The effects of osmotic agents on the morphology and integrity of the cell membrane

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INTRODUCTION

Cell membrane is the only cellular structure which is found in every single organism on Earth. Membrane is what separate and maintains the living properties of the cell. It defines the cell.

The main purpose of this lab is to learn about membranes and understand how they function. Such as understanding the semi-permeability of cell membranes. The term semi-permeability means a membranethat isselectivelypermeable and allows only certain molecules/ions to pass through. Moreover, this lab also enhances our knowledge on the effects of osmosis on cells. Osmosis is a term that describes the spontaneous movement of water across a semipermeable membrane from a region of lower concentration of solute to a region of a higher concentration of solute to make the solute concentration equal on both sides. Thus, with the knowledge of osmosis we can identify whether solutions are hypoosmotic, hyperosmotic or isosmotic by observing the effects on cells. In this lab I predict….

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Furthermore, this lab examines the effects of osmotic agents in the morphology of cell and observes membrane integrity by exclusion of Trypan blue, a polar and bulky dye molecule.

PART A: attached with the hardcopy of the lab.

PART B: 1. Please refer to Table 2.1 in the hardcopy of the lab 2. Please refer to Table 2.1 in the hardcopy of the lab 3. I also observed that the isotonic solutions of glucose and potassium chloride are of different concentration for blood cells but they are of same concentration for plant cells. The expected answer is that the isotonic solutions of both glucose and potassium chloride should be of different concentrations due to difference in the Van’t Hoff factor (i). Van’t Hoff factor is measure of the number of particles released by a substance when dissolved. Since Potassium chloride releases 2 particles ( K+ and Cl-) when dissolved and glucose only releases one particle when dissolved therefore the expected isotonic solutions of glucose and potassium chloride should be different. Based on the observation of Table2.1 my answer agrees to it when comparing isotonic solutions for blood cells but it doesnot agree for plant cells and that’s due to error in detection through microscope.

Exercise 2.2

4. Based on the experimental data and results from table 2.2, the identity of my Unknown 1 is Glycine and the identity of my unknown 2 is Methyl-β-cyclodextrin. The % blue cells of unknown 1 is 38% which is close to Glycine % blue cells which is 23%. On the other hand unknown 2 is % blue cells is 44.7% which is close to % blue cells of Methyl-β-cyclodextrin which is 78%. Thus, the identity of my Unknown 1 is Glycine and the identity of my unknown 2 is Methyl-β-cyclodextrin.

5. Based on the Trypan blue exclusion assay, Methyl-B-cyclodextrin is the compound which caused the most damage to the plasma membrane. Methyl-β-cyclodextrin is a compound that is used to severely deplete cells of cholesterol. Basically it removes cholesterol from membrane and disrupts membrane integrity. Thus, Methyl-β-cyclodextrin have disrupt the membrane integrity of the cells and damage the plasma membrane. As a result, the trypan blue easily entered the cell and stained it.

DISCUSSION AND CONCLUSIONS (15 marks total) Provide a discussion of your laboratory observations and results. Include the following: 1) A brief and concise (100-150 word) summary of your findings. Include the name of the assays that you performed, what you measured and the effects of any drugs or additional treatments that you performed. (5 marks)

In the first part of this lab, we identified whether the glucose and potassium chloride solutions are hypoosmotic, hyperosmotic or isosmotic by observing the effects on cell that whether the cells are shrinking or swelling. I also observed that the isotonic solutions of glucose and potassium chloride are of different concentration for blood cells but they are of same concentration for plant cells. The expected answer is that the isotonic solutions of both glucose and potassium chloride should be of different concentrations due to difference in the Van’t Hoff factor (i). Based on the observation of Table2.1 my answer agrees to it when comparing isotonic solutions for blood cells but it does not agree for plant cells and that’s due to error in detection through microscope.

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In the second part of the experiment, we examines the effects of osmotic agents in the morphology of cell and observes membrane integrity by exclusion of Trypan blue, a polar and bulky dye molecule. Based on table 2.2, %blue cells of HPMI are 0%, while %blue cells of Glycine are 23%, %blue cells of Methyl-β-cyclodextrin are 78% . By comparing my unknowns to the rest, I observed that the identity of my Unknown 1 is Glycine and the identity of my unknown 2 is Methyl-β-cyclodextrin. The % blue cells of unknown 1 is 38% which is close to Glycine % blue cells which is 23%. On the other hand unknown 2 is % blue cells is 44.7% which is close to % blue cells of Methyl-β-cyclodextrin which is 78%. Thus, the identity of my Unknown 1 is Glycine and the identity of my unknown 2 is Methyl-β-cyclodextrin.

Thus, this lab successfully identifies whether solutions are hypoosmotic, hyperosmotic or isosmotic by observing the effects on cells and successfully examines the effects of osmotic agents in the morphology of cell and observes membrane integrity by exclusion of Trypan blue.

2) Provide an answer to the following questions:

  1. Mammalian cells are very sensitive to salt concentration. However, there are organisms that thrive in very high-salt environments. What are they called and name one species at least? (2 marks)

The organisms that thrive in very high-salt environments are called Halophiles. The word Halophile itself describes its meaning as Halophile comes from a Greek word which means salt-loving. (https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/microbial-evolution-phylogeny-and-diversity-8/euryarchaeota-111/extremely-halophilic-archaea-575-7678/ ). Even though Halophiles are largely classified into Archaea domain, however there are alsohalophilic bacteria and somehalophilic eukaryotes such as theDunaliella salina, alga or the fungusWallemia ichthyophaga (https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/microbial-evolution-phylogeny-and-diversity-8/euryarchaeota-111/extremely-halophilic-archaea-575-7678/ ).

  1. What does methyl -cyclodextrin do to cell membranes? Based on this, explain the effect you would expect this compound to have on Trypan Blue exclusion. Does your observation agree with this expectation? (4 marks)

Cholesterol plays a very important role in cell membranes as it is involved in maintaining the permeability and fluidity of membrane. Basically it gives the cell membrane extra support by keeping the membrane fluid consistent. Methyl-β-cyclodextrin is a compound that is used to deplete cells of cholesterol. Basically it removes cholesterol from membrane and disrupts membrane integrity. Thus, I expect that Methyl-β-cyclodextrin would disrupt the membrane integrity of the cells and damage the plasma membrane. As a result, the trypan blue can easily enter the cell and stain it. Moreover, based on experimental data from table 2.2, 78% of cells with Methyl-β-cyclodextrin are stained will Trypan blue. Thus, my observation agree with this expectation. http://www.sciencedirect.com/science/article/pii/S0005273608000837

  1. Predict what each of the following treatments will do to cell permeability. Provide a brief (1-2 sentence) explanation for each of your predictions (1 marks each):
  • Acetone : Acetone is an organic solvent and organic solvents dissolve the lipids of a membrane. Acetone is one of the common solvents for different organic molecules and has a greater ability to dissolve lipids than some other organic solvents like methanol. Acetone dissolves lipids from cell membranes and makes them permeable to antibodies.
  • Methanol: Methanol is also an organic solvent which is used to dissolve a membrane’s lipids. However it is a weaker organic solvent compared to acetone. It also dissolves lipids from cell membranes and makes them permeable to antibodies.
  • Saponin: Sapnion is a detergent which interacts with membrane cholesterol, selectively removes it and leaves holes inside the membrane.
  • Triton X-100: This is also a detergent which interacts the membrane the same way as Sapnion and it also removes cholesterol. However there is also a disadvantage of using detergents like Triton X-100 because it is non-selective in nature and extracts proteins along the lipids.
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Bibliography

Swift Digital M10T-MCS-MP Phase Contrast Tablet Microscope. (n.d.). Retrieved May 18, 2015.

Retrieved from https://www.microscopeworld.com/p-2167-swift-digital-m10t-mcs-mp-phase-contrast-tablet-microscope.aspx

The Search engine I used is Google and the terms I used in search is phase contrast microscope with digital camera and the original reference is given below:

Swift Digital M10T-MCS-MP Phase Contrast Tablet Microscope. (n.d.). Retrieved May 18, 2015 Retrieved from https://www.microscopeworld.com/p-2167-swift-digital-m10t-mcs-mp-phase-contrast-tablet-microscope.aspx

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