Core Practical 5 – From Topic 3 (Voice of the Genome)
Aim: To be able to prepare and stain a root tip to observe the stages of mitosis under a microscope.
- Garlic roots
- Sharp knife
- 1moldm¯³ of hydrochloric acid
- Acetic alcohol
- Orcein ethanoic stain
- Ice-cold distilled water
- Water bath (60 ̊C)
- 2 watch glasses
- Test tube
- Test tube rack
- 2 pipettes
- Microscope slides
- Mounted needle
- Filter paper
- Microscope with x100 & x400 magnification
- Place a test tube of 2cm³ HCl (1moldm¯³) into a test tube rack in a 60 ̊C water bath.
- Cut off 1-2cm of a root tip from garlic root. Put the tip in a watch glass containing 2cm³ of acetic alcohol for at least 12 minutes.
- Remove the tip and then place into another watch glass containing 5cm³ of ice cold distilled water. Leave for 4-5 mins, and then remove and dry.
- Place the tip into the heated HCl for 5 minutes then repeat the process again by placing tips back into acetic alcohol etc. Tip: The tips will be very fragile at this point.
- Transfer 1 tip onto a microscope slide and cut 4-5mm from the growing tip (site of mitosis) and keep it.
- Gently break up (macerate) the root tip with a mounted needle.
- Add 1 small drop of orcein ethanoic stain and leave for 2 minutes.
- Add a coverslip and blot with filter paper.
- View under a microscope and identify the stages of mitosis.
Results & Calculations: The percentage of cells in each stage of mitosis can be observed and noted down. A Mitotic index can also be formed:
Number of cells containing visible chromosomes ÷ Total number of cells in the field of view
Conclusion: Mitosis consists of four different sections: prophase, metaphase, anaphase and telophase.
Prophase – Chromosomes condense (chromatids joined by centromere)
– Spindle fibres join to both centrioles
– Nuclear envelope breaks down
Metaphase – Centromeres attach to spindle fibres at the equator.
– Chromosomes line up
Anaphase – Centromeres split
– One chromatid from each chromosome is pulled to either end of the cell
Telophase – Chromosomes unravel
– Two nuclear envelopes reform
Cytoplasmic division then occurs where the cell surface membrane divides to form two daughter cells. Interphase then takes place (G1, S and G2 sub-phases). The cell cycle then repeats.
- Low resolution of microscope (systematic error) – use a more detailed microscope with a greater magnification
- Human error in counting numbers of cells (random error) – take a picture of the image in the lens as this should make counting easier
- Not enough time in the solutions to enable successful maceration or staining (systematic error) – leave for 5 minutes longer in future trials