Practical lesson #1: Beetroot Lab Report

Cell Homeostasis

Practical 1 - Effect of environmental conditions on beetroot
22nd January 2014

Aim: To investigate the factors affecting the cellular homeostasis of Beetroot cells

Materials:

1. Scalpel
2. Ruler
3. 5 test tubes & rack
4. Forceps
5. Distilled water
6. 25%, 50% alcohol
7. Beetroot (Beta vulgaris)
8. White tile (for cutting purposes)
9. 3 droppers (water, 25%, 50% alcohol)
10. Thermometer
11. Stop watch 
12. 1 beaker
13. Colourimeter

Procedure:

1. Set up a water bath by collecting hot water into a beaker provided in the lab. 
2. Use a ruler and scalpel to cut the cylinder of beetroot into 25 discs of 2mm each. 
3. Take 5 discs of beetroot and cut them into smaller pieces. 
4. Rinse the beetroot discs and pieces until the water is colourless.
5. Label and prepare 5 test tubes as follows:
   
   

   Tube	Content
   A	4 ml of water
   B	4 ml of 25% alcohol
   C	4 ml of 50% alcohol
   D	4 ml of hot water (90-100°C)
   E	4 ml of water with chopped beetroot

   
   
6. Place 5 discs of beetroot in tubes A-D and all the chopped beetroot in tube E using the forceps. 
7. Leave the tubes to stand in your test tube rack for 15 minutes.
8. Shake the tubes gently after 15 minutes and hold it against the white tile to note the colour. Record your observations in a table. 
9. Use the colourimeter to record the percentage of transmission of light
10. Decant a small amount of the liquid from each tube into a cuvette to measure the percentage of transmission. * Hold the cuvette only at the rough sides. Turn the cuvette so that the arrow is facing you when you insert it into the colourimeter.
11. Dispose of the content of the tubes after the experiment. 

Discussion questions:

• Why was it necessary to wash the beetroot slices thoroughly before using them int this exploration?

The coloured pigments that give the beetroot its red colour are located in the vacuoles of the cells, called betalain. When the beetroot is cut into discs or chopped (for tube E), many of the cells are cut mechanically, and these betalain are released from the vacuole, which is why the cutting tile would have red stains, caused by the pigments leaking out of the beetroot. These pigments need to be removed by rinsing them off as the experiment results will get affected. The change in colour of the surrounding solution must be due to the pigments leaving the cell rather than from the pigments that have already previously leaked out due to the cutting, as if the discs were not rinsed, the final colour of the solution would be darker than expected, which would lead to unreliable results.

• Identify the independent and dependent variables in this experiment. Which was the control set-up?

Independent variable(s): 1. Concentration of alcohol in solution 

                                       2. Temperature 

                                       3. Exposed surface area of beetroot

Dependent variable(s): 1. Percentage of light transmitted 

                                     2. Final colour of liquid 

Control set-up: Test tube A

• Construct a suitable table, with appropriate headings and units, to tabulate your data. 

Test tube	Contents	Description of liquid	Percentage transmission of light (%)
A	4 ml of water with 5 discs of beetroot	From colourless to pale pink	71.04
B	4 ml of 25% alcohol with 5 discs of beetroot	From colourless to pink	69.27
C	4 ml of 50% alcohol with 5 discs of beetroot	From colourless to dark pink	40.37
D	4 ml of hot water (90-100°C) with 5 discs of beetroot	From colourless to very dark pink	2.74
E	4 ml of water with chopped beetroot	From colourless to pink	58.66

• Explain, with reference to the tabulate data, the effect of different solutions in tubes A-C on the readings obtained in the experiment. You should make references to the knowledge you acquired from the lessons on cellular homeostasis.

Tube A (4 ml of water with 5 discs of beetroot):
The beetroots in tube contains a higher concentration of betalain pigments as compared to the surrounding diffusion. Hence the betalain will move down a concentration gradient, from the beetroot and diffuse through the tonoplast and cell membrane into the surrounding solution, causing the water in tube A to turn from colourless to pale pink, with a transparency of 71.04%.

Tube B/C (4 ml of 25%/ 50% alcohol with 5 discs of beetroot):
With alcohol in the solution, the alcohol, a fat solvent, interacts with the phospholipid bilayer and the breaks down the structure by dissolving the fats and proteins, leaving the cell membrane denatured. This leaves gaps in the membrane, making the membrane porous and the alcohol can then enter the cell easily through the cell. Similarly, the structure of the tonoplast is similar to the cell membrane, hence the membrane will also get denatured and porous, and the betalain then leave the cell quickly through these gaps in the membranes. The higher the concentration of alcohol, the greater the amount of damage done onto the membrane, the greater amount of betalain escaping the cell, finally resulting in a lesser percentage of transmission of light. The solution in tube B turned from colourless to a pink colour while the solution in tube C turned from colourless to a dark pink colour.Since tube C has higher concentration of alcohol in its solution than tube B, C has a lower transparency of light of 40.37% than tube B with a higher transparency of light of 60.27%. 

• Suggest an explanation for the observations of tube D & E.

Tube D (4 ml of hot water with 5 discs of beetroot):

The higher the temperature of the water, the greater the amount of betalain diffusing out of the beetroot, the lower of the transparency of the solution. When the beetroot is heated up by the hot water, the membrane gets disrupted and the phospholipids become more fluid due to the increasing vibrations of surrounding molecules caused by the gain in heat. When the proteins in the phospholipid bilayer is heated, the proteins (formed of coiled and folded strings of amino acids) will untangle and break apart due to the vibrations of the surrounding molecules. The proteins will span the membrane, forming holes that destroys the structure, and this repeats itself on the tonoplast, and the pigments can then spill out of the cell easily and causes the solution in tube D to turn from colourless to a very dark pink colour, with a low transparency of 2.74%. 

Tube E ( 4 ml of water with chopped beetroot):

The beetroot are chopped into smaller pieces instead of discs, which gives them a greater exposed surface area. The greater the exposed surface area, the greater the amount of diffusion, the lower the transparency of the solution. Hence, more betalain can diffuse out of the exposed ares of the chopped betalain, which makes the solution turn from colourless to a pink colour, with a transparency of 58.66%.

Photos of experiment:

Instructions on the board

Cuvettes A-E, according to chronological order starting from the left.