A eukaryotic cell is a cell that contains a karyon and many membranous and membrane-bound cell organs such as Golgi organic structure and endoplasmic Reticulum. Eukaryotic cells are found in eukaryotes, which are either unicellular or multicellular beings. They have a typical size of 10-100 i?m in diameter which is larger than prokaryotes. In add-on, their familial stuff is present in the karyon where it contains many additive chromosomes which are used for the synthesis of proteins and for cell reproduction. Eukaryotes can be found among animate beings, workss, Protozoa, algae and Fungi ( Yuen ) .
1.2 Nucleic acids
Nucleic acids are biological molecules that store all the familial information of the cell. They are responsible in commanding all the activities of the cell, straight or indirectly through the synthesis of proteins. They besides pass on the familial information from parent to offspring. There are two types of nucleic acid and they are deoxyribonucleic acid ( DNA ) and ribonucleic acid ( RNA ) . Both nucleic acids are supermolecules that exist as polymers called polynucleotides ( Arnold, 2010 ) .
1.2.1 Deoxyribonucleic acids ( Deoxyribonucleic acid )
Deoxyribonucleic acid is double-stranded and can be found in the karyon and cell organs such as chondriosomes and chloroplast. It is a long polymer made from reiterating units called bases. Nucleotide is the basic unit of DNA and RNA. A base is made up of a nitrogen-bearing base, a phosphate unit and a pentose sugar ( Figure 2 ) .
The bases in bases contain nitrogen atoms, therefore they are called nitrogen-bearing bases. The bases contain carbon-nitrogen heterocyclic rings. There are two types of nitrogen-bearing bases, pyrimidines and purines. A pyrimidine has a six -membered ring of C and N atoms. The members of the pyrimidine household include Cytosine ( C ) , Thymine ( T ) and Uracil ( U ) . A purine has a 5-membered ring fused to a 6-membered ring, hence purines are by and large larger than pyrimidines. The members of the purine household include Adenine ( A ) and Guanine ( G ) . The bases A, T, C and G are present in DNA ( Yuen ) .Sugars
Figure 1: 2′-deoxyribose sugar ( Carr, 2008 )
A pentose sugar is a sugar with 5 C atoms. Since the atoms in the bases are already numbered, the C atoms in the sugars contain a premier ( ‘ ) after the figure so as to distinguish between them. The numbering system of the C atoms in the sugar is 1 ‘ , 2 ‘ , 3 ‘ , 4 ‘ and 5 ‘ . At C 2 ‘ , the OH group is replaced with a H group, therefore the sugar is called 2’-deoxyribose ( Figure 1 ) . The base is linked to carbon 1 ‘ of the sugar by a glycosidic bond from N1 of the pyrimidines or N9 of the purines. This linkage forms a nucleoside ( Yuen ) .
Phosphodiester bondThe phosphate unit is attached to carbon 5 ‘ of the sugar by phosphodiester bonds. Phosphodiester bond is the linkage between the 3 ‘ C atom of one sugar molecule and the 5 ‘ C of another. It is formed when 2 phosphate molecules, called pyrophosphates, are cleaved, bring forthing energy for the phosphodiester linkage. The phosphate units are the grounds for the strong negative charge of nucleic acids. The sugar-phosphate anchor, together with the nitrogen-bearing base, forms a base which so joins together with more bases to organize a polynucleotide-DNA ( Yuen ) .
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Figure 2: Deoxyribonucleic acid construction ( Encyclopaedia Brittanica, 1998 )
1.2.2 Ribonucleic acids ( RNA ) Sugars
Figure 3: Ribose sugar ( Carr, 2008 ) RNA has the same construction as Deoxyribonucleic acid, but some of their constituents differ. RNA is single-stranded and can be found in the nucleole and in the cytol, where it exists as ribosomal RNA ( rRNA ) . At C 2 ‘ of the pentose sugar, an OH group is present alternatively of a H group, hence, the sugar is named ribose sugar ( Figure 3 ) . The bases A, C and G can be found in both DNA and RNA, but the 4th base is different. RNA contains the base Uracil alternatively of Thymine ( Yuen ) .
1.3 Methyl green-pyronin ( MGP )
Methyl green-pyronin is a mixture of green and ruddy dye which stains extremely polymerised DNA green and low molecular weight RNA rose-red. It is unable to stain depolymerised RNA and DNA. Methyl green contains cations which bind instead specifically to DNA while pyronin is reasonably specific for RNA with some binding to protein. The staining is possible as nucleic acids are negatively charged due to the phosphate units while the dye is positively charged. Therefore, the dye is able to stain DNA and RNA ( Heidcamp ) .
1.4 Ribonuclease ( RNase )
RNase is a nuclease that aids in the debasement of RNA into its smaller constituents by interrupting the phosphodiester bonds between the bases. In this manner, pyronin will be unable to stain RNA due to the depolymerisation of RNA. The digestion of RNA is of import as it is used as a control to observe whether the pyronin has stained RNA and non other compounds because pyronin does non look to be wholly specific to RNA. ( Jurand and Goel, 1975 )
1.5 White blood cells
White blood cells, besides known as leukocytes, are produced by the bone marrow and can be found in the whole organic structure, such as the blood and the lymphatic system. They exist as portion of the immune system and their function is to protect the organic structure against diseases and infections. There are two groups of white blood cells, granulocytes and agranulocytes ( Anissimov ) .
Granulocytes are white blood cells that are involved in different types of immune reaction. They contain nucleus of different forms and granules that carry digestive enzymes ( Bianco, 2000 ) . There are three types of granulocytes and they are basophils, neutrophils and eosinophils. Harmonizing to Liang, neutrophils are the most common type of white blood cells as they make up 54 % to 62 % of the leukocytes while basophils are the least common type as they comprise less than 1 % of the white blood cells. Eosinophils merely do up 3 % of the leucocytes.IndicationIndication
Figure 4: White blood cells ( Dugdale, 2009 ) Granulocytes
A neutrophil contains a nucleus with two to five lobes. Neutrophils are the first to get at the infection site where they ingest the bacteriums through the procedure known as phagocytosis. They are ephemeral, but plentiful, therefore, they are one of the organic structure ‘s chief defences against bacteriums ( Foster, 2010 ) .
A basophil contains many granules and a nucleus with two lobes. The granules release histamine which causes blood capillaries to be more permeable so that white blood cells can come in and contend off infection ( Foster ) . They besides release Lipo-Hepin which is an anti-clotting agent that prevents blood from coagulating so that white blood cells can go to the septic country without much obstruction ( Bianco, 2000 ) .
An eosinophil contains a nucleus with two lobes. Eosinophils are of import as they help to support the organic structure against infections and besides kill parasites ( Liang ) .
Agranulocytes are white blood cells that do non incorporate granules. There are two types of agranulocytes and they are monocytes and lymph cells. Harmonizing to Liang, monocytes make up 2 % to 8 % of the go arounding white blood cells while lymph cells make up 25 % to 33 % of the white blood cells.
Monocytes contain bean-shaped karyons and they play a function in the immune system. When monocytes enter a tissue, they can develop into macrophages which can assail any foreign stuff by phagocytosis so that it is unable to harm the organic structure. They are besides able to continue an antigen to let the organic structure to recognize the foreign stuff in future. In add-on, macrophages can besides consume septic cells so that the infection would non be passed on to other cells ( Smith, 2010 ) .
Lymphocytes contain big and circular karyon. They help the organic structure to separate its ain cells from foreign 1s. When they detect a foreign stuff, they will instantly let go of chemicals to kill it. There are two chief types of lymph cells and they are T cells and B cells. T cells are lymphocytes that migrate from the bone marrow to the Thymus and mature at that place while B cells are lymphocytes that mature in the bone marrow ( Campbell, et al. , 2008 ) . T cells map by attaching themselves to the infected cell and releasing chemicals that digest both antigen and infected cell. T cells can besides release a chemical that activates B cells, doing them to bring forth antibodies to contend the antigens ( Blackburn, 2011 ) .
The stuffs used were four frosted-end microscope slides, one lancet, two unfertile intoxicant swabs, one hair drier, five Coplin jars, two coverslips, one 37oC H2O bath, Kim wipes, one bright-field microscope, one forcep, 95 % ethyl alcohol, Carnoy fixative, distilled H2O, ribonucleinase ( RNase ) , methyl green-pyronin ( MGP ) , Permount® and submergence oil.
3.1 Preparation of blood vilifications
Two frosted-end microscope slides were cleaned exhaustively after they were dipped in 95 % ethyl alcohol and wiped with Kim Wipes® . Aseptic techniques were used to obtain the blood sample. After the custodies were cleaned with soap and H2O and dried, the tip of the in-between finger was cleaned with a unfertile intoxicant swab. After the fingertip had dried, it was lanced with a unfertile lancet and so squeezed. The first bead of blood was wiped off. The finger was squeezed once more and the bead of blood was placed near one terminal of the slide. A 2nd slide was held with its border about 30o angle on the first and brought towards the bead. After contact had been made, the bead spread along the border of the slide. The 2nd slide was pushed to the other terminal in one smooth action. The process was repeated to do a 2nd blood vilification. The lanced finger was cleaned with a unfertile intoxicant swab after the vilifications have been made.
3.2 Staining the blood vilifications with MGP
The blood vilifications were dried utilizing a hair drier. They were fixed onto the slides utilizing Carnoy fixative placed in a Coplin jar for 10 proceedingss. Then, the vilifications were placed in a Coplin jar filled with 95 % ethyl alcohol for a few seconds so as to desiccate them. After which, the vilifications were rinsed with distilled H2O for 2 proceedingss in a Coplin jar. The H2O on the slides was so drained off.
3.3 Treatment with RNase
The two slides incorporating blood vilifications were labelled. The slide labelled MGP was stained with MGP while the slide labelled RNase + MGP was pre-treated with RNase.
The enzyme intervention and all staining processs were done in Coplin jars. The Coplin jars were placed in the H2O bath. Forcepss were used to put slides in or take slides from Coplin jars. During the enzyme intervention, the slide labelled MGP was left on the lab bench. The slide labelled RNase + MGP was placed in a 0.1 % aqueous solution of RNase, with pH 6.5-7.0, at 37 oC for 15 proceedingss. Then, it was rinsed in distilled H2O for a few seconds in a Coplin jar. Both slides were placed in the methyl green-pyronin staining solution for 10 proceedingss. Each slide was so rinsed in distilled H2O for 2 to 3 seconds in a Coplin jar. They were rapidly drained and the dorsum of each slide was wiped with a Kim rub. They were so allowed to air-dry in a close perpendicular place.
3.4 Microscopic scrutiny of the slides
A bead of Permount® was added to each slide. A coverslip was so placed onto each slide and positioned such that it will cover the vilification. The coverslip was lowered easy to understate air bubbles and the extra liquid was gently pressed out with a paper towel. Each slide was so observed under high power ( 40X ) and high-octane oil submergence ( 100X ) .
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Figure 5: White blood cells stained with MGP at 100X magnification
The slide labelled MGP was stained green and rose-red. As shown in Figure 5, the green discolorations were in the form of a karyon and hence, it can be seen that DNA was present in the karyon. The rose-red discolorations were found about and in the green discolorations which indicate that RNA was present in the cytol and nucleole. Degree centigrade: Documents and SettingsOwnerDesktopIMG084.jpg
Figure 6: White blood cells pre-treated with RNase and stained with MGP at 100X magnification
The slide labelled MGP + RNase was stained visible radiation green merely. As shown in Figure 6, the light green discolorations were in the form of a karyon and hence, it can be seen that DNA was present in the karyon. Rose-red discolorations were absent as RNase was added to the slide.
From Figures 5 and 6, it can be seen that the karyon of each cell was different. Therefore, it was easy to separate granulocytes from agranulocytes. A granulocyte has a multi-lobed karyon while an agranulocyte has a big and circular karyon.
Methyl green-pyronin is a dye produced by blending methyl green and pyronin. Methyl green contains cations which bind sooner to DNA. Pyronin besides contains cations which bind instead specifically to RNA. In this experiment, blood was smeared onto two slides in which one of them was pre-treated with RNase. Both of the slides were so stained with methyl green-pyronin. For the slide that was non pre-treated with RNase, rose-red and green discolorations were observed. This indicates that DNA and RNA were present. In add-on, the green discolorations were in the form of a multi-lobed karyon and a big, circular karyon ( Figure 5 ) , which showed that DNA was present in the karyon and the cells can be differentiated into granulocytes and agranulocytes. Rose-red discolorations were seen in and around the green discolorations ( Figure 5 ) , which indicate that RNA was present in the cytol and nucleole.
For the slide that was pre-treated with RNase, light green discolorations were observed ( Figure 6 ) . This indicates the presence of DNA. The absence of rose-red discolorations suggests that RNA was non present. This was due to the action of RNase, which was used as a control to corroborate that pyronin had stained RNA. The fact that RNase degrades RNA and that rose-red discolorations were absent in RNase-treated slide but present in non-treated slide showed that RNA was present in Figure 5.
It has been known that Deoxyribonucleic acid is present in both nucleus and cytol, but green discolorations were non seen in the cytol as mitochondrial DNA was excessively little to be detected in this experiment.
The light green discolorations on the MGP slide might either be caused by the loss of methyl viridity during the staining process or due to the low concentration of DNA. The higher the strength of green and rose-red color observed, the higher the concentrations of DNA and RNA ( Yuen, 2010 ) . Methyl green might hold been lost when the slides were rinsed with distilled H2O. Therefore, it would be better to avoid rinsing the slides with distilled H2O or rinse the slides at a shorter clip.
To better this experiment, deoxyribonuclease ( DNase ) should besides be used. DNase is a nuclease that aids in the debasement of DNA into its smaller constituents by interrupting the phosphodiester bonds between the bases. In this manner, it has the same intent as RNase, which is to move as a control. One slide could be stained with MGP and the other with MGP + DNase. If the slide that was pre-treated with DNase contained merely rose-red discolorations, RNA is present. If the slide stained with MGP contained both rose-red and green discolorations, DNA and RNA are present. This proves that the green discoloration is DNA.
Xylene can besides be added to the slides for one minute. This will give a transparent background so that the cells would look clearer under the microscope. Since xylol is carcinogenic, it should be handled with attention.
For this experiment, it was necessary to take safety safeguards as bodily fluids were involved. Aseptic techniques should be used to obtain the blood sample. The tip of the finger should be cleaned with an intoxicant swab before lancing. The lanced finger should be wrapped with a plaster to forestall infection. In add-on, the lancet should non be used more than one time. All other waste should be treated as general waste and discarded into the waste bin with black disposal bags.
All in all, this experiment was successful as the nucleic acids were identified by the usage of MGP. It had besides confirmed that methyl green-pyronin discolorations DNA green and RNA rose-red ( Figure 5 ) . The ability of the enzyme, RNase, in degrading RNA was besides proved when it was used to corroborate the presence of RNA, in which rose-red discolorations were undetected in the RNase-treated slide. The different types of white blood cells, agranulocytes and granulocytes, could besides be distinguished as karyon of different forms could be seen through the discolorations ( Figure 5 or 6 ) .