Group 4 Project 2004 THEME 2004: OIL - BIOLOGY
Research question: What are the effects of detergents on the phospholipid bi-layer of blood cells?
Introduction
Lindsay, Natalia and Jack at work -Photo taken by Rachel
The Group 4 project this year is Oil, so we decided to examine membranes. Knowing that every day we use and are exposed to Soaps and Shampoos, we decided to investigate their effect on us. Considering that all the cells in our body are surrounded by the phospholipid bi-layer, in order to accurately analyze the effect that detergents have on our bodies, we decided to test the effect of the detergents on the phospholipid bi-layer. The bi-layer, containing mainly lipids, fit the overall theme of oils, considering that oils are lipids. Originally, the idea was to test the detergents on our skin; however we quickly realized how it would be impossible to gather concrete results from this method. So we chose blood next as our testing material, because it allowed us to get results that were easily readable with a colorimeter. This haveing been said, the actual aim of the experiment can be found on the next page.
Aim
Our aim is to find the effects different types of soaps and/or shampoos have on the membranes of pig’s blood. We will do this therefore by exposing the blood to our chosen detergents, and record the results using a colorimeter. The reason we chose to test on blood is for the pigments in the hemoglobin, and for the membranes surrounding the hemoglobin. This way, we can analyze the soap’s effect on phospholipids easily through the color. When the blood is exposed to the detergents, if there is an effect, the membranes surrounding the hemoglobin will break, releasing the red pigment. The degree of the soap’s effect depends on the hemolytic properties of the solution. Our solution will be hypotonic; hence the degree of the soap’s affect depends on how hypotonic it makes the solution. After placing the solution in a centrifuge to make the intact blood cells sink, we can gather quantitative results of absorption through the colorimeter. Therefore we would hypothesize that if we had a very low absorption for a solution; it would mean that the detergent did not affect the blood very much. On the other hand, if the detergent affected the blood heavily, the ending solution would be much redder and have therefore higher absorption.
Variables
* Controlled variables - We needed two controls, one with only blood and physiological saline and the other with blood, physiological saline and distilled water, in order to make sure that neither the distilled water nor the saline had any effect on our results.
* Independant variables - These are the different types of detergents we used. These are: liquid hand soap, shampoo, and bar soap.
* Dependent variables - These are the quantities of each of these soaps, and therefore altering the overall concentration of our solutions.
Methods
Diluted bar soap (side view)
Method 1: Preparation of bar soap solution (to be used for all experiments)
Equipment:
-Distilled water / -1× 50cm3 beaker-Knife / -Digital scale (brand?)
-Tissue paper / -Bar soap (brand?)
-Mortar and pestle / -25cm3 measuring cylinder
Method:
Using the knife scrape off soap in form of shavings.
Place the tissue paper on the scale, reset and then weigh out 2.5g of soap from the shavings made.
Transfer the soap to the mortar
Measure out 25cm3 of water with the measuring cylinder and add to the mortar
Using the pestle, crush and mix the soap with the water until it becomes a smooth, uniform liquid soap solution (with the least and smallest possible pieces of solid soap). Be careful not to agitate the mixture too much or it will become too foamy, and consequently it cannot be measured accurately.
Transfer the mixture to a 50cm3 beaker.
Diluted bar soap (view above)
Lindsay and Nat (talking about the bar soap): "Is this saliva or yoghurt?"
Lindsay (talking about the shampoo): "This looks like puss bursting out of skin!"
Natalia (talking about the liquid soap): "It’s cheap, 10 year old sour cream!"
Method 2: The effect of detergent on the lipid layer of red blood cell membranes
Equipment:
-Pig’s blood / -6× 50cm3 beakers-Distilled water / -Bar soap solution
-Centrifuge (brand) / -2× 10cm3 pipettes
-12 centrifuge test tubes / -25cm3 measuring cylinder
-Shampoo (Shampooing de Fruits pour Cheveux normaux ou gras_ Leader Price) / -12 bungs to fit test tubes
-Liquid soap (Crème Lavante à l’huile d’amande douce_ Leader Price) / -Physiological Saline
-Colorimeter for TI-83 (along with its required equipment, i.e. LabPro, test tubes and adaptator) / -1cm3 pipette
-Test tube rack / -Marker
Method:
Fill a 50 cm3 beaker with liquid soap. Do this for the Physiological Saline, bar soap solution, shampoo and distilled water.
Using a 1cm3 pipette measure out 0.1cm3 of pig’s blood and transfer to a test tube. Repeat this procedure for another 4 test tubes.
With a 10 cm3 pipette measure out 10 cm3 of Physiological Saline and transfer to one of the test tubes already containing blood in it. Repeat for the other 4 test tubes.
Using a second 10 cm3 pipette measure 2 cm3 of liquid soap and transfer to one of the test tubes containing blood and Physiological Saline, label the test tube. Rinse the pipette out a couple of times with distilled water; repeat the process changing the liquid soap for bar soap solution, shampoo, distilled water and Physiological Saline. The last two can be labelled control 1 and control 2.
Wait 2 minutes for the solutions to begin to react
Place bungs on the test tubes and shake until the mixtures have each a uniform colour and texture.
Remove bungs and place test tubes in centrifuge. Allow them to spin for 5 minutes at a speed of 6.5 units.
Set and start the colorimeter (explain how exactly?) transfer some of the solution of a test tube into a colorimeter test tube, making sure that fingers only get hold of one side of the tube. Place tube in colorimeter and note results. Repeat for the other 4 solutions taken out of the centrifuge.
Repeat steps 1-7, however, change the quantity of Physiological Saline to 9cm3 (instead of 10 cm3) and the variable solution to 3 cm3 (instead of 2 cm3). Repeat again for 11 cm3 Physiological Saline and 1 cm3 of variable solution.
Natalia shaking one of the test tubes in order to mix the components
Results
Data Collection
Raw data:
Series with variable combination of quantities / 12cm3 (±1cm3) Colorimeter reading for Solutions of Pig’s Blood, Physiological Saline and Variables (units?)
Liquid Soap Shampoo Bar Soap solution Control with Physiological Saline Control with Water
10cm3 Physiological Saline + 2cm3 of variable 1.067 2.153 0.017 0.971 1.142
9cm3 Physiological Saline + 3cm3 of variable 1.679 1.679 NO RESULT 1.063 1.092
11cm3 Physiological Saline + 1cm3 of variable 0.355 1.741 1.264 0.174 0.164
Observations valid for each of the series: liquid soap and bar soap solution turned solid after centrifuge. This ‘solid’ either fixed itself along one side of the test tube concerned, or on the surface forming a soap ‘lid’ on the solution.
In this case, the detergent solidified diagonally in a barrier-like form. The blood cannot pass through.
Data Processing
Combination of liquids in cm3 ± 1cm3 / Combination Percentage ±8.4%Physiological Saline ±0.5cm3 / Detergent ±0.5cm3 / Physiological Saline ± 4.2% / Detergent ±4.2%
11 / 1 / 91.67 / 8.33
10 / 2 / 83.33 / 16.67
9 / 3 / 75 / 25
Legend
1: Liquid Soap4:Physiological Saline Control
2: Shampoo5:Water Control
3:Bar Soap Solution (lack of results for standard deviation)
:Standard deviation
(The two soap results are zero, when we don’t have them)
Graph A:
Graph B:
Legend (corresponds to Graph A and B)1: Liquid Soap3:Physiological Saline Control
2: Shampoo4:Water Control
Legend
1: Liquid Soap 4:Water Control
2: Shampoo5: Bar soap solution
3:Physiological Saline Control
(A) One set of test tubes before centrifuge; (B) Another set of results after Centrifuge
Conclusion
Day 1:
Taking the first set of results into account (from the first day):
(1)
Liquid Soap / 1.067Control with Physiological Saline / 0.971
Shampoo / 2.153
Bar soap / Invalid sample - (0.017)
Control with water / 1.142
We see that the shampoo has a very high absorbance level compared to the other soap, (and indeed it gave a very dark brown solution compared to a relatively light one for liquid soap), probably because the other products included to the fabrication of shampoo break down the red blood cell’s phospholipid bi-layer more than the soap present in the shampoo, and so the liquid soap, unable to brake the membrane has a lower absorbance level. This might account as to why shampoo hurts the eyes more than soap (speaking out of strictly personal experience).
Day 2:
Taking the second and third set of results into account (from the second day):
(2)
Liquid Soap / 1.679Control with Physiological Saline / 1.063
Shampoo / 1.679
Bar soap / No reading
Control with water / 1.092
(3)
Liquid Soap / 0.355Control with Physiological Saline / 0.174
Shampoo / 1.741
Bar soap / 1.264
Control with water / 0.164
Now here we have very little consistency in the results: only the shampoo results stay high and constant in both takes, but the other results are too wide (between themselves and compared to day 1) for us to be able to grant much trust in the shampoo results. I dare not advance any conclusion for such wide, poor and insufficient results, the fact of using more detergent falling outside the expected trends of having more absorbency. The only claim we can make is as to the shampoo always having the greater absorbency of the set of results thus supporting a little our conclusion to day 1 concerning shampoo.
Evaluation
Day 1:
Now on the first day the only true problem was lack of time: we had an acceptable set of results (except for the bar soap) and we were not able to double or triple check them and so there is still a possiblility that the results could be freaks (and we have none for bar soap). Also, the blood and saline solution would not mix properly with the soap, even after having been in the Centrifuging machine and so there is a possibility that the soap was not in contact with the blood enough to make the experiment viable.
Day 2:
For starters we can say that the blood was very black and prone to clotting compared to the blood used on day 1 and seemed more dense (Indeed, the test tube for bar soap in our first set of results broke during centrifuging). Further examination proved that the blood was of a very different quality to the one used Day 1 and so the results cannot be satisfactorily compared between the days. The other great problem concerns the colorimeters that were used. We must have used at least 3 different TI-83 compatible colorimeters and two calculators only to find we would often get negative, and so completely incomprehensible, results. Only using different filters gave us any viable results, but we had to change filter for every individual solution and, often enough, colorimeters, even using a decade old manual version that would also give very unsatisfactory results (here again changing filters). So even if filters do not change the light absorbency much the fact that no two solutions were treated the same way makes us rather dubious looking at these already very untrustworthy results, who are also quite insufficient.
Lindsay (talking about the manual colorimeter):
"I feel like I’m building a time machine"
(1) Manual Colorimeter; (2) Lindsay's way of washing the pipette
General criticism of the method/Suggestions:
First looking at our method of making the soap solutions (especially the bar soap) we should have diluted them a lot more (both the shampoo and the liquid soap were not diluted at all) so as to permit the blood and saline to react better with the soap oils, a recurrent problem in the course of the experiment.
For our general method to get our results, we had no control or reference as to the origin of the physiological saline or of the blood so taking into these two factors we it is obvious we cannot grant too much credit to any of the results acquired using this method, and the fact that the colorimeters were not working properly shadows further these results but otherwise on the whole the experiment does give a clue as to what extent the oils in red blood cells are affected by the detergents present in soaps, and why it is not a good idea to swallow these.