The experiment was performed in order to determine the effect of different osmolarities and salinities on a cell such as the red blood cell or erythrocyte. It has been established that a cell maintains its size and shape based on the presence and concentration of solutes in the solution where the cell is situated in. In this particular experiment, a conductivity sensor was calibrated to 338 mOsm/kg connected to an SW500 interface. Calibration was performed using distilled water and a 1.1% sodium chloride (NaCl) solution.
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Using a pipettor, 5 ml of the prepared solutions of different osmolarities were pipetted into twelve labeled test tubes. Each test tube contained a different concentration of NaCl, expressed in grams per liter (g/L), such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% 1.1%. Red blood cells or erythrocytes at a constant volume of 20 ul were introduced to the constant volume of 5 ml. The control test tube did not contain any amount of NaCl hence this may be described as a solution with 0.0% NaCl and is actually 100% distilled water.
Twenty microliters (20 µl) of blood were added to each test tube and gently mixed by hand tapping to homogeneously mix the cell suspension. The top of the test tubes was covered with Parafilm in order to ensure that no cells or the suspension will be spilled during the rest of the experiment. After mixing the solutions with the red blood cells, the twelve test tubes were placed on a water bath for thirty minutes at 37oC because this is the normal temperature that the red blood cells are known to circulate and live in, just like the human body. The test tubes were kept at this temperature for 30 minutes because this duration of incubation is enough time for the red blood cells to undergo and show any responses to the different concentrations of NaCl. After the appropriate incubation period, the suspensions were again gently agitated to resuspend the red blood cells in the solution. The incubation period of 30 minutes generally results in the sedimentation or aggregation of the red blood cells at the bottom of the test tube. The red blood cells were then collected by centrifugation at 3,000 RPM for 5 minutes. Centrifugation can separate cells and organelles according to their molecular weight, wherein the heavier cells or organelles go to the bottom of the tube while the lighter organelles stay somewhere in the middle of the column of solution.
After centrifugation, using a transfer pipettor, the top portion of the solution on the test tube was transferred into a rectangular cuvette. This procedure was performed for each test tube. The rectangular cuvette was placed into a previously calibrated PASCO colorimeter for 30 seconds, in order to obtain a reading which reflects the extent of transmittance (% T). Spectrophotometric quantification of the cell suspensions and the control solution was performed using the fluorescence (green) filter with an absorbance wavelength of 565 nm. The amount of transmittance (% T) was recorded for all 12 solutions and the absorbance was computed as –log %T. The degree of hemolysis (% hemolysis) was calculated by dividing the absorbance of each suspension by the absorbance reading of the control test tube. The results of the calculations were graphed for analysis and presentation.