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Practical Two

Practical Two: Cell Physiology
Introduction
The plasma membrane surrounds the cell providing structure, protection as well as closely monitoring the exchange of substances which enables the cell to maintain normal functioning. Movement of these substances into and out of the cell is achieved through means of active and passive transport.

Definitions:
Active Transport: The ATP dependant absorption or secretion of solutes across a plasma membrane.

Crenation: Cellular shrinkage due to osmotic movement of water out of the cytoplasm.

Diffusion: Passive molecular movement from a higher concentration to a lower concentration.
Haemolysis: the breaking open of red blood cells, resulting in the release of hemoglobin into the surrounding fluid.
Hypertonic: Comparing two solutions, the solution with the higher osmolarity or the solution having a greater solute concentration than that of the cytosol.

Hypotonic: Comparing two solutions, the solution with the lower osmolarity or the solution having a lesser solute concentration than that of the cytosol.
Iso-Osmotic: Having the same or equal osmotic pressure. Where the total number of solutes in a solution is equal or the same as the total number of solutes in another solution.
Isotonic: A solution with osmolarity that does not result in water movement across plasma membrane. A solution with the same solute concentration as that of the cytosol
Kinetic Theory: Is the study of the behavior of molecules in various states of matter and how variants effect the molecular bonds.

Osmosis: The movement of water across a selectively permeable membrane in response to a difference in osmotic pressure.
Passive Transport: substance moves down the concentration gradient without the use of cellular energy – ATP.

Aim:
The purpose of this experiment is to demonstrate and understand the physiology of a cell and the membranes permeability through the means of osmosis and diffusion, and how the rate is effect by chemical and temperature stimulus.
Hypothesis:
Part A:
The hot water solution turns purple from the potassium permanganate crystal more rapidly than the cold water solution with the potassium permanganate crystal.

Part B:
The liquid level in the funnel stem with the solution containing protein, glucose and sodium chloride, remains at the same level as the glucose and protein molecules are too large to pass passively through the permeable membrane, resulting in the Labstix strip being positive once tested.
In the second solution containing sodium chloride, the molecules will permeate the tubbing creating a white precipitate and the fluid levels in the funnel will decrease.
Part C:
At 0.9 sodium chloride concentration, nothing will happen to the red blood cells.

At 2.0 sodium chloride concentration, it will result in the red blood cells shriveling up and an increase in distance between each cell. Lysis of cells.

At 0.5 sodium chloride concentration, the red blood cells with expand and the distance between the cells will decrease. Lysis of cells.

Part D:
0.4 – Complete lysis of red blood cells
0.5 – Severe hypertonic and some lysis
0.6 – Severe hypertonic
0.7 – Moderate hypertonic
0.8 – Mild hypertonic
0.9 – No movement/change
2.0 – Complete lysis of red blood cells
Method
As described in the College of Health and Biomedicine, RMB1518 Human Pathology 1 Laboratory Manual – VUT, Semester 1, 2018, Practical Number Two: Cell Physiology.

Results
Part A – Diffusion
TABLE 1: DIFFUSION
Description: Time (Minutes):
Start 00.00
Complete diffusion in hot water 00.333
Complete diffusion in cold water 90.00
lefttop
Figure 1: Rate of Diffusion with the Effect of Temperature: Complete diffusion occurred more rapidly in the hot water at 20secs (0.33333 minutes) when compared with the cold water solution which took 90mins for complete diffusion to occur.
Part B – Osmosis
TABLE 2 : THE PERMEABILITY OF CELLULOSE TUBING TO GLUCOSE, PROTEIN AND SODIUM CHLORIDE
Solutes Presence of Solute Over Time (Minutes)
Beaker Tubing
00.00 90.00 00.00 90.00
Glucose O O O O
Protein O O O O
Sodium Chloride O O O +
+ indicates solute is present
O indicates solute is not present
Table 2: Shows which solutes were present in each solution after a certain duration. We note NaCl was present in the tubing after 90mins. No solutes were detected in the beaker solution. Glucose and protein molecules were undetected in the tubing.
Part C – Red Blood Cells

Figure 2: Red blood cell in 0.9% sodium chloride

Figure 3: Red blood cell in 2.0% sodium chloride

Figure 4: Red blood cells changes after adding 0.5% sodium chloride.

Part D – Effects of saline concentration on cells
TABLE 3: OBSERVATIONS OF SALINE CONCENTRATION EFFECTS ON CELLS
Saline Concentration (%) 0.4% 0.5% 0.6% 0.7% 0.8% 0.9% 2.0%
Degree of Haemolysis ++ ++ + + + O O
O No haemolysis+ Moderate amounts of haemolysis++ Large amounts of haemolysis
Figure 5: Observations of Saline Concentration Effects on RBC’s demonstrates how different concentrations cause large, moderate or no amounts of haemolysis within a red blood cell.

Discussion
Part A – Diffusion
Part A, Figure 1 of the practical, observed the rate of diffusion when different temperatures were applied. The hotter the solution resulting in a more rapid diffusion, whilst the cooler the solution slowed down the rate of diffusion.
This is because when you apply heat to these molecules, you increase their movement resulting in a quicker rate of complete diffusion. When you endure the molecules to a cooler environment, you reduce the rate of movement and therefore, slowing the overall diffusion rate.

Part B – Osmosis
With reference to Table 2, the results show there was no diffusion of glucose, protein and sodium chloride (NaCl) through the cellulose tubing to the beaker containing distilled water.
When the tubing solution was testing for protein, glucose and sodium chloride, the results indicate there was sodium present within the tubing, however, glucose and protein were undetected.
Glucose and protein are large molecules which are unable to permeate through the cellulose tubing. Therefore, they were not detected when tested for in the beaker solution. However, both would be expected to be detected in the tubing solution.
Sodium chloride was undetected in the beaker solution, this reading was thought to be incorrect as the NaCl would have permeated the membrane (slight leakage) whilst the distilled water in the beaker, would have entered the tubing in response to the different concentration gradients. This is achieved through the process of osmosis and diffusion.
Sodium chloride levels may not have been detected at the second time mark as this is a slow process or the quantities may have been too diluted for accurate readings.
Volume in the funnel increased by 3.5cm, due to water molecules moving down the water concentration gradient to inside the tubing, causing the volume in the beaker to decrease and the volume in the funnel to increase.

Part C – Red Blood Cells
Red blood cells (RBC’s) were exposed to hypertonic solution of 2.0% NaCl, observations of RBC’s shriveling and shrinking up due to the movement of water molecules out of the cell is greater than the ones entering.
A hypotonic solution of 0.5% NaCl was added to RBC’s, resulting in the osmotic flow of water into the cell. This movement caused RBC’s to swell, increase in size and eventually rupturing (haemolysis) as water molecules are entering the RBC faster than they leave.
When a 0.9% NaCl (isotonic) solution was applied to RBC’s, no observations to the change of the RBC’s as there is no osmotic flow occurring. NaCl and water molecules enter and exit the cell at the same rate, allowing the RBC to maintain shape.
These findings were accurate with the original hypothesis.
Chemicals used in anesthetics change the property of the cell membrane by blocking channels. The blockages effect the sensitivity of neurons and muscle cells by inhibiting responsiveness to pain stimulus.

Part D – Effects of saline concentration on cells
Table 3 depicts the effect of different saline concentrations on RBC’s. It is observed concentrations of 0.4% and 0.5% NaCl had large amounts/complete haemolysis as the solution was clear.
Concentrations of 0.6%, 0.7% and 0.8% saline had moderate amounts of haemolysis, where the solution appeared mildly cloudy.
When RBS’s were exposed to the 0.9% and 2.0% NaCl, it was noted the solution turned severely cloudy indicating no haemolysis has occurred. This is due to 0.9% NaCl being a isotonic solution and no osmotic flow is occurring.
On the other hand, the 2.0% NaCl caused the saline solution to enter the cell, causing RBX’s to become crenated, although the membrane is severely effected we note no rupturing occurred.

The cell membrane controls the rate various substances enter or leave the cell by a selectively permeable membrane. This is achieved by; lipid diffusion, osmosis, passive transport, active transport and vesicles.
References
Sonmez, M., Ince, H., Yalcin, O., Ajdžanovi?, V., Spasojevi?, I., Meiselman, H., & Baskurt, O. (2018). The Effect of Alcohols on Red Blood Cell Mechanical Properties and Membrane Fluidity Depends on Their Molecular Size.

Vay, D. L., & Stafford-Brown, J. (2015). Anatomy & physiology: a complete introduction: teach yourself. Retrieved from https://ebookcentral.proquest.com(2018). Retrieved from https://sciencing.com/effect-temperature-process-diffusion-10046049.htmlDiffusion and Temperature. (2018). Retrieved from https://learn.concord.org/resources/762/diffusion-and-temperatureIsosmotic – Biology-Online Dictionary. (2018). Retrieved from https://www.biology-online.org/dictionary/IsosmoticMartini, F., Nath, J., & Bartholomew, E. (2014). Fundamentals of Anatomy & Physiology (9th ed., pp. 49-131). London: Pearson.

NOTE: All practical results were obtained from Victorian University RBM1518 student ‘Geoffrey’.Appendix
Part A calculations:
Minutes (X) = Seconds / 60X=20/60X=0.3333333