This experiment examines cell membrane permeableness, osmosis and membrane electromotive forces ; all of which are of import in understanding how cells are affected by their environment. The motion of H2O across membranes is of import for cell volume and therefore the volume of extracellular compartments. The mechanisms for solute conveyance are indispensable in keeping cell maps and homeostasis. Furthermore, ion conveyance across membranes generates membrane electromotive forces, which are of import in keeping osmotic balance. Previous related experiments have been conducted, for illustration de Wit and van Gastel ( 1969 ) investigated the relationships between cell age and osmotic breakability. Their findings concluded that younger cells are more immune to lysis and older cells were relatively more susceptible to immune hemolysis.
Naccache and Sha’afi’ ( 1973 ) found that cell construction was a prevailing factor in a cell’s permeableness to solutes. Separate 1 sought to mensurate the motion of H2O in response the add-on of assorted concentrations of NaCl. It was hypothesised that as the concentration of NaCl additions, so the sum of lysis of the ruddy blood cells would diminish. Separate 2 aimed to find how permeable ruddy blood cell membranes were to assorted solutes. It was hypothesised that as the lipid solubility of solutes ( Kether ) increases, as does the permeableness coefficient. Part 3 of the experiment aimed to bring forth and mensurate diffusion potencies across two different membranes of semi-permeability. It was hypothesised that as the electromotive force additions, as does the log of the concentration gradient. Method
Methods for Part 1 and Part 2 were performed as described in the SCIE1106 Lab Manual ( 3 ) . Small alterations were made in Part 3 of the experiment.
Data was processed through Microsoft Excel.ConsequencesAssorted colorss and phases of cell lysis was observed in Part 1. The 3 non-centrifuged trial tubings ( incorporating 0.01M, 0.09M and 0.15M of NaCl severally ) were all ab initio cloudy. The 0.01M tubing was observed to turn about immediately clear, whilst the 0.09M tubing merely became somewhat less cloudy. The 0.15M tubing remained opaque. The NaCl concentration ranged from 0.01M to 0.15M in the centrifuged trial tubing. The tubings were observed to be crystalline but showed changing strengths of coloring material from 0.01-0.07M. A diffuse pellet was observed from 0.07-0.15M. Figure 1 indicates that hemolysis per centum begins at above 100 % , at 0 millimeter NaCl concentration. A slow lessening can be observed between 0 to 85 millimeter as the concentration of NaCl additions. A crisp negative diminution of per centum hemolysis from 85 to 100 millimeter is shown, which finally plateaus when NaCl concentration reaches about 110 millimeters. Separate 2.The line of best tantrum is declarative of the straight relative relationship between lipid solubility ( Kether ) and corrected membrane permeableness.
Refer to Figure 2. Separate 3. The initial reading of 3.6mV was subtracted from the electromotive force readings. The concluding readings can be observed in Figure 3, where the gradients of the ruddy and bluish electrodes are close mirrors of each other. The bluish gradient starts at 47.6mV, 0.01 log concentration, increases to 50.5mV at 0.03 log concentration and eventually bit by bit declines to -21.3mV when the log concentration reached 10. Initially the ruddy electrode started at -56.7mV, 0.01 log concentration, increased to -44.8mV at 0.03 log concentration but so decreased somewhat to -43mV at 0.1 log concentration. The ruddy gradient so bit by bit increases until it reached 20.8mV when the log concentration reached 10. The blue and ruddy electrode gradients crossed waies at 1mV at log concentration of -1.3. A tendency was observed in the graph ; where the higher the dilution, the longer the suspension of electrodes was needed to obtain a comparatively stable reading. Discussion
It was hypothesised in Part 1 that as the concentration of NaCl increased, the sum of ruddy blood cell lysis would diminish. The hypothesis was supported by informations obtained, as shown in Figure 1. When analyzing the 0-85mM NaCl concentration, the lessening in per centum hemolysis can be attributed to the assorted ages of the ruddy blood cells, and therefore their different lysis threshold. Younger cells have a higher threshold for lysis ( de Wit & A ; van Gastel, 1969 ) and as the concentration increases so the figure of ‘younger’ cells that lyse lessenings as they have a higher opposition to swelling. Alternatively ‘older’ cells display lyse before, as shown in the steep bead from 85-100 millimeter. It should besides be noted that there is no alteration observed at the 150 millimeter as the osmolarity is the same as the ruddy blood cells. Percentage hemolysis remains low and changeless towards as NaCl concentration additions, as scallop begins.
In Part 2 it was hypothesised that as the lipid solubility of solutes ( Kether ) increases, as does the permeableness coefficient. The hypothesis was supported as shown in Figure 2, whereby both category and group informations indicate a straight relative relationship between Kether and the permeableness coefficient. The ascertained informations can be explained as lipid solubility is dependent on the molecule’s construction and that molecules incorporating more polar groups are more lipophobic and therefore are less lipid soluble. Glycerol, with a chemical expression of CH2OHCHOHCH2OH, has the lowest Kether due to the presence of three polar hydroxyl groups which give glycerin its high mutual opposition. Propylene glycerin, with a chemical expression of HOCH2CHOHCH3 groups has the highest Kether as it is much less polar than glycerin ; incorporating merely two polar hydroxyl groups and a longer C concatenation. Diethylene ethanediol ( ( HOHCH2CH2 ) 2O ) and ethylene ethanediol ( OHCHCH2CH2CHOH ) have similar chemical expression nevertheless the longer C concatenation in diethylene ethanediol makes it less polar.
There was a significant difference in the values for the corrected permeableness from group to group, which can be explained by the subjective conditions in which the hemolysis clip was measured. Mistakes in the reaction clip of experimenters, the distance from the trial tubing to the oculus and the subjectivity of what a solution looks like when ‘clear’ along with the exact clip at which the solution is ‘clear’ may hold all had an consequence on the values obtained by each group. The usage of the mean value of all group’s informations increased dependability nevertheless extra trials from each group should be taken in future experiments to increase the dependability of the consequences. In Part 3, it was hypothesised that as the electromotive force additions, as does the concentration of the gradient.
The hypothesis was supported as shown in Figure 3 whereby the greater the electrochemical gradient for the peculiar ion consequences in a greater rate of ion diffusion ( Beilharz, 2014 ) . With regard to their extracellular fluids, cytols of cells are typically have a negative charge. Therefore, the changing concentrations of solutions give rise to changing gradients that emphasise ion diffusion down the gradient. The bluish electrode ab initio showed a positive membrane electromotive force value which indicated its permeableness to anions. Conversely, the ruddy electrode displayed a negative electromotive force value and therefore was permeable to cations due to the positive ions moved out of the cell. During this portion of the experiment, we were unable to obtain accurate values due to a malfunction in the research lab equipment. More clip should hold been allotted as the stabilization for each reading took a significant ball of clip ( approx. 20 proceedingss ) . Appendixs
Figure 1. Osmotic breakability curve demoing the per centum haemoysis of undiluted coney ruddy blood cells in different concentrations of NaCl ( millimeter ) solution. It was assumed that 100 % haemylosis occurred in the ruddy blood cells of 10 millimeters NaCl solution. Figure 2. Presentation of the relationship between lipid solubility ( Kether ) and corrected permeableness values of assorted ethanediols derivative solutes for coney ruddy blood plasma membranes. The permeableness coefficient is graphed on a log-log graduated table against Kether. The corrected permeableness was calculated utilizing the average values of the category information. A line of best tantrum has besides been included. It is shown that there is an addition in the membrane potencies with increasing concentration gradient. Figure 3. Graph of the measuring of the corrected membrane electromotive forces ( millivolt ) between two electrodes with differing permeableness to positive and negative ions when placed in different dilutions of standard Thomson solutions. The bluish electrode and ruddy electrode diffusion potencies ( millivolt ) are plotted in their several coloring material. It is shown that there is an addition in membrane potencies with increasing concentration gradient.
Prince alberts, B. , Johnson, A. , Lewis, J. , Raff, M. , Roberts, K. , and Walker, P. ( 2008 ) Molecular Biology of the Cell 5th erectile dysfunction. ( New York, Garland Science ) . Beilharz, M. ( 2014 ) Molecular Biology of the Cell 106 ( SCIE1106 ) Unit Manual Semester 2 ( The University of Australia, Australia ) . Dudok de Wit, C. and van Gastel, C. ( 1969 ) . Red Cell Age and Susceptibility to Immune Haemolysis. Norse Journal of Haematology 6, 373-376 Naccache, P. , and Sha’afi, R.I. ( 1973 ) . Patterns of nonelectrolyte permeableness in human ruddy blood cell membrane. He Journal of General Physiology 62, 714-736