Plan an investigation to find out if there is a relationship between the concentration of phosphate ions in nutrient solutions and the number of cells dividing by mitosis in root tips

I need to understand what happens when root cells are introduced to phosphate ions of different concentrations. I will use garlic cloves, as they produce several roots and will use phosphate tablets to vary concentration of phosphate ions available.

Variables

Independent: different phosphate concentrations.

Dependent:  number of cells undergoing mitosis.

Fixed/ confounding: (kept constant to ensure a fair test is carried out, and that the only variable having an effect on the rate of mitosis is the phosphate concentration.)

For all experiments:

1. Light  – exposed to the same amount of light at all times (same time/same bench)
2. Temperature - carried out at room temperature.
3. Time- phosphate solution will be available to roots for 48 hours, ensuring fair results to be recorded for all tubes.
4. Nutrient solution – same amount will be used so results are not bias or disrupted.
5. Phosphate solution – amount used will be the same.

PREDICTION

The more phosphate present, the more mitosis taking place.

• High levels of phosphate allows more ATP to be produced so more energy is available. Mitosis is an ‘ active’ process, ATP will be needed by the energy consuming cells.

• Needed to activate nucleotides in cytoplasm so DNA can be replicated.  Insufficient amounts of phosphate means that DNA nucleotides can’t be made or activated, therefore no replication.

• More phosphate ions allows more nucleotides (DNA & RNA) to be produced, as they are a component of the backbone.

• Daughter nuclei produced will go through cytokinesis and phosphate ions will be needed to synthesise new membranes.

• Lack of phosphate means protein synthesis won’t occur or will be inefficient, vital enzymes needed during mitosis can’t be made and mitosis will not take place.

PRELIMINARY

“ Preparation & observation of the stages of mitosis in root tips.”

From this experiment I learnt how to prepare a squash slide to view under a microscope. I understood the consequences of adding too much acetic orcein (stained chromosomes too deeply.) I heated the hydrochloric acid by warming over a flame; I developed this for my method, as it was not an accurate technique. The temperature couldn’t be controlled and therefore could disrupt my results so I plan on using a water bath at a set temperature and a thermometer to ensure the temperature was maintained.

APPARATUS

• Garlic clove - roots

• Microscope – view specimen under magnification

• Nutrient solution - required for growth

• Phosphate tablets - vary concentrations

• Acetic orcein - stain chromosomes making them visible

• Slides & coverslips - prepare specimen for viewing

• Scalpel - cut root

• Watch glass - hold specimen

• Filter paper -mop excess liquids

• HCL (2.0 mol dm3)  - spread cells

• Thermometer - record temperature

• Waterbath -  control set temperature environment

• Test tubes -  use in water bath

• Glass rod - maceration

• Volumetric flasks - measure phosphate solutions

• Beakers -  hold end solution

METHOD

1. Label 7  250cm volumetric flasks 0-5 & 10
2. Add one phosphate tablet to flask 1 and so on respectively. (flask 0 no tablets are added)
3. Fill flasks with distilled water, read at eye level and ensure that meniscus is resting on the line.
4. Label 7  500cm beakers 0-5 & 10.
5. Fill beakers with 200cm nutrient solution.
6. Add phosphate solution to each beaker respectively. Contents of flask 0 poured into beaker 0 e.t.c.
7. Cut 7 root tips 2cm long from garlic clove and place one into each beaker.
8. Leave for 48 hours.
9. To 7 test tubes add 10cm of hydrochloric acid and place in water bath at 60°C.
10. Transfer root tips to each test tube for 5 minutes.
11. Place root tip ‘0’ onto slides, cut apical tip 3mm long. Discard the rest.
12. Macerate root using glass rod and spread out as thinly as possible.
13. Add 3 drops of acetic-orcein allow to stain for 2 minutes.
14. Place coverslip on slide and blot firmly using filter paper; press gently to spread cells.
15. View under microscope.
16. Count how many cells in the 4 main stages of mitosis for each root.
17. Record results.
18. Repeat steps 11-17 for roots 1-5 and 10
19. Record results.

(Using the results recorded in this table a graph can be plotted with x-axis “number of phosphate tablets used” and y-axis “ total number of cells in mitosis”, from this graph reliable and accurate conclusions can be drawn.)

JUSTIFICATION

• All other variables were kept constant and only phosphate level was varied, to understand the effects of the different concentrations on mitosis. This ensured that a fair test was carried out and that unbias and reliable results was recorded.

• Several ranges of phosphate concentration used, to easily note the effect on mitosis. On one solution there was no phosphate tablet used, this was the control of the experiment, and enabled me to understand what happened in the absence of phosphate. Solution 10 was used to understand the effects of excessive phosphate on mitosis.

• Volumetric flasks used to accurately ensure that volume of phosphate solutions was the same for all concentrations, ensuring accurate results.
•  Hydrochloric acid was used to break down calcium pectate of middle lamella allowing cells to spread out.

• Acetic orcein stained the chromosomes making them visible under the microscope.

• Roots were macerated to spread out.

• Water was removed to prevent a precipitate forming when stained.

SAFETY

• Do not point scalpel at face or others.

• Acetic orecin and hydrochloric acid are corrosive, use carefully.

• Don’t drop test tubes, glass may break of hot water splash.
• Wear laboratory jacket to protect clothing.

REFERENCES

•  Cambridge Biology 1
• Saps

WORD COUNT

987 words