Investigating the effect of temperature on cell membrane permeability Free Essay Example

To investigate the effect of temperature on cell membrane permeability, I am going to use beetroot due to its pigment properties. The pigment is a strong colour, which means that I can easily measure, using a colorimeter, how much pigment is released at certain temperatures.

During this practical, I will need to apply controls and a variable. The variable being temperature, and the controls being:

* Disc thickness

* The amount of water used when heating

* The measure of solution put into the cuvette

* The time temperature is maintained

If these factors were not controlled, results would be likely to produce false reading, due to too many variables.

Apparatus

Cuvette

Colorimeter

Beetroot disc

Water bath

Beaker

Tripod

Bunsen burner

Boiling tube

Cork borer

Forceps

Safety glasses

Thermometer

Stop clock

Scalpel

Pipette

Method

1. Put on safety goggles as a safety precaution

2. Switch on colorimeter to allow it to warm up and stabilise, ready for when it is needed

3. Prepare living specimen of beetroot using core borer, to get small discs of the same diameter

4. Using a scalpel, cut the cylinder of beetroot into discs of same thickness for fairness

5. Wash beetroot in water for 24 hours to rid the pigment produced from damaged cells during preparation

6. Place 3 discs of beetroot in a boiling tube using forceps to prevent contamination ready for heating

7. Place 3cm3 of water into the boiling tube for the beetroot pigment to leak into during heating

8. Place boiling tube in beaker of water on a tripod over a bunsen burner to heat to required temperature

9. Heat beetroot at 20 oc, using a thermometer to measure the temperature and maintain for 2 minutes

10. When heat reaches the required temperature, remove the boiling tube from the water bath using tongs because the tube will be hot.

Work quickly to allay cooling of the solution.

11. Using a pipette, fill a cuvette 3/4 full of beetroot pigment solution.

Be careful to handle only the frosted or rigid sides of the cuvette to prevent ‘polluting’ the clear sides

12. Set the colorimeter range to transmission and place a cuvette with pure water in place (reference cuvette) before depressing the ‘press to zero’ button to reset the colorimeter

13. Select a filter (green 604) on the colorimeter – because this is the closest colour match to that of the solution [see sheet titled “why use a colorimeter?”] and place cuvette in with clear sides facing left and right to allow light to pass through

14. Compare colour given from colorimeter and record results

15. Repeat investigation at other temperatures (20, 35, 45, 55, 65, 75, 85, 95 oc) allowing 1 minute for thermometer to cool between temperature variables, recording results at each stage

Diagram

Results

Temperature (oc)

% of light transmission

Test 1

Test 2

Test 3

Average % of light transmission

20

98%

97%

98%

35

96%

92%

94%

45

81%

80%

81%

55

74%

76%

75%

65

71%

50%

56%

59%

75

23%

30%

27%

85

14%

19%

17%

95

17%

14%

16%

For a temperature of 65oc, I made a third repeat because the first and second tests were so far apart.

Analysis and conclusion

The trend shown clearly depicts a correlation between percentage of light transmission and temperature applied to the specimen (see graph and table below).

The product moment correlation co-efficient (PMCC) is a measure of how strong the correlation is between two variables. A positive value indicates a positive correlation and the higher the value, the stronger the correlation. Similarly, a negative value indicates a negative correlation and the lower the value the stronger the correlation.

Hence, Ex represents the sum of the temperatures. Ey represents the sum of the average results. Ex2 is the sum of all the temperatures squared (20�)+(35�)+(45�)+55�)+(65�)+(75�)+(85�)+(95�). Similarly, Ey2 is the sum of all the results squared. Exy is Ex multiplied by Ey (20*98)+(35*94)+(45*81)+(55*75)+ (65*59)+(75*27)+(85*17)+(95*16) i.e.: the temperature multiplied by the result.

Sxx = Ex2 – (Ex)2 /number of results i.e.: 8

Syy = Ey2 – (Ey)2 /number of results i.e.: 8

Sxy = Exy – ((Ex)(Ey) /8)

The PMCC demonstrates that the results show a very strong positive relationship between temperature and light transmission. Likewise, the graph also shows a positive correlation – a pattern that quickly became apparent early in the investigation.

As the results show more beetroot pigment can leak out at higher temperatures, I can construe that cell membrane becomes more ruptured as temperature increases.

This brings me to conclude that in beetroot, as temperature increases, light transmission decreases. This means that the more betalaine (beetroot pigment) is released at higher temperatures, making the solution more concentrated, allowing less light transmission.

Why use a colorimeter?

A colorimeter entails a light source, a filter to select the most appropriate wavelength, a photocell to interpret the light intensity and a digital display.

A colorimeter gives the percentage of light which can pass through the solution.

The most suitable filter to use can be selected by choosing the one with the closest colour to match that of the solution being tested.

Filters are used to give light from a specific region. The ideal filter is the one which selects the range of wavelengths most strongly absorbed by the specimen.

I chose the filter to use by using the following tables:

Beetroot in a colorimeter

The pigment betalaine can be divided into betacyanins and betaxanthins based upon its molecular structure.

Betacyanins generally appear red to red violet in colour – they absorb in the 535-550nm range.

Betaxanthins generally appear yellow in colour (absorb in the 475-480nm range). They cause colour in flowers, fruits and sometimes vegetative organs. They are found in the vacuole and they are water-soluble.

What happens to a cell membrane when it is heated?

When beetroot is heated, the cell membranes are disrupted. A biological membrane is made of a phospholipid bilayer. These are formed because the phospholipids that make up the bilayer have a polar, hydrophilic (water-loving) head and a hydrophobic (water-hating) tail. The tails pack together, exposing only the polar heads to the water. The most effective way of doing this is to create two layers one atop of the other, with the fatty acid tails towards each other. This is the phospholipid bilayer. The water around and within the compartments formed by the phospholipid bilayers is also crammed with protein (the cytoplasm).

When something is heated, it is given energy. Molecules start to spin and vibrate faster. Thus, the water will expand. This will have a disruptive effect on any membrane in its path. Lipids become more fluid as temperature goes up (like butter) so the membranes become more fragile.

Proteins are formed of coiled and folded strings of amino-acids, held together by hydrogen bonds and disulphide bridges. If you heat them too much, they will vibrate vigorously, causing them to untangle and break apart. When this happens to the proteins spanning a lipid membrane, they will form holes that will destroy the delicate structure. Now, any pigments in the inner part of the cell, will spill out.

Beetroot pigment (betalaine) is used commercially as a food dye. As it is heat sensitive and may change colour, it can only be used in ice-cream, sweets and other confectionary, where temperature change will not occur. It is both cheap and has no known allergic side effects.

Evaluation

Had I more time, I would have done three repeats for each temperature tested in order to obtain more reliable results, but as it was only the results for 65 oc were greatly different, therefore I decided that to repeat this a third time would be sufficient.

Limitations

A notable limitation of the apparatus used in my experiment, is the use of a bunsen burner as a crude heat source, whereas a Thermo-water bath would have been better suited to maintaining a constant temperature. Marginal variance regarding temperature may have caused small anomalies, as seen in the table for test 1 (85 oc, 14%).

Another problem which would have proved to have affected the results is the technique by which the beetroot discs were cut. The discs were hand cut, which means that they are not all of uniform size. For this reason, the amount of pigment in each disc will not be consistent either. A natural variation of the pigment contained in each disc will influence my results further. If one disc of beetroot contains more pigment than the next, then the experiment would not have been a fair one.

My tests where consistent, with one exception which was rectified when repeated. This suggests that my method had a dependable output. The use of a colorimeter provided consistency and accuracy over a visual colour check. I can conclude this because the line of best fit on the graph is predominantly smooth.

However, I believe the reliability and precision of data has proved adequate to support my conclusion that as temperature increases, light transmission decreases. The limitations noted were minimal in regard to the resources available. Overall, I believe that they didn’t greatly corrupt the expected trend.