General Observation and Analysis of Animal and Plant Cells

Abstract

The main aim of this experiment is to observe the animal and plant cells, the division of plant cells and to be able to define the unknown sample by given materials. In general, the experiment had major three parts. During the first part of it, two samples of cells were examined under the microscope. Since almost all sample cells are transparent, there were used basic dyes which helped to colorize the specimen due to the chemical reactions such as ionization. As an animal cells human’s oral cavity was used to get the oral epithelial cells, and for the plant cells the onion epidermal was examined. All of them were applied the stained basic dyes according to their cell component differences in colours. Due to the enlarged resolutions of microscopes, the distinction between animal and plant was identified.

The second part of the experiment was about the plant cell division. It aimed to observe the multicellular organisms’ cell division, which are known as eukaryotes and be able to distinguish each phase. For this part also an onion epidermal was taken as the specimen. After being stained for some period of time with the same basic dye, it was examined under a microscope. There were found different phases of cell division. However, it was hard to observe all phases of cell division. Since it is obvious, that any life stops after some time of being cut from life resources, we mostly could see only the last stage of cell division.

In the third part of an experiment we worked with proteins. The aim was to identify the unknown sample according to the curve which is a straight line. The equation for this curve was calculated by examining several solutions with different indexes of concentration where DW and BSA made one solution proportionally.

To conclude, all the living organisms are structured from cells which have certain distinctions for different types, and the proteins are the basic molecules for cells to be formed.

Introduction

Formation of cell was the crucial step in the origin of live, it contains complex proteins and other biological molecules which are enclosed and surrounded by a membrane. So, cell can be defined as the basic unit of life, which can maintain and replicate itself. According to its structure, cell can be classified as eukaryotes and prokaryotes. Prokaryotes are the first organisms which are unicellular that contain genetic information and other biological structures inside the membrane. Eukaryotes are multicellular organisms which are formed later than prokaryotes and have nuclei and other internal organelles that the first ones are lack of that. Nucleus is the largest intracellular organelle that contains chromosome. [1] Because of the transparency of cells, for the animal and plant cells methylene blue and aceto-orcein dyes were used respectively. These allowed nucleuses to be visible.

Figure 1. – Animal and Plant Cells

Cell division is a process by which a parent cell duplicates its genetic data and then splits into two equivalent cells. Cell division plays detrimental roles in the growth and repair of tissues in multicellular organisms, as well as in the reproduction of all organisms. There are two types of cell division: asexual reproduction and sexual reproduction. First, is about where the cell is divided into two cells identical to their parent cell, in other words, it clones itself. The second, sexual reproduction is where daughter cells may have considerable genetic changes due to the fusion of specialized cells named gametes. Prokaryotes due to their simple structure have cell division made up from the binary fission. In eukaryotes, there are two types: mitosis and meiosis. Mitosis has its identical genetic information in their daughter cells, while in meiosis diploid number of chromosomes become haploid in daughter cells. Their cell cycle in general can be viewed in three stages: interphase, mitotic phase and cytokinesis. During the first stage the cell prepares to mitosis division by getting enough resources, in mitotic cycle it is divided into two different cells, and on the final phase two cells are completely formed. [2]

Figure 2.- Mitosis (from left to right: parent, prophase, metaphase, anaphase, 2 daughter cells)

Figure 3.- Meiosis (from left to right: parent, prophase 1, metaphase 1, anaphase 1, telophase 1, 4 daughter cells)

Formation of nucleic acids was crucial step for the evolution of life, they are molecules that could reproduce themselves and maintain the data for the synthesis, or manufacture, of large molecules with complex but stable shapes. These large, complex molecules were proteins. Proteins were essential for the cell to origin, from then build blocks of cells. They can considered as molecules that manage the chemical reactions in cells and contribute much of an organism’s structure. [3]

Materials and Methods

For the first part of experiment there were needed light microscope, slide glass, cover glass, cotton swab, tissue paper, yellow tip, pipette, methylene blue (1% solution), onion, razor blade, aceto-orcein solution. Oral cavity of human was rubbed to obtain the oral epithelial cells, then rubbed onto the slide glass and added methylene blue since the cell is not visible, so it could be colored after being stained for 5 to 10 minutes. The cover glass was put over the slide glass without having any bubbles in order to have a clear image, then the oozing dye was absorbed by the tissue paper. The light microscope was used with three objective lenses to get the image of cells correctly, it helps to view the objects with the 0.2 ㎛ resolution. After the oral epithelial cells, the onion cell was examined with almost the same procedure. Except, its little fresh piece was snapped and teared off in order to get as thinner spread to observe it, also the aceto-orcein dye was used to make the cells visible, it gave them red color.

The second part experiment needed root apex of the onion, isaac of rye, slide glass, cover glass, aceto-orcein, razor, dissection needle, filter paper, optical microscope, pipettes and tips. The root tip of onion was cut with a razor and placed on the slide glass, tissue stained with a 20uL of aceto-orcein and the onion root tip was smashed with a dissection needle. Sample was stained for 5-10 minutes and added a drop of a dye, cover glass was put over the sample and the extra dye was absorbed by the tissue paper. In the end the sample was observed under the microscope. Alongside with the onion root tip, the ear of rye was examined under the same condition with the same manner.

The final part of experiment was done by using the Bradford assay solution (5x), BSA (2 mg/ml), unknown sample, tubes, pipette, DW and spectrophotometer. 2uL of 5x Bradford assay solution was mixed with 8uL of DW in order to get the 1x Bradford assay solution. Then 6 samples were prepared which made up mixes of 980 uL of 1x Bradford assay solution each with 20 uL combination of DW starting from 20 uL with each sapmle decreasing and with the increasing BSA from 0 to 16 uL as shown below.

BSA (2 mg/ml)

1 ul

2 ul

4 ul

8 ul

16 ul

DW

20 ul

19 ul

18 ul

16 ul

12 ul

4 ul

Bradford assay solution (1x)

980 ul

980 ul

980 ul

980 ul

980 ul

980 ul

Figure 4. – Sample solutions

Also, 2uL of unknown sample was mixed with 18uL of DW and 980 ul of 1x Bradford assay solution. In order to regulate the spectrophotometer, the blank solution was used to adjust the first value to zero. All 7 samples tubes were inverted and incubated for at least 5 minutes in room temperature. After their optical densities were measured at the wavelength of 595 nm (600 nm on a spectrophotometer) with the help of spectrophotometer. The obtained values were lately used to plot a graph with the protein concentration on the x-axis and O.D. values on the y-axis, and to get the equation.

Results

The figures provided below are the results of the first experiment, depiction of animal and plant cell.

Sample

Onion epidermal cell

Oral epithelial cell

Figure 5

A. Own slide

B. Prepared slide

C. Own slide

D. Prepared slide

[image: ]400x

[image: ]

400x

[image: ]

400x

[image: ]

400x

Due to the basic dye, the plant cell and its nucleus could be observed. Plant cells are enclosed with the membrane and have nucleuses inside which is deep red color absorbed from aceto-orcein.

Due to the methylene blue dye, the animal cell as its nucleus were visible. However, longer time is needed for the dye to be absorbed better in order to have a vivid and precise resolution. We can observe that animal cells have irregular shapes while plant cell have certain well formed shapes. This is due to their structural differences.

The following figures demonstrate the outputs of the second experiment.

Figure 6.- Meiosis of the ear of rye (400x)

During the experiments it was difficult to define each phase, since the process happens very quickly and the light microscope is a bit weaker to see observe the chromosomes. However, in the ear of rye, in the figure below we can barely see the prophase because it is the stage when chromosomes are most visible; metaphase since chromosomes are gathering at equatorial line; telophase where 4 cells are being divided; and cytokinesis where the cytoplasm is being divided into four to become daughter cells by letting us know that this is meiosis. It was difficult to catch the anaphase which is one of the hardest phases to observe due to its short period.

Phase

Prophase

Metaphase

Telophase

Cytokinesis

Figure 7

On the below picture we can observe the telophase and cytokinesis where cell is being split into two daughter cells which is a property of mitosis. The instant cell division process makes it difficult to observe all the stages. For these two cells to be seen the aceto-orcein dye was used.

Figure 8.- Mitosis of onion epidermal (400x)

The following figure shows a graph plotted from the results of the third experiment.

Figure 9.- Graph of O.D. vs P.C.

From the obtained values we plotted a scatter plot and drew a line that would suit best to these values. According to the calculations we assume the equation to be y=0.04*x+0.0184 and R^2=0.9814. The concentration of an unknown sample was 0.011, we put this value as y and calculated x, then divided x into 2 since the solution was 2 uL. All in all, x is 0,0925.

Discussion

As it was stated in the introduction, nucleus is the biggest organelle inside a cell, it has chromosomes. Prokaryotes are single cellular which means their structure is lot simpler. The biggest difference between prokaryotes and eukaryotes that the last one have nucleus where the most of DNA is concentrated. Actually, eukaryotes have their origin from prokaryotes. They might have been evolved in order to adapt in the harsh environment 2.5 billion years ago when there was no oxygen and air was toxic. Due to the evolutionary changes and needs they developed new organelles like nucleus. [4]

In the first experiment, it was mentioned that animal cell shapes have unordered shape when plant cells have mostly homogeneous rectangular shaped structures. First, we should consider the differences in their cellular structure. Plant cells have comparatively huge vacuole, cell wall, and chromosome. The chromosome is needed for plants because they do not get sugar like animals get it from food to convert into energy. Even though animal cell has vacuoles, they are considerably tiny in contrast with the plant cell’s vacuole which takes more than a half of cell’s area. Finally, we should consider the role of the cell wall that surrounds the membrane. Inner organelles that are lack in animal cells make plant cell less emptier and the cell wall helps to maintain the outer layer. Consequently, all of these distinctions make the plant cells regular shaped. [5]

In the cell cycle the prophase is when the chromosomes are clearly seen. During this phase the chromosomes become more tightly coiled and compact that they can be certainly seen with a light microscope after staining with special dyes. The longest stage in life cycle is the interphase when cell prepares for mitosis, it spends more time in this phase carrying out its specialized functions. Then in order goes prophase where chromosomes start to gather to the centre which takes longer time than following phases; in metaphase they line up in the equator; anaphase manages divided daughter chromosomes to move away to two side; telophase starts after anaphase where chromosomes become less compact and there become two nuclei in a cell. The final stage is cytokinesis where cytoplasm splits which happens quickly. There are some differences between meiosis and mitosis. In the beginning both parent cells have 2n number of chromosomes, and during the prophase in meiosis homologous chromosomes pair up and cross over which does not happen in mitosis. In metaphase, individual chromosomes line up at the equatorial plate in mitosis, and crossed homologous pairs in meiosis. Centromeres separate in mitosis, and homologs in meiosis. There are two identical daughter cells with 2n chromosomes in mitosis after the telophase, and in meiosis after the telophase, two daughter cell undergo the meiosis 2, and overall there are four haploid daughter cell which have genetic differences but same n number of chromosomes. As it can be observed from the results of the experiment there are two daughter cells in mitosis and four in meiosis in cytokinesis. To see this processes with microscope we needed aceto-orcein is a positively charged red basic dye which pulls negatively charged tissue part by giving them a colour. Since, plant cell has green compartments like chloroplast, the red dye is more efficient to use. [6]

Accurate protein quantification is crucial to all experiments related to proteins studies in a multitude of investigation since they play a main role in cell formation as a complex biological molecule. By knowing the concentration of protein of known sample we can derive a concentration of an unknown sample in way of plotting its graph and calculating the formula. This part of the experiment is simple, however, the work with samples of considerably tiny sizes increases the possibility of error occurrence. Since, the liquids have surface tension property they tend to come out from the pipette in lesser amount than needed or initially were taken, moreover, the experiment mostly contained combinations of different solutions, so one error facilitated another error. This can be reduced by repeating the experiment for several times and taking the average measure. Another error can occur from the apparatus and appliances which error can be reduced, except by replacing them with the more powerful instruments. Also, while plotting the graph the equation is calculated as the best suitable equation which means it is not precise, this in consequence, rises another error. There are another types of experiments aimed to quantify the protein. One of them is due to the absorption of the UV lights. There are aromatic amino acids in protein which give a property to absorb the UV light, this produces image where proteins concentration can be seen. This is faster procedure than Bradford assay, however, most error prone since requires extremely pure protein solution and exact coefficient of absorption. Third method foul be the bicinchoninic acid (BCA), it is a certain chromogenic reagent for Cu1+ and in the next phase of the reaction, two BCA molecules react with one Cu1+ ion. The amount of Cu2+decreased is a function of protein concentration that can be determined with a spectrophotometer by a color change of the sample solution from blue into purple, which absorbs light. The absorbance is directly proportional to the amount of protein in the solution and it can be calculated by comparing with a known protein sample. This method is least error prone and can be used for wider range of proteins, but incompatible with detergents. [7]

To sum up, all the parts experiment achieve their aims and statements about the cell structure and its division were observed, also protein concentration was determined.

References

1. Hillis, Savada, Hill & Price. (2013). Principles of Life (Second Edition). Chapter 1.1.
2. Hillis, Savada, Hill & Price. (2013). Principles of Life (Second Edition). Chapter 7.1.
3. Hillis, Savada, Hill & Price. (2013). Principles of Life (Second Edition). Chapter 1.1.
4. Hillis, Savada, Hill & Price. (2013). Principles of Life (Second Edition). Chapter 1.1.
5. Plant vs animal cells. Retrieved from: https://www.khanacademy.org/science/high-school-biology/hs-cells/hs-plant-vs-animal- cells/a/hs-plant-vs-animal-cells-review
6. Hillis, Savada, Hill & Price. (2013). Principles of Life (Second Edition). Chapter 7.2.
7. Protein Quantitation. Retrieved from https://www.labome.com/method/Protein-Quantitation.html