The cell cycle is the series of events that takes place in a cell leading to its division and duplication (replication). In cells without a nucleus ( prokaryotic cells e.g. bacteria), the cell cycle occurs through a process termed binary fission . In cells with a nucleus ( eukaryotes ), the cell cycle can be divided in two brief periods: interphase —during which the cell grows, accumulating nutrients needed for mitosis and duplicating its DNA —and the mitosis (M) phase, just after which the cell splits itself into two distinct cells, often called "daughter cells". The cell-division cycle is a vital process by which a single-celled fertilized egg develops into a mature organism, as well as the process by which hair , skin , blood cells , some internal organs are renewed and wounds are healed

Figure 1

Diagram - Cell division.

The cell cycle consists of four distinct phases: G 1 phase , S phase (synthesis), G 2 phase (collectively known as interphase ) and M phase (mitosis). M phase is itself composed of two tightly coupled processes: mitosis, in which the cell's chromosomes are divided between the two daughter cells, and cytokinesis , in which the cell's cytoplasm divides in half forming two distinct cells.

Figure 2

Diagram - Schematic of the cell cycle. outer ring: I = Interphase , M = Mitosis ; inner ring: M = Mitosis , G ₁ = Gap 1 , G ₂ = Gap 2 , S = Synthesis ; not in ring: G ₀ = Gap 0/Resting . [1]

Table – Phases of the cell cycle (the details of the G and S phases are not required but are included to give an overview).

Figure 3

Diagram – Allium (Onion) cells in the different cycle of mitosis.

Growth – Living tissue grows by mitosis e.g. bone and skin.

Repair - Damaged and worn-out tissues are replaced with new cells by mitosis.

Asexual reproduction - Single-celled (unicellular) organisms and bacteria often reproduce asexually by mitosis. Organisms like amoeba are able to split from a single individual into two and therefore can reproduce without a mate and sexual reproduction.

In mitosis at the end of prophase the chromosomes appear as X-shaped threads. Each thread is in fact a chromatid and they are joined in the centre at a point called the centromere. There are two of each chromosome and the full set of chromosomes is often shown with the complimentary or homologous chromosomes paired up.

Figure 4

Introduction

Talking about what chromosomes do during mitosis could be very interesting, but seeing them for yourself adds an extra dimension.

Video - Preparing Microscope Slides

http://www.youtube.com/watch?v=GHnndVuaync

Lesson Organisation

The allium/onoin roots need to be prepared 1-10 days in advance of the lesson. Some practitioners report that cutting the root tips around noon makes a difference to the mitotic index, so you may want your technician to cut and ‘fix’ the tips in ethanoic alcohol rather than ask your students to carry out this step. If you have access to a video microscope it is worth capturing some images as this procedure can be frustrating.

Method

1. Pour approximately 5 ml. methanol-acetic acid  fixative  into a small beaker. Place 2-3 mm length onion root tip into the fixative. Incubate at 60 C for 15 mins. 2. At the end of the fixative incubation period, pour off the fixative into a waste beaker. Be careful not to pour off your fixed onion root tip. Now add approximately 5 ml. 1 M HCl to your fixed onion root tip to partially  hydrolize  the cells. Incubate at 60 C for  exactly 10 mins.  3. At the end of the hydrolysis incubation period, pour off the 1 M HCl into a waste beaker. Be careful not to pour off your fixed/hydrolyzed onion root tip. Now add approximately 1 ml. Feulgen stain to your fixed/hydrolyzed onion root tip in order to stain  the chromosomes. Bath your onion root tip in the Feulgen stain for 20-30 mins. to allow the Feulgen stain to penetrate the chromosomes.4. To make a slide of your stained onion root tip, transfer your onion root tip from the beaker to a microscope slide and add a small drop of 45% acetic acid.  Do not allow the onion root tip to dry out during the subsequent steps. Add 45% acetic acid if you notice your specimen is drying out. 5. Pulverize your onion root tip into a fine pulp on the microscope slide by tapping it with a glass rod. Try to produce as fine a pulp as possible to prevent large cell clumps which will not be useful for microscopic examination.6. Now place a microscope cover slip on top of your pulverized onion root tip. Put two layers of paper towel on top of the microscope cover slip and press down hard enough to squash the root but not enough to break the cover slip. This should result in the onion root tip cells from forming a monolayer which is ideal for microscopic examination.7. Using the scanning objective focus on the onion root tip cells and identify a cell undergoing mitosis by looking for pink-staining bodies (chromosomes) within the cell.8. Switch to the low-magnification10X objective and fine-focus.9. Switch to the high-magnification 40X objective and fine-focus. At this magnification you should be able to identify cells in several stages of mitosis. Identify a specific stage of mitosis and go on to step 10.10. Add a drop of oil to your prepared slide and switch to the oil-immersion 100X objective and fine-focus. Repeat steps 9 and 10 for all four stages of mitosis. As part of your Lab Exit Quiz you will be asked to: 1) show your prepared onion root tip slide to a lab instructor under the microscope.2) identify several stages of mitosis on your prepared onion root tip slide.

Mitotic Index

Figure 5

The duration of each stage of mitosis has been recorded and the data (see table below) could be used to compare the observed frequencies of the different stages as recorded by students.

Task – Look at Cells 1 -5. Decide which stage of mitosis each cell is in. For each cell describe the features that make you think it is in this stage.

Figure 6

1. 1. Why do we study the root tip to find mitosis instead of any other part of the onion plant? We study the root tip because it is growing therefore cells are dividing rapidly. This makes it the best part of the plant to observe various stages of mitosis.

2. Based on you data what can you infer about the relative length of time an onion root-tip cell spends in each stage of the cell cycle? Most cells are in interphase because most time is spent in this phase.

3. Based on your understanding of the structure of the chromosome, why might it take longer to complete prophase than the other phases of nuclear division? Prophase is the longest phase of mitosis because the chromosomes have to coil up into organized bodies. It takes a long time for the chromatin to coil or condense into chromosomes.

4. How do you account for the differences between the slides made by different groups? Possible answers: Not all lab groups had the same slide so there can be differences among the growth rates of the plants that were used to prepare the slide. The groups may have been looking at different areas of the root. Some groups may not have followed the instructions as carefully as others.

5. If you examined cells in the Zone of Differentiation (Zone of Maturation) would you expect to get similar results? No Why or why not? These cells are starting to specialize into mature tissues. They are no longer meristematic cells.

6. Why did we use the pie chart to graph the data? The pie chart was used because the data represented the parts of a whole and it is relatively easy to show proportions of the whole event.

http://bealbio.wikispaces.com/Genetics

Description: Gives a detailed description of the steps involved in mitosis with animated videos and narrative voice-over. Short multiple choice quiz provided containing the following multiple choice questions:

Table 2

1					Which of the following events do NOT occur in prophase of mitosis?
Figure 7	A) Figure 8	DNA condenses to form chromosomes
Figure 9	B) Figure 10	nuclear membrane breaks down
Figure 11	C) Figure 12	nucleolus breaks down
Figure 13	D) Figure 14	chromosomes are replicated
Figure 15	E) Figure 16	mitotic spindle begins to form
2					The mitotic spindle fibers attach to chromosomes via special structures termed
Figure 17	A) Figure 18	centrioles.
Figure 19	B) Figure 20	asters.
Figure 21	C) Figure 22	centromeres
Figure 23	D) Figure 24	centrosomes.
Figure 25	E) Figure 26	keratins.
3					Which of the following statements about spindle fibres during anaphase is TRUE?
Figure 27	A) Figure 28	those attached to chromosomes elongate, while those that are unattached shorten
Figure 29	B) Figure 30	those attached to chromosomes shorten, while those that are unattached elongate
Figure 31	C) Figure 32	both attached and unattached microtubules shorten
Figure 33	D) Figure 34	both attached and unattached microtubules elongate
Figure 35	E) Figure 36	both attached and unattached microtubules elongate at first and then shorten
4					Centromeres divide during metaphase.
Figure 37	A) Figure 38	True
Figure 39	B) Figure 40	False
5					Cytokinesis in plant cells occurs by means of a cleavage furrow.
Figure 41	A) Figure 42	True
Figure 43	B) Figure 44	False

Mitosis Slides

Identify the stage of mitosis for each of the onion root-tip slides below (most stages are represented more than once).

Table 3

	Identify stage			Identify stage
Figure 45	__i)_________________________		Figure 46	__ii_________________________
Figure 47	______iii_____________________		Figure 48	__________________iv_________
Figure 49	______________v_____________		Figure 50	____vi_______________________

Practice locating each of the stages of mitosis in the following slides of the onion root tip. Each picture contains at least one cell at each stage of mitosis (and some stages are represented by multiple cells).

Figure 51

(vii)

Answers

Answers to (vii)

The numbered arrows indicate cells at various stages of mitosis (most of the rest of the cells are in interphase). The key to the stages is to the right of each figure. You will not be asked to distinguish early from late; this is provided to help clarify the more ambiguous stages.

Table 4

Figure 52

Key: late Prophase/early Metaphase Metaphase Telophase early Telophase early Telophase Metaphase Prophase Anaphase late Anaphase