Biology I Practical 6: Observe mitosis in garlic root tips

LAURA ORTEGA 12G

Practical 6: Observe mitosis in garlic root tip

Objective

Have ability to prepare a temporary slide of a garlic root tip to observe the process of mitosis.

Be able to recognise the stages of mitosis in the dividing cells.

Associate hazards related to the procedure.

Safety

·         Eye protection worn

·         Gloves worn

·         HCL is IRRITANT ( diluted)  at 2 mol dm-3

·         Causes severe burns ·         Routes of Entry: Skin, eyes, inhalation and ingestion. ·         First-Aid Information: Inhalation: corrosive action on mucous membranes.  Eyes: Contact rapidly causes severe damage  Skin: Severe and rapid corrosion from contact  Ingestion: Do Not Induce Vomiting! Severe and rapid corrosive burns of the mouth, gullet and gastrointestinal tract will result if swallowed.

·         Ethanoic acid is CORROSIVE

·         Produces burns. In case of accident or discomfort should go immediately to doctor. ·         For microscopy, chromosome staining ·         Composition: Orcein 2.0g, Acetic Acid 55ml and Water 55m

· Cederwood Oil is IRRITANT

·         Irritating to eyes, respiratory system and skin. In case of contact rinse immediately with abundant water and seek medical advice. (wear eye protection and protective clothing)          ·         Use this because: “If air is between the light source and the oil immersion lens, the light is scattered by the air molecules. Cedar oil or other immersion oils have the same refractive index as the glass lenses and keeps the light from being scattered thus better illumination of the specimen being observed.”

Equipment:

·         Garlic clove with growing root tip

·         Glass slide and coverslips

·         Scissors

·         Two dissecting needles

·         Paper towels

·         Microscope

·         White tile

·         Transparent plate

·         Fine forceps

·         Stop clock

·         ETHANOIC ORCEIN STAIN

·         Cederwood Oil

·         prepared beaker containing hydrochloric acid (1 mol dm–3)

·         (HCL) pre-heated to constant temperature in a water bath at 60 °C.

·         Distilled water

·         Beakers

Procedure:

1.       Put a bottle of 1 mol. dm^-3 of Hydrochloric Acid in a water bath with 60*C , and let it warm itself.

2.       Put inside the garlic clove with the roots for 5 minutes. 

3.       Take the garlic out, hold and cut 3/4 roots of about 5 to 10mm.

4.       Put the roots in a transparent plate (any base) and add distilled cold water with pipette and mix to wash away the HCL.

5.       Slightly dry the roots by placing them in paper towel.

6.       Place the roots in the white tile and tease them apart with the needle to spread the cells (maceration). Move this to the glass slide. After that cover the whole root with the Ethanoic Acid.

·         Put the ethanoic acid with an interval of 2 minutes each, to see how stain is affected by different time periods. (the first one will have 8 mins and the last 2 mins)

7.       At an angle of 45* put the cover slip on top, and wrap the slide with several layers of paper towel and press hard to squash the tissues.

8.       Examine under the microscope on low power to identify the meristem. ( small, square, no obvious vacuole, found in rows)

9.       Position the microscope at an appropriate field view, move to high power (x400) and identify stages of mitosis and interphase, count the cells. Add a few drops of the cederwood oil to surface of coverslip when using the high power.

10.   Record data and draw diagrams

Results: Diagram and Pictures

Figure A: First field view, of low power of 4/0.1 160/0.17, on the 2 minute stain slide

It requires high power to see the cells in detail. Put it helps to see it this way to have a whole picture of what the garlic clove is. There are a few air bubbles as a result of difficulty in placing the coverslip exactly at 45 degrees.

Figure B: 4 minute slide, at medium power ( 100/1.25) I learnt that the more time there was stain in the slide, the better it showed on the microscope field, this is because the stain has deepened more into the cells. Staining the cells red, to observe them. In my experiment the 6 minute slide was better than the 8 minute slide, this was an anomaly. We repeated the experiment a second time and as predicted the longest one with the stain showed the better.

This being the highest power (x400)

didn’t show the stages of mitosis or interphase. The only thing deducted is

they are all under interphase. So my partner

and myself when to test another slide, under another microscope with higher resolution. 

	Interphase : As the cells are dividing and creating new DNA material. No visual change in chromosomes as they are uncoiled filling the nucleus.
	Prophase(Mitosis) : Chromosomes are still coiled up , the nucleolus is breaking down
	Metaphase (Mitosis) -this is the harder to see, but I think this shows metaphase as the chromatins are lined up but not coiled and yet not splitting up.
	Anaphase(Mitosis) The centromeres are split up to become new chromosomes, they are going opposite sides.(opposite poles)
	Telophase (Mitosis) : nuclear membrane is formed around the two sections of chromosomes , its not cytokinesis as there is no division of cytoplasm yet.
	Cytokinesis : It shows is more than the telophase as there is a division of the cytoplasm, with a separation occurring here. There are starting a cellulose walls with the middle lamella , Golgi vesicles move aside.

		(used a small section of Figure D, as there where many cells to count from)
Cells Under Interphase	50
Cells Under Mitosis	6
Field View in Class
Interphase	70
Mitosis	12

Analysis of results:

·         More interphase occurs than mitosis this is prooved because out of 70/82 cells were taking interphase and only 12 mitosis.

·         Our resolution of the slides was not defined enough to

Mitotic index :=	number of cells containing visible chromosomes ( mitosis)
	Total number of cells in the field of view

Figure D	In Class (taken measurements)
6	7
50	82
12 %	8.5%

: The mitotic index using ethanoic acid stain suggests that only 13% ( average between two) of the cells in our view were undergoing mitosis and the rest are still interphase. This percentage is related to the time taken of the cells in each stage of mitosis. The greater the percentage the longer they take in that stage.

Conclusion:

Mitosis has four stages, prophase, metaphase, anaphase and telophase. Different slides will have different times of mitosis completed on them. Overall the longest stage is interphase as it has the highest percentage, most cells spent longer there because of cell growth, replication of the chromosomes and other activities. The greater the percentage the more time spent in each stage. Although the size cell during interphase is smaller in comparison to the ones undertaking mitosis, and the biggest was telophase and cytokinesis. ( As in telophase the cell is dividing into two daughter cells)

The slides that had the ethanoic acid for the longest time period, the stain was absorbed more and they displayed more clearly when under field in the microscope.

The use of garlic roots to observe mitosis ,was a good choice as the stages in plant growth can be observed on the meristem , easier than animal cells. As meristem is always dividing mitosis, every stage can be observed clearly.