Method
- Make up the saline solutions in the following concentrations using pipettes; carefully measure out the volumes of both sodium chloride and water to give all the concentrations required and place in separate test tubes.
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Shake the bottle from which the blood sample will be taken and carefully measure out 0.1cm3 of blood using micropipette and test tube. Note each test tube must have a separate stopwatch designated to it so the experiment is as fair as possible and stopwatch must be started as soon as the blood sample is introduced to the solution (s)
Stirring of blood sample is very important to ensure uniform distribution of the cells
Note: Allow a 180second gap between adding blood to each test tube, this is so there is sufficient time at the end of the 1,800 seconds to transfer sample into the cuvette and take a colorimetric reading. If a gap is not left the blood could be left in solution longer than the other samples which could result in unfair and anomalous results that do not agree with the hypothesis.
- Leave test tube at room temperature for 1,800 seconds (30 minutes) to allow haemolysis to occur. Mix the sample regularly using glass rod, this is to ensure the blood is mixed equally within the saline solution and prevent the red blood cells from clumping together and at the bottom of the test tube.
- Once 1,800 seconds have elapsed transfer the sample from test tube to cuvette.
- Set colorimeter at filter 590nm and zero colorimeter using a blank (cuvette containing distilled water)
- Place cuvette in colorimeter and measure the absorbance reading for each test tube.
- Repeat steps 2-6 for 0.1, 0.0.5, 0.7, 0.9 and 1.0 sodium chloride concentrations.
In order to determine suitable concentrations that will give me measurable results different concentration of sodium chloride were tried. As is known red blood cells are most suited to a concentration of 0.9% sodium chloride which is isotonic. I decided to use concentrations, which ranged from 1.0 to 0 % sodium chloride concentrations, as I believed that the results produced would be easily measurable and also sodium chloride concentration within the human body very rarely fluctuates outside this range. This range would also give me the opportunity to discuss the importance of maintaining a concentration as close as possible to 0.9% for the correct function of the human body.
(Concentrations I have decided to use are shown in table below)
Sodium Chloride Concentration
Preliminary test results
As a result of preliminary test results I decided to carry out my experiment with the concentration I had used in my trial run. Rom then results obtained it was quite evident that as the sodium chloride concentration decreased from the isotonic concentration of 0.9% there was an decrease in the absorbance readings obtained which implied that haemolysis of red blood cells increased.
Method:
I measured out the sodium chloride solution and distilled water in the ratios below to give me the concentration required. Both distilled water and sodium chloride were measured accurately using pipettes. Once the concentration required had been made I allocated an individual stop clock to each of the seven test tubes, the primary reason for this was to avoid confusion as well to make the experiment fair.
The bottle containing the blood was stirred and mixed for 30 second to ensure uniform distribution of cells; once the sample had been mixed I carefully measured 0.1cm3 of the blood and placed it in the test tube containing the 0% sodium chloride solution. And immediately started the stopcock, I allowed a two minute gap before adding the blood to the next test tube containing 0.1% sodium chloride (blood was mixed again)
This was repeated for the remaining five test tubes at concentration of 0.3, 0.5, 0.7, 0.9 and 1.0%.
The test tubes were stirred using a glass rod at regularly intervals to allow the cells to spread throughout solution.
As soon as the 1,800 seconds were completed the first sample from the 0.0% solution was transferred to a clean cuvette, the colorimeter was zeroed using a blank cuvette containing distilled water only and the cuvette containing the sample from the 0.0% solution was placed in the colorimeter and an absorbance value taken and recorded.
This was repeated for all the solutions containing varying concentration of sodium chloride and 0.1cm3 of blood, each sample was placed in a clean dry cuvette and colorimeter zeroed before obtaining the absorbance reading this was to avoid any mistakes.
Once all the absorbance values had been obtained the test was repeated a further two times so I could take an average of the absorbance values. The values were recorded in a results table and the mean calculated, once I had calculated the mean a graph was produced.
As a control experiment I set up one test tube containing no sodium chloride.
Risk assessment
This experiment is low risk; there are no toxic or harmful chemicals to be used. Despite using non-risk chemicals sensible precautions need to be taken during the experiment. This includes wearing gloves when handling blood sample as well as safety goggles and lab coats. Also any practical work needs to be carried out in a space where there is plenty of room to work safely without the risk of spillage of samples.
If spillage of blood sample occurs, workbench is to be cleaned immediately using disinfectant
Variables
Independent variables
I will be varying the concentration of the sodium chloride solution that I will be putting into individual test tubes during the period of 1800 seconds
The sodium chloride solution will be range from 0 % - 1.0 %. This will enable me to tabulate and produce a graph of results showing the affect of varying sodium chloride solution on haemolysis.
Controlled variables
I will be controlling the time that the 0.1cm3 blood sample remains in the saline solution(s) before the colorimetric analysis is carried out. This will be done by
Allocating an individual stopwatch for each test tube (7 in total) .the stopwatch will be started as soon as the blood sample is introduced to saline solution for a period of 1,800 seconds after which sample will be transferred to a cuvette so colorimetric analysis can be carried out.
All samples will be measured accurately using pipettes for sodium chloride solution and distilled water; a micropipette will be used to accurately measure 0.1cm3 of blood.
All test tubes for the duration of 1,800 seconds will be placed in test tubes at room temperature and will also be stirred regularly
Picture shows two test tubes, test tube1 shows no signs of haemolysis as this blood sample has introduced to an isotonic solution where the water potential in the saline solution sample is equal to that with in the erythrocytes. Therefore there is “no net movement of water” from the saline solution through selectively permeable plasma membrane into the cells causing cells to swell and eventually burst.
Test tube on right shows signs of haemolysis; sample has changed from characteristic colour of dark murky red which is due to the red blood cells having an opaque disc structure that allow very little transmission of light. Blood sample in test tube 2 has been introduced into a hypotonic solution where the water potential within the cell is greater than the water potential outside it, therefore water molecule pass through the plasma membrane into the cell causing lysis. This is shown in test tube where there is a lot more transmission of light and at the bottom of test tube haemoglobin can be seen.
Results
Conclusions
Main Trends and Patterns
My results show that sodium chloride concentration quite clearly has a major affect on the haemolysis of red blood cells.
The data shown in both the graphs and results table show a clear pattern which is increased haemolysis as the concentration of sodium chloride decreases from 0.9-0.1 %.
The raw data shows that haemolysis is at its greatest at a concentration of 0.1 with an absorbacnce reading of only 20% meaning 80% of light had passed through solution, and at its least at a concentration between 0.9 and 1.0 % which is very close to the isotonic value of 0.9% where the absorbance reading was 81%. This suggests that the red blood cells are very much more suited to a concentration of 0.9% and at this concentration there is very little haemolysis if any at all.
The graph shows very clear relation with regards to sodium chloride concentration and absorbance. There is one anomalous result which has been highlighted in both the results table and the graph, this occurred at a concentration of 0.3% and shows an absorbance reading of 49.3% which is strange as I would have expected this reading to be a lot lower especially considering at a concentration o 0.5% the absorbance reading was 47.3%. As the water potential of the 0.3% solution is less negative than that of the 0.5% solution the absorbance reading at this concentration should have been a lot lower as more water molecules would have passed through the plasma membrane into the cell where the water potential is more negative resulting in more cells haemolysing and the solution becoming less cloudy as haemoglobin is released.
Another anomalous result is at 0% concentration which shows an absorbance reading of 32%
This is a lot higher than would expect and may have been due to an error on my part when taking the absorbance reading or possibly adding some sodium chloride solution to this test tube by mistake.
I believe this anomalous result may have occurred as a result of either me not correctly calibrating the colorimeter using the blank cuvette containing distilled water prior to the colorimeter test at 0.3% sodium chloride concentration.
Explanation of results
The red cell is enclosed in a thin membrane that is composed of chemically complex lipids, proteins, and carbohydrates in a highly organized structure. Extraordinary distortion of the red cell occurs in its passage through minute blood vessels, many of which have a diameter less than that of the red cell. When the deforming stress is removed, the cell springs back to its original shape. The red cell readily tolerates bending and folding, but, if appreciable stretching of the membrane occurs, the cell is damaged or destroyed. The membrane is freely permeable to water, oxygen, carbon dioxide, glucose, urea, and certain other substances, but it is impermeable to haemoglobin.
There are three types of solution these are isotonic, hypotonic and hypertonic.
Isotonic – Water potential within red blood cells and plasma (Solutions) are equal. Therefore both water potentials are in equilibrium. This is shown on the graph at a concentration of 0.9%
Hypertonic – Water potential outside cell is less negative than water potential within cell so water molecules leave the cell causing cell to shrink.
Hypotonic- Water potential outside cell is more negative than water potential within cell, so water molecules enter the cell by osmosis resulting in haemolysis.
The whole concept of water potential is an interesting one and is vital for our very existence, without osmotic gradients and water potentials our bodies would not be able to survive as they would have no way of increasing or decreasing the concentration of fluids within the body.
Research into haemolysis is important as there are many factors which can haemolyse cells and hospitals are constantly trying to reduce haemolysis when blood transfusions are taking place. Haemolysis
Experimental Limitations.
The most important experimental limitation was the freshness of the blood sample, when blood has been removed from its source it begins to breakdown and haemolysis occurs naturally. Although the sample I was given was said to be a fresh sample there would have been cells within the sample which had already haemolysed. Taking this into account although my results support my hypothesis I am not very confident that the results obtained were as accurate as they could have been.
Clearly further repeat results would have provided me with more accurate results however I believe an experiment such as this should be carried using a different method, the method which I believe should be used is one where a haemocytometer is used, a small volume of the solution could be pipette onto a slide and the number of cells visible counted. Although this is a time consuming process the results obtained would be a lot more accurate. Centrifuging the blood samples would have also been a better way of carrying out this experiment, as I was measuring percentage absorbance (turbidity) this is not always as accurate as the results should be.
Overall this investigation did provide me with evidence that supports my initial hypothesis that decreasing sodium chloride concentration increases haemolysis.
The importance of maintaining a concentration as close as possible to 0.9% is vital to lead a healthy life, if the concentration with the plasma begins to decrease this could be life threatening as red cells haemolyse the oxygen carrying capacity of the red cells would be reduced, this would have a knock on affect to the entire body as the demand for oxygen is not being met. Also if the concentration of sodium chloride increases this can result in the biconcave shape of red cells being lost reducing surface area of red blood cell.
Bibliography
Listed below are textbooks, websites that I have used during my investigation
- Molecules and Cells , John Adds, Erika Larkcom, Ruth Miller
- Exchange and Transport energy and Ecosystems.
- Collins AS Biology: Unit 1 Molecules and Cells
- Collins AS Biology Exchange, Transport and Reproduction.
- Essential A Level Biology
Websites:
Guidance was also taken from my biology teachers, Steve Beesley and Linda Cooper