Hemolysis Lab free essay sample

Organisms also have white blood cells, also referred to as leukocytes, which combat foreign antibodies in the immune system. White blood cells are complex in structure, and in contrast to red blood cells, have a nucleus. They include such cells as lymphocytes, monocytes, eosinophils, neutrophils and basophils. While some cells such as lymphocytes make antibodies, others attack foreign objects, such as leukocytes, and others have several support jobs that help the immune system perform more efficiently. The immune system also consists of platelets. They are produced in the bone marrow of animals by megakaryocytes (bone marrow cells) which continuously go into the blood system and help clot blood (Barrilleaux 2012). Cell membranes are composed of a phospholipid bilayer, making them hydrophobic. Membranes have many functions, most importantly holding the cytoplasm and organelles. Cell membranes often contain protein channels that allow substances to enter the cell (Bowe et al. , 1997). Cell membranes are selectively permeable, meaning that some substances and chemicals can enter the cell, but not others. Most often, hydrophobicity and size determines permeability rates (Barrilleaux 2012). If too much of a substance rushes into the cell, then they create an osmotic imbalance, meaning that the pressure inside the cell compared to outside the cell differs so much that the cell membrane bursts. This process is called hemolysis (Ivanov 1999). Hemolysis is the process in which red blood cells are disrupted. The cells then release their cytoplasm and organelles. Since the cells are microscopic, we cannot view one cell undergoing hemolysis by the naked eye, however we can view a solution of them undergoing hemolysis without any specific equipment. However you can also view a specific number of cells using a phase contrast microscope, which will not only magnify the cells, but also shows depth and contrast (Barrilleaux 2012). We can also measure hemolysis by a spectrophotometer. A spectrophotometer measures how much light is absorbed by the solution. If a solution is more turbid (cloudy) then it will have a higher absorbance. Throughout this experiment, we wanted to test the membrane permeability of mammalian red blood cells by using hemolysis. We would view it under phase contrast microscopes, spectrophotometers and our eyes. We don’t know what the exact partition coefficients are yet of all the chemicals we will be testing. We will test the membrane permeability of 12 different chemicals, and our hypothesis is that they will differ by their molecular composition, structure, size and whether or not they are ionic. Barrilleaux, A. (2012). Cells and Heredity Laboratory Manual. (pp. 90). New Orleans, LA: Loyola University. Bowe, C. L. , Mokhtarzadeh, L. , Venkatesan, P. , Babu, S. , Axelrod, H. R. , Sofia, M. J. , Karkarla, R. , Chan T. Y. , Kim, J. W. , Lee, H. J. Amidon, G. L. Choe, S. Y. , Walker, S. , Kahne, D. (1997). Design of Compounds that Increase the Absorption of Polar Molecules. Proceedings of the National Academy of Sciences of the United States of America, 94, 2218-12223. Ivanov, I. T. (1999). Low pH-Induced hemolysis of erythrocytes is related to the entry of the acid into cytosole and oxidative stress on cellular membranes. Biochimica et Biophysica Acta-Biomembranes, 1415, 349-360. Ree ce, J. B. , Urry, L. A, Cain, M. L. , Wasserman, S. A. , Minorsky, P. V. , Jackson, R. B. (2011). Membrane Structure and Function. Wilbur, B, (9th ed. ) Campbell Biology (pp. 125-142). San Francisco, CA: Pearson Education. Materials and Methods: Spectrophotometry: After setting the Genosys spectrophotometer to measure the absorbance of light, we set the wave length to 540 nanometers. We pipetted 1. 2 mL of . 3M glycerol into a cuvette and blanked the machine. We then mixed 3 ml of . 3M glycerol and 10 ul of whole blood [1] in a test tube, covered it with parafilm and then inverted the tube to mix the solution adequately. We then pipetted 1. mL of the blood/glycerol solution into a new cuvette, put it in the spectrophotometer and recorded the absorbance for a time of ‘zero’. We then repeated these steps with . 15M NaCl. We blanked 1. 2 mL of a . 15M solution, and then mixed 3mL of the . 15M solution and 10 ul of horse blood in a test tube. We covered the test tube with parafilm and inverted the mixture, we then pipetted 1. 2 mL of the mixture out and into a new cuvett e. We measured the absorbance for a time of ‘zero’. We then simultaneously measured the absorbance of the glycerol/blood mixture and the NaCl/blood mixture every minute for 30 minutes. Basic Contrast Microscopy: We cleaned two glass slides with alcohol and put them aside. We then combined 1 mL of . 15M NaCl and 10ul of whole horse blood in a microcentrifuge and immediately transferred 10 mL of the mixture to the clean glass slide, added a cover slip, recorded the start time and watched the cells under 400x bright 4field microscopy and recorded what we observed. We then switched to 400x phase contrast microscopy and also recorded what we saw periodically and noted any change. We then repeated the same procedure for . 3M glycerol. We added 1 mL of a . M glycerol solution and 10ul of horse blood into a separate microcentrifuge and instantly pipetted 10 ul of the mixture onto another clean glass slide, covered with a coverslip, recorded the start time and viewed under 400X phase contrast microscopy. We watched the slide for 14 minutes and recorded and drew how many cells were in our viewing area. We stopped recording what we saw when cells were no longer visible. Turbi dty: We predicted which chemicals would take a long time (longer than an hour) to turn clear, so we tested those chemicals first. We put 3mL of each chemical in a separate test tube, mixed it with 10 ul of whole horse blood, and documented how much time passed until the mixture turned clear. We then rated it on our own scale of one through five of how turbid it was at time zero. We started with putting NaCl in a test tube and then KCl in another test tube, and then so on ammonium chloride, ammonium acetate, sodium acetate, glucose, sucrose, ethylene glycol, ethanol, glycerol, glycine, and then methanol. After each test tube was labeled with which chemical was inside, we added the horse blood and recorded how long it took the mixture to turn clear. We repeated some of the mixtures, such as ammonium acetate and ammonium chloride because we documented the time incorrectly. We then put the chemicals on a chart in order of how long it took (in minutes) for the turbid mixtures to clear up. Results: Hemolysis: Spectrophotometry: In our results of our spectrophotometry, we recorded the absorbance of each mixture and discovered that our . 3M Glycerol and blood mixture level of absorption initially increased insignificantly and then flattened out for the duration of the experiment at . 355 nanometers. Simultaneously, we recorded the NaCl/blood mixture and it decreased extremely gradually, with the exception of one discrepancy in the middle of the experiment (Figure 1). Phase Contrast: We observed roughly 100 red blood cells using 400X bright field microscopy at the commencement of our experiment for . 15M NaCl/whole blood (Figure 2). We then viewed the red blood cell/NaCl mixture using 400X phase contrast and viewed the same amount of cells, except this time they were mainly small black dots clustered around each other (Figure 3). We witched from using the bright field microscopy to phase contrast microscopy because phase contrast shows depth and has a clearer picture. We then did the same procedure with a . 3M glycerol/blood solution. The start time was 4:45. We observed the first slide using 400X phase contrast microscopy. The start image and it indicates that there were roughly 100 cells (Figure 4). Figure 5 shows what was happening at 4:50; there were roughly 50 cells left and the ghosts of the cells were clearly visible. Figure 6 shows at 4:51 that approximately 30 cells were left, and they were disappearing at an extremely quickly. Figure 7 shows that at 4:55 10 cells were left. Almost all the cells were gone. Figure 8 shows that all the cells have disappeared and only ghosts were left at 4:59 P. M. After the experiment was concluded, figure 9 compared the number of red blood cell mixtures over time. Also, if this experiment was done again, and water was substituted for . 15M NaCl, then the red blood cell would swell and burst because the water is a hypotonic solution compared to the red blood cell. Membrane Permeability: Turbidity: Some chemicals, such as ethylene glycol, glycerol and methanol changed instantaneously from turbid to clear. Others such as NaCl, KCl, sodium acetate, glucose, sucrose and glycine did not change from turbid. Table 10 shows that chemicals reacted differently with the 10 ul horse blood in both how turbid it was at the start of the experiment, and how long it took each chemical to turn completely clear. Figure 11 demonstrates the relationship of time-to-turbidity loss (based on our relative scale of 1-4 we determined at the beginning of each chemical experiment) to each chemical that did change turbidity. Discussion: During this experiment, we fulfilled the objectives in which we wanted to test membrane permeability and test chemicals and whether or not they cause hemolysis. We discovered that NaCl, KCl, sodium acetate, glucose, sucrose and glycine do not cause hemolysis because they are not hypotonic solutions; however, ammonium chloride, ammonium acetate, ethylene glycol, ethanol, glycerol and methanol are hypotonic solutions. In which case the red blood cell has lower pressure than the outside of the red blood cell, so the solution rushes in causes the red blood cell to lose its cytoplasmic inside. Some chemicals and solutions cause hemolysis quicker because they are much smaller in atomic size and mass compared to large molecules that cannot permeate the red blood cell membrane as easily, which slows down hemolysis (Bowe et al. , 1997). Our control (. 15M NaCl) are consistent throughout our experiment. They didn’t cause hemolysis in the spectrophotometer, phase contrast, and with the test tubes. With every one of our other chemicals, we could use NaCl as baseline to refer to, and to see whether or not that chemical was causing hemolysis or if it was an isotonic solution. There were a few issues in the data gathering category; we had to repeat the turbidity test tube experiment for two chemicals because we marked down the wrong start time. We also had trouble viewing . 3M glucose and blood solution under phase contrast, because our microscope was not set up correctly initially, so we had to keep adjusting. We had to gather the data from another group. Every method we used to view hemolysis, whether it be the spectrophotometer, microscope, or our eyes, each had its benefits and downfalls. The spectrophotometer allowed for absorption to be measured better than our eyes and microscope. However, we couldn’t actually see it unless we took the cuvette out of the spectrophotometer. Our eyes were a good way to actually view turbidity without an additional object. It was helpful to actually see the experiment going on in front of you, it allows an additional perspective of envisioning the experiment later on, because it’s easier to actually think about what is happening in the experiment. The microscopes are the best at actually viewing the hemolysis on an extremely small scale. Overall, it was important to view hemolysis with each data collecting instrument, whether it be the spectrophotometer, microscope or eyes. Each had a separate purpose and each came in handy when interpreting the results. It was a great experiment and I thoroughly enjoyed getting â€Å"hands on† training, and also it was vital to view hemolysis and the chemicals that cause hemolysis. Our hypothesis is accepted because the smaller the molecular composition, the quicker the red blood cell membrane was permeated. Also, other scientific articles such as Design Compounds That Increase the Absorption of Polar Molecules and Low pH is Related to the Entry of the Acid Into Cytosole and Oxidative Stress on Cellular Membranes support our hypothesis.