Exercise No. 2: Microscopic Observations of Mitotic Divisions - PDF

EXERCISE INSTRUCTIONS 2 - MOLECULAR BIOLOGY / Medicine Faculty / Academy of Silesia / Sem. 1 / 2024/25 Exercise no. 2. Microscopic observations of mitotic divisions of meristematic tissue cells of Allium cepa onion using immersion. Theoretical part A student: ✓ knows the construction of an optic...

EXERCISE INSTRUCTIONS 2 - MOLECULAR BIOLOGY / Medicine Faculty / Academy of Silesia / Sem. 1 / 2024/25 Exercise no. 2. Microscopic observations of mitotic divisions of meristematic tissue cells of Allium cepa onion using immersion. Theoretical part A student: ✓ knows the construction of an optical microscope, ✓ knows the phases of mitosis. Construction of an optical microscope. The microscope is one of the basic devices in a biological laboratory. Two systems are combined in the construction of the microscope: optical and mechanical. The optical system consists of two interconnected parts: lighting and magnifying. The first one is used for optimal illumination of the observed object (preparation), while the second one is used for two-stage magnification of its image. The mechanical system ensures proper positioning of the individual elements of the optical system. In the microscope, the resulting image is simple, magnified and virtual. Page 1 of 9  EXERCISE INSTRUCTIONS 2 - MOLECULAR BIOLOGY / Medicine Faculty / Academy of Silesia / Sem. 1 / 2024/25 OPTICAL ELEMENTS OF THE MICROSCOPE The light source – a mirror was this element in simple microscopes, nowadays it is a built-in light bulb with a reflector. Lighting should be adapted to the magnification, use weak light intensity with low magnifications, with high - strong. The condenser - it is a set of 2-3 lenses strongly concentrating light forming a cone sufficient to illuminate the field of the preparation in question. The diaphragm regulates the amount of light entering the condenser. The lenses - elements that magnify the image. They create a real image of the preparation. They collect the light coming from the object and create its magnified intermediate image when viewed through the eyepiece(s) of the microscope. We distinguish dry (air) and wet (immersion) lenses. Dry lenses magnify up to 60 times, immersion lenses 90 to 150 times. The immersion lenses require the lens to be immersed in a liquid that is applied between the lens and the preparation, i.e. in the space between the cover glass of the preparation and the tip of the lens. Immersion oil is commonly used, which can also be applied directly to the specimen without the use of a coverslip. The use of immersion can increase the resolution of the microscope up to 0.16 µm. The use of immersion prevents the scattering of light rays that refract after passing through the basic and cover glass (optically denser environment) to the optically rarer environment, which is air. The lens with immersion liquid is called immersion lens. Attention! The immersion lens is labelled OIL or IMMERSION OIL. The ocular lens are made of a series of lenses and magnify the image like a magnifying glass. They create a virtual image of the preparation. They usually magnify from 2-30 times. The magnification of the eyepiece is indicated on the frame. The magnification of a microscope is equal to the product of the magnification of the objective lens and the magnification of the eyepiece, and can be up to 2000 times. The resolving power of the microscope is defined as the minimum distance of two objects at which these objects can be seen separately. This size depends on the lens used and can be about 0.25 µm that is 250 nm for an optical microscope. MECHANICAL ELEMENTS OF THE MICROSCOPE The base and arm ensure the rigidity of the structure. The mechanical stage is used to fix the preparation and move it horizontally in the X and Y axes, which changes the field of view. Page 2 of 9  EXERCISE INSTRUCTIONS 2 - MOLECULAR BIOLOGY / Medicine Faculty / Academy of Silesia / Sem. 1 / 2024/25 The coarse adjustment knob (macrometer screw) - used to set the distance between the preparation and the objective lens. Depending on the design, the screw raises/lowers the stage or objective lenses. The fine adjustment (micrometer screw) - used for focusing. The nose piece (revolver) - this is a rotating microscope disk in which the objective lenses are placed. Its rotation allows you to easily change the lens and thus the magnification used. The head (tube) is the space between the lens and the eyepiece where the image is formed. The mechanical system of the condenser allows you to adjust the position of the condenser vertically. Obligatory literature for the test Bruce Alberts, Dennis Bray, Karen Hopkin, Essential cell biology. Sixth edition. New York: W. W. Norton & Company, 2023, Chapter 1. Cells: The Fundamental Units of Life “Seeing cell structure” pages: 6-11, panel 1-1 pages: 12-13. “The Eukaryotic cell”, pages: 18-27 and Chapter 18. The Cell- Division Cycle, pages: 635-667. A student: ✓ describes the course of successive stages of interphase and mitosis in eukaryotic cells, ✓ characterizes structural modifications of chromatin in the course of the cell cycle, ✓ explains the mechanism of replication in the S phase and chromosome condensation in mitosis, ✓ recognizes the phases of mitosis in microscopic preparations, draws and describes the picture of the phases of mitosis. CELL CYCLE The cell cycle is an ordered sequence of events that occur in the cell between successive mitotic divisions (i.e. during the interphase). The main stages of the cell cycle are the following phases: G1, S and G2, and the M phase, i.e. mitosis. The G1 and G2 phases (Engl. gap) are stagnation phases in which the processes that prepare the cell for DNA replication (G1) or mitosis (G2) take place. In the S phase, DNA replication takes place leading to the doubling of each chromatid (this prepares the cell for the next division). The tasks of the cell cycle are: exact doubling of DNA content in chromosomes,, precise distribution of DNA copies in genetically identical cells. This division plays a very important role in the growth and regeneration processes of organisms. It also enables vegetative reproduction and an increase in the number of eukaryotic, single-celled Page 3 of 9  EXERCISE INSTRUCTIONS 2 - MOLECULAR BIOLOGY / Medicine Faculty / Academy of Silesia / Sem. 1 / 2024/25 individuals. The eukaryotic cell cycle usually occurs in four phases The cell-cycle control system ensures that key processes in the cycle occur in the proper sequence. B. Alberts. Essential cell biology. Fifth edition. New York: W. W. Norton & Company, 2019 Interphase is the stage of the cell cycle that covers the period between successive cell divisions (mitosis). This is the longest stage of the cell cycle (except for very intensively dividing cells). Interphase is the period of the most active metabolic changes. During interphase, the chromosomes are in an non-condensed state and are not visible under the microscope as separate entities. This enables intensive gene expression and full metabolic activity of the cell. The following can be distinguished in the interphase: phase G1 -the pre-DNA synthesis phase. This is the period between the M phase and the beginning of the synthesis phase. During this time, the mass and volume of the cell increase intensively, phase S - the phase of DNA synthesis, during which the cell replicates nuclear DNA (the DNA content increases from 2n to 4n), phase G2 - the pre-division phase, which is the period between the end of the S phase and the beginning of the M phase. Chromosomes condense. Elements for the construction and operation of the mitotic apparatus are synthesized. The G1 and G2 phases are breaks in the cell cycle during which the cell "checks" whether the conditions, both internal and external, are suitable for entering the next phase (S or M phase). Both of these phases (G1 and G2) give the cell extra time to grow and multiply its cytoplasmic cell organs. There is also the G0 phase, known as the resting phase. Cells in the G0 phase do not divide but retain their remaining metabolic functions. The entry of cells into the G0 phase can be caused, among others, by: the end of the cell differentiation process, lack of nutrients as well as damage to the genetic Page 4 of 9  EXERCISE INSTRUCTIONS 2 - MOLECULAR BIOLOGY / Medicine Faculty / Academy of Silesia / Sem. 1 / 2024/25 material. The quiescent state of the cell can last from several days to several months. Mitosis is the division of a cell nucleus into two daughter nuclei. It takes place in the somatic cells of eukaryotic organisms. Each daughter nucleus receives the same set of chromosomes, identical to what it was in the parent nucleus before cell division. A nucleus with a diploid number of chromosomes (2n) after mitosis gives two nuclei also with a diploid number of chromosomes. The M phase consists of karyokinesis / mitosis (division of the cell nucleus) and cytokinesis (division of the cytoplasm). The following phases can be distinguished in karyokinesis, or mitosis: prophase - chromatin condenses, chromosomes begin to be visible, doubled chromosomes consist of two closely linked sister chromatids, a mitotic spindle is formed outside the nucleus between two centrosomes, which begin to move away from each other, prometaphase begins with the breakdown of the nuclear envelope, chromosomes can attach to the spindle microtubules through their kinetochores and begin to move, metaphase - chromosomes align in the equatorial plane of the spindle, and paired kinetochore microtubules on each of the sister chromatids attach them to opposite spindle poles, kinetochores of all chromosomes are aligned in the midplane between the two spindle poles, anaphase - sister chromatids separate synchronously and daughter chromosomes are formed, which move to opposite poles of the cell; kinetochore microtubules shorten and the spindle poles move away from each other, owing to these processes the chromosomes separate, telophase - daughter chromosomes reach the spindle poles; the nuclear envelope is reassembled around each set of chromosomes; two new nuclei are formed. The division of the cytoplasm begins with the assembly of the contractile ring. Cytokinesis - the cell is divided into two parts by a contractile ring made of actin and myosin filaments. This ring tightens in the cell and creates two daughter cells, each of which has one cell nucleus. Page 5 of 9  EXERCISE INSTRUCTIONS 2 - MOLECULAR BIOLOGY / Medicine Faculty / Academy of Silesia / Sem. 1 / 2024/25 PRACTICAL PART A student: uses an optical microscope fluently and is able to adjust the image of the preparation using various lenses, uses immersion, documents work with a microscope by making drawings of selected image fragments, uses theoretical knowledge about the phases of mitosis for practical purposes, e.g. finding and interpreting selected fragments of a specimen image. Exercise 1 Describe the structure of an optical microscope - indicate the optical and mechanical parts and state their applications. Exercise 2 Find the image of the selected preparation, focus using the 4x magnifying lens. Observe the same image of the preparation using immersion oil. Copy the selected part of the image faithfully in the fields below. The image of the selected preparation, focus The same image of the preparation using using the 4x magnifying lens. immersion oil. Page 6 of 9  EXERCISE INSTRUCTIONS 2 - MOLECULAR BIOLOGY / Medicine Faculty / Academy of Silesia / Sem. 1 / 2024/25 Exercise 3 Find all phases of mitotic division of the apical meristem cells of the onion Allium cepa L. root. then draw images under the microscope on the exercise card. PROPHASE METAPHASE ANAPHASE TELOPHASE Page 7 of 9  EXERCISE INSTRUCTIONS 2 - MOLECULAR BIOLOGY / Medicine Faculty / Academy of Silesia / Sem. 1 / 2024/25 Exercise 4 Microscopic photos show the phases of mitotic cell division. Draw the individual phases of mitosis for 3 pairs of chromosomes, including chromosomes with double and single chromatids occurring in the phases presented in the exercise card. PROPHASE METAPHASE ANAPHASE TELOPHASE Images of mitosis phases from an optical microscope using oil immersion. Page 8 of 9  EXERCISE INSTRUCTIONS 2 - MOLECULAR BIOLOGY / Medicine Faculty / Academy of Silesia / Sem. 1 / 2024/25 Exercise 5 Enter the phase of cell nuclear division during or just before/after mitosis: 1. 2 ……………………………………………………….…………………………………………………… 3. 4 ……………………………………………………….…………………………………………………… 5.……………………………………………………….. 6. …………………………………………………………. 7 8.……………………………………………………….…………………………………………………………… Page 9 of 9