • process of nuclear division (division of genetic material) • Cytokinesis • Process of dividing cytoplasm/cell
The Cell Cycle • The life of a cell is divided into three stages known as the cell cycle: 1. Interphase: cell carries out normal functions and prepares to divide 2. Mitosis: nucleus divides splits into two 3. Cytokinesis: cell and contents divide into two daughter cells.
Interphase • This phase consist of the G1,S, and G2 phases of the cell cycle. • The chromatin is diffuse. • It may not look like much is going on here, but there is a lot of activity because the cell must prepare for Mitosis: • protein synthesis, • DNA synthesis, • replication of other cellular structures.
Mitosis • There are 4 main phases: • Prophase, • Metaphase, • Anaphase, • Telophase. • Cytokinesis (division of the cytoplasm) follows and one cell becomes two.
Mitosis: Prophase Major processes during this phase: • Chromosomes condense and form visible bodies • Chromosomes become thicker, shorter, and easily visible when stained under the light microscope. • Two “sister chromatids” join near their middle at a structure called the centromere. • The nucleolus and the nuclear membrane disappear. The mitotic apparatus the spindle, begins to organize within the cell
Mitosis: Metaphase • Chromosomes become aligned at midpoint or equator between poles of the cell • are at their thickest and shortest structure. • They are easily identified as two longitudinally double sister chromatids.
Mitosis: Anaphase • The centromere replicates and splits • The sister chromatids separate and are pulled to opposite sides of the cell
Mitosis: Telophase • Chromosomes now uncoil • Nuclear envelope reappears and surrounds the chromosomes Cytokinesis • The cytoplasm and all its contents are divided between the 2 daughter cells (cytoplasmic division) • membrane creates between the 2 new daughter cells • In plants, such as the onion root tip cells, this is seen as the formation of a cell plate
Mitosis in Root Tip • In a growing plant root, the cells at the tip of the root are constantly dividing to allow the root to grow. • Because each cell divides independently of the others, a root tip contains cells at different stages of the cell cycle. • This makes a root tip an excellent tissue to study the stages of cell division
Materials • Slides & cover slips • Microscope • Fresh onion root tips • Fixative ( methanol-acetic acid 3:1 v/v) • Forceps • 1 M HCl • Razor blade • Stain • Paper towel, or absorbent paper
Method • Cut 2-3 mm of onion root • Use forceps to transfer an onion root tip into the cup of HCl. • Leave for 4 minutes • Transfer the root tip to the cup containing fixative and leave it for 4 minutes. • Then place the root tip on a slide. • Cover the root tip with a few drops of stain for 2 minutes • Cover the root tip with one to two drops of 45% acetic acid • Put a cover slip over the root, put a paper towel or other absorbent paper and with your thumb firmly press on the cover slip.
Observe your preparation under the low power (X10) of a microscope • Search the slide to find cells in various stages of cell division, once you have located cells in division, change to high power (X40) & try to observe several stages of division. • Record the number of cells in each stage. Count at least three full fields of view. You should have counted over 200 cells. • Record your data in the table • Calculate the percentage of cells in each phase and record in the table
Mitotic index • A measure for the proliferation status of a cell population. • It is defined as the ratio between the number ofcellsinmitosisand the total number of cells.
Interphase Prophase Metaphase Anaphase Telophase