INTERNATIONAL EDUCATION CENTRE (INTEC)

UiTM Section 17 Campus 40200 Shah Alam Selangor Darul Ehsan. 

TEL: 603-55227000

TITLE: OBSERVING MITOSIS

NAME: NUR AMALINA BT ZOLKEFLEE

SID NUMBER: 2010843164

CLASS: 11M2

DATE OF EXPERIMENT: 24TH JANUARY 2011

LECTURER’S NAME: MISS FATHIAH ABDULLAH

PARTNER’S NAME: FATIN NABILAH BT MOHD NASIR

NUR AIN SOFIA BINTI HARITH

SITI NASHUHA BINTI OSMAN

1|Page
 OBJECTIVES

Basically, our experiment is done to prepare some slides of actively dividing plant

tissues. The other purpose of this experiment is to observe the stages of the cell cycle in

living tissue and to consider the duration of the stages of mitosis in relation to the whole

cell cycle. Through this experiment also, we can develop certain experimental skills,

namely working safely, the use of microscopes, and producing valid results and recording

results. Not just that, we are also taught to calibrate an eyepiece graticule and use it to

measure the size of the cells.

INTRODUCTION

 BACKGROUND INFORMATION

Genetic information of human, plants and animals reside in unique structure

called chromosomes. For example, each human cell posses 46 chromosomes while

onion cell posses 8 chromosomes. All cells must replicate their DNA in order to

pass it to the next generation of cells in the whole body. During DNA replication,

two strands of DNA separate and for each strand, new complementary DNA is

produced thus yielding two identical DNA molecules. After DNA replication, the

process followed is mitosis which is important to ensure that each daughter cell

receives one copy of each replicated chromosome. During mitosis, the

chromosomes pass through several stages like prophase, metaphase, anaphase and

telophase. The division is considered as complete once the cell undergo

cytokinesis.

2|Page
 Figure 1 shows the cell cycle that includes interphase, mitosis and cytokinesis.

Source: http://mysite.cherokee.k12.ga.us/personal/gregg_schumaker/site/Important
%20Class%20Documents/1/Growth%20and%20Heredity/Observing%20Mitosis
%20Lab.pdf

The first stage in mitosis is prophase. During prophase, chromatin condense to form

highly condensed chromosomes. Chromosomes are visible at high magnification through

light microscope. The two strands of non- sister chromatids are joined at one region

called centromere. The microtubules from cytoplasm form three dimensional structure

called spindle fibre which are formed between two poles. The centrioles move around

nuclear envelope and locate themselves at opposite sides of the cell. Nuclear envelope

and nucleolus break down.

3|Page
Figure 2 shows some events that take place during prophase.

Source: http://chuck16.wordpress.com/2009/04/30/phases-of-mitoses/prophase-
late__civyrosejpg/
During metaphase, the centromeres of chromosomes line along the metaphase plate or

equatorial plane, an imaginary line that is equidistant from centrosome poles. When this

arrangement has been completed, the cell has reached the end of metaphase.

Figure 3 shows how the chromosomes arrange themselves at metaphase plate during

metaphase.

Source : http://www.sparknotes.com/biology/cellreproduction/mitosis/section2.rhtml

4|Page
 The third stage in mitotic division is anaphase. During this stage, the centromeres split.

The spindle shorten to pull the two halves of each centromere in opposite directions. One

chromatid of aech chromosome is pulled to each of the poles. Anaphase ends when the

separated chromatids reach the poles and the spindle breaks down.

Figure 4 shows the events occur during anaphase.

Source: http://www.sparknotes.com/biology/cellreproduction/mitosis/section2.rhtml
The last stage in mitotic division is telophase. The events taking place during telophase

is a reverse process of prophase. Chromosomes unravel and nuclear envelope reforms, so

that two sets of genetic information become enclosed in separate nuclei.

Figure 5 shows the events take place during telophase.

Source: http://www.sparknotes.com/biology/cellreproduction/mitosis/section3.rhtml

5|Page
 Cell cycle consists of three main stages which are interphase, mitosis and

cytokinesis. Cytokinesis is also known as cytoplasmic division. During cytokinesis, plant

cells divide due to formation of walls between two daughter cells. New cell wall material

is brought by microtubule system forming the phragmoplast, a complex organelle

consisting microtubules and actin filaments.

Figure 6 shows the sequence taking place during cytokinesis.

Source: http://www.bms.ed.ac.uk/research/others/smaciver/Cyto- topics/Plant

%20Cytokinesis.htm

Overall, mitosis is very crucial in order to maintain genetic consistency. This is

because the daughter cells are made to be ganetically identical to each other and parent

cell. Can you imagine to have different colours of skin? This probably happen if mitosis

do not occur as our genetic information is not restored in producing new cells. Genetic

consistency is achieved by DNA replication prior to nuclear division and during the

arrangement of the chromosomes on the spindle fibre and the separation of chromatids to

the poles. Mitosis is also important to animals and plants that practice asexual

reproduction. For example starfish can regrow a completely new body from a fragment of

its body. Furthermore, mitosis is also significant in replacing our dead cells sepecially

6|Page
our skin cells which are easily rubbed by the friction. So, in order to maintain in good

condition, mitosis will occur to replace those old cells.

In this experiment, we are using onion roots due to several specialities that it owns.

Firstly, the onion roots are easily grewn in large numbers. Therefore, no ethical issues

arise here as it is not an endangerd population and it will be much cheaper to be compared

to other type of plants. Secondly, cells at the tip of roots are actively dividing and thus

many cells will be in stages of mitosis. The tips can be prepared in a way that allows them

to be flattened or microscopes slide(‘squashed’) so that chromosomes of individual cells

can be observed. The chromosomes of onion cells can be stained to make them more

easily observable and as the chromosoes are large and become very dark when stained,

therefore the onion root cells is the most suitable to be used in this experiment. There are

three cellular regions at the tip of onion root. The first one is root cap. It contains cells

that cover and protect the underlying growth region as the root pushed through the soil.

The second region is the region of cell division(meristem). Here, the cells are actively

dividing but not increasing in size while the third region is the region of cell elongation

where the cells increase in size but not dividing.

Figure 7 shows the three regions at the tip of onion root

Source: http://www.excellup.com/InterBiology/morphologyplant.aspx

7|Page
 In order to get an accurate result, we have to make sure that we know how to use the

microscope correctly because our result is only depending on microscope. What we see

through the microscope indicate how successful our experiment is. Firstly, you have to

put your microscope on flat surface. Then, switch on the microscope’s light source and

then adjust the diaphragm to the largest hole diameter to allow the greatest amount of

light passing through. Use the lowest power objective (usually 4x for 40x magnification).

Place the slide on the stage and adjust the large coarse focus knob until specimen is in

focus. After that, adjust the small fine focus knob until specimen is clearly in focus. Then,

adjust the diaphragm to get the best lighting. Rotate the objective lens to 10x objective for

100x magnification. Refocus and view the specimen carefully. Repeat this step by using

40x objective. It is an optional for you to use the 100x objective for 1000x magnification.

You need to put 1-2 drops of immersion oil over the slide coverslip before viewing it at

highest power.

Figure 9 shows a microscope

Source : http://www.hometrainingtools.com/how-to-use-a-microscope/a/1120/

8|Page
 In this experiment also, we are using toluidine blue instead of carbol fuschin.

Toluidine blue is a polychromatic dye that absorbs different colours depending on the

nature of its chemical binding with components of the tissues. At pH 4.4, it will bind to

pectins in cell walls and colour them pink. Compounds containing benzene rings such as

lignin will be coloured green. At lower acidic pH, toluidine blue gives only blue or only

green colour. At higher basic pH (11.1) the stained tissue will be coloured dark pink since

most pectins will be charged and hinder the green colour of lignin.

EXPERIMENTAL HYPOTHESIS

Mitosis occurs in onion root tip and it is easily observable. By observing onion

meristem cells under microscope, we can see the interphase, the four stages of mitosis

that is prophase, metaphase, anaphase and telophase and cytokinesis clearly. The most

time spent in a stage is in prophase and the least time spent in a stage is anaphase. All the

stages can be differentiated by observing the chromosomes in nucleus that is the

chromosomes’ position within the nucleus. If only long strand can be seen, it indicates the

cell is undergoing interphase. If the chromosomes scattered in the nucleus, it indicates the

prophase stage while if the chromosomes arrange themselves at the centre which is

known as metaphase plate, it shows metaphase stage. On the other hand, if the spindle

looked shorter and like pulling the chromosomes to the pole, it means that the cell is

undergoing anaphase. The telophase stage can be observed when you can see two sets of

chromosomes in a cell. Cytokinesis is the easiest stage to be detected; that is when you

can see cell plate formed in between the two sets of chromosomes.

9|Page
 MATERIALS AND APPARATUS

MATERIALS: carnoy fixative, holding solution(70% ethanol), HCl acid( 18%), toluidine

blue.

APPARATUS : microscope slides, coverslips, small beakers, watch glasses, blades,

Forceps, filter paper, compound microscope.

METHOD

Preparing the sample

1. Sample of onion root cells is obtained from the holding solution.

2. Two cups of solutions are prepared. The first cup contains HCl acid while the

second one contains carnoy solution.

3. By using a forcep, an onion root tip is transferred into HCl acid solution for four

minutes.

4. Then, the root tip is transferred into carnoy solution for four minutes.

5. By using a blade, 1 mm of the root tip is cut and is put on microscope slide.

6. A few drops of toluidine blue is dropped into the root tip and is left for two

minutes. Then, a few drops of water is put onto the root tip to dilute the

concentrated toluidine blue.

7. By using a filter paper, the stain surround the root tip is blotted away.

8. Then, a cover slip is put over the specimen.

9. The slide is covered with a paper tissue and the cover slip is pressed with the

thumb.

10. Then, the slide is observed under microscope.

10 | P a g e
Preparing the microscope

1. A stage micrometer is slide on the stage of microscope. The smallest division on

the stage micrometer is measured.

2. By using low power objective, the microscope is focused on stage micrometer.

The eyepiece is rotated and the slide is moved.

3. The number of divisions on eyepiece graticule is counted and is made equivalent

to the smallest division on stage micrometer and hence the length that one

eyepiece division is calculated.

4. Step 3 is repeated for medium and high power objectives. The cell size can be

measured.

RESULTS AND DATA

Figure 10 shows the drawing of overlapping cell walls.

What we can see from the microscope is only overlapping cell walls.

11 | P a g e
Figure 11 shows Nucleus is hardly seen due to compact cell wall.

Source: http://mrswolfgang.wikispaces.com/Animal+and+Plant+Cells+-+Bizousky,
+Byerly

Stage of cell cycle Number of cells in each stage

Interphase Cannot be observed
Prophase Cannot be observed
Metaphase Cannot be observed
Anaphase Cannot be observed
Telophase Cannot be observed

Table 1 shows number of cells in each stage

DISCUSSION

Based on our result, we found that our result is not valid at all due to some errors

that we encounter that we will discuss in detail in sources of error. After discussing with

other groups that manage to conduct their experiment successfully, we found that each

stage in cell cycle can been observed clearly under microscope. Here is some pictures

showing how chromosomes behave in each stage of mitotic division and interphase.

12 | P a g e
Figure 12 shows all the cells that can be observed under microscope using low power

objective.

Figure 13 shows the chromosomes behaviour using higher power objective.

Source: http://www.practicalbiology.org/areas/advanced/cells-to-systems/cell-
division/investigating-mitosis-in-allium-root-tip-squash,121,EXP.html

Through our discussion too, we found that our hypothesis made is accepted. That is

most of the time in mitotic division is spent in prophase while the least time spent is in

anaphase.

13 | P a g e
Table 2 shows number of cells in each stage.

Source: http://www.practicalbiology.org/areas/advanced/cells-to-systems/cell-
division/investigating-mitosis-in-allium-root-tip-squash,121,EXP.html

In calibrating the eyepiece graticule, we found that the smallest division on the stage

micrometer equals to 100 micrometer. The eyepiece is rotated and the slide is moved to

superimpose the scales of eyepiece graticule and stage micrometer. Number of division

on eyepiece graticule is found to be three divisions equal to 1 smallest division of the

stage micrometer. Therefore, each division on eyepiece graticule is equal to 33.33

micrometer.

Figure 14 shows the stage micrometer and eyepiece graticule scale.

14 | P a g e
 VALIDITY AND RELIABILITY

It is obvious that our experimental result is not valid. This is because we could not see

any stages in the cells. Our group only manage to see the overlapping cell walls. But then,

our experimental result is quite reliable because we have been repeated this experiment

for three times but unfortunately, we obtained the same results. So, we come into

conclusion that there might be errors due to apparatus because our experimental result is

constantly the same.Not just that, this experiment is a time critical experiment. This is

because a few steps in this experiment is constraint with time. As we have been repeated

this experiment three times, our result obtained can be considered as quite reliable but not

valid.

SOURCE OF ERROR

There are lots of errors that we have analysed. The first one comes from microscope.

Our group’s microscope has been contaminated by the emulsion oil. The emulsion oil can

damage the structure of the cell. The lens of the microscope are not being wiped out first

before we are using it. Secondly, the intensity of toluidine solution used is too low until

the nucleus of the specimen cannot be observed under microscope. It makes us difficult to

trace the stage easily. Thirdly, we assumed that the root tip has not been fully immersed

in HCl and carnoy solution which later interrupt the image observed under microscope.

Next, the duration for the root tip to be immersed in HCl acid and carnoy solution is not

exactly four minutes. As the root tip has been cut in a large size and not exactly 1 mm, it

cause the specimen to overlap each other. Not just that, the specimen might be damaged

due to high pressure exerted by the thumb on it.

15 | P a g e
 SAFETY MEASURES AND PRECAUTION

There are a few safety measures that must be taken into consideration in order to make

sure that the experiment is going on smoothly without any accidents. First, we must wear

the lab coat to prevent any stain onto our cloth. For example, toluidine blue can stain your

cloth if you do not wear lab coat. We have to make sure the concentration of HCl acid

used is not too concentrated because it might be dangerous to be handled. Do not eat and

drink throughout the experiment because the biology lab still keep the hazardous

chemical substances even we are not using it. We must also be very careful when

handling the lab apparatus like beakers and cavity slide which tend to break easily and

will harm our safety. The microscope is fragile and light bulbs can get so hot, so we have

to be careful. Ethanoic ethanol is corrosive, so wear eye protection (goggles). Not just

that, take care with scalpels and always carry them on a white tile to prevent any injury

occur during lab session. And always keep the laboratory in clean state.

LIMITATIONS

There are a few limitations that prevent us from getting a very acurate and precise

result. This is due to lack in time. Each step takes at least 2-3 minutes to be done. As there

are many steps of them; overall, it takes about 30 minutes to prepare each specimen.

Furthermore, it is not easy to observe the specimen under microscope. It takes about 10

minutes for us to actually see the specimen. Basically, this experiment consume a lot of

time. Next, the microscopes provided are not enough to accommodate all of us. For

example, as our microscope is not in good condition, we have to use other group’s

microscope as there is no extra microscope provided. We have to wait to use a particular

microscope. This consume more and more time. Furthermore, this is our first experience

16 | P a g e
in conducting mitosis experiment. We are lacking in skill especially the skill to cut the

root tip.

MEDICATION AND FURTHER WORKS

In order to get a more accurate result, a few suggestions had came across. The first one

is we have to wipe the lens before we are using it to prevent any immersion oil that might

be used by the previous group. We have to make sure that the concentration of toluidine

blue is just fine( neither too dilute nor too concentrated) so that we can see the nucleus

clearly. We have also to make sure that the root tip is fully immersed in HCl acid and

carnoy fixative so that the structure of the cell can be clearly observed. Moreover, we

have to increase our alertness in recording time so that the duration taken for the root tip

to be immersed is actually as required. From what I have read some tips from example

reports, they are suggesting us to squash the root tip using blunt pencils. This could be a

good alternative.

CONCLUSION

After we have done this experiment, finally we came into conclusion that our

experiment is not success to prove what had been stated in hypothesis that is the most

time spent is in prophase and the least time spent is in anaphase. Therefore hypothesis is

rejected and in order to verify this statement, we must undergo this experiment one more

time by using different microscope, slides and other apparatus and material to overcome

any lacking during conducting this experiment. However through long discussion with

other group members, we can conclude that mitotic stages of onion root tip can be

observed with a light microscope and the most time spent is in prophase while the least

time spent is in anaphase.

17 | P a g e
 REFERENCES AND BIBLIOGRAPHY

 FROM INTERNET
 2011. Available from: http://www.practicalbiology.org/areas/advanced/cells-to-
systems/cell-division/investigating-mitosis-in-allium-root-tip-
squash,121,EXP.html. Accessed on 1st February 2011
 2011. Available from : http://en.wikipedia.org/wiki/Mitosis. Accessed on 1st
February 2011
 2011. Available from : http://www.123helpme.com/preview.asp?id=156102.
Accessed on 1st February 2011
 2011. Available from : http://www.experiment-resources.com/validity-and-
reliability.html. Accessed on 1st February 2011
 2011. Available from: http://staff.jccc.net/pdecell/celldivision/oniontip.html.
Accessed on 1st February 2011
 FROM BOOK
 Angela,H. et al . 2008. Voice of Genome. In Salters-Nuffield Advanced Biology
or Edexcel AS Biolog, p.116-117. London. Edexcel Pearson.

18 | P a g e