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Nucleic Acids has been a topic well researched on and also has a lot of attention given by the researchers and scientists alike. It encodes our genes, proteins, enzymes and also controls cell division.
Friedrich Miescher discovered Nuclein (DNA); James and Francis proposed the double helix structure of DNA, all these discoveries have made way to more exciting discoveries such as chromosomes.
However, for this experiment, we will be focusing on the appearance of nucleic acids and the position of the acids (DNA and RNA). The Nucleic Acids will be stained with Methyl Green Pyronin (MGP).
Carnoy Fixative will be used to fix the white blood cells that will be taken from the blood smears and microscopic examination will be view under power magnification of 40X and also high power oil immersion 100X.
Actual results will be compared to the theoretical expectations to show that the hypothesis is correct and proves the experiment a success.
Definition of Nucleic Acids
Nucleic Acids, as the name suggests, are found within the nucleus of an Eucaryotic cell. There are two types of Nucleic acids namely, Deoxyribonucleic Acid (DNA); and Ribonucleic Acid (RNA). DNA codes for all proteins and enzymes. DNA is always present in the nucleus and it is only visible when the cell is undergoing meiosis and mitosis. RNA is only present when there is a need to code for proteins.
There are three types of RNA, messenger RNA (mRNA); ribosomal RNA (rRNA);and transfer RNA (tRNA).
rRNA combines with polypeptides to form Ribosomes. tRNA mediates the transfer of information from DNA to be translated into polypeptides. mRNA undergoes a second stage of gene expression and serves as a platform for the final product which is a polypeptide.
Background Information On Nucleic Acids
Friedrich Miescher, a young Swiss doctor, is the first to isolate Nuclein (now called Nucleic Acids) in 1869. He noted that â€œenzymes capable of breaking down proteins were unable to degrade it, and it could not be extracted by strong organic solvents (Dahm, 2008). The analysis of its elementary composition held another surprise for him (Dahm, 2008). Besides containing the elements carbon, oxygen, hydrogen and nitrogen, which are known to be very abundant in proteins, the molecule did not contain sulfur and it did harbor large quantities of phosphorus (Dahm, 2008). The latter was a very unusual finding because virtually no other organic molecules containing phosphorous were known at the time (Dham, 2008). This result finally convinced Miescher that he had discovered a fundamentally new type of cellular substance (Dahm, 2008).
Nearly 90 years later, James Watson and Francis Crick in 1953 proposed that DNA has a Double Helix structure (NLM, 2008). This revolutionary discovery, helped to propel how genes encode traits and also proteins and enzymes.
Objectives and Hypothesis
Hypothesis of Experiment
The experiment will visualise the placing of the DNA and RNA using Methyl-Green Pyronin (MGP) in leucocytic cells. The Methyl Green-Pyronin procedure uses the high net negative charge of nucleic acids(Heidcamp,2003). Methyl green is a cation which binds rather specifically to DNA and thus serves as a convenient means of staining nuclei in both f ixed material and living cells. Pyronin, a red dye, is fairly specific for RNA with some binding to protein(Heidcamp,2003). Therefore, the nucleus situated near the centre of the cell will be stained green and the RNA will be stained red. Two slides will be prepared and one will be served as a control for the experiment.
Objectives of Experiment
This experimentâ€™s aim is to be able to come to a conclusion that white blood cells can be classified under two headings; agranulocyte and granulocyte. Visualising the unique placing of the various nucleic acids namely DNA and RNA and be able to describe the appearance of agranulocyte and granulocyte.
Materials and Methods
Materials used in the Experiment
Materials present and would be used in the experiment:
Methyl Green Pyronin (MGP)
Methods of Experiment
Prepare a blood smear
Two frosted-end microscope slides were cleaned thoroughly by dipping in 95% ethanol and wiping with Kim Wipes Â®. Aseptic techniques were used to obtain the blood sample. After cleaning the hands with soap and water, and drying them, the tip of the ring or middle finger was cleaned with a sterile alcohol swab. When the fingertip has dried, lance it with a sterile lancet. The lancet should never be used more than once and should be discarded in the SHARPS CONTAINER.
The finger was squeezed and a drop of blood was placed near one end of the slide. A second slide with its edge about 30o angle on the first was held and brought it towards the drop. After contact was made spread the drop along the edge of the slide, push the second slide to the other end in one smooth action. The procedure was repeated to make a second slide.
Stain the blood swear with MGP
A hair dryer was used to dry the blood smear. The blood smear was fixed by dipping the slides into a Coplin jar filled with Carnoy Fixative for 10 minutes. The slides were dehydrated with alcohol for a few seconds in a Coplin jar. Slides were dipped, for a few seconds, in distil water in another Coplin jar to rinse off excess alcohol.
The slides were labelled with MGP and RNases+MGP.
Treatment with RNase
Place the slides labelled RNase +MGP in a 0.1% aqueous solution of RNase, pH 6.5-7.0, at 37oC, for 30 minutes. The enzyme treatment and all staining procedures are to be done in the Coplin jars. The Coplin jars are placed in the water bath. Use forceps to place slides in or remove the slides from the jars. During the enzyme treatment, the slide labelled MGP can be left on the laboratory bench.
Staining with MGP
Rinse the slide that undergone RNase treatment with distil water for a few seconds. Place both slides into the Coplin jar in the MGP staining solution for 30 minutes.
After staining, rinse the slides in distilled water for 2 to 3 seconds in another Coplin jar. Air-dry the slides in a vertical position.
Microscopic examination of the slides
Add a drop of Permount and a coverslip. Position the coverslip so that it will cover the smear. Lower the coverslip slowly to minimise air bubbles and then gently press out the excess liquid with a paper towel. Observe each slide under dry high power(40X) and high-power oil immersion(100X).
(Adapted from CP2037 Cell Biology Practical Manual Experiment 2: Identification of nucleic acids in eukaryotic cells)
Results and Discussions
Comparison of theoretical and actual results
The results collected from the actual experiments tallies with the theoretical results, therefore the it justifies the hypothesis. The MGP stains the DNA green and the RNA rose red. The centre of the white blood cells stained green and the cytosol stained rose-red. The stain also enabled the identification of granulocytes and agranulocytes.
The slide tagged with MGP+RNase only had the nucleus being stained green and leaving the cytosol spared from the stain. The slide that does not have the enzyme treatment shows both stains. Appearance also differs as shown below.
Figure.1 and 2 of agranulocyte and granulocyte under microscopic observation.
Fig.1 shows a granulocyte under microscopic examination. [http://medsci.indiana.edu/a215/virtualscope/docs/chap7_1.htm accessed on 1st August 2010]
Fig.2 shows agranulocyte under microscopic examination. [http://human.freescience.org/htmx/monocyte.php accessed on 1st August 2010]
The agranulocyte shows a S-shaped nucleus and the granulocyte shows an almost round shaped nucleus.
Errors of Experiment
The experiment had errors. First of all, the smearing technique might be wrong or done wrongly, therefore causing the white blood cells to be scraped off or damaged. The usage of hair dryer to dry the smear might also denature the cell structure thus causing the sample to be unable to be used for microscopic observation.
During the staining procedures, the white blood cells may have float off into the solution and thus reducing the number for observation. The lost of white blood cells could have occurred throughout the experiment as the procedures is staining intensive, therefore losing the cells to the solution.
Microscopic examination of the cells after staining, the microscope might be poorly adjusted, resulting in the inability to view the MGP stained cells.
Safety Aspect in the Laboratory
Gloves were worn throughout the whole experiment and also when doing the necessary staining. Fingers were swabbed with alcohol before and after lancing it to get a drop of blood. All the materials that had made contact with blood or any bodily fluids were thrown away into the BIOHAZARD BAG that was situated at every laboratory bench. All the other waste is disposed as general waste into a black trash bag.
The samples displayed good stains that enabled the identification of the region that is rich in DNA and RNA. The MGP stained the DNA green and RNA rose-red, therefore be able to differentiate the locations of these nucleic acids. The staining also enabled the differentiation of agranulocyte and granulocyte and also to be able to recognise the appearance of the two different types of white blood cells. Many of the cells were well fixed and preserved, therefore slides were kept for future references. The objectives of the experiment was met and the hypothesis proven correct. The experiment was a success in general.