Introduction structure


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Introduction structure

  2. 2. Office Hrs. MW 11-12; STC 239 Tue 10-1; NEWDL 312 Independence Rd. Laboratory M/W 12-3 Lecture MW 3-4:15
  3. 3. Syllabus
  4. 5. Dasek et al. 2000
  5. 6. Human Pathogens <ul><li>1415 known human pathogens </li></ul><ul><li>61% are zoonotic </li></ul>
  6. 7. Wild Animals Involved in MPXV Transmission (USA, 2003) Gambian Giant Rat (Crycetomys sp.) Prairie Dog (Cynomis sp.)
  7. 8. Ghana TX WI IL * IA 15/4 09/04 Entry and Distribution Route of Crycetomys sp . and Cynomys sp. Involved in MPXV Outbreak (USA, 2003) Source: MMWR 52 (23), CDC, 2003. 15/4
  8. 9. Index case in Marshfield, 26 May 2003: Disseminated lesions.
  9. 10. Distribution of MPXV Cases in the USA WI IL IN OH KS MO Number of cases = 87 WI = 38 IN = 24 IL = 19 OH = 4 KS = 1 MO = 1 Source: MMWR 52 (23), CDC, 2003.
  10. 11. Microbiology <ul><li>Has been defined as the study of organisms and agents too small to be seen clearly by the naked eye – that is, the study of microorganisms </li></ul>
  11. 12. Scope and Relevance <ul><li>Microorganisms are exceptionally diverse, are found almost everywhere, and affect human society in countless ways. Modern microbiology is a large discipline with many different specialties: medicine, agricultural & food sciences, ecology, genetics, and biochemistry. </li></ul>
  12. 13. Microbiology
  13. 14. Microbiology
  14. 15. Microbiology <ul><li>Spontaneous Generation </li></ul><ul><li>Molecular Biology/Genetics </li></ul><ul><li>Fermentation </li></ul><ul><li>Infectious Disease </li></ul>
  15. 17. Infectious Disease - History <ul><li>1798 Jenner, smallpox vaccine </li></ul><ul><li>1835-1844 Bassi, silkworm disease </li></ul><ul><li>1847-1850 Semmelweis </li></ul><ul><li>1849 Snow, cholera </li></ul><ul><li>1861 Pasteur disproves spontaneous generation </li></ul><ul><li>1867 Lister antiseptic surgery </li></ul>
  16. 20. Infectious Disease - History <ul><li>1876-1877 Koch anthrax is caused by Bacillus anthracis </li></ul><ul><li>1881 Pasteur develops anthrax vaccine </li></ul><ul><li>1884 Koch’s postulates published </li></ul><ul><li>Autoclave developed </li></ul><ul><li>Gram stain developed </li></ul><ul><li>1885 Pasteur develops rabies vaccine </li></ul>
  17. 21. Infectious Disease - History <ul><li>1887 Petri dish developed </li></ul><ul><li>1890 Von Behring prepares antitoxins for diphtheria and tetanus </li></ul><ul><li>1899 Ross shows that mosquitoes carry malaria </li></ul><ul><li>1910 Ehrlich – magic bullet </li></ul><ul><li>1923 – Bergey’s Manual 1 st edition </li></ul>
  18. 22. Infectious Disease - History <ul><li>1929 Fleming discovers penicillin </li></ul><ul><li>1935 Domagk discovers sulfa drugs </li></ul><ul><li>1975 Lyme Disease </li></ul><ul><li>1983 HIV </li></ul><ul><li>1986 hepatitis B vaccine – genetically engineered </li></ul>
  19. 23. DISEASE Occurrence of Disease
  20. 24. Signs & Symptoms of Disease <ul><li>Fever </li></ul><ul><li>Fatigue </li></ul><ul><li>White Blood Cell Count </li></ul><ul><li>Pain </li></ul><ul><li>Muscle Aches </li></ul><ul><li>Blood Pressure </li></ul>
  21. 25. Molecular Biology <ul><li>1941 Beadle and Tatum, one gene-one-enzyme hypothesis </li></ul><ul><li>1944 Avery shows that DNA carries information during transformation </li></ul><ul><li>1952 Hershey and Chase viral infection of bacteria </li></ul><ul><li>1953 Watson & Crick </li></ul>
  22. 26. Molecular Biology - History <ul><li>1961 Jacob & Monod propose the operon model of gene regulation </li></ul><ul><li>1961-1966 Nirenberg et al. elucidate the genetic code </li></ul><ul><li>1970 Arber & Smith – restriction endonucleases </li></ul><ul><li>2000 Human genome sequenced </li></ul>
  23. 27. Fermentation - History <ul><li>1857 Pasteur shows that lactic fermentation is due to a microorganism </li></ul><ul><li>1897 Buchner prepares yeast extract that ferments </li></ul>
  24. 28. Spontaneous Generation <ul><li>1799 Spallanzani attacks spontaneous generation </li></ul><ul><li>1861Pasteur disproves spontaneous generation </li></ul>
  25. 29. Kingdoms <ul><li>Animal </li></ul><ul><li>Plant </li></ul><ul><li>Fungi </li></ul><ul><li>Protista </li></ul><ul><li>Prokaryotic </li></ul>
  26. 30. Classification <ul><li>Kingdom </li></ul><ul><li>Phylum </li></ul><ul><li>Class </li></ul><ul><li>Order </li></ul><ul><li>Family </li></ul><ul><li>Genus </li></ul><ul><li>species </li></ul>
  27. 32. Infectious Agents / Microrganisms <ul><li>Bacteria </li></ul><ul><li>Fungi </li></ul><ul><li>Prions </li></ul><ul><li>Protozoans </li></ul><ul><li>Helminths </li></ul>
  28. 33. Toxins – Exo and Endotoxins
  29. 49. Prokaryotes <ul><li>Greek – before a nucleus </li></ul>
  30. 51. Prokaryotes / Eukaryotes <ul><li>There are a number of differences between bacterial cells and plants, animals, fungi and protozoans. </li></ul>
  31. 52. Prokaryotes / Eukaryotes Size of cell 1-10 u m diameter 10-100 u m diameter Nucleus No nuclear membrane True nucleus Membrane bound organelles absent present Flagella 2 protein building blocks complex
  32. 53. Prokaryotes / Eukaryotes Glycocalyx Slime layer Absent Cell Wall Usually present Complex When present, simple Plasma membrane No CHO and lack sterols Sterols and CHO present Cytoplasm No cytoskeleton cytoskeleton
  33. 54. Prokaryotes / Eukaryotes Ribosomes Small size 70s Large 80s; small 70s Chromosome Circular, lacks histones Linear, with histones Cell division Binary fission mitosis Sexual reproduction No meiosis meiosis
  34. 55. Bacterial Classification <ul><li>Cellular Characteristics </li></ul><ul><li>Morphology – cell shape, cell size, arrangement of cells, arrangement of flagella, capsule, endospores </li></ul>
  35. 59. Cellular Characteristics <ul><li>Staining Reactions – Gram stain, acid-fast stain </li></ul>
  36. 74. Cellular Characteristics <ul><li>Growth and nutritional characteristics – appearance in liquid culture </li></ul>
  37. 75. Cellular Characteristics <ul><li>Growth and nutritional characteristics–colonial morphology </li></ul>
  38. 77. Cellular Characteristics <ul><li>Growth and nutritional characteristics pigmentation </li></ul>
  39. 79. Cellular Characteristics <ul><li>Growth and nutritional characteristics energy sources, C, N sources, </li></ul>
  40. 80. Cellular Characteristics <ul><li>Growth and nutritional characteristics fermentation products </li></ul>
  41. 81. Cellular Characteristics <ul><li>Growth and nutritional characteristics modes of metabolism </li></ul>
  42. 83. Cellular Characteristics <ul><li>Biochemical Characteristics – cell wall constituents, pigment biochemicals, storage inclusions, antigens, RNA molecules </li></ul>
  43. 84. Cellular Characteristics <ul><li>Physiological and Ecological Characteristics – temperature range and optimum </li></ul>
  44. 85. Cellular Characteristics <ul><li>Physiological and Ecological Characteristics oxygen relationships </li></ul>
  45. 86. Cellular Characteristics <ul><li>Physiological and Ecological Characteristics – pH tolerance range </li></ul>
  46. 87. Cellular Characteristics <ul><li>Physiological and Ecological Characteristics –salt requirement and tolerance </li></ul>
  47. 89. Cellular Characteristics <ul><li>Genetic Characteristics- DNA G + C </li></ul><ul><li>DNA hybridization </li></ul>
  48. 90. Fimbriae & Pili <ul><li>G- bacteria have short, fine, hairlike appendages that are thinner than flagella and not involved in motility </li></ul>
  49. 91. Fimbriae & Pili <ul><li>slender tubes composed of helically arranged protein subunits and are about 3 to 10 nm in diameter and up to several u m long </li></ul>
  50. 92. FIMBRIA (s) FIMBRIAE (pl) <ul><li>Composition varies, contain protein </li></ul><ul><li>Tendency to stick to each other and surfaces </li></ul><ul><li>Bacterial attachment in aqueous environments </li></ul><ul><li>Role in colonization  infection </li></ul>
  51. 94. PILUS (s) PILI (pl) <ul><li>Hollow, non-helical (9-10nm dia) </li></ul><ul><li>Filamentous appendages </li></ul><ul><li>Thinner than flagella, more numerous </li></ul><ul><li>Example F-pilus ( SEX PILUS ) </li></ul><ul><li>entry of genetic material during conjugation </li></ul><ul><li>GRAM -VE BACTERIA ONLY </li></ul>
  52. 98. E.M Pili on E. coli – N. gonorrhea
  53. 99. Pili & Fimbriae <ul><li>Some types of fimbriae attach bacteria to solid surfaces such as rocks in streams and host tissues </li></ul><ul><li>Pili – about 1 to 10 per cell, differ from fimbriae: are larger (9 to 10 nm in diameter), they are genetically determined by sex factors or conjugative plasmids and are required for bacterial mating </li></ul>
  54. 100. Pili & Fimbriae <ul><li>Some bacterial viruses attach specifically to receptors on sex pili at the start of their reproductive cycle </li></ul>
  55. 101. Flagella & Motility <ul><li>Most motile bacteria move by use of flagella, threadlike, locomotor appendages extending outward from the plasma membrane and cell wall. </li></ul><ul><li>Slender, rigid structures, about 20 nm across and up to 15 to 20 u m long </li></ul>
  56. 102. Flagella - Arrangements <ul><li>Monotrichous </li></ul><ul><li>Amphitrichous </li></ul><ul><li>Lophotrichous </li></ul><ul><li>Peritrichous </li></ul>
  57. 103. Flagellar Ultrastructure <ul><li>Filament </li></ul><ul><li>Hook </li></ul><ul><li>Basal body </li></ul>
  58. 106. ARRANGEMENT OF FLAGELLA <ul><li>POLAR - at one or both ends </li></ul><ul><li>MONTRICHOUS - single e.g., Vibrio sp. </li></ul><ul><li>LOPHOTRICHOUS - small tufts at same site </li></ul><ul><li>e.g., Pseudomonas sp. </li></ul>
  59. 107. <ul><li>AMPHITRICHOUS - at both poles, e.g., Spirillum sp. </li></ul><ul><li>LATERAL </li></ul><ul><li>PERITRICHOUS - surrounding entire cell, e.g., Proteus sp. </li></ul>
  60. 108. MOTILITY <ul><li>CHEMOTAXIS: movement towards/away from chemicals </li></ul><ul><li>MAGNOTAXIS: orientation of movement in magnetic field </li></ul><ul><li>Aquaspirillium magnetotacticum - Magnetosomes </li></ul><ul><li>(Fe 3 O 4 crystalline magnetic iron oxide) </li></ul><ul><li>PHOTOTAXIS: Differences in light intensity </li></ul><ul><li>THERMOTAXIS: heat </li></ul>
  61. 109. <ul><li>Rotation of flagellar motor: reversible </li></ul><ul><li>Clockwise (CW) or Counterclockwise (CCW) </li></ul><ul><li>Smooth swimming/running motion </li></ul><ul><li>Motor rotates CCW direction </li></ul><ul><li>Flagella sweep around cell (in common axis) </li></ul>
  62. 110. <ul><li>Tumbling motion </li></ul><ul><li>Motor reverse (CW direction) </li></ul><ul><li>Flagella disperse </li></ul>
  63. 112. Flagellar Synthesis <ul><li>Complex process – involving at least 20 – 30 genes </li></ul><ul><li>Flagellin subunits are transported through the filament’s hollow internal core. </li></ul><ul><li>When they reach the tip, the subunits spontaneously aggregate so that the filament grows at its tip rather than at the base </li></ul><ul><li>Self - assembly </li></ul>
  64. 113. The Mechanism of Flagellar Movement <ul><li>The filament is in the shape of a rigid helix, and the bacterium moves when this helix rotates </li></ul>
  65. 114. The Mechanism of Flagellar Movement <ul><li>Act like propellers on a boat </li></ul>
  66. 115. The Mechanism of Flagellar Movement <ul><li>E. coli rotates 270 r.p.s., Vibrio alginolyticus averages 1,100 r.p.s. </li></ul>
  67. 116. <ul><li>Distance and speed: </li></ul><ul><li>20-90  m/sec </li></ul><ul><li>Equivalent to: </li></ul><ul><li>6ft human running 5 body lengths/second </li></ul>
  68. 117. AXIAL FILAMENTS <ul><li>Modified flagellum </li></ul><ul><li>Long thin microfibril, inserted into a hook, entire structure enclosed in periplasmic space </li></ul><ul><li>ENDOFLAGELLUM </li></ul>
  69. 119. CELL SURFACE <ul><li>3 Basic Layers - </li></ul><ul><li>GLYCOCALYX </li></ul><ul><li>CELL WALL </li></ul><ul><li>CELL MEMBRANE </li></ul><ul><li>collectively termed CELL ENVELOPE </li></ul>
  70. 120. Glycocalyx <ul><li>Is a network of polysaccharides extending from the surface of bacteria and other cells </li></ul><ul><li>Aids in bacterial attachment to surfaces of solid objects in aquatic environments or to tissue surfaces in plant and animal hosts </li></ul>
  71. 121. GLYCOCALYX <ul><li>External mucilaginous layer </li></ul><ul><li>Surrounds cell </li></ul><ul><li>Shows organisation </li></ul><ul><li>SLIME LAYER - abundant, easily washed off </li></ul><ul><li> (poorly organised) </li></ul><ul><li>CAPSULE - abundant, not easily washed off </li></ul>
  72. 122. Capsule <ul><li>Well organized and not easily washed off </li></ul><ul><li>Composed of polysaccharides, but may be constructed of other materials </li></ul><ul><li>i.e. Bacillus anthracis has a capsule of poly-D-glutamic acid </li></ul><ul><li>Visible with the light microscope </li></ul><ul><li>Resist phagocytosis </li></ul>
  73. 126. Capsules <ul><li>Contain a great deal of water – protect cell from desiccation </li></ul><ul><li>Exclude viruses </li></ul><ul><li>Exclude most hydrophobic toxic substances </li></ul>
  74. 127. FUNCTIONS <ul><li>Provide protection (drying) </li></ul><ul><li>Block attachment of bacteriophages </li></ul><ul><li>Antipathogenic (inhibit engulfment of pathogenic bacteria by WBC’s) Contributes to VIRULENCE or INFECTIVE ABILITY </li></ul><ul><li>Promote attachment to surfaces </li></ul><ul><li>Streptococcus mutans : adheres to teeth ( GLUCAN ), DENTAL CARIES </li></ul>
  75. 128. Complement Activation <ul><li>Some capsules prevent formation of C3 convertase on the bacterial surface </li></ul>
  76. 129. Host response – Antibody <ul><li>Subvert this type of protective host response by having capsules that resemble host polysaccharides. </li></ul>
  77. 130. CELL WALL <ul><li>Important in bacterial characteristics </li></ul><ul><li>Determines shape </li></ul><ul><li>Provides support/rigidity </li></ul>
  78. 131. STRUCTURE <ul><li>Composed of PEPTIDOGLYCAN (MUREIN) </li></ul><ul><li>Insoluble, porous </li></ul><ul><li>Cross-linked polymer (glycan), provides strength and rigidity </li></ul><ul><li>N -acetyl Muramic Acid (NAM) </li></ul><ul><li>N -acetyl Glucosamine (NAG) </li></ul><ul><li> 1-4 glycosodic bonds </li></ul>
  79. 132. <ul><li>Differences in structure - basic principles of </li></ul><ul><li>GRAM STAIN REACTION </li></ul><ul><li>Christian Gram 1884 </li></ul><ul><li>Differential stain : ability of eubacterial cells to retain dye </li></ul><ul><li>(crystal violet) after discolouration with 95% ethanol </li></ul><ul><li>Cells retain stain: Gram +ve (thick cell walls) PURPLE </li></ul><ul><li>Cells lose stain: Gram -ve (thin cell walls) RED </li></ul>
  80. 137. Peptidoglycan Structure <ul><li>Peptidoglycan or murein is an enormous polymer composed of many identical subunits. </li></ul>
  81. 138. Peptidoglycan Structure <ul><li>Most G- cell wall peptidoglycan lacks the peptide bridge. </li></ul>
  82. 139. Gram Negative Cell Walls <ul><li>More complex than the G+ cell walls. </li></ul><ul><li>Peptidoglycan 5-10% of the wall weight. </li></ul><ul><li>Braun’s lipoprotein – a small lipoprotein covalently joined to the underlying peptidoglycan and embedded in the outer membrane by its hydrophobic end. </li></ul><ul><li>Lipopolysaccharides (LPSs) </li></ul>
  83. 141. Lipopolysaccharide Structure <ul><li>Known as ENDOTOXIN </li></ul><ul><li>Complex molecule: </li></ul><ul><li>Inner most LIPID (Lipid A), achors LPS to outer membrane </li></ul><ul><li>Polysaccharide portion (external to Lipid A) known as O-antigen </li></ul><ul><li>O-polysaccharide long repeating sequence of sugars </li></ul>
  84. 142. LPSs <ul><li>Contain both lipid and carbohydrate </li></ul><ul><li>Consist of three parts: lipid, the core polysaccharide and the O side chain </li></ul>
  85. 143. LPSs <ul><li>The lipid A region contains two glucosamine sugar derivatives, each with three fatty acids and phosphate or pyrophosphate attached. </li></ul>
  86. 144. LPSs <ul><li>Lipid A is buried in the outer membrane and the remainder of the LPS projects from the surface. </li></ul>
  87. 145. LPS <ul><li>The core polysaccharide is joined to lipid A. In Salmonella it is constructed of 10 sugars, many of them unusual in structure. </li></ul>
  88. 146. LPS <ul><li>The O side chain or O antigen is a short polysaccharide chain extending outward from the core. It has several peculiar sugars and varies in composition between bacterial strains. </li></ul>
  89. 147. LPS <ul><li>G- bacteria can rapidly change the nature of their O side chains to avoid detection. </li></ul><ul><li>Contributes to the negative charge of the bacterial surface </li></ul><ul><li>LPS helps stabilize membrane structure </li></ul><ul><li>Lipid A is toxic – endotoxin </li></ul><ul><li>Serves as a protective barrier </li></ul>
  90. 148. LPS – protective barrier <ul><li>Prevents or slows entry of bile salts, antibiotics, and other toxic substances. </li></ul>
  91. 149. Porin Proteins <ul><li>Cluster together and span the outer membrane to form a narrow channel through which molecules smaller than about 600 to 700 daltons can pass. </li></ul><ul><li>. </li></ul>
  92. 150. Porin Proteins <ul><li>Larger molecules such as vitamin B 12 must be transported across the outer membrane by specific carriers. The outer membrane also prevents the loss of constituents like periplasmic enzymes. </li></ul>
  94. 154. Periplasmic Space <ul><li>Contains enzymes involved in peptidoglycan synthesis and the modification of toxic compounds that could harm the cell. </li></ul>
  95. 155. Periplasmic Space <ul><li>Gram – contains many proteins that participate in nutrient acquisition. Ex. hydrolytic enzymes attacking nucleic acids and phosphorylated molecules, and binding proteins involved in transport of materials into the cell. </li></ul><ul><li>Denitrifying and chemolithoautotrophic bacteria – electron transport proteins in periplasm </li></ul>
  96. 159. CELL/CYTOPLASMIC MEMBRANE <ul><li>4-5nm thick </li></ul><ul><li>composed 1 o phospholipids 30-40% </li></ul><ul><li>and protein 60-70% </li></ul><ul><li>Phospholipid bilayer: </li></ul><ul><li>Polar heads </li></ul><ul><li>(outwards into aqueous phase - membrane surface) </li></ul><ul><li>Fatty acyl tails </li></ul><ul><li>(inwards - semi/liquid phase at interior) </li></ul>
  97. 160. FLUID MOSAIC MEMBRANE Davison & Danelli
  98. 161. <ul><li>ALSO CONTAINS: </li></ul><ul><li>PROTEINS </li></ul><ul><li>INTEGRAL - removed by destruction </li></ul><ul><li>i.e., Detergents </li></ul><ul><li>PERIPHERAL - loosely attached, easily removed </li></ul><ul><li>i.e., Osmotic shock </li></ul>
  99. 163. FUNCTION <ul><li>Transport - control nutrients </li></ul><ul><li>Oxidative phosphorylation (Respiration) </li></ul><ul><li>Secretion - discharge of metabolic products </li></ul><ul><li>Anchoring DNA (during cell division) </li></ul><ul><li>Metabolism - enzyme sites </li></ul>
  100. 164. INTERNAL CONTENTS <ul><li>Cell Material divided into </li></ul><ul><li>PROTOPLASM </li></ul><ul><li>Granular appearance </li></ul><ul><li>Site of biochemical activity </li></ul><ul><li>Water 70-80% </li></ul><ul><li>acts as solvent for nutrients, sugars, Aa’s & salts </li></ul>
  101. 165. <ul><li>CHROMATIN AREA </li></ul><ul><li>no distinct membrane enclosed nucleus </li></ul><ul><li>no mitotic apparatus </li></ul><ul><li>BACTERIAL CHROMOSOME </li></ul><ul><li>Typically single circular strand of DNA ( CHROMATIN BODY ) </li></ul><ul><li>Exception Streptomyces & Borrelia sp (Linear) </li></ul><ul><li>Rhodobacter sphaeroides (2 separate chromosomes) </li></ul><ul><li>all genes are linked </li></ul><ul><li>Aggregated in one area ( NUCLEOID ) </li></ul>
  102. 166. Bacterial Chromosome
  103. 167. <ul><li>PLASMIDS </li></ul><ul><li>Additional to chromosome </li></ul><ul><li>1 or more, small circular macromolecules of DNA </li></ul><ul><li>Capable of self-replication </li></ul>
  104. 168. <ul><li>Types: </li></ul><ul><li>Fertility (F-plasmid): genes for mating in conjugation </li></ul><ul><li>Resistance (R-plasmids): antibiotics, metals </li></ul>
  105. 169. <ul><li>Virulence factor: enterotoxin, fimbriae, antibiotic production </li></ul><ul><li>Colicinogenic (col-plasmids): gene for protein ( COLICINS ) toxic to closely related bacteria (eliminates competitors) </li></ul><ul><li>Transformation (Ti-plasmids): plant microbiology (formation of crown gall tumors) Agrobacterium </li></ul><ul><li>Metabolic: utilization of camphor, toluene </li></ul>
  106. 170. OTHER FEATURES <ul><li>RIBOSOMES </li></ul><ul><li>Located in Protoplasm </li></ul><ul><li>RNA/PROTEIN bodies </li></ul><ul><li>Composed of 2 sub units (70S) </li></ul><ul><li>Svedberg Units </li></ul><ul><li>Sites of Protein Synthesis </li></ul>
  107. 171. <ul><li>MESOSOMES: </li></ul><ul><li>Extensive invaginations (infoldings) of cyto membrane </li></ul><ul><li>Continuous with membrane </li></ul><ul><li>Function NOT KNOWN </li></ul><ul><li>Corynebacterium parvum </li></ul>
  108. 172. <ul><li>INCLUSIONS/VACUOLES </li></ul><ul><li>compensate for poor availability of nutrients </li></ul><ul><li>present in Protoplast </li></ul><ul><li>VOLUTIN GRANULES/METACHROMATIC ( coloured ) </li></ul><ul><li>composed of POLYPHOSPHATE </li></ul><ul><li>energy rich storage structures </li></ul>
  109. 173. <ul><li>VOLUTIN </li></ul><ul><li>i.e., POLY-  -HYDROXYBUTYRATE </li></ul><ul><li>serve as carbon and energy source </li></ul><ul><li>METACHROMATIC </li></ul><ul><li>i.e., Aquatic bacteria - colored crystals </li></ul><ul><li>( Blue or Red dyes) </li></ul><ul><li>e.g., Corynebacterium </li></ul>
  110. 174. Cyanobacterium: Microcystis (12,600x) <ul><li>Gas vacuoles - blue </li></ul><ul><li>Storage granules - red </li></ul>