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UNIVERSITI TEKNOLOGI MARA (UiTM)CAWANGAN NEGERI SEMBILAN KAMPUS KUALA PILAHBIO 411: CELL BIOLOGYLABORATORY REPORTPRACTICAL : 2- CELL DIFFERENTIATIONSTUDENT’S NAMES :CLASS GROUP : ASLECTURER’S NAME :1 TITLECell Differentiation1 OBJECTIVES To identify examples of specialised cells. To become familiar ...

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UNIVERSITI TEKNOLOGI MARA (UiTM) CAWANGAN NEGERI SEMBILAN KAMPUS KUALA PILAH

BIO 411: CELL BIOLOGY LABORATORY REPORT PRACTICAL

: 2- CELL DIFFERENTIATION

STUDENT’S NAMES : CLASS GROUP

: AS201

LECTURER’S NAME :

1.0 TITLE Cell Differentiation

1.1 OBJECTIVES 1. To identify examples of specialised cells. 2. To become familiar with the principle of blood smears and Giemsa staining techniques. 3. To be familiar with the proper operation of a microscope for observing the slides. 4. To familiar with the laboratory technique to estimate a sperm concentration using a haemocytometer. 5. To locate actively dividing tissues in plant 6. To familiar with a chromosome staining technique 7. To identify stages of mitosis in plant cells. 8. To record and label the structures observed. 9. To translate the observed technique in a written laboratory report.

2.0 INTRODUCTION In biology, staining techniques is important because it can enhance visualization of the cell or certain cellular components under microscope. Staining cell also can highlight metabolic process and differentiate between live and dead cells in sample. Staining techniques for examine blood smear is Leishman staining. Leishman Stain consists of Methylene blue, and Eosin dye, prepared in Alcohol medium and diluted with buffer or distilled water during staining. This stain use to stain components of blood in range of shade between red and blue. Leishman stain is vital to observe and differentiate nuclear and cytoplasmic morphology cells of blood under the microscope. For example, RBCs, platelets, WBCs as well as for the parasites. After staining with Leishman Stain the colour of chromatin is purple and nucleoli is light blue. The colour of cytoplasm by Leishman Stain are different for each components. Erythrocytes is pink, reticulocytes is dark blue, lymphocytes is blue, monocytes is grey blue, neutrophils is bluish pink and basophils is blue. The staining techniques for sperm cell is differential staining. There are two types of staining techniques for sperm cell under differential staining which are Sperm Blue techniques and

Rapidiff staining or also known as Diff-Quik techniques. When sperm cell stained using Sperm Blue techniques, it had the closets head sperm morphology to those unstained sperm. Silver nitrate staining consider as universal because it used to reveal the cell structure more details. Rapidiff staining techniques is a fast and simple techniques which used silver nitrate solution to stain the spermatozoa. Silver nitrate is one of the alkaline dye and used to identify acidic chromatin protein and nucleolus in mitotic chromosomes. Rapidiff staining techniques stain the nucleoli during mitosis to show the boundary between acrosomal region and post-acrosomal region. For the acrosomal region it stain lighter while for post-acrosomal region stain darker. The staining techniques is important to observe mitosis in onion root tips. Plant cells are glued together by a middle lamella of pectins. During staining we used hydrochloric acid to break down the pectin that hold the cell together. The cell will spread out and separated into a layer, then we can identify the different stages mitosis in the onion root tips cell. Acetic orcein use to stain the chromosomes dark red and fix the cells, stopping mitosis. Further, acetic orcein stain gives better definition of chromosome structure.

2.1 MATERIALS - Blood smear preparation and staining 

Blood sample



Glass slides



Leishman’s stain



Distilled water



Dropper



Petri dish



Compound light microscope



Immersion oil



Cotton



Rubbing alcohol



Pricking device

2.2 METHOD - Blood smear preparation and staining 1. Two glass slides was taken and cleaned with 90% alcohol. 2. A finger was disinfected with alcohol and pricked with a pricking device.

3. A drop of blood was placed at the center of one corner of one of the glass slide. 4. Using a second pre-cleaned glass side, the drop of blood was touched while the slide was inclined at a 45 degree angle. Then, the inclined slide was gently and briskly moved towards the other end of the slide in order to obtain the smear. 5. The smear was allowed to air dry for 1 minute. 6. The slide was placed in a petri dish with the blood smear facing upward. 7. Several drops of the Leishman's stain was added to the blood smear and covered with a petri dish. 8. Twice the volume of distilled water was added to the stain. The water and the stain was allowed to properly mixed and kept aside for 10 minutes. 9. The slide was drained and washed with distilled water. 10. The slide was air dried while kept in an inclined position. 11. The slide was observed under a compound microscope.

2.3 MATERIALS - Calculate sperm concentration using a haemocytometer 

Haemocytometer



3% Saline



Minishaker



Petri dish



Straw



Filter paper



Distilled water

2.4 METHOD - Calculate sperm concentration using a haemocytometer 1. A few drops of 3% Saline was added to the sperm sample. 2. The mixture was made sure to be mixed thoroughly using a minishaker. 3. The cover slip was fixed in place over the counting chambers after the side was moistened to prevent the cover slip from further movement. 4. 10 microliters of sample was aspirated and the pipette tip was placed close to the edge of the cover slip. 5. The sample was slowly ejected. It was repeated on the opposite side.

6. It was made sure to not overflood the chamber and the cover slip was made sure to not move. 7. The haemocytometer was placed in a humid chamber containing moistened filter paper and two straws. 8. Once loaded, the sample was allowed to settle aside for 3 – 5 minutes. 9. The haemocytometer was observed under a microscope.

2.5 MATERIALS - Mitosis in Onion Root tip Experiment 

Onion



Beakers



Toothpicks



Carnoy’s fluid



70% ethanol



1N HCl



Acetocarmine stain



Glass slides



Cover slips



Blade



Petri dish



Tiny vials



Bunsen burner



Blotting paper



Dropper



Forceps



Scissors



Compound light microscope



Immersion oil

2.6 METHOD - Mitosis in Onion Root tip Experiment 1. An onion was taken and fixed on a beaker containing tap water using toothpicks.

2. The base of the onion was made sure to touch the water level. It was kept for a couple of days. 3. Once the roots have grown, one or two centimeters of the root tips were cut out and transferred into a tiny vial containing Carnoy's fluid. The root tips were left in the fluid for 48 hours. 4. A few root tips were taken from Carnoy's fluid and transferred onto a petri dish containing 1N HCl. 5. The petri dish was gently warmed on a flame for 5 seconds. The root tips were exposed in the acid for 2 minutes. 6. The root tips were rinsed in distilled water several times. 7. A few drops of acetocarmine stain was added onto a new petri dish. 8. The root tips were transferred into the petri dish containing acetocarmine stain. 9. The stain was gently warmed on a flame for 5 seconds. 10. The root tips were left in the stain for 5 - 10 minutes. 11. A drop of water was added onto a clean glass slide. 12. The root tips were transferred onto the glass slide. 13. A milimetre of the root tips were removed using a blade and the rest are discarded 14. A cover slip was gently placed on the glass slide. 15. The cover slip was gently tapped a few times by using the blunt end of a forcep until the root tips were properly squashed. 16. The slide was observed under the compound microscope.

3.0 RESULT EXPERIMENT1: Blood smear preparation and staining

Figure 1- Blood smear under the microscope.

Figure 2- Types of Eosinophils observed

Figure 3- Types of Lymphocytes observed.

Figure 4- Types of Monocyte observed

Figure 5- Types of Neutrophils observed

EXPERIMENT2: Sperm concentration calculation using a haemocytometer

Formula: Sperm Concentration (Number of sperm/mL) = Average both sides × 5 × Dilution factor × 10 000 Sample name

Side

Mick

1 2

Counts 1 58 49

2 43 42

3 37 45

4 49 47

5 42 52

Calculation Sum 229 235

Average 232

232 × 5 × 400 × 10000 =4640million/ml

EXPERIMENT3: Mitosis in onion root tip

Figure 6- Root tip of Onion

1) Interphase: Cells in resting phase are easily identified by their prominent nuclei. 2) Prophase: Chromosomal condensation results in thick rope-like chromosomes, which begins to assemble into chromatids and centromers. 3) Metaphase: Chromosome allign along metaphase/ equatorial plate. 4) Anaphase: Chromosomes with seperated sister chromatids begin to move away from the equatorial plate, towards opposite poles in equal numbers. Sister chromatids will reach the opposite poles in equal numbers. 5) Telophase: Chromosome almost fully decondensed to form original chromatin

Figure 7- Stages of mitosis in the root tip of Onion

4.0 DISCUSSION Blood cell is biconcave and disc-shaped which appear as though a donut without a hole in the center, this shape allows the cell to effectively carry oxygen molecule (The Editors of Encyclopaedia Britannica, 2020). For mammals, overall weight of the red cells reduce by absence of the nucleus. Prior to this, it allow the cell to move faster as they transport oxygen. This cell rich in haemoglobin, a pigment composed of four hemmes that make the cell red colour (The Editors of Encyclopaedia Britannica, 2020). Hemoglobin in the cell carry 4 molecules of oxygen because there are four hemes attach to a single protein to form a polypeptide chain (The Editors of Encyclopaedia Britannica, 2020). This structure helps the cell to carry oxygen and transport it to other body cells. In the blood smear, the white blood cells composed has assortment type of blood cells such as neutrophil,

eosinophil, basophil, monocyte, and lymphocytes. Neutrophils, eosinophils, and basophils are granulocytes since the presence of granules in these cells distinguish them from agranulocytes that lack granules.

The most abundant cell in a white cell is the Neutrophil which function as phagocytoses bacteria before hydrolyzing them and secrete a range of proteins that have antimicrobial effects as well as tissue remodelling potential (Sapkota, 2020). Eosinophils which appear spherical with acidophilic refractive granules are proinflammatory mediators so they function in allergy, parasite eradication, and chronic inflammation (Wen, T., & Rothenberg, M. E. 2016). Another granulocyte is basophils, basophils can regulate the behaviour of T cells and control the type of secondary immune responses (Chirumbolo, S., Bjørklund, G., Sboarina, A., & Vella, A. 2018). Monocytes fight certain infections and help other white blood cells eliminate dead or damaged tissues, destroy cancer cells, and regulate immunity against foreign substances (Mary Territo, 2020). Lymphocytes are divided into two types which are T lymphocytes and B lymphocytes. T lymphocytes function to destroy damaged or infected cells while B lymphocytes produce antibodies that destroy specific antigens. T lymphocytes capacity to devastating harms or contaminated cells while B lymphocytes produce antibodies that annihilate explicit antigen (R Luz Elena Cano and H. Damaris E. Lopera. 2013)

For sperm cells, the structure has three parts consist different of head, midpiece, and tail. 10% of the entire cell is head and the nucleus takes up 65% of the sperm’s head and consists of 23 chromosomes (Bingbing Wu1,2, Hui Gao1, 2020). The chromosome will unite with the female gamete once it enters the cell egg and makes up 46 chromosomes (Bingbing Wu1,2, Hui Gao1, 2020). The head is also made up of acrosome and acrosomal cap. The outer membrane of acrosome borders the plasma membrane while the inner acrosomal membrane borders the nuclear membrane. Lysosomal enzymes in acrosome function to degrade the thick membrane of the egg and it is involved in the recognition of oocytes to be fertilized (Khawar, M. B., Gao, H., & Li, W. 2019). The centriole is located between the head and the midpiece of sperm. This structure is involved in the formation of sperm aster and zygote which play important roles in the movement of pronuclear in combination with the female genome (Tomer Avidor-Reiss, Alexa Carr, Emily Lillian Fishman,, 2020). 80 percent of the entire length of sperm made up of sperm tails and it is divided into several parts including connecting piece, midpiece, principal piece,

and end piece (Kumar, N., & Singh, A. K., 2020). The connecting piece connects the flagellum to the sperm head (Kumar, N., & Singh, A. K., 2020). The midpiece has tightly packed mitochondria that provide the energy required for propulsion as the cell travels towards the female gamete and mitochondria play role in control cell death or known apoptosis (Kumar, N., & Singh, A. K., 2020). The principal piece or known as axial filament and end piece help generate the waveform that allows for movement.

For the onion root tip, the plant cell has a fixed shape and closely resembles a rectangle because of the presence of the cell wall. This characteristic proves that onion root tips are plant cells due to the presence of the cell wall. Unlike animal cells, the onion root tip cells are positioned next to each other. Perhaps the fixed shape allows them to form such an arrangement. The process of mitosis in onion root tip is divided into five stages start from prophase, prometaphase, metaphase, anaphase, and telophase (Jain, 2015). During prophase, the chromosomes supercoil, formation of the fibers of the spindle apparatus begin between centrosomes at the pole of the cells (Jain, 2015). The nuclear membrane disintegrates and freeing the chromosomes into the surrounding cytoplasm. Next is prometaphase, during this stage some of the fibers attach to the centromere of each pair of sister chromatids and they begin to move toward the center of the cell (Jain, 2015). During metaphase, the chromosomes come to rest along the center plane of the cell (Jain, 2015). During anaphase, the centromeres split and the sister chromatids begin to move toward the opposite poles of the cell (Jain, 2015). During the last stage which is telophase, the chromosomes at either end of the cell begin to cluster together and form new nuclear membrane (Jain, 2015). Cytokinesis occurs during this stage, leading to two separate cells (Jain, 2015).

The stain used to differentiate human and bacterial cells is Giemsa Stain and contains a mixture of Azure, Methylene blue, and Eosin dye (Rijal, 2019). Giemsa stain is specific for the phosphate groups of DNA and it will attach at high amounts of adenine-thymine bonding (Rijal, 2019). Azure and eosin are acidic dye stains cytoplasm and granule, methylene blue acts as the basic dye and stains the acidic components like the nucleus of the cell (Rijal, 2019). In Giemsa stain, we used methanol as a fixative to not allow any further change in the cells and make them adhere to the glass slide (Rijal, 2019). Aceto-carmine behaves like a basic dye. It stains both the nucleus, cytoplasm and used for the rapid staining of fresh unfixed chromosomes (Rijal, 2019).

To stain chromosomes while keeping the cytoplasm colourless, the sample should be first treated with formaldehyde then, hydrolyzed with HCl at 60 °C with the correct hydrolyzing time (Lakna, 2018). Hydrochloric acid can breaks down the cellulose walls of plant cells which are held together by pectins such as calcium pectate (Lakna, 2018). Finally, it can be treated with acetocarmine. Acetocarmine produces large dye aggregates in weakly acidic conditions (pH 45) (Lakna, 2018). They bind permanently to the nucleoprotein component of chromatin. That why chromatin can be visualized by treating it with the two dyes. Acidic substances are removed from all parts of the cell except the nucleolus and its derivatives (Lakna, 2018). The residual acidity, which is dependent on formaldehyde for its resistance to hydrolysis, may well be due to phospholipids in the nucleolus. For this reason, the method may be specific for this class of compounds. Considerable confirmation, however, is required to justify the technic being called a histochemical one. On the other hand, the method appears to be capable of distinguishing accurately between nucleoli and chromosomes.

In the field of medical laboratory and agronomic staining techniques have been applied for diagnosing infectious disease (Thairu, Yunusa. and Nasir, Idris. and Usman, Yahaya, 2014). For instance, the Gram stain was used to differentiate gram-positive and gram-negative bacteria. The main benefit of a gram stain is that it helps doctor learn if we have a bacterial infection, and determines what type of bacteria are causing it (Thairu, Yunusa. and Nasir, Idris. and Usman, Yahaya, 2014). This help a doctor to determine an effective treatment plan. Staining is the only method to detect the presence of an organism in the clinical specimen and it provides rapid and cost-effective information for preliminary diagnosis of infectious diseases (Thairu, Yunusa. and Nasir, Idris. and Usman, Yahaya, 2014). Acid-fast staining is used primarily to detect Mycobacterium tuberculosis and other severe infections. Many infectious agents grow slowly on culture media or may not grow at all.

5.0 CONCLUSION In conclusion, we observed that white blood cell in blood smear composed a lot of blood cells. These blood cell including neutrophil, eosinophil, basophil, monocyte and lymphocyte play important roles for immunity system. For the sperm cell, we observed that the structure of sperm

consist of head, midpiece and tail part. The head part made up of acrosome and acrosomal cap, the midpiece part composed of centriole. The tail divided into several parts including connecting piece, midpiece, principal piece, and end piece. Differences in sperm head dimensions after the application of different staining techniques are due to the fixatives and chemical reagents used in the procedure. This lead to many type of staining techniques can be used to stain sperm cells. During the onion root tips experiment we observed that mitosis in plant cell divide into five stage start from prophase, prometaphase, metaphase, anaphase, and telophase. Each stage of the process is vital and have their own function to produce two daughter cells. Lastly, we discovered that Aceto-carmine used as a basic dye for Giemsa staining to stain both the nucleus, cytoplasm and used for the rapid staining of fresh unfixed chromosomes. Hence staining techniques is important so we can observe the cell more detail under the microscope.

6.0 REFERENCES Bingbing Wu1,2, Hui Gao1. (2020).The coupling apparatus of the sperm head and tail. doi:https://doi.org/10.1016/j.mce.2020.110987 Brian J. Ford and Robert R. Shannon. (20 August, 2020). Microscope instrument. Retrieved 30 August, 2020, from https://www.britannica.com/technology/microscope Cano RLE, L. H.-V. (18 July, 2013). Introduction to T and B lymphocytes. (A. F. Bedside, Ed.) R...