Organisms also have white blood cells, also referred to as leukocytes, which mamba 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, monocot’s, sinkholes, interruptions and basophilic. 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 mastectomy’s (bone marrow cells) which continuously go into the blood system and help clot blood (Barbarically 2012). Cell membranes are composed of a phosphoric bilateral, 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 (Oboe et al. , 1997). Cell membranes are selectively permeable, meaning that some substances and chemicals can enter the cell, but not others.
Most often, hydrophobic and size determines permeability rates (Barbarically 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 ell differs so much that the cell membrane bursts. This process is called hemolytic (Vivian 1999). Hemolytic 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 hemolytic by the naked eye, however we can view a solution of them undergoing hemolytic 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 We can also measure hemolytic 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 absorbency. Throughout this experiment, we wanted to test the membrane permeability of mammalian red blood cells by using hemolytic. 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 ND whether or not they are ionic. Barbarically, A. (2012). Cells and Heredity Laboratory Manual. (up. 90). New Orleans, LA: Loyola University. Oboe, C. L. , Mainstreamed, L. , Ventilates, P. , Baby, S. , Exalted, H. R. , Sofia, M. J. , Karakul, R. , Chain T. Y. , Kim, J. W. , Lee, H. J. Maiden, G. L. Echo, S. Y. , Walker, S. , Kahn, 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. Vivian, l. T. (1999). Low pH-launched hemolytic of erythrocytes is related to the entry of the acid into systole and oxidative stress on cellular membranes. Biochemical et Biophysics Acta-Boomerangs, 1415, 349-360. Erect, J. B. , URI, L. A, Cain, M. L. , Wassermann, S. A. , Minority, P. V. , Jackson, R. B. (2011).
Membrane Structure and Function. Wilbur, B, (9th De. ) Campbell Biology (up. 125-142). San Francisco, CA: Pearson Education. Materials and Methods: Spectrophotometer: After setting the Genomes spectrophotometer to measure the absorbency of light, we set the wave length to 540 manometers. We pipettes 1. 2 ml of . MM glycerol into a cavetti and blanked the machine. We then mixed 3 ml of . MM lechery and 10 LU of whole blood  in a test tube, covered it with paraffin and then inverted the tube to mix the solution adequately. We then pipettes 1. Ml of the blood/glycerol solution into a new cavetti, put it in the spectrophotometer and recorded the absorbency for a time of ‘zero’. We then repeated these steps with . MM Niacin. We blanked 1. 2 ml of a . MM solution, and then mixed ml of the . MM solution and 10 LU of horse blood in a test tube. We covered the test tube with paraffin and inverted the mixture, we then pipettes 1. 2 ml of the mixture out and into a new cavetti. We measured the absorbency for a time of ‘zero’. We then simultaneously measured the absorbency of the glycerol/blood mixture and the Nasal/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 . MM Nasal and 1 OLL of whole horse blood in a microelectronic 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 xx bright field microscopy and recorded what we observed. We then switched to xx phase contrast microscopy and also recorded what we saw periodically and tote any change. We then repeated the same procedure for . MM glycerol. We added 1 ml of a . M glycerol solution and 1 OLL of horse blood into a separate microelectronic and instantly pipettes 10 LU of the mixture onto another clean glass slide, covered with a coveralls, recorded the start time and viewed under XX 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. Turbidity: We predicted which chemicals would take a long time (longer than an hour) to urn clear, so we tested those chemicals first.
We put ml of each chemical in a separate test tube, mixed it with 10 LU 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 Nasal in a test tube and then KICK in another test tube, and then so on ammonium chloride, ammonium acetate, sodium acetate, glucose, sucrose, ethylene glycol, ethanol, glycerol, glycogen, and then methanol. After each test tube was labeled with which chemical was inside, we added the horse blood and corded 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: Hemolytic: In our results of our spectrophotometer, we recorded the absorbency of each mixture and discovered that our . MM Glycerol and blood mixture level of absorption initially increased insignificantly and then flattened out for the duration of the experiment at . 355 manometers. Simultaneously, we recorded the
Nasal/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 XX bright field microscopy at the commencement of our experiment for . 1 MM Nasal/whole blood (Figure 2). We then viewed the red blood cell/Niacin mixture using 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 switched from using the bright field microscopy to phase contrast microscopy because phase entrant shows depth and has a clearer picture.
We then did the same procedure with a . MM glycerol/blood solution. The start time was 4:45. We observed the first slide using XX phase contrast microscopy. The start image and it indicates that there were roughly 1 00 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 one.
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 . MM Nasal, 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 Nasal, KICK, sodium acetate, glucose, sucrose and glycogen did not change from turbid. Table 10 shows that chemicals reacted differently with the 10 LU 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 employees. We discovered that Nasal, KICK, sodium acetate, glucose, sucrose and glycogen do not cause hemolytic 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 cytoplasm inside.
Some chemicals and solutions cause hemolytic quicker because they are much smaller in atomic size and mass compared to large molecules that cannot eremite the red blood cell membrane as easily, which slows down hemolytic (Oboe et al. , 1997). Our control (. MM Nasal) are consistent throughout our experiment. They didn’t cause hemolytic in the spectrophotometer, phase contrast, and with the test tubes. With every one of our other chemicals, we could use Nasal as baseline to refer to, and to see whether or not that chemical was causing hemolytic 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 . MM 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 hemolytic, 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 cavetti 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 hemolytic on an extremely small scale.
Overall, it was important to view hemolytic 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 “hands on” training, and also it was vital to view hemolytic and the chemicals that cause hemolytic. 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 Systole and Oxidative Stress on Cellular Membranes support our hypothesis. For future experiments, we could substitute other mammalian red blood cells, such as monkey or rabbit, compared to horse red blood cells, to see if their red blood ells react differently with the 12 chemicals we tested. We could also change the concentration or temperature of the 12 chemicals we used in the turbidity experiment.