• Syed Ibrahim

Introduction

The plasma membrane is vital as it defines the boundary between cells and their environment (Heidcamp et al., 2014). Plasma membranes are crucial in maintaining electrochemical gradients, controlling material exchange, and allowing signal transduction (Zhao et al., 2004). The purpose of this lab was to identify isoosmotic solutions by examining the effect of osmotic pressure on the plasma membrane of blood cells and Elodea guard cells; as well as to understand cell viability and membrane integrity using the Trypan blue exclusion assay.

To examine the effects of osmosis, animal blood cells and guard cells from Elodea leaves were examined in this lab. Plasma membranes are found in both animal cells and plant cells; however, cell walls are only present in plant cells (Freeman et al., 2011). It was expected that these cells would shrink in hypertonic solutions, stay the same size in isotonic solutions, and swell (in the case of Elodea guard cells) or even burst (in the case of animal blood cells) in hypotonic solutions (Heidcamp et al., 2014). Since glucose has a van’t Hoff factor of 1 while potassium chloride (KCl) has a van’t Hoff factor of 2, it was predicted that 0.15M glucose and 0.07M KCl would be the isoosmotic solutions for both the animal blood and the Elodea guard cells. Concentrations greater than the isoosmotic solution were expected to be hyperosmotic, while concentrations less than it were expected to be hypoosmotic.

Viable cells have intact plasma membranes (Heidcamp et al., 2014). In this lab macrophage cells, a type of leukocytes, were treated with various compounds and the effect of these compounds on the membrane of the cells was examined using Trypan blue, which differentially stains viable and non-viable cells based on whether the cells have an intact plasma membrane (Freeman et al., 2011; Strober, 2011). Of the three known compounds, it was predicted that hepes-buffered RPMI (HPMI) would damage the membrane the least as it contains vitamins and supplements, and it was predicted that methyl β-cyclodextrin (MβCD) would damage the cell the most as it removes cholesterol from plasma membranes (LifeTechnologies, 2013; Rodal et al., 1999).

Results

The following three equations were used in calculations. Equation 1 was used to calculate osmolarity, in order to determine the osmotic effect of a particular solution, (Heidcamp et al., 2014). Equation 2 was used to calculate osmotic pressure and Equation 3 was used to convert Celsius temperature to kelvin temperature (Heidcamp et al., 2014; USMA, 2012).

Where:

= osmolarity

= van’t Hoff factor

= molar concentration

Where:

= temperature

= temperature

Where:

= osmotic pressure (kPa)

= van’t Hoff factor

= molar concentration

= ideal gas constant = (Chieh, 2002)

= temperature

Part A: Lab 2 Report Sheets

Please refer to attached sheets.

Part B: Answers to Assigned Questions

1. Based on the observations of my colleagues and myself, 0.15M glucose and 0.15M potassium chloride (KCl) were the isotonic solutions for the Eloda guard cells, while 0.15M glucose and 0.035M potassium chloride (KCl) were the isotonic solutions for the animal blood cells.

2. Solutions with solute concentrations greater than the isotonic solution were hypertonic, while concentrations less than it were hypotonic. Based on the observations from the lab, 0.6M glucose and 0.3M glucose were hypertonic for both the plant and blood cells, while 0.07M glucose and 0.035M glucose were hypotonic for the plant and blood cells. Additionally, 0.6M KCl, and 0.3M KCl, were hypertonic for the plant cell, while 0.07M KCl and 0.035M KCl were hypotonic for the plant cell. As well, 0.6M KCl, 0.3M KCl, 0.15M KCl, and 0.07M KCl were hypertonic for blood cells while there were no observed hypotonic KCl solutions for blood cells.

3. It is expected that the isotonic solutions of glucose and KCl will have the same osmolarity. Based on Equation 1, osmolarity is the product of the van’t Hoff factor and molar concentration. Since glucose has a van’t Hoff factor of 1 while potassium chloride (KCl) has a van’t Hoff factor of 2, it is expected that glucose will have double the molar concentration of KCl to obtain the same osmolarity in the isotonic solutions. The observations from this lab reveal that for plant cells, glucose and KCl had the same concentrations for isotonic solutions; while for animal cells, glucose had approximately four times the molar concentration of KCl for isotonic solutions. This may have occurred due to the subjective nature of classifying tonicity, and since these observations were split up amongst four groups, their subjective impressions may have been different. Ideally, one group to do all four sets, but due to limited time in the lab, this was not possible.

4. Tween-20 is a non-ionic type of surfactant that can be used solubilizing agent for membrane proteins (Iwahashi et al., 1991; Sigma, 2003). Bovine serum albumin (BSA) is a protein that adsorbs onto negatively charged portions of the membrane bilayer, forming temporary gaps in the membrane, thus increasing the permeability of the membrane (Tsunoda et al., 2001). Based on this information, Tween-20 would likely cause more damage on the membrane bilayer, resulting in more blue cells (after the Trypan blue exclusion test). Therefore, the odd unknown is most likely Tween-20, and the even unknown is most likely BSA.

Search Engine: Web of Science

Search Terms: Tween 20 [sorted by relevance]

Reference: Iwahashi, K., Tsubaki, M., Miyatake, A., Miura, S., Hosokawa, K., & Ichikawa, Y. (1991). Catalytic properties of cytochrome P-450scc from bovine and porcine adrenocortical mitochondria: Effect of tween20 concentration. The Journal of Steroid Biochemistry and Molecular Biology, 38(6), 727-731.

Search Engine: Google

Search Terms: Tween-20

Reference: Sigma. (2003). Tween-20 (P5927) Product Information. SigmaAldrich. Retrieved October 6, 2014, from https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/p5927pis.pdf

Search Engine: Web of Science

Search Terms: “bovine serum albumin” [sorted by relevance]

Reference: Tsunoda, T., Imura, T., Kadota, M., Yamazaki, T., Yamauchi, H., Kwon, K. O., et al. (2001). Effects of lysozyme and bovine serum albumin on membrane characteristics of dipalmitoylphosphatidylglycerol liposomes. Colloids and Surfaces B: Biointerfaces, 20(2), 155-163.

5. Based on the Trypan blue exclusion assay observations recorded in Table 2.2, unknown 1 (most likely Tween-20) caused the most damage to the plasma membrane as it had the highest percentage of blue cells (60%). Tween-20 belongs to the class of polyoxyethylene sorbate combounds (Boxtel et al., 1990). Among many functions, these compounds solubilize the proteins and lipids found in the plasma membranes, thereby lysing the membranes (Boxtel et al., 1990). This allows Trypan blue to enter the lysed membranes, making them blue.

Discussion and Conclusions

Summary of findings

In the first part of this lab, the effects of osmosis were examined on animal blood cells and Elodea guard cells. It was determined that isoosmotic solutions for the Eloda guard cells were 0.15M glucose and 0.15M potassium chloride (KCl), while the isoosmotic solutions for the animal blood cells were 0.15M glucose and 0.035M KCl. Solutions with solute concentrations greater than the isotonic solution were hypertonic, while concentrations less than it were hypotonic. It was expected that the isotonic solutions of glucose and KCl would have the same osmolarity, since neither of them can easily diffuse across the membrane without the use of a channel or transport protein (Heidcamp et al., 2014) . Based on Equation 1, osmolarity is the product of the van’t Hoff factor and molar concentration. Since glucose has a van’t Hoff factor of 1 while potassium chloride (KCl) has a van’t Hoff factor of 2, it was expected that glucose would have double the molar concentration of KCl to obtain the same osmolarity in the isotonic solutions. The results from this lab do not reflect this theoretical expectation. This may have occurred due to the subjective nature of classifying tonicity. Ideally, one group should to do all four sets of observations for Table 2.1, but due to limited time in the lab, this was not possible.

In the second part of this lab, membrane integrity and permeability was examined using the Trypan blue exclusion. Normally Trypan blue is not permeable in living cells. However, if the plasma membrane is not intact, Trypan blue is able to enter the cell, staining it blue (Heidcamp et al., 2014; Strober, 2011). Once treated with the specific compounds (such as glycine or Tween-20), the Trypan blue exclusion assay was performed on the RAW macrophage cells. Afterwards, the number of blue (non-viable) and non-blue (viable) cells were counted and their respective percentages were calculated. Glycine is smallest of the twenty common amino acids, and nearly all (97%) of treated cell remained viable (Freeman et al., 2011). Methyl β-cyclodextrin (MβCD) is a compound that removes cholesterol from plasma membranes, making the membrane more permeable, and it resulted in only 55% viable cells (Rodal et al., 1999). Hepes-buffered RPMI (HPMI) is a medium that contains vitamins and supplements that can be used for cell growth, and nearly all (98%) of treated cells remained viable (LifeTechnologies, 2013). In addition to these three compounds, the cells were treated with two unknowns. The odd unknown resulted in relatively few (40%) viable cells, while the even unknown resulted in many (85%) viable cells. Tween-20 belongs to a class of detergents which are known to solubilize membrane proteins, thereby lysing the membranes (Boxtel et al., 1990; Iwahashi et al., 1991). Bovine serum albumin (BSA) is a protein that forms temporary gaps in the membrane, thus increasing the permeability of the membrane (Tsunoda et al., 2001). Based on the literature, Tween-20 would cause more damage on the plasma membrane resulting in fewer viable cells. Therefore, the odd unknown was determined to be most likely Tween-20, and the even unknown was most likely BSA.

Answer to questions

1. An organism that thrives in high-salt environments is known as a halophile, such as Halomonas meridian (James et al., 1990; Ventosa, 1998).

Search Engine: Web of Science

Search Terms: halophile

Reference: James, S., Dobson, S., Franzmann, P., & Mcmeekin, T. (1990). Halomonas meridiana, a New Species of Extremely Halotolerant Bacteria Isolated from Antarctic Saline Lakes. Systematic and Applied Microbiology, 13(3), 270-278.

Search Engine: Web of Science

Search Terms: halophile

Reference: Ventosa, A., Nieto, J., & Oren, A. (1998). Biology of Moderately Halophilic Aerobic Bacteria. Microbiology and Molecular Biology Reviews, 62(2), 504-544.

2. Methyl β-cyclodextrin (MβCD) is a compound that removes cholesterol from plasma membranes (Rodal et al., 1999). Thus it is expected, that MβCD makes the plasma membrane more permeable, and allows Trypan blue to enter the cell. The results from this lab agree with this, as a relatively large percentage (45%) of cells treated with MβCD was stained blue in the Trypan blue exclusion assay.

Search Engine: Web of Science

Search Terms: methyl beta cyclodextrin

Reference: Rodal, S. K., Skretting, G., Garred, O., Vilhardt, F., Deurs, B. V., & Sandvig, K. (1999). Extraction of Cholesterol with Methyl-beta -Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles. Molecular Biology of the Cell, 10(4), 961-974.

3. The predictions are below:
   1. Acetone is an organic solvent that solubilizes lipids from the plasma membrane (Jamur & Oliver, 2010). High concentrations of acetone can disrupt lipid packing in the membrane, thereby increasing membrane fluidity and permeability (Posokhov & Kyrychenko, 2013). Therefore is predicted that acetone will increase cell permeability and allow some of the Trypan blue into the cell, staining some cells blue.
   2. Methanol is an organic solvent that dissolves lipids from the plasma membrane (Jamur et al., 2010). Methanol can increase the rotational mobility of membrane lipids, increasing its fluidity (Joo et al., 2012). Similar to acetone, it is expected that methanol will increase cell permeability and allow some of the Trypan blue into the cell, staining some cells blue.
   3. Saponin is a detergent that selectively removes cholesterol from the plasma membrane, resulting in small holes in the membrane (Jamur et al., 2010). This would act in a manner similar to MβCD, likely causing the cell to be permeable to Trypan blue and staining many cells blue.
   4. Triton X-100 is a non-ionic detergent that non-selectively solubilizes proteins from the plasma membrane (Jamur et al., 2010). This would act in a manner similar to Tween-20, likely causing the cell to be highly permeable to Trypan blue and staining a large percentage of them blue.

Search Engine: PubMed

Search Terms: cell membrane permeability

Reference: Jamur, M. C., & Oliver, C. (2010). Permeabilization of cell membranes. Immunocytochemical Methods and Protocols, 588, 63-68.

Search Engine: Web of Science

Search Terms: effect of acetone on membranes

Reference: Posokhov, Y. O., & Kyrychenko, A. (2013). Effect of acetone accumulation on structure and dynamics of lipid membranes studied by molecular dynamics simulations. Computational Biology and Chemistry, 46, 23-31.

References

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2008).Molecular Biology of the Cell(5th ed.). New York: Garland Science.

Boxtel, R. M., Lambrecht, R. S., & Collins, M. T. (1990). Effect of polyoxyethylene sorbate compounds (Tweens) on colonial morphology, growth, and ultrastructure of Mycobacterium paratuberculosis. Apmis, 98(7-12), 901-908.

Chieh, C. (2002). The Ideal Gas Law. Ideal Gas. Retrieved October 5, 2014, from http://www.science.uwaterloo.ca/~cchieh/cact/c120/idealgas.html

Freeman, S., Harrington, M., & Sharp, J. (2011). Biological Science (Canadian ed.). Toronto: Pearson Canada.

Heidcamp, W., Antonescu, C., Botelho, R., & Victorio-Walz, L. (2014).Laboratory Manual: Cell Biology – BLG311(Fall 2014 ed.). Toronto: Ryerson University.

Iwahashi, K., Tsubaki, M., Miyatake, A., Miura, S., Hosokawa, K., & Ichikawa, Y. (1991). Catalytic properties of cytochrome P-450scc from bovine and porcine adrenocortical mitochondria: Effect of tween20 concentration. The Journal of Steroid Biochemistry and Molecular Biology, 38(6), 727-731.

James, S., Dobson, S., Franzmann, P., & Mcmeekin, T. (1990). Halomonas meridiana, a New Species of Extremely Halotolerant Bacteria Isolated from Antarctic Saline Lakes. Systematic and Applied Microbiology, 13(3), 270-278.

Jamur, M. C., & Oliver, C. (2010). Permeabilization of cell membranes. Immunocytochemical Methods and Protocols, 588, 63-68.

Joo, H., Jang, H., Yun, I., Bae, S., Chung, I., Bae, M., et al. (2012). The Effect of Methanol on the Structural Parameters of Neuronal Membrane Lipid Bilayers. The Korean Journal of Physiology & Pharmacology, 16(4), 255.

LifeTechnologies. (2013). RPMI 1640 Medium, HEPES. Cell Culture & Transfection Reagents. Retrieved October 6, 2014, from http://www.lifetechnologies.com/order/catalog/product/22400089#productDetailPage

Posokhov, Y. O., & Kyrychenko, A. (2013). Effect of acetone accumulation on structure and dynamics of lipid membranes studied by molecular dynamics simulations. Computational Biology and Chemistry, 46, 23-31.

Rodal, S. K., Skretting, G., Garred, O., Vilhardt, F., Deurs, B. V., & Sandvig, K. (1999). Extraction of Cholesterol with Methyl-beta -Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles. Molecular Biology of the Cell, 10(4), 961-974.

Sigma. (2003). Tween-20 (P5927) Product Information. SigmaAldrich. Retrieved October 6, 2014, from https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/p5927pis.pdf

Strober, W. (2011). Trypan blue exclusion test of cell viability. Current Protocols in Immunology, 21(3B), A.3B.1–A.3B.2.

Tsunoda, T., Imura, T., Kadota, M., Yamazaki, T., Yamauchi, H., Kwon, K. O., et al. (2001). Effects of lysozyme and bovine serum albumin on membrane characteristics of dipalmitoylphosphatidylglycerol liposomes. Colloids and Surfaces B: Biointerfaces, 20(2), 155-163.

USMA. (2012). Metric System Temperature. U.S. Metric Association. Retrieved October 6, 2014, from http://lamar.colostate.edu/~hillger/temps.htm

Ventosa, A., Nieto, J., & Oren, A. (1998). Biology of Moderately Halophilic Aerobic Bacteria. Microbiology and Molecular Biology Reviews, 62(2), 504-544.

Zhao, Y., Zhang, W., Kho, Y., & Zhao, Y. (2004). Proteomic Analysis of Integral Plasma Membrane Proteins. Analytical Chemistry, 76(7), 1817-1823.