A eucaryotic cell is a cell that contains a nucleus and many membranous and membrane-bound organelles such as golgi body and endoplasmic reticulum. Eucaryotic cells are found in eucaryotes, which are either unicellular or multicellular organisms. They have a typical size of 10-100 ïm in diameter which is larger than procaryotes. In addition, their genetic material is present in the nucleus where it contains many linear chromosomes which are used for the synthesis of proteins and for cell replication. Eucaryotes can be found among animals, plants, protozoa, algae and fungi (Yuen).
1.2 Nucleic acids
Nucleic acids are biological molecules that store all the genetic information of the cell. They are responsible in controlling all the activities of the cell, directly or indirectly through the synthesis of proteins. They also pass on the genetic 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 macromolecules that exist as polymers called polynucleotides (Arnold, 2010).
1.2.1 Deoxyribonucleic acids (DNA)
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DNA is double-stranded and can be found in the nucleus and organelles such as mitochondria and chloroplast. It is a long polymer made from repeating units called nucleotides. Nucleotide is the basic unit of DNA and RNA. A nucleotide is made up of a nitrogenous base, a phosphate unit and a pentose sugar (Figure 2).
The bases in nucleotides contain nitrogen atoms, thus they are called nitrogenous bases. The bases contain carbon-nitrogen heterocyclic rings. There are two types of nitrogenous bases, pyrimidines and purines. A pyrimidine has a six -membered ring of carbon and nitrogen atoms. The members of the pyrimidine family include Cytosine (C), Thymine (T) and Uracil (U). A purine has a five-membered ring fused to a six-membered ring, therefore purines are generally larger than pyrimidines. The members of the purine family 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 carbon atoms. Since the atoms in the bases are already numbered, the carbon atoms in the sugars contain a prime (') after the number so as to differentiate between them. The numbering system of the carbon atoms in the sugar is 1', 2', 3', 4' and 5'. At carbon 2', the OH group is replaced with a H group, thus 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' carbon atom of one sugar molecule and the 5' carbon of another. It is formed when 2 phosphate molecules, called pyrophosphates, are cleaved, producing energy for the phosphodiester linkage. The phosphate units are the reasons for the strong negative charge of nucleic acids. The sugar-phosphate backbone, together with the nitrogenous base, forms a nucleotide which then joins together with more nucleotides to form a polynucleotide-DNA (Yuen).
Figure 2: DNA structure (Encyclopaedia Brittanica, 1998)
1.2.2 Ribonucleic acids (RNA)Sugars
Figure 3: Ribose sugar (Carr, 2008) RNA has the same structure as DNA, but some of their components differ. RNA is single-stranded and can be found in the nucleoli and in the cytoplasm, where it exists as ribosomal RNA (rRNA). At carbon 2' of the pentose sugar, an OH group is present instead of a H group, therefore, the sugar is named ribose sugar (Figure 3). The bases A, C and G can be found in both DNA and RNA, but the fourth base is different. RNA contains the base Uracil instead of Thymine (Yuen).
1.3 Methyl green-pyronin (MGP)
Methyl green-pyronin is a mixture of green and red dye which stains highly 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 rather specifically to DNA while pyronin is fairly 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)
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RNase is a nuclease that aids in the degradation of RNA into its smaller components by breaking the phosphodiester bonds between the nucleotides. In this way, pyronin will be unable to stain RNA due to the depolymerisation of RNA. The digestion of RNA is important as it is used as a control to detect whether the pyronin has stained RNA and not other compounds because pyronin does not seem to be entirely specific to RNA. (Jurand and Goel, 1975)
1.5 White blood cells
White blood cells, also known as leucocytes, are produced by the bone marrow and can be found in the whole body, such as the blood and the lymphatic system. They exist as part of the immune system and their role is to protect the body 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 shapes and granules that carry digestive enzymes (Bianco, 2000). There are three types of granulocytes and they are basophils, neutrophils and eosinophils. According to Liang, neutrophils are the most common type of white blood cells as they make up 54% to 62% of the leucocytes while basophils are the least common type as they comprise less than 1% of the white blood cells. Eosinophils only make 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 arrive at the infection site where they ingest the bacteria through the process known as phagocytosis. They are short-lived, but plentiful, thus, they are one of the body's main defenses against bacteria (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 enter and fight off infection (Foster). They also release heparin which is an anti-clotting agent that prevents blood from clotting so that white blood cells can travel to the infected area without much blockage (Bianco, 2000).
An eosinophil contains a nucleus with two lobes. Eosinophils are important as they help to defend the body against infections and also kill parasites (Liang).
Agranulocytes are white blood cells that do not contain granules. There are two types of agranulocytes and they are monocytes and lymphocytes. According to Liang, monocytes make up 2% to 8% of the circulating white blood cells while lymphocytes make up 25% to 33% of the white blood cells.
Monocytes contain bean-shaped nucleus and they play a role in the immune system. When monocytes enter a tissue, they can develop into macrophages which can attack any foreign material by phagocytosis so that it is unable to harm the body. They are also able to preserve an antigen to allow the body to recognise the foreign material in future. In addition, macrophages can also ingest infected cells so that the infection would not be passed on to other cells (Smith, 2010).
Lymphocytes contain large and round nucleus. They help the body to distinguish its own cells from foreign ones. When they detect a foreign material, they will immediately release chemicals to kill it. There are two main types of lymphocytes and they are T cells and B cells. T cells are lymphocytes that migrate from the bone marrow to the thymus and mature there while B cells are lymphocytes that mature in the bone marrow (Campbell, et al., 2008). T cells function by attaching themselves to the infected cell and secreting chemicals that digest both antigen and infected cell. T cells can also secrete a chemical that activates B cells, causing them to produce antibodies to fight the antigens (Blackburn, 2011).
The materials used were four frosted-end microscope slides, one lancet, two sterile alcohol swabs, one hair dryer, five Coplin jars, two coverslips, one 37oC water bath, Kim wipes, one bright-field microscope, one forcep, 95% ethanol, Carnoy fixative, distilled water, ribonuclease (RNase), methyl green-pyronin (MGP), Permount® and immersion oil.
3.1 Preparation of blood smears
Two frosted-end microscope slides were cleaned thoroughly after they were dipped in 95% ethanol and wiped with Kim Wipes®. Aseptic techniques were used to obtain the blood sample. After the hands were cleaned with soap and water and dried, the tip of the middle finger was cleaned with a sterile alcohol swab. After the fingertip had dried, it was lanced with a sterile lancet and then squeezed. The first drop of blood was wiped away. The finger was squeezed again and the drop of blood was placed near one end of the slide. A second slide was held with its edge about 30o angle on the first and brought towards the drop. After contact had been made, the drop spread along the edge of the slide. The second slide was pushed to the other end in one smooth action. The procedure was repeated to make a second blood smear. The lanced finger was cleaned with a sterile alcohol swab after the smears have been made.
3.2 Staining the blood smears with MGP
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The blood smears were dried using a hair dryer. They were fixed onto the slides using Carnoy fixative placed in a Coplin jar for 10 minutes. Then, the smears were placed in a Coplin jar filled with 95% ethanol for a few seconds so as to dehydrate them. After which, the smears were rinsed with distilled water for 2 minutes in a Coplin jar. The water on the slides was then drained off.
3.3 Treatment with RNase
The two slides containing blood smears were labelled. The slide labelled MGP was stained with MGP while the slide labelled RNase + MGP was pre-treated with RNase.
The enzyme treatment and all staining procedures were done in Coplin jars. The Coplin jars were placed in the water bath. Forceps were used to place slides in or remove slides from Coplin jars. During the enzyme treatment, 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 minutes. Then, it was rinsed in distilled water for a few seconds in a Coplin jar. Both slides were placed in the methyl green-pyronin staining solution for 10 minutes. Each slide was then rinsed in distilled water for 2 to 3 seconds in a Coplin jar. They were quickly drained and the back of each slide was wiped with a Kim wipe. They were then allowed to air-dry in a near vertical position.
3.4 Microscopic examination of the slides
A drop of Permount® was added to each slide. A coverslip was then placed onto each slide and positioned such that it will cover the smear. The coverslip was lowered slowly to minimise air bubbles and the excess liquid was gently pressed out with a paper towel. Each slide was then observed under high power (40X) and high-power oil immersion (100X).
4. ResultsC:\Users\Fujitsu User\Desktop\IMG085.jpg
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 stains were in the shape of a nucleus and therefore, it can be seen that DNA was present in the nucleus. The rose-red stains were found around and in the green stains which indicate that RNA was present in the cytoplasm and nucleoli. C:\Documents and Settings\Owner\Desktop\IMG084.jpg
Figure 6: White blood cells pre-treated with RNase and stained with MGP at 100X magnification
The slide labelled MGP + RNase was stained light green only. As shown in Figure 6, the light green stains were in the shape of a nucleus and therefore, it can be seen that DNA was present in the nucleus. Rose-red stains were absent as RNase was added to the slide.
From Figures 5 and 6, it can be seen that the nucleus of each cell was different. Thus, it was easy to distinguish granulocytes from agranulocytes. A granulocyte has a multi-lobed nucleus while an agranulocyte has a large and round nucleus.
Methyl green-pyronin is a dye produced by mixing methyl green and pyronin. Methyl green contains cations which bind preferably to DNA. Pyronin also contains cations which bind rather 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 then stained with methyl green-pyronin. For the slide that was not pre-treated with RNase, rose-red and green stains were observed. This indicates that DNA and RNA were present. In addition, the green stains were in the shape of a multi-lobed nucleus and a large, round nucleus (Figure 5), which showed that DNA was present in the nucleus and the cells can be differentiated into granulocytes and agranulocytes. Rose-red stains were seen in and around the green stains (Figure 5), which indicate that RNA was present in the cytoplasm and nucleoli.
For the slide that was pre-treated with RNase, light green stains were observed (Figure 6). This indicates the presence of DNA. The absence of rose-red stains suggests that RNA was not present. This was due to the action of RNase, which was used as a control to confirm that pyronin had stained RNA. The fact that RNase degrades RNA and that rose-red stains 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 DNA is present in both nucleus and cytoplasm, but green stains were not seen in the cytoplasm as mitochondrial DNA was too small to be detected in this experiment.
The light green stains on the MGP slide might either be caused by the loss of methyl green during the staining procedure or due to the low concentration of DNA. The higher the intensity of green and rose-red colouration observed, the higher the concentrations of DNA and RNA (Yuen, 2010). Methyl green might have been lost when the slides were rinsed with distilled water. Therefore, it would be better to avoid rinsing the slides with distilled water or rinse the slides at a shorter time.
To improve this experiment, deoxyribonuclease (DNase) should also be used. DNase is a nuclease that aids in the degradation of DNA into its smaller components by breaking the phosphodiester bonds between the nucleotides. In this way, it has the same purpose as RNase, which is to act 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 only rose-red stains, RNA is present. If the slide stained with MGP contained both rose-red and green stains, DNA and RNA are present. This proves that the green stain is DNA.
Xylene can also be added to the slides for one minute. This will give a transparent background so that the cells would appear clearer under the microscope. Since xylene is carcinogenic, it should be handled with care.
For this experiment, it was necessary to take safety precautions 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 alcohol swab before lancing. The lanced finger should be wrapped with a plaster to prevent infection. In addition, the lancet should not be used more than once. 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 use of MGP. It had also confirmed that methyl green-pyronin stains DNA green and RNA rose-red (Figure 5). The ability of the enzyme, RNase, in degrading RNA was also proved when it was used to confirm the presence of RNA, in which rose-red stains were undetected in the RNase-treated slide. The different types of white blood cells, agranulocytes and granulocytes, could also be distinguished as nucleus of different shapes could be seen through the stains (Figure 5 or 6).