Microscopes and Cell Biology - Lab Report Example

LAB REPORT INTRODUCTION TO MICROSCOPY Every single one cell and a lot of organisms are too minute to be premeditated using the naked eyeball. Microscopes enlarge specimens and formulate them to emerge bigger than they accurately are. This allows individuals to precisely study and depict cells and infinitesimal organisms. A microscope has very many parts that play major roles in the magnification of specimen. These parts are build together to ensure that they work together to achieve magnification. The microscope has two systems of lenses which are known as the ocular and the objective lens for greater magnification1. The oculars are also identified as the eyepiece lens and are the ones that a person looks into so as to see magnified specimen and are usually of 10X or 15 X magnifications. The objectives are the lens closest to the object and are usually of different magnification powers. The objective lens is held together by a revolving nose piece that is able to rotate in an easy manner enabling the change of the different lens powers. These powers are of three or four different types and are mostly four, ten, forty, and a hundred times. For complete magnification, both the eyepiece and the objective lens powers are multiplied. The eyepiece and the objectives are connected by a type of tube that is usually straight in shape. This tube is connected and supported to the base by a part identified. For the microscope to function, it is important to have an illuminator that is described as a steady light source that is usually about one hundred and ten volts. In most cases, the illuminator is usually used in place of a mirror that is used to reflect light from an external light source. The specimen are placed on slides and then placed on a platform commonly known as the stage and is usually at the bottom part of the microscope. It is important to understand that the specimen require to be held in place as the magnification takes place. The most common of all microscopes have movable stages that can move right, left, top or bottom. A condenser lens is used to direct light rays from an illumination source to the specimen that is bound to get examined. Condenser lenses are most useful at the highest powers (400X and above).  Microscopes with in stage condenser lenses render a sharper image than those with no lens (at 400X).  If your microscope has a maximum power of 400X, you will get the maximum benefit by using a condenser lenses rated at 0.65 NA or greater.  0.65 NA condenser lenses may be mounted in the stage and work quite well.  A big advantage to a stage mounted lens is that there is one less focusing item to deal with.  If you go to 1000X then you should have a focusable condenser lens with an N.A. of 1.25 or greater.  Most 1000X microscopes use 1.25 Abbe condenser lens systems2.  The Abbe condenser lens can be moved up and down.  It is set very close to the slide at 1000X and moved further away at the lower powers.   Methods The appropriate method to focal point a microscope is to begin with the lowly power objective lens and as one is observing from a specific region, eccentric the lens downward as near to the sample as possible devoid of moving it. After that, glance using the eyepiece lens and spot increasingly until the specimen is sharp.  In case there happens to appear some problems during the focus, a repeat of the whole process is supposed to be done. As soon as the image is sharp with the low power lens, an individual should be able to adjust in the next power lens and do slight changes with the focus handle.  If the microscope is fitted with a fine focus adjustment, rotating it would be enough to achieve great images.   Materials used Compound microscope Glass slides Cover slips Eye dropper Beakers Scissors Tooth picks Iodine or Methylene Blue Plant or algae specimens Procedure Microscope Handling 1. Take the microscope and carry it using two hands, whereby one on the arms holds the base and the other the arm 2. Someone from every grouping has to re-visit the microscope storeroom so as to safely deliver the microscope. 3. So as to work fast, a single person should be able to collect the devices to be used in the experiment like a pair of scissors, newsprint, a slide, and a cover slip. 4. The next step involves removal of the dust cover and proper storage. 5. Scrutinize the microscope and provide the purpose for each of the parts. Conclusion The magnification of the given specimen is calculated by multiplying the power of the eyepiece lens with that of the objective lens used. After the experiment, switch off the microscope so as to return it to its storeroom. Position the low power objective in place and lower the body tube. Envelop the scope with the dust cover and place it in its usual place3. LAB REPORT OBSERVING CHROMOSOMES UNDERGOING MITOSIS IN ONION ROOT TIPS Introduction Mitosis is best observed in cells that always have rapid growth as in the onion root cell tips. This is because the root contains a special growth region commonly referred to as the apical meristem whereby high percentage of cells are believed to undergo mitosis4. Onion cells are in general rectangular in shape where they mostly range from a quarter to a half millimeter. To be able to observe chromosomes properly, it is essential to ensure that the microscope used is of high quality and has high magnification power. This is because the observation of the chromosomes in the nucleus requires that one has their ultimate count. Materials Light microscope Slides Onion Clarke’s fluid Bijou tubes Eppendorf tubes Petri dishes Distilled water Clock 1M Hydrochloric acid Aceto-orcein Acetic acid Incubator Forceps Cover slips Pencil Procedure 1. Cut off about 2 mm of the root tip from an onion. 2. In a bijou tube, fix the root tips in Clarke’s fluid and wait for about ten minutes. 3. Remove the root tip from the fluid and place it in a Petri dish that has pure distilled water. 4. Put one millimeter of1M Hydrochloric acid in an Eppendorf tube and then place a root tip to incubate at sixty degrees Celsius for about five minutes. 5. Pour contents of tube into a petri dish and carefully pick out the root tips with forceps and place them in an eppendorf tube containing aceto-orcein solution and leave in dark for about ten minutes. 6. After the ten minutes, place the root tip on a slide in a drop of forty five percent acetic acid and cover with a cover slip. It is important to note that the above procedures are meant to soften the roots so that they can spread adequately on a microscope slide. 7. Squash the softened, stained root tip by lightly tapping on the cover slip with a pencil, whereby the results expected are that the root tip spreads out as pink mass. Preparing the root tip squash Transfer a root to the center of a clean microscope slide and add a drop of water. Using a razor blade cut off most of the unstained part of the root, and discards it. Cover the root tip with a cover slip, and then carefully push down on the cover slide with the wooden end of a dissecting probe. Push hard, but do not twist or push the cover slide sideways. The root tip should spread out to a diameter about 0.5 – 1 cm. 8. Using light microscopy of about X400, view the chromosomes at their different stages of mitosis. Observations of onion root tip squash. Scan the microscope under the 10x objective. Look for the region that has large nuclei relative to the size of the cell; among these cells will be found cells displaying stages of mitosis. Examples are shown in the figure to the right. Switch to the 40X objective to make closer observations. Since prophase and pro-metaphase are difficult to distinguish, classify all these at cells as prophase. 9. Make at least two drawings showing the different stages of mitosis using the X400 magnification. It is important to include the dimensions of the cells undergoing mitosis during the various stages. Conclusion The root cells undertake nuclear duplication through a series of steps that splits the nucleus into divided nuclei at differing sides. It is necessary to note that when a cell undergoes division, the process can be referred to as cytokinesis. If the cells used for this experiment were not from the root tip, the majority of the cells would be in the interphase stage and hence produce inaccurate results. The stages that the observer should be keen to note include prophase, metaphase, anaphase, and telophase. Prophase is a lengthy phase, anaphase is the least prolonged, while mitosis is just one section of a cell’s life. The highest time of a cell’s life (75% to be exact) is usually in interphase, a phase just prior to prophase5. During this phase, DNA duplication takes place. Prophase involves the first signs of cell division with a thickening of the chromatin threads until the chromatin is condensed to chromosomes. In metaphase, the chromosomes shift to the center of the spindle and the centromere attaches to the spindle. During anaphase, the chromatids are separated and moved to opposite ends of the poles. The final stage, telophase, involves the condensation of the chromosomes and the formation of a new nuclear envelope. Following telophase, cytokinesis may occur and the cytoplasm will be divided into two cells. LAB REPORT CELLULAR LOCATION OF METABOLIC ENZYMES Summary It is important to always have an idea of the cellular location of some important enzymes contained in the liver cells. The liver has a lot of mitochondrion which are needed the production of the amount of energy needed in the organ. INTRODUCTION Eukaryotic cells contain several membrane-bound compartments called organelles that perform specialized biological functions. Sub cellular compartmentalization allows the cell to maintain different environments that bring enzymes and substrates into physical proximity, participating in compartment-specific processes. In different cells, group of enzymes interact in various ways ensuring that the product achieved constitutes the substrate for a different enzyme. These types of reactions are referred to as multi enzyme system. Revealing the sub cellular localization of proteins within membrane-bound compartments is of a major importance for inferring protein function. Though current high-throughput localization experiments provide valuable data, they are costly and time-consuming, and due to technical difficulties not readily applicable for many Eukaryotes. Physical characteristics of proteins, such as sequence targeting signals and amino acid composition are commonly used to predict sub cellular localizations using computational approaches. Recently it was shown that protein–protein interaction (PPI) networks can be used to significantly improve the prediction accuracy of protein sub cellular localization. However, as high-throughput PPI data depend on costly high-throughput experiments and are currently available for only a few organisms, the scope of such methods is yet limited. Method Work rapidly or the fractions will lose enzymatic activity. Conduct all procedures on ice. Once the mitochondria have been isolate, two members of each lab group should proceed with the assay of Succinate Dehydrogenase while the other member continues to repurify the nuclear fraction and isolate the microsomes. Tissue Preparation 1. Obtain one of the beakers containing the liver tissues (prepared by the Instructor). The weight of the liver is written on the side of the beaker, record it. 2. Using a pair of scissors, mince the tissue. 3. Decant and discard the pink solution, including the floating material. 4. Tissue Wash: This step removes any enzymes released into the buffer as a result of physical tissue damage. These released enzymes could have been solubilized into the final Homogenization Buffer, where they could register as free cytoplasmic enzymes in our subsequent assay. a. Submerge the liver tissue in fresh Homogenization Buffer. b. Continue to mince and rinse the tissue until the Buffer is nearly colorless Tissue Homogenization 5. Transfer about one-half (~2 gm) of the tissue to a glass dounce homogenizer on ice. Do not use a tight-fitting ground-glass homogenizer in this step since the organelle membranes may disrupt partially. 6. Add 18 ml of ice-cold Homogenization Buffer to the homogenizer. Note: the Calcium in the buffer is used here to stabilize the organelle membranes. 7. Homogenize 3-5 strokes until a homogeneous homogenate is made. Keep the homogenizer on ice. Caution: Excessive grinding or heating can damage and inactivate sub cellular fractions. 8. Homogenize with a teflon Potter-Elvehjem homogenizer to ensure total tissue disruption. 9. Transfer the homogenate into a plastic 50-ml JA-20 tube on ice. 10. Repeat steps 5-9 (combining all homogenates) until all the minced liver tissue has been homogenize Isolation of Mitochondria Obtain the supernatant from step 12 in a JA-20 tube on ice. Centrifuge at 5,000 x g (for the JA-20 this is 6.5K) for 15 min. at 0ºC. If you are pressed for time: centrifuge at 8.0K for 10 min at 0ºC. The pellet contains the crude mitochondrial fraction. The supernatant contains microsomes, membrane fragments, ribosome’s, cytoplasmic enzymes, etc. Without disturbing the mitochondrial pellet, carefully decant the post-mitochondrial supernatant (into a graduated cylinder. Measure and record its volume, then pour the supernatant into a clean JA-20 tube on ice. Swirl the tube to mix and aliquot 4.0 ml to a test tube on ice for use as a "Negative Control" in the Succinate Dehydrogenase assay. With the remainder of the supernatant, the lab group member who isolated the nuclei should proceed to Isolation of the Microsomes To continue with mitochondrial isolation, add 20 ml of ice-cold Homogenization Buffer to the crude mitochondrial pellet. a. Using a teflon homogenizer, re-suspend the pellet in the buffer. b. Pour the suspension back into the JA-20 tube, add another 20 ml of buffer, mix, and centrifuge at 234,000 x g (this is 14K for the JA-20 rotor) for 10 min. at 0ºC. The pellet contains the washed mitochondria. c. Without disturbing the mitochondrial pellet, decant and discard the supernatant. Add 5.0 ml of ice-cold Homogenization Buffer to the mitochondrial pellet. Using a teflon homogenizer, re-suspend the pellet in the buffer6. Using a 10 ml pipette, measure the total volume of mitochondria and record it below and in the Aliquot the remainder of the suspension into two glass test tubes on ice. Label the tubes Mitochondria and store them at 20°C. References Borowick, Jerome N. lab report. Chicago: Prentice Hall, 2000. D., Andrew. Mitosis: Methods and Protocols (Methods in Molecular Biology). Chicago: Humana Press, 2009. Dawes, Clinton J. Introduction to biological electron microscopy: Theory and Techniques. Washington: Ladd Research Industries, 1988. Egerton, R.F. Physical Principles of Electron Microscopy. New York: Springer publishers, 2010. French, Marvin L. The Human Cell. New York: Marvin L French publishers, 2011. Gilbert, Scott F. Developmental Biology. Columbia: Sinauer Associates, 2010. Hayat, M. A. Introduction to Biological Scanning Electron Microscopy. Chicago: University Park Press, 1992. Matsudaira, Paul. Molecular Cell Biology. New York: W. H. Freeman, 1999. Najjar, V.A. Enzyme Induction and Modulation. New York: Springer, 2000. R.C, Elizabeth. All About Mitosis and Meiosis: Life Science. Washington: Teacher Created, 2008. Schrader, Franz. Mitosis: The movements of chromosomes in cell division. Columbia: Columbia University Press, 1944. Sullivan, Mary. All About Mitosis and Meiosis (Mission: Science) . New York: Compass Point Books, 2010. Read More