Cell Biology: Centrifugation and Cell-Free Systems
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Questions and Answers

What is the general meaning of the term 'cell'?

  • Small particle of matter
  • Small chamber-like structure
  • Small living organism
  • Small room, a hut (correct)

    • Who introduced the term 'nucleus' in 1833?

  • Schwann
  • Schleiden
  • Leeuwenhoek
  • R.Brown (correct)

    • What is the smallest unit of protoplasm capable of independent existence?

  • Cytoplasm
  • Nuclear region
  • Protoplasm
  • Cell (correct)

    • What is the term for the living substance of plants and animals?

    <p>Protoplasm</p> Signup and view all the answers

    Who made the first microscopic observations of protozoa, bacteria, and sperm?

    <p>Leeuwenhoek</p> Signup and view all the answers

    What is the term for the fluid located between and around cells?

    <p>Tissue fluid</p> Signup and view all the answers

    What is the main benefit of using cell-free systems in molecular biology?

    <p>To study the molecular mechanisms involved in cellular processes</p> Signup and view all the answers

    What is the function of centrifugation at high speeds (100 000 x g for 30 mins) in cell fractionation?

    <p>To pellet ER, golgi apparatus and other membrane fragments</p> Signup and view all the answers

    Who isolated myofibrils from skeletal muscle cells that contract upon the addition of ATP in 1949?

    <p>The researcher's name is not specified</p> Signup and view all the answers

    What is the function of centrifugation at very high speeds (300 000 x g for 3hrs) in cell fractionation?

    <p>To pellet ribosomes</p> Signup and view all the answers

    What is the definition of cells in the context of medical biology?

    <p>All of the above</p> Signup and view all the answers

    What are the components of mammalian tissue?

    <p>Cells and intercellular or extracellular substances</p> Signup and view all the answers

    In what year was the first cell-free system to carry out protein synthesis (in vitro translation) developed?

    <p>1954</p> Signup and view all the answers

    What is the function of intercellular or extracellular substances in mammalian tissue?

    <p>To support and nourish cells</p> Signup and view all the answers

    What is the main application of microdissection techniques in cell culture?

    <p>To isolate selected cells from tissue slices</p> Signup and view all the answers

    What type of cells can give rise to all cell types in the body?

    <p>Embryonic stem cells</p> Signup and view all the answers

    What is the primary use of hybrid cells?

    <p>To produce specialized cells for therapy</p> Signup and view all the answers

    Which of the following is an application of cell culture?

    <p>To understand the process of morphogenesis</p> Signup and view all the answers

    What is the result of cell fusion?

    <p>Membrane merging and cytoplasmic mixing of two cell types</p> Signup and view all the answers

    What is the significance of embryonic stem cells?

    <p>They can give rise to all cell types in the body</p> Signup and view all the answers

    Which organism is considered the most frequently used single cell eukaryote?

    <p>Saccharomyces cerevisiae (yeast)</p> Signup and view all the answers

    What is the primary advantage of using Escherichia coli in research?

    <p>It is easy to culture and manipulate</p> Signup and view all the answers

    What is the primary use of microdissection techniques in cell culture?

    <p>To isolate selected cells from tissue slices</p> Signup and view all the answers

    What is the significance of embryonic stem cells?

    <p>They can give rise to all cell types in the body</p> Signup and view all the answers

    What is the result of cell fusion?

    <p>Membrane merging and cytoplasmic mixing of two cell types</p> Signup and view all the answers

    Which of the following organisms is the most well understood in biology?

    <p>Escherichia coli</p> Signup and view all the answers

    What is the primary application of cell culture?

    <p>To study embryonic organs</p> Signup and view all the answers

    Which of the following is an application of hybrid cells?

    <p>To produce specialized cells for therapy</p> Signup and view all the answers

    What is the significance of Saccharomyces cerevisiae (yeast) in biology?

    <p>It is the most frequently used single cell eukaryote</p> Signup and view all the answers

    What is the benefit of using embryonic stem cells in medical research?

    <p>They can give rise to all cell types in the body</p> Signup and view all the answers

    What is the size of a typical animal cell?

    <p>20-30µm</p> Signup and view all the answers

    What is the oldest technique for examining living cells?

    <p>Examination of living cells under a microscope</p> Signup and view all the answers

    Who developed the first tissue culture in 1907?

    <p>Ross Harrison</p> Signup and view all the answers

    What is the purpose of culturing cells in solutions?

    <p>To keep cells alive for a prolonged period</p> Signup and view all the answers

    What is the classical culture media used in cell culture?

    <p>Blood serum</p> Signup and view all the answers

    What is the benefit of using cell culture?

    <p>To study the behavior of cells under controlled conditions</p> Signup and view all the answers

    What is the result of culturing fragments of spinal cord in lymphatic fluid?

    <p>Axons grow out</p> Signup and view all the answers

    What is the purpose of using embryo juice in cell culture?

    <p>To stimulate growth</p> Signup and view all the answers

    What is the primary purpose of a laminar-flow hood in a cell culture laboratory?

    <p>To provide a clean bench for cell culture</p> Signup and view all the answers

    What is the significance of pH 7.4 in cell culture?

    <p>It is indicated by the phenol red indicator dye</p> Signup and view all the answers

    What is the main advantage of using cell culture?

    <p>It allows for the study of cells in a controlled environment</p> Signup and view all the answers

    What is a characteristic of primary cell cultures?

    <p>They have a limited life span</p> Signup and view all the answers

    What is a characteristic of cell lines?

    <p>They can grow indefinitely in culture</p> Signup and view all the answers

    What is the purpose of using growth factors in cell culture?

    <p>To promote cell growth and division</p> Signup and view all the answers

    What is the significance of anchorage-dependent cells in cell culture?

    <p>They must be cultured while attached to a solid or semi-solid substrate</p> Signup and view all the answers

    What is the purpose of using a CO2 incubator in cell culture?

    <p>To regulate the gas content of the culture</p> Signup and view all the answers

    What is the purpose of cell fractionation?

    <p>To study certain organelles from a cell</p> Signup and view all the answers

    How are cells disrupted in cell fractionation?

    <p>Using both osmotic shock and ultrasonic vibration, or forced through a small orifice, or ground up</p> Signup and view all the answers

    What is the result of carefully applying disruption procedures in cell fractionation?

    <p>Leaving organelles such as nuclei, mitochondria, the Golgi apparatus, lysosomes, and peroxisomes largely intact</p> Signup and view all the answers

    What is the suspension of cells called after disruption?

    <p>Homogenate</p> Signup and view all the answers

    What is cell fractionation?

    <p>The separation of homogeneous sets of organelles from a larger population of cells</p> Signup and view all the answers

    What are the methods used in cell fractionation?

    <p>Homogenization, differential centrifugation, and density gradient centrifugation</p> Signup and view all the answers

    What is the purpose of differential centrifugation in cell fractionation?

    <p>To separate organelles according to their specific gravity</p> Signup and view all the answers

    What is the final step in cell fractionation?

    <p>Collection of fractions</p> Signup and view all the answers

    What is the purpose of using low-speed centrifugation in cell fractionation?

    <p>To separate intact cells from the medium</p> Signup and view all the answers

    What is the result of homogenization in cell fractionation?

    <p>Breakdown of cell membranes and liberation of organelles and cellular structures</p> Signup and view all the answers

    What is the purpose of using filters in cell fractionation?

    <p>To remove insoluble tissue</p> Signup and view all the answers

    What is the purpose of using a cold isotonic buffer in cell fractionation?

    <p>To stop enzyme reactions, osmosis, and pH changes</p> Signup and view all the answers

    What is the purpose of using high-speed centrifugation in cell fractionation?

    <p>To separate subcellular components</p> Signup and view all the answers

    What is the result of differential centrifugation?

    <p>Isolation of pure fractions of subcellular components</p> Signup and view all the answers

    What is the purpose of using ultracentrifugation in cell fractionation?

    <p>To isolate pure fractions of subcellular components</p> Signup and view all the answers

    What is the result of grinding tissue in a blender in cell fractionation?

    <p>Breakdown of cell membranes and liberation of organelles and cellular structures</p> Signup and view all the answers

    What is the main purpose of centrifugation in cell fractionation?

    <p>To separate components that differ greatly in size</p> Signup and view all the answers

    What is the term for the process of moving components in a mixture through a salt solution at different rates during centrifugation?

    <p>Velocity sedimentation</p> Signup and view all the answers

    What is used to protect the bands from convective mixing during centrifugation?

    <p>A shallow gradient of sucrose</p> Signup and view all the answers

    What type of instrument is used to achieve a finer degree of separation during centrifugation?

    <p>Ultracentrifuge</p> Signup and view all the answers

    What is the purpose of layering the homogenate as an arrow band on top of a dilute salt solution during centrifugation?

    <p>To achieve a finer degree of separation</p> Signup and view all the answers

    What is the method of disrupting cells that uses enzymes such as lysozyme and chitinase?

    <p>Enzymatic disruption</p> Signup and view all the answers

    Study Notes

    Cell Biology

    • Cells are the basic morphological and functional units of the body, and are too small to be seen with the naked eye.
    • Development of cell biology had to await the development of a magnifying device, such as the light microscope.
    • The term "Cellula" was first used by Robert Hook in 1665 to describe small chamber-like structures observed in cork under a microscope.

    Cell Components

    • Protoplasm is the living substance of plants and animals, comprising the nuclear region and cytoplasm.
    • The smallest unit of protoplasm capable of independent existence is the cell.

    Cell Theory

    • Cells are the fundamental units of both structure and function in all living organisms.
    • All living organisms are composed of cells and their products.
    • Cells arise only from preexisting cells.

    Cell Culture and Applications

    • Microdissection techniques allow selected cells to be isolated from tissue slices.
    • Applications of cell culture include:
      • Embryonic organs can be cultured.
      • Study of cancer cells.
      • Cell to virus relations.
      • Cytogenetic research.
      • Cell-cell interactions.
      • Cell nutrition.

    Stem Cells

    • Embryonic stem cells are the most promising cell lines from a medical point of view.
    • These cells can proliferate indefinitely and give rise to all cell types in the body.
    • ES cells might be used to replace and repair damaged mature human tissues.

    Model Organisms

    • Escherichia coli is the best understood cell in biology.
    • Saccharomyces cerevisiae (yeast) is the most frequently used single cell eukaryote.
    • Studies with bacteria and yeast established the basic principles of molecular biology.

    Cell Fusion

    • Cell fusion is the process of membrane merging and cytoplasmic mixing of two cell types.
    • Hybrid cells are produced by fusion of animal cells.
    • Examples include human cell and mouse cell fusion.

    Microdissection Techniques

    • Allow selected cells to be isolated from tissue slices
    • Applications include:
      • Embryonic organ culture
      • Study of cancer cells
      • Cell-virus relations
      • Cytogenetic research
      • Cell-cell interactions
      • Cell nutrition

    Embryonic Stem Cells

    • Most promising cell lines for medical applications
    • Derived from inner cell mass of early mouse embryos
    • Can proliferate indefinitely
    • Can give rise to all cell types in the body
    • Can be used to replace and repair damaged mature human tissues
    • Potential therapeutic applications:
      • Muscular dystrophy
      • Parkinson's disease
      • Type I diabetes

    Model Organisms

    • Escherichia coli (E. coli): best understood cell in biology
    • Saccharomyces cerevisiae (yeast): most frequently used single-cell eukaryote
    • Studies with bacteria and yeast established basic principles of molecular biology

    Cell-Cell Fusion

    • Hybrid cells produced by fusion of animal cells
    • Cell fusion: membrane merging and cytoplasmic mixing of two cell types
    • Examples: human cell and mouse cell fusion

    Cell Size and Number

    • Human body composed of trillions of cells
    • Size of a typical animal cell: 20-30 µm
    • Largest cell: human egg (150-200 µm)
    • Smallest cell: granular cells of cerebellum (4-5 µm)

    Methods for Examining Cells

    • Examination of the cell as a whole (without disruption)
    • Examination of living cells (fresh tissues)
    • Examination of killed and preserved tissues and cells
    • Fractionation of cells and analyzing their molecules

    Cell Culture

    • Isolating cells and growing them in culture
    • Living cells can be suspended in an appropriate liquid (e.g. saline solution)
    • Prolonged study of living cells can be made by culturing them in solutions containing necessary nutrients
    • First tissue culture (1907): Ross Harrison, isolated fragments of spinal cord of a frog

    Culture Conditions

    • Medium supplying essential nutrients (amino acids, carbohydrates, vitamins, minerals)
    • Growth factors
    • Hormones
    • Gases (O2, CO2)
    • Regulated physico-chemical environment (pH, osmotic pressure, temperature)
    • Most cells are anchorage-dependent and require a solid or semi-solid substrate

    Primary Cell Culture and Cell Lines

    • Primary cell culture: derived from normal animal cells, limited life span, divide 25-50 times, and then stop dividing (cell senescence)
    • Cell lines: eukaryotic cell lines, widely used for experiments, transformed cells, grow indefinitely in culture (immortal cells)

    Fractionation of Cells and Analyzing their Molecules

    • Cell fractionation is a method to study certain organelles from a cell, involving the disruption of cells and isolation of organelles and macromolecules in pure form.
    • Cells can be disrupted in various ways, such as osmotic shock, ultrasonic vibration, or grinding up.

    Cell Disruption Methods

    • Chemical: alkali, organic solvents, detergents
    • Enzymatic: lysozyme, chitinase
    • Physical: osmotic shock, freeze/thaw
    • Mechanical: sonication, homogenization, French press

    Steps of Subcellular Fractionation

    • Homogenization
    • Differential centrifugation
    • Further separation and purification by density gradient centrifugation
    • Collection of fractions
    • Analysis of fractions

    Centrifugation

    • Centrifugation is the first step in most fractionations, separating components that differ greatly in size.
    • Low speed centrifugation is used to separate intact cells from medium.
    • High speed centrifugation can be used to separate subcellular components.
    • When a centrifugal force is applied to an aqueous mixture, components of larger size and density will sediment faster.

    Density Gradient Centrifugation

    • A finer degree of separation can be achieved by layering the homogenate as an arrow band on top of a dilute salt solution that fills a centrifuge tube.
    • The various components in the mixture move as a series of distinct bands through the salt solution, each at a different rate, in a process called velocity sedimentation.

    History of Mitochondria Isolation

    • 1934: First isolation of mitochondria from liver by Bensley
    • 1937: First chemical analysis of mitochondria

    Isolation of Components of Living Cells by Differential Centrifugation

    • Organelles and macromolecules can be separated by ultracentrifugation.
    • Pure fractions of nucleus, nucleolus, mitochondria, lysosomes, microsomes, and ribosomes can be isolated.
    • This method has made it possible to obtain knowledge on the molecular composition of cell components.

    Method of Differential Centrifugation

    • Cut tissue in an ice-cold isotonic buffer to stop enzyme reactions, osmosis, and pH changes.
    • Grind tissue in a blender to break open cells.
    • Filter to remove insoluble tissue.
    • Centrifuge filtrate at low speeds (1000 X g for 10mins) to pellet nuclei.
    • Centrifuge at medium speeds (10 000 x g for 30 mins) to pellet mitochondria.

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    Description

    This quiz covers the principles of centrifugation in cell biology, including the separation of cellular components and the study of cell-free systems. It also touches on the history of cell-free systems and their role in understanding cellular processes.

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