The Method
Sheep blood was used to measure the percentage of hemolysis in the red blood cells (RBCs). The experiment was initiated by placing the blood sample in a high concentration of sodium chloride solution to measure the osmotic pressure. The osmoregulation process was almost evident as the osmotic balance took place with the erythrocytes seemingly more flaccid than when we started the experiment. The electrolyte solution of sodium chloride was the best fit for this solution due to the dissolution of its ions in the water, thereby generating a condition of osmotic balance. The conductance of each of the NaCl soln was measured by calibrated conductivity sensor. The calibration of the Pasco 10X conductivity sensor in distilled water and later in 1% NaCl solution which was labeled as solution 10 was to help with getting accurate reading. The resulting conductance data measured from calibration probe was recorded and the process repeated twice for accuracy.
Solutions conductance test
We used 12 test tubes labeled 1-11 for this test with a control test as well. The 11 test tubes were filled with 5ml solutions of NaCl of different concentrations. The control test tube was filed with 5ml, 0% concentration of the Sodium chloride solution. 20 μl of the sheep blood was added to each of the 12 test tubes using different micropipettes to avoid contamination. The tubes capped tightly, gently shaken and allowed to rest for observation.
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Incubation and Centrifugation
The experimental 12 tubes were put in holder rack containing water bath at 37 0C for a period of 30 minutes after which they were removed, changes observed and recorded. The test tubes under incubation were mixed for centrifugation test where they were placed at 3000 revolutions per minute rate (RPM) for five minutes after which they were removed for observation.
Transmittance Percentage Measure
Colorimeter was used to measure the hemolysis percentage in each of the tubes, including the control test. Fist point was to calibrate the colorimeter using the deionized water. The calibration occurred in cuvette and test for transmission for Green 565nm. 0.5cm of the solution was carefully removed from each tube and placed in a calorimeter for measurement. Each tube’s percentage transmittance (%T) was recorded for analysis.
Calculations
The recorded transmittance rate from each test tube was used to calculate the absorbance rate. The formula for calculating absorbance rate was:
Absorbance=_log (%T/100)
Calculation for hemolysis was done using the control test by the formula
% Hemolysis= (Absorbance tube X/Absorbance tube C) * 100
