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MICROBIAL CELL                           BIOLOGY                               Biology 120 Lecture 3                      ...
LECTURE OUTLINE        • Observation of               Microorganisms        • Size, Shape and               arrangement of...
OBSERVATION OF         MICROORGANISMS        • Units of • 1000 nm = 1               Measure                     µm        ...
OBSERVATION OF           MICROORGANISMS     • Simple Microscope (1   • Compound Microscope            lens)              (...
OBSERVATION OF           MICROORGANISMS        • RECALL: Microscopy!         • Resolution         • Refractive Index      ...
TYPES OF                        MICROSCOPES                               • Brightfield                                vers...
TYPES OF MICROSCOPES                          • Phase Contrast                           • accentuates diffraction of     ...
TYPES OF MICROSCOPES                          • Fluorescence                           • fluorescent substance absorbs     ...
TYPES OF MICROSCOPES                             • Measures sound waves                               that are reflected ba...
ELECTRON MICROSCOPY        • Transmission Electron Microscope (Res 2.5nm)        • Scanning Electron Microscope (Res 20nm)...
PREPARING SPECIMENS: WET                           MOUNT                                 • Wet Mount                      ...
WET MOUNT                                    Parungao-Balolong 2011Tuesday, July 5, 2011
STAINING SPECIMENS       •Stained        •dead        •fixation (heat or ethanol)        •Smear       • Simple       • Diff...
SMEAR                        PREPARATION                            FROM                        COLONIES OR               ...
FIXATION       • Process by which the internal and external structures              of cells and microorganisms are preser...
TYPES OF FIXATION       •Heat-fixation                   •Chemical fixation       • By flaming air-dried           • Used to ...
STAINING BACTERIA                                     Acid-Fast staining                 Simple staining                  ...
SIZE, SHAPE &        ARRANGEMENT OF CELLS        • Average Size:                 • Environmental               0.2um-1.0um...
SIZE, SHAPE &        ARRANGEMENT OF CELLS                        Parungao-Balolong 2011Tuesday, July 5, 2011
SIZE, SHAPE &        ARRANGEMENT OF CELLS                        Parungao-Balolong 2011Tuesday, July 5, 2011
SIZE, SHAPE &        ARRANGEMENT OF CELLS                        Parungao-Balolong 2011Tuesday, July 5, 2011
SIZE, SHAPE &        ARRANGEMENT OF CELLS                        Parungao-Balolong 2011Tuesday, July 5, 2011
SIZE, SHAPE &        ARRANGEMENT OF CELLS                        Parungao-Balolong 2011Tuesday, July 5, 2011
SIZE, SHAPE &        ARRANGEMENT OF CELLS            Stella sp.   Haloarcula sp.                                          ...
FUNCTIONAL ANATOMY      OF PROKARYOTES        • Basics of Prokaryotes         • One circular chromosome not enclosed in a ...
FUNCTIONAL ANATOMY      OF PROKARYOTES                        Parungao-Balolong 2011Tuesday, July 5, 2011
FUNCTIONAL ANATOMY      OF PROKARYOTES   Plasma               Selectively permeable barrier, mechanical   Membrane        ...
PLASMA MEMBRANE     Phospholipid        bilayer     Peripheral        proteins     Integral proteins     Transmembrane...
PLASMA MEMBRANE      Membrane is as          viscous as olive          oil.      Proteins move to          function.    ...
PLASMA MEMBRANE        Selective permeability allows passage of some molecules        Enzymes for ATP production       ...
PLASMA MEMBRANE    Damage to the membrane by alcohols,        quaternary ammonium (detergents),        and polymyxin anti...
PLASMA MEMBRANE    EUBACTERIA           Ester linkage           Weaker linkage    ARCHAEBACTERIA           Ether link...
MEMBRANE               TRANSPORT: RECALL                            Simple diffusion:                             Movemen...
MEMBRANE               TRANSPORT: RECALL                            Parungao-Balolong 2011Tuesday, July 5, 2011
MEMBRANE           TRANSPORT: OSMOSIS                                                               Osmosis: The moveme...
MEMBRANE               TRANSPORT: RECALL                            Parungao-Balolong 2011Tuesday, July 5, 2011
INCLUSION BODIES    Metachromatic granules      Phosphate reserves    (volutin)                  Corynebacterium diptheria...
INCLUSION BODIES                                     Parungao-Balolong 2011Tuesday, July 5, 2011
RIBOSOMES              16S ribosomal DNA =                   prokaryotes              18S ribosomal DNA =                 ...
FUNCTIONAL ANATOMY      OF PROKARYOTES   Nucleoid             Localization of Genetic Material                        (DNA...
PERIPLASMIC SPACE/                    PERIPLASM                             • Space between the                           ...
CELL WALL  Prevents osmotic lysis  Made of peptidoglycan (in bacteria)                                         Parungao-...
PEPTIDOGLYCAN (PG)    Polymer of disaccharide       N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)    Linked b...
PEPTIDOGLYCAN (PG)                            Parungao-Balolong 2011Tuesday, July 5, 2011
PEPTIDOGLYCAN (PG)      Thick peptidoglycan      Teichoic acids      In acid-fast cells, contains mycolic acid         ...
PEPTIDOGLYCAN (PG)    Thin peptidoglycan    No teichoic acids    Outer membrane                            Parungao-Bal...
TEICHOIC ACIDS : G+     Lipoteichoic acid links        to plasma membrane     Wall teichoic acid links        to peptido...
OUTER MEMBRANE : G-     Lipopolysaccharides, lipoproteins, phospholipids     Forms the periplasm between the outer membr...
CELL WALLS & GRAM STAIN      Crystal violet-iodine crystals form in cell.      Gram-positive            Alcohol dehydra...
CELL WALLS & GRAM STAIN                        Parungao-Balolong 2011Tuesday, July 5, 2011
GRAM + VS GRAM -                                     Parungao-Balolong 2011Tuesday, July 5, 2011
GRAM + VS GRAM -                                     Parungao-Balolong 2011Tuesday, July 5, 2011
WHEN GRAM STAIN NOT APPLICABLE     Mycoplasmas            Lack cell walls            Sterols in plasma               me...
TARGETING THE CELL WALL         Lysozyme digests disaccharide in peptidoglycan.         Penicillin inhibits peptide brid...
PROTOPLASTS                                  Parungao-Balolong 2011Tuesday, July 5, 2011
FUNCTIONAL ANATOMY      OF PROKARYOTES     Capsules and Slime Layers   Resistance to phagocytosis,                        ...
FIMBRIAE & PILI    Fimbriae allow        attachment    Pili are used to        transfer DNA from        one cell to anot...
FIMBRIAE & PILI                                     Parungao-Balolong 2011Tuesday, July 5, 2011
THE F PLASMID                                   Parungao-Balolong 2011Tuesday, July 5, 2011
ENDOSPORE     Resting cells     Resistant to desiccation,         heat, chemicals     Bacillus, Clostridium     Sporul...
Parungao-Balolong 2011Tuesday, July 5, 2011
Parungao-Balolong 2011Tuesday, July 5, 2011
ENDOSPORE RESISTANCE     15% of spore dry weight consists of dipicolinic acid complexed         with calcium ions     DP...
OUTSIDE THE WALL:                   GLYCOCALYX       Outside cell wall       Usually sticky             A capsule is ne...
OUTSIDE THE WALL:                   GLYCOCALYX                        Capsule Staining                                    ...
OUTSIDE THE CELL                        WALL : FLAGELLA   Outside cell wall   Made of chains of       flagellin   Attac...
OUTSIDE THE CELL                        WALL : FLAGELLA                                     Parungao-Balolong 2011Tuesday,...
OUTSIDE THE CELL                        WALL : FLAGELLA                                     Parungao-Balolong 2011Tuesday,...
MOTILITY    Rotate flagella to run or tumble    Move toward or away from stimuli (taxis)    Flagella proteins are H ant...
MOTILITY                                   Parungao-Balolong 2011Tuesday, July 5, 2011
MOTILITY                                   Parungao-Balolong 2011Tuesday, July 5, 2011
CYTOPLASM    Cytoplasm is the substance inside the       plasma membrane.     Nuclear area (nucleoid)                   ...
FUNCTIONAL ANATOMY       OF EUKARYOTES        • Observation of               Microorganisms        • Size, Shape and      ...
FUNCTIONAL ANATOMY OF      EUKARYOTES                        Parungao-Balolong 2011Tuesday, July 5, 2011
PROKARYOTES VERSUS                  EUKARYOTES                             Parungao-Balolong 2011Tuesday, July 5, 2011
PROKARYOTES VERSUS                  EUKARYOTES                             Parungao-Balolong 2011Tuesday, July 5, 2011
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Transcript of "Bio 120 lecture 3 2011 2012"

  1. 1. MICROBIAL CELL BIOLOGY Biology 120 Lecture 3 Reference: Chapter 3 and 4 TORTORA Parungao-Balolong 2011Tuesday, July 5, 2011
  2. 2. LECTURE OUTLINE • Observation of Microorganisms • Size, Shape and arrangement of Cells • Functional Anatomy of Prokaryotes • Functional Anatomy of Eukaryotes Parungao-Balolong 2011Tuesday, July 5, 2011
  3. 3. OBSERVATION OF MICROORGANISMS • Units of • 1000 nm = 1 Measure µm • 1 µm = 10–6 m • 0.001 µm = 1 = 10 –3 mm nm • 1 nm = 10 –9 m = 10–6 mm Parungao-Balolong 2011Tuesday, July 5, 2011
  4. 4. OBSERVATION OF MICROORGANISMS • Simple Microscope (1 • Compound Microscope lens) ( more than 1 lens) • Magnification differsTuesday, July 5, 2011 Parungao-Balolong 2011
  5. 5. OBSERVATION OF MICROORGANISMS • RECALL: Microscopy! • Resolution • Refractive Index • Immersion Oil Parungao-Balolong 2011Tuesday, July 5, 2011
  6. 6. TYPES OF MICROSCOPES • Brightfield versus Darkfield Parungao-Balolong 2011Tuesday, July 5, 2011
  7. 7. TYPES OF MICROSCOPES • Phase Contrast • accentuates diffraction of light that passes through the specimen • Differential Interference Contrast • uses two beams of light Parungao-Balolong 2011Tuesday, July 5, 2011
  8. 8. TYPES OF MICROSCOPES • Fluorescence • fluorescent substance absorbs UV light and emit visible light • Confocal • laser illuminates each plane in a specimen (3D image) • *** fluorochromes Parungao-Balolong 2011Tuesday, July 5, 2011
  9. 9. TYPES OF MICROSCOPES • Measures sound waves that are reflected back from an object • For microbial cells on surface Parungao-Balolong 2011Tuesday, July 5, 2011
  10. 10. ELECTRON MICROSCOPY • Transmission Electron Microscope (Res 2.5nm) • Scanning Electron Microscope (Res 20nm) Parungao-Balolong 2011Tuesday, July 5, 2011
  11. 11. PREPARING SPECIMENS: WET MOUNT • Wet Mount • viable • heat fixing not done Parungao-Balolong 2011Tuesday, July 5, 2011
  12. 12. WET MOUNT Parungao-Balolong 2011Tuesday, July 5, 2011
  13. 13. STAINING SPECIMENS •Stained •dead •fixation (heat or ethanol) •Smear • Simple • Differential • Special Parungao-Balolong 2011Tuesday, July 5, 2011
  14. 14. SMEAR PREPARATION FROM COLONIES OR BROTH CULTURE Parungao-Balolong 2011Tuesday, July 5, 2011
  15. 15. FIXATION • Process by which the internal and external structures of cells and microorganisms are preserved and fixed in position • Inactivates enzymes that might disrupt cell morphology • Toughens cell structures to prevent changes during staining and observation • Usually microbes are killed when fixed Parungao-Balolong 2011Tuesday, July 5, 2011
  16. 16. TYPES OF FIXATION •Heat-fixation •Chemical fixation • By flaming air-dried • Used to protect fine smear gently cellular substructure; morphology of larger, • Adequately preserves more delicate microbes overall morphology but not structures within • Penetrates cells and cells react with cellular components (proteins and lipids) • Example: alcohol, formaldehyde, acetic Parungao-Balolong 2011Tuesday, July 5, 2011
  17. 17. STAINING BACTERIA Acid-Fast staining Simple staining Capsule staining Flagella staining Gram staining Parungao-Balolong 2011Tuesday, July 5, 2011
  18. 18. SIZE, SHAPE & ARRANGEMENT OF CELLS • Average Size: • Environmental 0.2um-1.0um x Factors 2-8um • pleiomorphism • Shape Based on: • devoid of cell wall • Genetics • monomorphism • uniqueness for each bacteria Parungao-Balolong 2011Tuesday, July 5, 2011
  19. 19. SIZE, SHAPE & ARRANGEMENT OF CELLS Parungao-Balolong 2011Tuesday, July 5, 2011
  20. 20. SIZE, SHAPE & ARRANGEMENT OF CELLS Parungao-Balolong 2011Tuesday, July 5, 2011
  21. 21. SIZE, SHAPE & ARRANGEMENT OF CELLS Parungao-Balolong 2011Tuesday, July 5, 2011
  22. 22. SIZE, SHAPE & ARRANGEMENT OF CELLS Parungao-Balolong 2011Tuesday, July 5, 2011
  23. 23. SIZE, SHAPE & ARRANGEMENT OF CELLS Parungao-Balolong 2011Tuesday, July 5, 2011
  24. 24. SIZE, SHAPE & ARRANGEMENT OF CELLS Stella sp. Haloarcula sp. Parungao-Balolong 2011Tuesday, July 5, 2011
  25. 25. FUNCTIONAL ANATOMY OF PROKARYOTES • Basics of Prokaryotes • One circular chromosome not enclosed in a membrane • no histones • no organelles • Cell walls with peptidoglycans • Divides via Binary Fission Parungao-Balolong 2011Tuesday, July 5, 2011
  26. 26. FUNCTIONAL ANATOMY OF PROKARYOTES Parungao-Balolong 2011Tuesday, July 5, 2011
  27. 27. FUNCTIONAL ANATOMY OF PROKARYOTES Plasma Selectively permeable barrier, mechanical Membrane boundary of cell, nutrient and waste transport, location of many metabolic processes (respiration, photosynthesis), detection of environmental cues for chemotaxis Gas Vacuole Buoyancy for floating in aquatic environments Ribosomes Protein synthesis Inclusion Bodies Storage of carbon, phosphate and other substances Parungao-Balolong 2011Tuesday, July 5, 2011
  28. 28. PLASMA MEMBRANE  Phospholipid bilayer  Peripheral proteins  Integral proteins  Transmembrane proteins Parungao-Balolong 2011Tuesday, July 5, 2011
  29. 29. PLASMA MEMBRANE  Membrane is as viscous as olive oil.  Proteins move to function.  Phospholipids rotate and move laterally. Parungao-Balolong 2011Tuesday, July 5, 2011
  30. 30. PLASMA MEMBRANE  Selective permeability allows passage of some molecules  Enzymes for ATP production  Photosynthetic pigments on foldings called chromatophores or thylakoids; chlorosomes; photosynthetic lamellae green sulfur bacteria purple sulfur bacteria purple non-sulfur bacteria Parungao-Balolong 2011Tuesday, July 5, 2011
  31. 31. PLASMA MEMBRANE  Damage to the membrane by alcohols, quaternary ammonium (detergents), and polymyxin antibiotics causes leakage of cell contents. Parungao-Balolong 2011Tuesday, July 5, 2011
  32. 32. PLASMA MEMBRANE  EUBACTERIA  Ester linkage  Weaker linkage  ARCHAEBACTERIA  Ether linkage  Stronger linkage  NOTE: POINT OF LINKAGE = POINT OF BREAKDOWN Parungao-Balolong 2011Tuesday, July 5, 2011
  33. 33. MEMBRANE TRANSPORT: RECALL  Simple diffusion: Movement of a solute from an area of high concentration to an area of low concentration.  Facilitative diffusion: Solute combines with a transporter protein in the membrane. Parungao-Balolong 2011Tuesday, July 5, 2011
  34. 34. MEMBRANE TRANSPORT: RECALL Parungao-Balolong 2011Tuesday, July 5, 2011
  35. 35. MEMBRANE TRANSPORT: OSMOSIS    Osmosis: The movement of water  Osmotic pressure: The pressure needed to stop the movement of water across the membrane. Parungao-Balolong 2011Tuesday, July 5, 2011
  36. 36. MEMBRANE TRANSPORT: RECALL Parungao-Balolong 2011Tuesday, July 5, 2011
  37. 37. INCLUSION BODIES Metachromatic granules Phosphate reserves (volutin) Corynebacterium diptheria Polysaccharide granules Energy reserves Example: Glycogen Lipid inclusions (PHBs) Energy reserves Mycobacterium, Bacillus, Azotobacter Sulfur granules Energy reserves Corynebacterium diptheria Carboxysomes Ribulose 1,5-diphosphate carboxylase for CO2 fixation Nitrifying Bacteria Gas vacuoles/Gas vesicles Protein covered cylinders Magnetosomes Iron oxide (destroys H2O2) Aquaspirillum magnetotacticum Parungao-Balolong 2011Tuesday, July 5, 2011
  38. 38. INCLUSION BODIES Parungao-Balolong 2011Tuesday, July 5, 2011
  39. 39. RIBOSOMES 16S ribosomal DNA = prokaryotes 18S ribosomal DNA = eukaryotes Parungao-Balolong 2011Tuesday, July 5, 2011
  40. 40. FUNCTIONAL ANATOMY OF PROKARYOTES Nucleoid Localization of Genetic Material (DNA) Periplasmic Contains hydrolytic enzymes and Space binding proteins for nutrient processing and uptake Cell Wall Gives bacteria shape and protection from lysis in dilute solutions Parungao-Balolong 2011Tuesday, July 5, 2011
  41. 41. PERIPLASMIC SPACE/ PERIPLASM • Space between the plasma membrane and the outer membrane • 40% in G- minimal in G+ • Loose PG (murein) and some proteins • For Gram + counterpart are exoenzymes Parungao-Balolong 2011Tuesday, July 5, 2011
  42. 42. CELL WALL  Prevents osmotic lysis  Made of peptidoglycan (in bacteria) Parungao-Balolong 2011Tuesday, July 5, 2011
  43. 43. PEPTIDOGLYCAN (PG)  Polymer of disaccharide N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)  Linked by polypeptides Parungao-Balolong 2011Tuesday, July 5, 2011
  44. 44. PEPTIDOGLYCAN (PG) Parungao-Balolong 2011Tuesday, July 5, 2011
  45. 45. PEPTIDOGLYCAN (PG)  Thick peptidoglycan  Teichoic acids  In acid-fast cells, contains mycolic acid Parungao-Balolong 2011Tuesday, July 5, 2011
  46. 46. PEPTIDOGLYCAN (PG)  Thin peptidoglycan  No teichoic acids  Outer membrane Parungao-Balolong 2011Tuesday, July 5, 2011
  47. 47. TEICHOIC ACIDS : G+  Lipoteichoic acid links to plasma membrane  Wall teichoic acid links to peptidoglycan  May regulate movement of cations  Polysaccharides provide antigenic variation Parungao-Balolong 2011Tuesday, July 5, 2011
  48. 48. OUTER MEMBRANE : G-  Lipopolysaccharides, lipoproteins, phospholipids  Forms the periplasm between the outer membrane and the plasma membrane.  Protection from phagocytes, complement, and antibiotics  O polysaccharide antigen, e.g., E. coli O157:H7  Lipid A is an endotoxin  Porins (proteins) form channels through membrane. Parungao-Balolong 2011Tuesday, July 5, 2011
  49. 49. CELL WALLS & GRAM STAIN  Crystal violet-iodine crystals form in cell.  Gram-positive Alcohol dehydrates peptidoglycan CV-I crystals do not leave  Gram-negative Alcohol dissolves outer membrane and leaves holes in peptidoglycan. CV-I washes out Parungao-Balolong 2011Tuesday, July 5, 2011
  50. 50. CELL WALLS & GRAM STAIN Parungao-Balolong 2011Tuesday, July 5, 2011
  51. 51. GRAM + VS GRAM - Parungao-Balolong 2011Tuesday, July 5, 2011
  52. 52. GRAM + VS GRAM - Parungao-Balolong 2011Tuesday, July 5, 2011
  53. 53. WHEN GRAM STAIN NOT APPLICABLE  Mycoplasmas  Lack cell walls  Sterols in plasma membrane  Archaea  Wall-less or  Walls of pseudomurein (lack NAM and D amino Parungao-Balolong 2011Tuesday, July 5, 2011
  54. 54. TARGETING THE CELL WALL  Lysozyme digests disaccharide in peptidoglycan.  Penicillin inhibits peptide bridges in peptidoglycan.  Protoplast (complete) is a wall-less cell.  Spheroplast (partial) is a wall-less Gram-positive cell.  L forms (reversible) are wall-less cells that swell into irregular shapes.  Protoplasts and spheroplasts are susceptible to osmotic lysis. Parungao-Balolong 2011Tuesday, July 5, 2011
  55. 55. PROTOPLASTS Parungao-Balolong 2011Tuesday, July 5, 2011
  56. 56. FUNCTIONAL ANATOMY OF PROKARYOTES Capsules and Slime Layers Resistance to phagocytosis, adherence to surfaces Fimbriae and Pili Attachment to surfaces, bacterial mating Flagella Movement Endospore Survival under harsh environmental conditions Parungao-Balolong 2011Tuesday, July 5, 2011
  57. 57. FIMBRIAE & PILI  Fimbriae allow attachment  Pili are used to transfer DNA from one cell to another Parungao-Balolong 2011Tuesday, July 5, 2011
  58. 58. FIMBRIAE & PILI Parungao-Balolong 2011Tuesday, July 5, 2011
  59. 59. THE F PLASMID Parungao-Balolong 2011Tuesday, July 5, 2011
  60. 60. ENDOSPORE  Resting cells  Resistant to desiccation, heat, chemicals  Bacillus, Clostridium  Sporulation: Endospore formation  Germination: Return to vegetative state Parungao-Balolong 2011Tuesday, July 5, 2011
  61. 61. Parungao-Balolong 2011Tuesday, July 5, 2011
  62. 62. Parungao-Balolong 2011Tuesday, July 5, 2011
  63. 63. ENDOSPORE RESISTANCE  15% of spore dry weight consists of dipicolinic acid complexed with calcium ions  DPA directly involved in spore heat resistance  GENERA: • Clostridium • Bacillus • Sporolactobacillus • Sporosarcina Parungao-Balolong 2011Tuesday, July 5, 2011
  64. 64. OUTSIDE THE WALL: GLYCOCALYX  Outside cell wall  Usually sticky  A capsule is neatly organized  A slime layer is unorganized and loose  Extracellular polysaccharide allows cell to attach  Capsules prevent phagocytosis Parungao-Balolong 2011Tuesday, July 5, 2011
  65. 65. OUTSIDE THE WALL: GLYCOCALYX Capsule Staining Parungao-Balolong 2011Tuesday, July 5, 2011
  66. 66. OUTSIDE THE CELL WALL : FLAGELLA  Outside cell wall  Made of chains of flagellin  Attached to a protein hook  Anchored to the wall and membrane by the basal body Parungao-Balolong 2011Tuesday, July 5, 2011
  67. 67. OUTSIDE THE CELL WALL : FLAGELLA Parungao-Balolong 2011Tuesday, July 5, 2011
  68. 68. OUTSIDE THE CELL WALL : FLAGELLA Parungao-Balolong 2011Tuesday, July 5, 2011
  69. 69. MOTILITY  Rotate flagella to run or tumble  Move toward or away from stimuli (taxis)  Flagella proteins are H antigens (e.g., E. coli O157:H7) Parungao-Balolong 2011Tuesday, July 5, 2011
  70. 70. MOTILITY Parungao-Balolong 2011Tuesday, July 5, 2011
  71. 71. MOTILITY Parungao-Balolong 2011Tuesday, July 5, 2011
  72. 72. CYTOPLASM  Cytoplasm is the substance inside the plasma membrane.  Nuclear area (nucleoid) Parungao-Balolong 2011Tuesday, July 5, 2011
  73. 73. FUNCTIONAL ANATOMY OF EUKARYOTES • Observation of Microorganisms • Size, Shape and arrangement of Cells • Functional Anatomy of Prokaryotes • Functional Anatomy of Eukaryotes Parungao-Balolong 2011Tuesday, July 5, 2011
  74. 74. FUNCTIONAL ANATOMY OF EUKARYOTES Parungao-Balolong 2011Tuesday, July 5, 2011
  75. 75. PROKARYOTES VERSUS EUKARYOTES Parungao-Balolong 2011Tuesday, July 5, 2011
  76. 76. PROKARYOTES VERSUS EUKARYOTES Parungao-Balolong 2011Tuesday, July 5, 2011
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