Bio 22 Lab Cell Transport Experiment

Diffusion, Dialysis and Osmosis Group 2 Asaad, Baldovino, Benuyo, Dee, Evangelista, Garcia, Vicuña

Biology 22 Lab 3B August 22, 2014

Abstract Cell is the basic unit of life and all living organisms are made up of cells. In order for the cell to sustain life, the cell must maintain internal stability. To do this, the cell makes use of a semi permeable membrane that acts as a regulator for the materials that pass through the cell. The materials may pass through the cell either passively by physical processes such as diffusion or actively by physiological processes such as active transport. Five different activities were done to predict the movement of the materials to the differentially permeable membranes; to discuss the importance and functions of the cell membrane; and to know the factors that affecting the rate of diffusion of the materials to the cell membrane. Using the methods in the activities, results were tabulated for each of the activities. Quantitative results show that the distilled water, the chloride test and potassium permanganate has the fastest diffusion or dialysis in their respective groups. For the osmosis, the water goes to the sucrose filled test tube. Also, different concentrations of NaCl solution affect the red blood cells differently. It was found out that the rate of diffusion is affected by the concentration and molecular weight of the substance. Substances move from higher areas of concentration to lower areas of concentration. Also, diffusion rate is lower if the substance has a higher molecular weight.

Introduction According to the cell theory, all organisms are made up of cells. It is the most basic unit of life. In fact, there are various single-celled life forms. More complex organisms, such as plants and animals, are multicellular; they have specialized cells that work together to maintain life. In response to environmental changes, cells need to maintain internal stability to sustain life. The cell regulates its composition, using a limiting semipermeable membrane, in order to preserve constant condition despite the changes in its environment. This semi-permeable membrane is referred to as cell or plasma membrane. The cell membrane, in response to different environmental changes, acts as a barrier to some particular substances while at the same time, allowing the passage of other materials. By controlling the passage of different materials, the cell can manipulate its own composition. Passive movement of materials is possible through different processes such as diffusion. Osmosis and dialysis are the two forms of diffusion processes. Physiological processes such as active transport, phagocytosis, and pinocytosis can possibly move the materials actively.

Method A. Diffusion in water

Two test tubes were obtained, one halffilled with tap water and the other half-filled with distilled water. The two test tubes were then labeled properly. In each of the two test tubes, two crystals of potassium permanganate (KMnO4) crystals were dropped using a pair of forceps. The test tubes were then placed on a rack so as to prevent any disturbance. The dissolution of the potassium permanganate crystals in each test tube was then observed. This dissolution is indicated by the dispersal of a purplish, light color. The time when the color had completely dispersed in the water in both test tubes were recorded. B. Diffusion in Colloid Three test tubes filled with hardened 5% gelatin solution were obtained. The three test tubes were then added with 1 mL of each of the following reagents simultaneously: (1) potassium permanganate, (2) methylene blue, and (3) congo red. The time it took for the stains to reach the bottom of each test tube was recorded. At five-minute intervals, the distance travelled by each stain through the gelatin was recorded. The same procedure was done for one and a half hours. C. Dialysis

In a small test tube, 3 mL of calcium chloride (CaCl2) solution and 2 mL methylene blue were combined. The mouth of the test tube was then sealed with a dried longganisa skin and a rubber band. It was made sure that there were no leaks on the seal. Half of the test tube was then immersed, with the sealed mouth facing down, in a beaker filled with about 30 mL of distilled water. The test tube was held vertically and it was made sure that the sealed mouth does not touch the bottom of the beaker. A test tube holder was used to hold the test tube in place. For every 30 seconds, 2-4 drops of water were aspirated from the beaker. A drop of it was placed in two separate test tubes. A drop of silver nitrate was then added to the first test tube whereas a drop of ammonium oxalate was added to the second test tube. These were done to test the presence of chloride and calcium ions respectively, which would be indicated by a white, cloudy precipitate. The same procedure was repeated until the precipitate became evident in both test tubes. The time when the precipitate became evident in both test tubes was recorded. It was also observed if the methylene blue would diffuse into the beaker. D. Osmosis A small test tube was filled with 5 mL of a 30% sucrose solution. The mouth of the test tube was then sealed with longganisa skin and a rubber band. It was made sure that there were no leaks on the seal. The test tube was then inverted and the level of the liquid inside the test tube was noted. The test tube was then immersed, with the sealed mouth facing down, in a beaker filled with about 60 mL of distilled water. The test tube was held vertically and it was made sure that sealed mouth does not touch the bottom of the beaker. A test tube holder was used to hold the test tube in place. The test tube was left immersed in this position for about two and a half hours. Afterwards, it was observed whether the level and weight of the liquid inside the test tube increased. E. Osmosis in Red Blood Cell Three clean microscope slides were prepared and labeled A, B, and C respectively.

Three drops of blood was extracted from a female volunteer by pricking her index finger with a sterile lancet. It was made sure the skin of the volunteer’s index finger was swabbed first with alcohol before doing the procedure. A drop of blood was placed on each of the three slides. The three slides were then added with a drop of each of the following solutions: (1) 0.07 M NaCl to the blood on slide A, (2) 0.15 M NaCl to the blood on slide B, and (3) 0.30 M NaCl to the blood on slide C. The blood and the NaCl solutions were mixed thoroughly, and the mixture was smeared evenly on the surface of the slides. The slides were focused at 400X using three different microscopes. With the aid of an ocular micrometer, the diameter of the cells in each slide was measured for every five minutes for forty minutes. Possible changes in the shape of the cells in each slide were observed.

Results A. Diffusion in Water The KMnO4 crystals diffused faster in the test tube half-filled with distilled water. In the test tube half-filled with tap water, the diffusion was slow, marked by the formation of a nebula-like structure suspended at the middle of the test tube (it disappeared eventually). The nebula-like structure was not observed in the test tube with dH2O since the purple color quickly scattered throughout the entire tube.

B. Diffusion in Colloid Results are shown through in a tabular format: Table 4.1 Distance travelled by Potassium permanganate, Methylene Blue and Congo Red. Time (min.)

Distance Travelled (cm) KMnO4

5

1.0

Methylene Blue 0.3

Congo Red

10

1.4

0.5

0.3

15

1.5

0.6

0.3

0.2

20

1.6

0.7

0.3

25

1.9

0.8

0.3

30

2.1

0.9

0.4

35

2.1

0.9

0.4

40

2.2

0.9

0.4

Based on the data, it can be said that the KMnO4 travelled the farthest distance, followed by methylene blue, and lastly by congo red.

C. Dialysis Table 4.2 Results of Dialysis of Methylene Blue, Calcium and Chloride Ions Time (min) 1.5 2.5 3.5

Chloride Test

Calcium Test

Methylene Blue

✔ ✔

4.5 5.5

✔

6.5

For the chloride test, the cloudy precipitate started to appear after 2.5 minutes, while for the calcium test, it took 5.5 minutes. The methylene blue diffused into the beaker after 3.5 minutes. D. Osmosis Some of the water in the beaker set-up was transferred to the test tube containing 30% sucrose. Initially, the water in the beaker was filled with 60 mL, then after 2 hours, 10 mL of the water in the beaker shifted to the sucrosefilled test tube.

E. Osmosis in Red Blood Cell

Table 4.3 Size of Red blood Cells under different concentrations of NaCl NaCl Concentration (M) Time (min.)

0.07 M

0.15 M Cell size in um

0.30 M

5

2

3

3

10

2

3

3

15

4

3

3

20

4

3

3

25

4

3

3

30

4

3

3

35

5

3

3

40

4

3

4

Based on the acquired data above, it can be said that under a NaCl concentration of 0.07 M, the size of the RBC increased, under the NaCl concentration of 0.15 M, the size of the RBC remained constant, whereas under the NaCl concentration of 0.30 M, the size of the RBC increased.

Discussion Diffusion is the process in which molecules move from a more concentrated solution to a less concentrated solution. That is why in experiments A and B, it was seen that the molecules of the solutes in the different test tubes diffused. However, it was observed in the results that the different substances showed different times of diffusion with one diffusion faster than the others. For experiment A, the test tube filled with distilled water diffused faster than the test tube filled with tap water. The reason for this is that distilled water is purer than tap water because tap water has impurities such as bacteria, minerals etc. This makes tap water more concentrated than distilled water. Because of this, KMnO4 was able to diffuse faster in distilled water. The concentration of a substance is a major factor in the speed of diffusion. This can be observed the experiment B in which diffusion is observed in colloidal materials. Unlike water,

these substances have colloidal suspensions that affect the rate of diffusion. In the experiment, three reagents namely potassium permanganate (KMnO4), methylene blue, and congo red were simultaneously added into three separate test tubes containing gelatin. After forty minutes, it was observed that among the three reagents potassium permanganate was the one to diffuse the farthest. Compared to the two other reagents, potassium permanganate has the smallest and lightest molecules which it faster and easier for it to diffuse. When it comes to the diffusion of colloids, the molecular weight of the chemical is a primary factor. The smaller and lighter the molecular weight of a chemical, the faster it will diffuse. Dialysis involves separation of substances in a solution by their unequal diffusion through their semipermeable membranes. In the experiment, it was observed that a cloudy precipitate formed. This happened due to the reaction between the ions and their reactants. The chloride ions diffused faster than the calcium ions because chloride ions have smaller molecules compared to the calcium ions. Methylene blue was observed to have diffused into the beaker. However, the molecules of methylene blue are very large that the membrane of the dried longganisa skin will not allow the passage of the molecules. The methylene blue should not have diffused into the beaker. The dried longganisa skin is considered as a semipermeable membrane because it allowed smaller molecules such as the chloride ions and calcium ions to pass through and prevented larger molecules from passing. Osmosis is the process in which water permeates through a cell through its membrane. Water is the one that diffuses in this process. Water is transported from substances with higher amounts of water to substances with lower amounts of water. That is why in experiment D, some of the water transferred to the test tube filled with 30% sucrose since there is a lower concentration of water in the test tube than in the beaker filled with distilled water. Osmosis is also observable in experiment E. In this experiment, it can be determined that the setup with 0.15 M NaCl is an isotonic solution

because there was no change in the size of the cell. This means that the concentration between the cells and the solution are the same – 0.15 NaCl. On the other hand, the setup with 0.07 M NaCl is a hypotonic solution because it caused the cells to get bigger. This means that the solution has greater amounts of water than the cell. Lastly, the setup with 0.30 M is a hypertonic solution because the solution has lesser amounts of water than the cell. However, results showed that the 0.30 M setup caused the cells to become bigger when theoretically it should have caused it to shrink.

Conclusion and Recommendation/s Based on the result of the experiment, it can be concluded that the rate of diffusion of a substance is affected by its concentration and molecular weight. Substances move from areas of higher concentration to areas of lower concentration just like what happened to potassium permanganate when it diffused faster in distilled water which happens to have a lower concentration than tap water. When it comes to the molecular weight of substances, a substance with lower molecular weight like potassium permanganate (158.03 g/mol) has a higher diffusion rate compared to a substance with higher molecular weight like Congo red with a molecular weight (696.66 g/mol). On the other hand, methylene blue which happens to have molecular weight of 319.85 g/mol has a slightly lower diffusion rate compared to potassium permanganate but faster than congo red. The rate of diffusion is faster in water compared to gelatin which is a colloid because water has more spaces to allow the spread of molecules compared to colloids where space is limited which also limits movement of molecules therefore the rate of diffusion is slow.

In dialysis, which refers to the diffusion of solutes across a selectively permeable membrane, the diffusion of substances with smaller molecules occurs faster just like what happened to chloride ions which started to appear ahead of the calcium ions. In osmosis, which is defined as the diffusion of water from a hypotonic solution or low concentration of solute to a hypertonic solution or high concentration of solute, there should be a balance on the ionic concentrations (isotonic solution) on both sides of the cell membrane for the cells to survive. This was shown in experiment E in the 0.15 M NaCl, where the size of the RBC remained constant. Therefore this is the isotonic solution which is needed for the cell to survive. On the other hand, results showed that the 0.30 M NaCl setup caused the size of the RBC to become bigger when theoretically it should have caused it to shrink. Perhaps, there was an unknown mistake in the conduction of the experiment in the 0.30 M NaCl setup. The researchers then recommend the repetition of the procedure for the 0.30 M NaCl setup to further validate the results.

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