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All eukaryotic cells have some features that are in common. These include nucleus, cytoplasm, cell membrane, mitochondria, endoplasmic reticulum, Golgi apparatus, ribosomes, cytoskeleton and centriole (National Center for Biotechnology Information (NCBI) 2004).
According to NCBI (2004), eukaryotes cells can be found in fungi, animals, and plants as well as some unicellular organisms. Eukaryotic cells have a length of 10 to 100 micrometers and a size of 10 times a prokaryotic cell. The membrane-bound compartment of the eukaryotic cells is where specific metabolic activities take place. Eukaryotic organisms also have other specialized, membrane-bounded structures, called organelles, which are small structures within cells that perform dedicated functions. But the most important feature is the nucleus as it is a membrane-delineated compartment that houses the eukaryotic cell's DNA.
White Blood Cells
According to Eldridge (2009), white Blood Cells or leukocytes are an immune cell which protects the body by defending the body against infectious disease and foreign materials.
Figure 2 - Two types of Leukocytes. (http://www.ecc-book.com/)
There are two main types of leucocytes as shown in figure 2 - granulocytes and agranulocytes. Granulocytes contain granules of enzymes and are classified as neutrophils, basophils or eosinophils while agranulocytes lack granules of enzymes and are classified as monocytes or lymphocytes. Leucocytes can be classified by staining with haematoxylin-eosin and then classify according to their resultant colours and appearances. Granulocytes have multi-lobe nucleus while agranulocytes appears in a single, large nucleus when observed under light microscope. Due to the stain that was used in this practical, we can only group them as granulocytes and agranulocytes but unable to distinguish between neutrophils, basophils, eosinophils, monocytes or lymphocytes (Yuen, 2010).
Nucleic acids are macromolecules found in all cells and viruses essential for life. The functions of nucleic acids have to do with the storage and expression of genetic information. There are two classes of nucleic acids which consist of deoxyribonucleic acid (DNA) which encodes the information the cell needs to make proteins and ribonucleic acid (RNA) which comes in different molecular forms that participate in protein synthesis (Brody 2010). Nucleic acids are made up of nucleotides linked by phosphodiester bonds. Each nucleotide consists of three components: nitrogenous base, pentose sugar and phosphate group.
Deoxyribonucleic Acid (DNA)
Figure 3 - A DNA double helix structure. (NES Health 2011)
"DNA is the chemical name for the molecule that carries genetic information in all living things." (Austin 2010) Genetic information is used to develop and give function to all known living organisms (with the exception of RNA viruses). Most DNA is found within the cell nucleus whereas there are small amount of DNA found in white blood cells occurs generally in the nucleus as linear chromosomes and in the cytoplasm. DNA has two strands of antiparallel nucleotides which joined together by hydrogen bonds and formed a double helix structure as shown in figure 3. Each strand composed of alternating phosphate groups and pentose sugar with four chemical bases adenine, cytosine, guanine and thymine attached to it (Scovell 2011).
Ribonucleic Acid (RNA)
Ribonucleic acid (RNA) is a molecule similar to DNA. Unlike DNA, RNA is single-stranded. An RNA strand has a backbone made of alternating phosphate groups and ribose sugar with four chemical bases adenine, uracil, cytosine, and guanine attached to it (Biesecker 2010).
RNA molecules are synthesized from DNA templates in a process known as transcription which converts genetic information from genes into the amino acid sequences of proteins. . It is convenient to divide RNA molecules into the three functional classes, all of which function in the cytoplasm (Scovell 2011).
According to Scovell (2011), there are 3 types of RNA which includes:
188.8.131.52 Messenger RNA: encodes amino acid sequence of polypeptide or protein
184.108.40.206 Transfer RNA: brings amino acids to ribosome during translation
220.127.116.11 Ribosomal RNA: bind with ribosomal proteins to make up ribosomes, the organelles that translate the mRNA
Methyl Green Pyronin
DNA has special affinity with Methyl green, which stains DNA green, and pyronin for RNA, which stains rose red. The higher the amount of DNA and RNA in the sample, the higher the intensity of green and rose red colouration observed, the higher the concentrations of DNA and RNA, respectively (Yuen, 2010).
The methyl green-pyronin uses high net negatively charge of nucleic acids. Methyl green is a cation which binds rather specifically to DNA and hence it acts as an appropriate means of staining nuclei in both fixed material and living cells. Pyronin, a red dye, is rather specific for RNA with some binding to protein (Heidcamp 2010).
RNase and DNase-Treated Control slides
To confirm the staining specificity for RNA, the usual way is to prepare a control slide. "Control slides are important in interpreting the results of methyl green-pyronin staining, since the procedure is readily susceptible to artifact. One or both of the nucleic acids should be removed either enzymatically or by acid extraction". (Heidcamp 2010, exercise 2.4)
A slide stained with the methyl-green pyronin mixture is known as an untreated slide and a slide pre-treated with ribonuclease (RNase), an enzyme that catalyses the degradation of RNA, is known as control slide. RNase control is required to confirm the structures stained with pyronin on the untreated slide as they contain RNA. The same theory applies to DNase-treated slides, but in this experiment it is confirm that the observed nucleic acid is RNA in the DNase-treated slide and the untreated slide contain DNA (Yuen 2010).
2. Material and Methods
The following materials were used in this experiment: two frosted slides, 95% ethanol, sterile lancet, sharp box, sterile alcohol swab, cotton wool, Coplin jar containing carnoy fixative, Coplin jar containing 95% ethanol, Coplin jar containing distilled water, 0.1% aqueous solution of RNase, water bath, methyl green-pyronin, Permount, coverslip and microscope.
2.2 Preparation of blood smears
First two frosted-end microscope slides were cleaned thoroughly by dipping in 95% ethanol and wipe with Kim Wipes. This was done to disinfect the slides and prevent contamination. Aseptic techniques should be observed when obtaining blood sample. Next wash hands with soap and water, and dry them. After washing, the tip of the middle finger was cleaned with a sterile alcohol swab. The fingertip was lanced with a sterile lancet, squeezed, and first drop of blood was wipes away to avoid dilution with tissue fluid. The lancet should only be used once and discard the lancet into the sharp box.
Figure 4 - Making a blood smear. (http://www.tpub.com)
The finger was squeezed again and a drop of blood was placed near one end of the slide. After the required blood was obtained, pressure was applied with a sterile cotton wool to the lanced finger. A second slide was hold with an edge of about 30ï‚° angle on the first and place it towards the drop. The blood drop will then spread along the edge of the slide; the second slide was pushed to the other end in one smooth action. A second blood smear was made by repeating the procedure.
2.3 Stain the blood smear with MGP
The blood smears were then dried by using a hair dryer. A dried blood smear will be dark brown in colour. The dried smears were placed in a Coplin jar containing carnoy fixative for 10 minutes to fix the smears. After fixing, the smears were transferred to another Coplin jar containing 95% ethanol for few seconds so to dehydrate the smears. After dehydrating, the smears were placed in a Coplin jar with distilled water for 2 minutes to rinse off the unwanted chemicals on the slides. Lastly, a paper towel was used to wipe off the excessive water on the slides gently and the slides are ready to be used.
The 2 stained slides were then labelled with initials. Other than the initials, one slide was labelled MGP and the second slide was labelled RNase + MGP where it will be pre-treated with RNase.
2.4 Treating slides with RNase
The slide labelled with RNase + MGP was placed in a 0.1% aqueous solution of RNase with a pH of 6.5 -7.0 in a Coplin jar. The Coplin jar was then place in a water bath at 37Â°C for 15 minutes. A forceps was used to place and removed the slide from the jar. After 15 minutes, the slide was removed from the jar and was dipped into another Coplin jar containing distilled water for a few seconds.
The both slides (MGP and RNase + MGP) were placed in the methyl green-pyronin staining solution for 10 minutes. After 10 minutes, the slides were then rinsed in distilled water for 2-3 seconds in a Coplin jar. Lastly, the slides were removed and Kimwipe was used to wipe the back of each slide, the slides were being air dried in a near vertical position.
2.5 Microscopic examination of slides
In this experiment, Permount was used, a drop of Permount was added onto the slide and the coverslip was slowly lowered so as to cover the smear and to minimise air bubbles. The excess liquid was gently press out with paper towel. Each slide was then observed under microscope of 40X and 100X.
Results and Discussion
3.1 Slide stain with MGP
Figure 6 - Granulocytes under microscope stain with MGP
Figure 7 - Agranulocytes under microscope stain with MGP
DNA has special affinity with methyl green which gives a green stain that indicates the presence of DNA, while RNA has special affinity with pyronin which gives a rose red stain indicates the presence of RNA. The higher the intensity of green and rose red stain, the higher the concentration of DNA and RNA, respectively.
The green stain of DNA is in the shape of multi lobe nucleus where it is known as granulocytes (shown in figure 4) and single large nucleus where it is known agranulocytes (shown in figure 5). Therefore from this experiment it can be seen that granulocytes and agranulocytes were present and they can be distinguish by their different nucleus shape. The blood smear also has high concentration of DNA and RNA as the intensity of green and rose red was high.
3.2 Slide stain with MGP & RNase
Figure 9 - Agranulocytes under microscope stain with MGP & RNase
Figure 8 - Granulocytes under microscope stain with MGP & RNase
Another slide which was treated with MGP was then pretreated with ribonuclease (RNase). RNase is an enzyme that catalyses the degradation of RNA. From figure 6 & 7, DNA was present as light green stains were observed. DNA appears in a lighter green as some methyl green might be washed off during the pre-treatment with RNase or maybe the intensity of DNA was lower in this blood smear. Granulocytes and agranulocytes are also distinguished by their different nucleus shape.
RNA was not present as rose red stains were not observed, due to the treatment of RNase. Since RNase was used as a control, the absence of rose red stains on the RNase treated slide indicates that the rose red stain present on the MGP treated slide was RNA.
In this experiment, two slides of blood smear were treated with MGP & MGP + RNase. For the slide that was treated with MGP, DNA was stained green while RNA was stained rose red. Green stain of DNA was being observed to distinguish granulocytes and agranulocytes with the differences in nucleus shape under the light microscope. While for the slide that was treated with MGP + RNase, only DNA was observed as RNA was being degraded. DNA was stained light green. Since RNA was not observed in the MGP + RNase slide, it indicates that the rose red stain observed on the MGP slide was RNA as RNA appear rose red when stain with MGP. The experiment was successful as all the objectives were being met.