BIOLOGY LAB REPORT

TITLE : OBSERVING MITOSIS

PREPARED BY :

I/C NUMBER :

STUDENT ID :

GROUP :

LECTURER’S NAME :

PRACTICAL DATE :

SUBMISSION DATE :

OBJECTIVE

To prepare some slides of actively dividing plant tissue

To observe the stages of the cell cycle in living tissue

To consider the duration of the stages of mitosis in relation to the whole cell cycle

To develop certain experimental skills, namely working safely, the use of

microscopes, producing valid results and recording results.

INTRODUCTION

MITOSIS(2)

Cell is known as unit of life. These cell divides either mitotically or meiotically , which is

based on its location. Mitotic cell division take place in somatic cells which means all the

cells in the body except the germ cells as they divide through another process called

meiosis. Basically, mitosis is the process where an eukaryotic cell separates the

chromosome in its cell nucleus into two identical sets in two separate nuclei which is known

as two identical daughter cells. Mitosis doubles the number of cells without changing the

genetic content. Mitosis which is known as M phase occurs after interphase which has

three stages namely G1 phase , S phase and G2 phase. M phase is generally followed by

cytokinesis which bring means of dividing the cytoplasm into two cells containing roughly

equal shares the cellular components.

Figure 1 : The roles of stages in Interphase(1) Figure 2 : Interphase(1)

Mitotic phase or M phase generally have four stages namely prophase, metaphase,

anaphase and telophase. Some of stages can be even divided as early phase and late phase.

Cytokinesis generally come together at the end of telophase stage. Cytokinesis in plant cell

occur as the cell plate forms, thus dividing the daughter cell. Cell cycle is control by control

chemical( cyclins). These cyclin build up and attach to enzymes forming cyclin-dependent

kinases(CDKs), which means they add phosphate group to phosphorylates, changing shape

and bring next stage of cell cycle. Mitosis is significant as they provide cell replacement,

regeneration, and involve in asexual reproduction.

During Prophase

Figure 3 : General prophase stage(1)

Figure 4 : Early and late prophase(1)

During Metaphase

Figure 5 : Metaphase(1)

During Anaphase

Figure 6 : Anaphase(1)

Figure 7 : Early and late anaphase(1)

During Telophase

Figure 8 : Telophase(1)

Cytokinesis

Figure 9 : Cytokinesis in animal cell(1) Figure 10 : Cytokinesis in plant cell(1)

Figure 11 : Mitosis is completed(1)

Figure 12 : In summary of Mitosis(1)

ONION CELL TIP(3)

Figure 13 : Onion cell tip(1)

Onion roots are used to view mitosis as the roots are easy to grow in large number, the cells

at the root tip are actively dividing resulting in many cells that will be in mitosis stage, the

tips can be squashed during preparation on microscopes slide so that individual

chromosome can be observed and the presence of choromosomes that can be stained to

make them more easily observable. There are three cellular regions near root tip of an

onion. The root cap contains cells that cover and protect the underlying growth region as

the root pushed through the soil.

Second region of cell division known as meristem is where cells are actively dividing but not

increasing significantly in size. And the third region is cell elongation, where the cell are

increasing in size, but not dividing. Since each cell has only eight chromosomes so it is

relatively easy to see them once they condensed.

HYDROCHLORIC ACID(4)

In order to see the chromosome inside the cells, the cells must be separated and spread out

into a layer that is ideally just one cell thick. Plant cells such as onion cells are glued

together by a middle lamella of pectins. Hydrochloric acid will break down the pectins that

hold the cell together.

TOLUIDINE BLUE SOLUTION(5)

Toluidine blue is a polychromatic dye which is widely used in testing for lignin, a complex

organic molecule that bond to cellulose fibres which strengthens and hardens the cell wall

in the plants. TB is quite useful to stain fixed tissue but it only provides minimal information

about the chemical make up of a tissue or organ. It has a strong affinity for the granules in

mast cells, one of the wandering cells of connective tissues and also often requested as a

specific stain for mast cell tumours. In this experiment, toluidine blue was used to stain the

cells to make the chromosomes being shown up so they will be easier to be observed under

the microscope later.

AIM

To investigate mitosis in the cells of onion root tip

PROBLEM STATEMENT

How do the cells in onion root tip divide ?

HYPOTESIS

Mitotically dividing cells are larger than normal cells.

The higher the percentage of dividing cells in one phase, the longer in time the cells

spend on the phase the phases.

APPARATUS

Watchglasses, glass slide, micrometer slide, beakers, coverslips, forceps, microsope,

stopwatch, safety goggles, dropper

MATERIALS

Onion root tips, carnoy fixative, holding solution (70% ethanol), 6 moldm-3 hydrochloric acid

(18 %), toluidine blue stain , carbon fuchsin, distilled water, paper towels

PROCEDURE

1. Two watchglasses labeled “ HCl “ and “ Carnoy” were prepared.

2. 6 moldm-3 hydrochloric acid and carnoy fixative were added in each watchglasses by

using dropper enough to cover the bottom of the watchglasses.

3. An onion root tip was transferred into the “ HCl “ watchglass from holding solution (70%

ethanol) using a forceps. It was then left for 4 minutes (time taken using stopwatch).

4. The root tip then transfer using forceps into “ Carnoy “ watchglass for another 4

minutes ( time taken using stopwatch)

5. The root tip was then taken out using a pair of forceps and was placed on a glass slide.

6. The root tip was then stained with a few drops off toluidine blue ( or carbol fuschin ) for

2 minutes ( time taken using stopwatch).

7. The excess stain surrounding the root tip was blotted way or soaked up gently and

carefully using tissue paper.

8. One or two drops of distilled water was dropped on the root tip when the stain has

been blotted away.

9. A cover slip was lowered gently to cover the root tip so that no bubble is trapped inside

the cover slip. Lateral movement of the coverslip was avoided

10. The cover slip was then pressed firmly using thumb carefully with putting paper towel

on it to spread the root tip into a single layer cells making the cells more easily and

clearly viewable.

11. The preparation slide was observed under the microscope ( X 100 magnification). The

cells that undergoing different stages of mitosis was searched and identified.

12. The following were counted :

i. The total number of cells in microscopic field

ii. The total number of cells containing visible chromosome

13. The mitotic index of the slide was calculated by using the following formula :

14. The size of cells that undergoing each stages of mitosis was calculated by comparing the

scalee on the eyepiece graticule with the micrometer slide.

15. Tables were drawn to summarise the results.

RESULTS

Stage of mitosis Number of cells Percentage of cells (%)

( number of cells / total cells

with chromosomes counted)

X 100%

Interphase 95 95 /120 X 100% = 79.167

Prophase 17 17 /120 X 100% = 14.167

Metaphase 1 1 /120 X 100% = 0.833

Anaphase 6 6 /120 X 100% = 5.000

Telophase 1 1 /120 X 100% = 0.833

Table 1: Stage of mitosis corresponding to their percentage of cells

25 / 120 = 0.208

Figure 14 : Onion cell under microscope(x 400 magnification)

ANAPHASE

METAPHASE INTERPHASE

TELOPHASE

Table 2 : The illustration of the stages in cell cycle

CALCULATION

Calibration of the eye piece graticule per unit :

Magnification (X 400)

100 eyepiece graticule = 25 stage microscale units

1 eyepiece graticule = 0.25 stage microscale units

= 0.25 X 0.01 X 1000

= 2.5μm

Magnification (X 100)

100 eyepiece graticule = 100 stage microscale units

1 eyepiece graticule = 1 stage microscale units

= 1 X 0.01 X 1000

= 10 μm

Magnification (X 40)

40 eyepiece graticule = 100 stage microscale units

1 eyepiece graticule = 2.5 stage microscale units

= 2.5 X 0.01 X 1000

= 25 μm

Formula :

Size of cell = Number of units in eyepiece graticule x 0.025 μm

Stage of mitosis Number of units in eyepiece

graticule

Size of cell in each stage

(µm)

Interphase 14 14 x 0.025 = 0.35

Prophase 0 0 x 0.025 = 0.00

Metaphase 1 1 x 0.025 = 0.025

Anaphase 1 1 x 0.025 = 0.025

Telophase 1 1 x 0.025 = 0.025

Table 3 : Size of cell in each stage of the cell cycle (x 400 magnification)

Stage of mitosis corresponding to their percentage of cells

INTERPHASE

PROPHASE

METAPHASE

ANAPHASE

TELOPHASE

Pie Chart 1 : Stage of mitosis corresponding to their percentage of cells

DISCUSSION

ANALYSIS OF DATA

The experiment is started with onion root tip that is already prepared by laboratory

assistant , dipped into HCL solution ( to break down the middle lamella), and then into carnoy

fixative solution continued with a few drops of toluidine blue .The stain is blotted and the tip is

covered with few drops of water. It is then covered with coverslip and pressed gently using

thumb forming single layer of cell. From low power to high power of a microscope, the cell is

observed and the mitotic rate is recorded.

The preparation of onion root tip freezes the mitosis process at one point, thus, the

higher the percentage of the cell in one stage , the higher the time spent on that stage. Based

on table one, interphase recorded the highest number of cell followed by anaphase, metaphase

and telophase. Cytokinesis is not identified as it is not a mitosis part, but incidence that hppen

after mitotic phase. This shows that the time spent on interphase is the longest . Basically,

when a group of cell is dividing rapidly, high proportion of the cell undergoes mitosis while non-

dividing group of cell stays in interphase stage of the cell cycle. Mitotic index is used to

calculate amount of cell division occurring in a tissue .

Figure 14 in my result section shows the image of onion root tip cell under microscope

(x 400 magnification). In this field of view, as stated in table 3 , 3 cells can be viewed ( have

visible chromosomes) out of 17 cells. The non-visible chromosome cell either can be in

interphase or cytokinesis. From these observation, mitotic index can be calculated giving result

of 0.43.

The number of cells which undergo different stages of mitosis and their corresponding

percentages are tabulated in Table 1. The table shows that the interphase stage has the highest

number of cell in the field view which is 95 cells and thus gives 79.167 % of the total cells. This

is followed by prophase stage which gives 17 cells thus record 14.167 % of the total cells. Then

both metaphase and telophase stage have 1 counted cells and thus make up 1.667 % of the

total cells respectively. Another 5% is the anaphase. These data can be illustrated in pie chart as

in the Pie Chart 1.

Stage of mitosis corresponding to their percentage of cells is shown in another form ,

pie chart. The Pie Chart 1 shows that the interphase has taken the largest portions. Hence, it

shows that cells spend the longest time in interphase during the cell cycle compared to the

other phases in mitosis. During interphase, the cell undergo three subphases namely G1 phase.

S phase and G2 phase. During all three subphases, the cells growth by producing proteins and

cytoplasmic organelles. Hence, it is clear that interphase spend the longest period of time in the

cell cycle as the cells increase in mass and size, replicate their DNA and carry out normal

activities. Meanwhile, among the other four phases in the M phase, the prophase is the longest

periods spend by the cells followed by anaphase. Metaphase and telophase are the least time-

consuming by the cells. This is because, during the prophase, the chromosomes coil and

condense , the centrioles begin to separate and start to form the mitotic spindle. Hence, these

processes take a longer time compared to the other stages in the mitotic (M) phase.

Table 3 shows sizes of cell in each different cell stage. The sizes of the cells are

increasing from the interphase to the telophase. The sizes of the cells that are undergoing

interphase are the smallest compare to the other stages which is 0..025 μm. This is because

during this stage, the chromosomes in the cells are uncoiled and are not divided. Since there is

no prophase stage cell in viewed field, thus the size of the cell is cannot be justified. But, the

overall study suggest that the prophase stage has the second largest size of cell and followed by

metaphase , anaphase and telophase. The activities that happen inside of the cells during

different stages results in the size of the onion root tip cell. The sizes of the cells are calculated

by comparing the scale on the eyepiece graticule and the scale on the micrometer slide.

FURTHER STUDY

Other than using the onion root tips, the same experiment can also be carried out by

using different type of cells such as garlic cells or cheek cells. Beside using toluidine blue stain,

orcein ethanoic stain can also be used as the other choice of stain to conduct the experiment.

In some preparation, only few dividing cell is observed. This is because maybe a wrong

section is cut from the root which is not the root tip thus actively dividing apical meristem of

the root is not being viewed. There is also the possibility that the root used no longer have

dividing cell due to X- ray or heavy metal emanation.

As stated earlier in the introduction, cellulose walls of the plant cells are held together

by cement called middle lamella. Treatment with hydrochloric acid breaks this wall and this is

essential as it allows cells to separate and produce one cell thick preparation, making viewing of

individual cells possible.

The result is reliable if repeated measurement which almost have similar values.

Besides, the length of the cell is measured three times to obtain a mean value. Next, the

eyepiece graticule is used with full care and it has been correctly calibrated using stage

micrometer and under supervision of lecturer..

EVALUATION

Limitation and improvement

Several limitation are found in this experiment. Onion root tips are very fragile and

easily damage. The presence of xylem in the onion root tip makes the maceration more

difficult. Thus, to prevent misleading results, the end of root tips were not cut. The onion root

tips should be handle with extra care. The onion root tips were prepared earlier by the

laboratory assistant and placed in holding solution (70% ethanol). Cutting the wrong end of

onion root tip will give arise to inaccurate results, thus it was not carried out .

Since the prepared specimen are very thin ( one cell layer thick) , it enable us to be

clearly observed under the microscope. But extra care have to be taken as the cover slip is

pressed. It should be pressed gently co that the pressure will spread the cells into single layer.

Lateral movement were avoided and only one direction is used to give pressure to make sure

that the samples were not damaged and prevent them from giving overlapping result.

Another limitation is the time constriction. In order to obtain percentage of cells in

different stages, one have to calculate the number of cells in different region of onion root tip.

However, it is not possible to be carried out as we only have limited time. Thus, eash student

manage to get only one field of view only. To overcome this limitation, the observation is

shared and observed to get better idea on mitotic process.

Validity and reability

Since , looking at only one slide of onion root tip is not enough to get accurate result as

different region of onion root tips have different rate of dividing or mitotic phase. Thus, every

student prepare onion root tip each and the best result among those specimen were chose.

This give rise to accurate reading as a lot of sample specimen is used. This ensures the validity

of the result.

Extra care was taken when lowering the glass slide co that no air bubbles will trap inside

the slide. Presence of air bubble inside the slide will inhibit observation process under

microscope. In order to prevent this phenomenon, excess toluidine blue stain solution was

removed by using tissue paper. This guarantee the validity and reability of data.

SAFETY PRECAUTION

In order to avoid any accident or injury during the experiment in laboratory, the

precautionary steps should be taken and applied. Wearing lab coat and a pair of suitable shoes

are compulsory when conducting an experiment in the lab at all times to protect the skin and

clothing from chemical substance. This is to ensure that no chemical solutions such as toluidine

blue solution is spilled to our skin and clothing as it will stain badly. Furthermore, the glassware

such as beakers and boiling tubes should be handled with full care because they are fragile. Not

only that, sharp objects such as forceps must be used properly to prevent any contamination

and injuries. Avoid consuming any solution that used in this experiment because they might be

contaminated. Clean and dry microscope slides , cover the microscope and place the apparatus

back in the places after finishing the experiment to prevent any accident and to maintain the

good state of the apparatus.

CONCLUSION

A dividing cell undergo mitosis which contain 4 phases namely prophase, metaphase, anaphase

and telophase. The higher the percentage of dividing cells in one phase, the longer in time the

cells spend on the phase. The hypothesis is accepted.

REFERENCES

1. http://www.google.com.my/search?q=mitosis&hl=en&safe=off&biw=1366&bih=681&pr

md=imvns&tbm=isch&tbo=u&source=univ&sa=X&ei=jrSWTubiAs6mrAedmdjuAw&ved=

0CFEQsAQ

2. http://en.wikipedia.org/wiki/Mitosis

3. http://www.docstoc.com/docs/3665608/Mitosis-in-Onion-Root-Tips

4. Edexcel AS Biology – Practical 3.1- Ann Fullick, Pearson Company, 2008

5. http://en.wikipedia.org/wiki/Tolonium_chloride