Laboratory Activity 1, Cell Morphology and Ultrastructure PDF

24/09/2024 Laboratory Activity 1 Cell Morphology...

24/09/2024 Laboratory Activity 1 Cell Morphology and Ultrastructure DN-DEN 121L 1 1 Learning Objectives: At the end of this laboratory activity, the students are expected to: 1. Identify key features of a cell. 2. Differentiate the cellular morphology of bacterial, plant, and animal cells. 3. Enumerate various cell ultrastructure that allow the cell to function. 2 2 1  24/09/2024 Wet Mount Method In a wet mount, the specimen is placed in a drop of water or other liquid held between the slide and the cover slip by surface tension. This method is commonly used to view microscopic organisms. Advantage: quick preparation 3 3 Microscope Slide Staining Microscope cell staining is a technique used to enable better visualization of cells and cell parts under the microscope. By using different stains, a nucleus or a cell wall are easier to view. Cells may also be stained to highlight metabolic processes or to differentiate between live and dead cells in a sample. Any basic dyes, such as methylene blue, crystal violet, malachite green, or safranin work well. 4 4 2  24/09/2024 Some Basic Dyes 1. Methylene blue stain - a solution used to highlight certain cell parts of animal, bacteria, and blood tissue specimens. This stain is a 1% aqueous solution, and it's especially useful for microscope slide preparation. 2. Iodine - Iodine binds to starch, making a blue-black color; gives color to the epidermal cells, making them more readily visible and observable. 3. Wright stain- hematologic stain that facilitates the differentiation of blood cell types. It is classically a mixture of eosin (red) and methylene blue dyes. 4. Giemsa staining solution- composed of methylene blue, azure and eosin; commonly used in hematology, histology, cytology and bacteriology 5 5 A. Preparation of Wet Mount of Cheek Epithelial Cells www.studyrankers.com 6 6 3  24/09/2024 Normal Saline Solution for Cheek Cells Purpose: to expose the cells to a solution similar to the one they are exposed to while in the mouth Importance: 1. Osmotic Balance: preventing cells from bursting or collapsing 2. Cell Integrity: preserves the structural integrity of the cells 3. Staining Aid: facilitates the staining process 7 7 Role of Methylene Blue in Cheek Cell Staining 1. Staining Nuclei 2. Enhancing Contrast 3. Cell Visualization https://urbanenjoy.com/en/?w=what-organelles-would-be-visible-in-a- cheek-cell-uu-BqdYa2F2 8 8 4  24/09/2024 Preparation of Plant Tissue Specimen Slide https://x.com/EducatingMiss/status/11163577 14978975744 9 9 Iodine for Onion Cells 1. Staining Cell Walls 2. Enhancing Contrast http://www.microscopy- uk.org.uk/mag/indexmag.html?http://www.microscopy- uk.org.uk/mag/artoct11/wd-onion9.html 10 10 5  24/09/2024 Bacterial cells Living bacteria are almost colorless, and do not present sufficient contrast with the water in which they are suspended to be clearly visible. The purpose of staining is to increase the contrast between the organisms and the background so that they are more readily seen in the light microscope. In a simple stain, a bacterial smear is stained with a solution of a single dye that stains all cells the same color without differentiation of cell types or structures. One example dye use is methylene blue, a basic stain. Basic stains, having a positive charge, bind strongly to negatively charged cell components such as bacterial nucleic acids and cell walls. 11 11 Bacterial cells: Gram staining What is the principle of gram staining? Gram staining differentiates bacteria by the chemical and physical properties of their To gather more information about First developed by the Danish bacteriologist cell walls by detecting peptidoglycan, bacteria, gram Hans Christian Gram which is present in the cell wall of Gram- staining can be used. in 1882 positive bacteria. Gram-negative cells also contain peptidoglycan, but a very small layer of it that is dissolved when the alcohol is added. 12 https://bio.libretexts.org/ 12 6  24/09/2024 13 13 14 https://www.technologynetworks.com/immunology/articles/gram-positive-vs-gram-negative-323007 14 7  24/09/2024 Prepared Escherichia coli Staphylococcus aureus Proteus vulgaris Bacterial Slides Cell Shape Rod cocci rod Cell Size 0.5 μm in 0.5 – 1.0 μm in 0.4–0.8 μm in diameter diameter diameter Notable Cell wall, Cell wall, capsule Flagella, cell structures flagella, villi, wall, capsule, capsule fimbriae 15 www.eiscolabs.com 15 CELL FEATURE ANIMAL CELL PLANT CELL BACTERIAL CELL Cell Wall Absent Present Present Plasma Membrane Present Present Present Nucleus Present Present Absent Ribosomes Present Present Present Mitochondria Present Present Absent Chloroplasts Absent Present Present Vacuoles Absent Present Absent Lysosomes Present Present Absent Cytoplasm Present Present Present Flagella/Cilia Present Absent Present Cell Size 10–20 μm 10-100 μm 0.5–5.0 μm 16 16 8  24/09/2024 Laboratory Activity 2 Observation of Mitotic Phases from Meristem and Calculation of the Mitotic Index DN-DEN 121L 17 17 Learning Objectives: At the end of this laboratory activity, the students are expected to: 1. Identify the stages of mitosis in meristem cells using a microscope. 2. Calculate the mitotic index by counting cells in different mitotic phases. 3. Observe chromosomal events during the various phases of cell division in meristem tissues. 18 18 9  24/09/2024 Mitosis Mitosis term was coined by Walter Flemming in 1882. The term mitosis is derived from the Greek word ‘mitos’ meaning warp thread. Mitosis is a type of cell division that results in two identical daughter cell with same set of chromosomes as parental cells. It results in increase in the number of cells at given time and space, i.e., proliferation. It is needed for general growth of living entities and replacement of damaged cells in the case of injuries or wound healing. 19 19 Why onion root tip for lab experiment? The onion roots grow rapidly in large number. The cells at the tip are actively dividing and therefore all cell cycle stages are easy to visualize in single preparation. The onion tips are easy to prepare for on microscopic slide. The slide preparation can be stained to make them more observable. Thies & Grossman, 2006; DOI: 10.1201/9781420017113.ch5 20 20 10  24/09/2024 Why onion root tip for lab experiment? There are 3 zones (regions) near the tip of the onion root. 1. Region of cell division: where size is not significantly increasing; aka meristem 2. Region of elongation: cells are increasing in size, but not dividing. Due to cell elongation, roots are lengthened. 3. Region of maturation: It is also called as the region of differentiation or root-hair zone. Cells mature into the various types of primary tissues. L.S. of root apex showing terminal root cap and sub apical meristem. 21 21 Observation Interphase: densely stained homogenously granular nucleus; distinct nuclear boundary; nucleolus may also be observed Prophase: chromatin appears as a network of fine threads; nuclei may or may not be visible. Metaphase: nuclear boundary is not seen; chromosomes are thick and arranged at the equatorial plane of the cell Anaphase: chromatids separation can be seen; chromosomes appear to be moving apart Telophase: chromosomes observed at two poles as chromatin mass; nuclear membrane appears to form on both the chromatin mass at the poles. Cytokinesis: The cell plate begins to form at the middle after telophase, which are extending outwards to Mitosis, Teaching biology, Biology classroom ultimately reach the margin of the cells and divide the https://www.pinterest.com/pin/747456869374725427/ cell into two. 22 22 11  24/09/2024 Cytokinesis Cytokinesis is the process by which one cell physically separates into two daughter cells at the end of each division cycle. Cytokinesis: cytos means cell and kinesis mean division s The cytokinesis is of two types, one that occurs in the plant cell is cell plate formation and the other in the animal cell is embryonic cleavage. https://bio.libretexts.org/ 23 23 Mitosis Mitosis is the phase of the cell cycle where the nucleus of a cell is divided into two nuclei with an equal amount of genetic material in both the daughter nuclei. DNA condenses into chromosomes during prophase, chromosomes line up during metaphase and separate during anaphase, and separate nuclei form during telophase. The major purpose of mitosis is for growth and to replace worn out cells. 24 24 12  24/09/2024 Mitotic index Mitotic Index (MI) is defined as a ratio of the total number of dividing cells (n) and the total number of cells (N) in a particular focus chosen randomly under the microscope. Therefore, By randomly selecting 5 to 10 foci, mitotic index can be estimated. 25 25 Mitotic index Calculate MI: https://ib.bioninja.com.au/standard-level/topic-1-cell-biology/16-cell-division/mitotic-index.html 26 26 13  24/09/2024 Mitotic index Calculate MI: https://ib.bioninja.com.au/standard-level/topic-1-cell-biology/16-cell-division/mitotic-index.html 27 27 Importance of Mitotic Index The mitotic index can be used to examine cell proliferation in primary root tissue. The mitotic index provides a measure of the capacity of cells to divide and of the rate of cell division. It is used to identify the sites of growth within a tissue and to determine which cell types are dividing. Interpretation: In general, for plant cells the mitotic index decreases with increasing distance from the root cap junction. 28 28 14  24/09/2024 Guide Questions Aside from the mitotic phases (PMAT), what other notable observations or events did you notice on the given slide? 1. Many cells may at interphase stage. 2. The degree of chromatin condensation varies 3. Variations in cell size and shape, which could indicate differences in cell type, function, or stage in the cell cycle. 4. Staining Variations: The intensity of staining can provide clues about cellular components; for instance, darker staining might indicate more abundant RNA or DNA. 5. Signs of Apoptosis: In some cases, some cells are undergoing programmed cell death, which can present as cell shrinkage, fragmentation, or the presence of apoptotic bodies. 29 29 Guide Questions What is the Mitotic Index? What does a high or low mitotic index indicate? What is the medical significance of the mitotic index? A high mitotic index indicates a greater number of actively dividing cells, often associated with aggressive tumor behavior, while a low mitotic index suggests slower growth and potentially less aggressive tumors(Clahsen et al., 1999)(Kroese et al., 1990). Medical Significance of the Mitotic Index 1. Prognostic Indicator: MI serves as a crucial prognostic factor in various cancers, including breast cancer, where it correlates with disease-free survival(Clahsen et al., 1999). 2. Tumor Classification: In uterine smooth muscle tumors, accurate MI assessment aids in proper classification, impacting treatment decisions(Veras et al., 2009). 3. Comparative Analysis: The MI can be compared with apoptotic indices to evaluate tumor behavior, as seen in studies of melanoma and basal cell carcinoma(Mooney et al., 1995). 30 30 15  24/09/2024 End of Discussion 31 31 16