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  1. 1. ‫‪MICROBIOLOGY‬‬ ‫•‬ ‫•‬ ‫•‬ ‫•‬ ‫اعداد‬ ‫أ. فريد ابو العمرين‬ ‫ماجستير العلوم الطبية المخبرية‬ ‫رئيس قسم الفيروسات والمناعة والهيماتولوجي‬ ‫مستشفى الشفاء‬ ‫4 :‪Lec‬‬ ‫1‬
  2. 2. Burton's Microbiology for the Health Sciences Chapter 4. Microbial Diversity Part 1: Acellular and Procaryotic Microbes
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  4. 4. Acellular Microbes • Viruses – Complete virus particles are called virions. – Most viruses are from 10 to 300 nm in diameter. – Viruses infect humans, animals, plants, fungi, protozoa, algae and bacterial cells. 4
  5. 5. Viral structure • A typical virion consists of a genome of either DNA or RNA, surrounded by a capsid (protein coat) which is composed of protein units called capsomeres. • Some viruses (enveloped viruses) have an outer envelope composed of lipids and polysaccharides. 5
  6. 6. Viruses have 5 properties that distinguish them from living cells: 1. They possess either DNA or RNA – living cells possess both. 2. They are unable to replicate on their own. 3. Do not divide by binary fission, mitosis, or meiosis. 4. They lack the genes and enzymes necessary for energy production. 5. They depend on the ribosomes, enzymes, and metabolites of the host cell for protein and nucleic acid production. 6
  7. 7. Viruses are classified by: • Type of genetic material (either DNA or RNA) • Shape and size of capsid • Number of capsomeres • Presence or absence of an envelope • Type of host it infects • Disease it produces • Target cell(s) • Immunologic/antigenic properties 7
  8. 8. virus DNA Single-strand 8 Double-strand RNA Single-strand Double-strand
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  12. 12. Herpesviruses acquiring their envelopes as they leave a host cell’s nucleus by budding. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins 12
  13. 13. FIGURE 4-4. Virus particle becoming enveloped in the process of budding from a host cell. 13
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  15. 15. Comparative sizes of virions, their nucleic acids, and bacteria. 15
  16. 16. Bacteriophages  Viruses that infect bacteria are known as bacteriophages.  There are two categories of bacteriophages: virulent bacteriophages and temperate bacteriophages.  Virulent bacteriophages always cause the lytic cycle, which ends with the destruction of the bacterial cell. 16
  17. 17. Lytic process A partially lysed cell of Vibrio cholerae with attached virions of phage CP-T1. 17
  18. 18. Bacteriophages. The bacteriophage T4 is an assembly of protein components. 20 facets, filled with DNA Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins 18 Viral DNA enters the cell through the core.
  19. 19. Multiplication of animal viruses Animal viruses escape from their host cells either by lysis of the cell or budding. Viruses that escape by budding become 19 enveloped viruses.
  20. 20. Latent Virus Infections – Viral infections in which the virus is able to hide from a host’s immune system by entering cells and remaining dormant. – 20 Herpes viral infections are examples.
  21. 21. Antiviral Agents – – Antibiotics are not effective against viral infections. Antiviral agents are drugs that are used to treat viral infections. – These agents interfere with virus-specific enzymes and virus production by disrupting critical phases in viral multiplication or inhibiting synthesis of viral DNA, RNA, or proteins. 21
  22. 22. Acellular Microbes, cont. • Oncogenic Viruses – – Viruses that cause cancer. Examples include Epstein-Barr virus, human papillomaviruses, and HTLV-1. • Human Immunodeficiency Virus (HIV) – – – – 22 The cause of acquired immunodeficiency syndrome (AIDS). It is an enveloped, single-stranded RNA virus. The primary targets for HIV are CD4+ cells. CD4+ cells = T-helper cells = WBC
  23. 23. Human Immunodeficiency Virus (HIV) 23
  24. 24. Viroids and Prions • Viroids and Prions (smaller and less complex infectious particles than viruses) – Viroids • Viroids are short, naked fragments of single-stranded RNA, which can interfere with the metabolism of plant cells. • Viroids are transmitted between plants in the same manner as viruses. 24
  25. 25. Prions – Prions are small infectious proteins that cause fatal neurologic diseases in animals; examples: Scrapie, Bovine Spongiform Encephalopathy (“Mad Cow Disease”) and Creutzfeldt-Jacob disease. – Of all pathogens, prions are the most resistant to disinfectants. 25
  26. 26. Bacteria
  27. 27. The Domain Bacteria Characteristics • Bacteria are divided into 3 major phenotypic categories: 1. Those that are Gram negative and have a cell wall 2. Those that are Gram positive and have a cell wall 3. Those that lack a cell wall (Mycoplasma spp.) • 27 Characteristics of bacteria used in classification and identification include: cell morphology, staining reactions, motility, colony morphology, atmospheric requirements, nutritional requirements, biochemical and metabolic activities, enzymes that the organism produces, pathogenicity, and genetic composition.
  28. 28. Cell Morphology • There are 3 basic categories of bacteria, based on shape: – – – 28 Cocci (round bacteria) Bacilli (rod-shaped bacteria) Curved and spiral-shaped bacteria
  29. 29. Cocci • Cocci is round bacteria may be seen singly or in pairs (diplococci), chains (streptococci), clusters (staphylococci), packets of 4 (tetrads), or packets of 8 (octads). • The average coccus is about 1 µm in diameter. • Some cocci have “coccus” in their name. 29
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  31. 31. Morphologic Arrangements of Cocci Gram-positive Staphylococcus aureus in clusters. 31 Streptococcus mutans illustrating cocci in chains.
  32. 32. Diagram Showing Various Forms of Bacteria That Might be Observed in Gram-Stained Smears 32
  33. 33. Bacilli • Bacilli – – 33 They may occur singly, in pairs (diplobacilli), in chains (streptobacilli), in long filaments, or branched. – – – – Often referred to as rods; may be short or long, thick or thin, and pointed or with curved or blunt ends. An average sized bacillus is 1 x 3 µm. Extremely short bacilli are called coccobacilli. Examples of medically important bacilli: Escherichia, Klebsiella, and Proteus spp, Pseudomonas, Haemophilus, and Bacillus spp.
  34. 34. Curved, Spiral-Shaped Bacteria • Curved and Spiral-Shaped Bacteria – Examples of curved bacteria: • Vibrio spp. • Campylobacter spp. • Helicobacter spp. – Examples of spiral-shaped bacteria: • Treponema spp. • Borrelia spp. 34
  35. 35. Staining Procedures • Three Major Categories of Staining Procedures 1. 2. Simple stains Structural staining procedures • • Spore stains • 3. Capsule stains Flagella stains Differential staining procedures • 35 Gram and acid-fast staining procedures
  36. 36. Staining Procedures, cont.  Bacterial smears must be fixed prior to staining  The fixation process serves to: 1. 2. 3.  kill organisms, preserve their morphology, shape anchors the smear to the slide The two most common types of fixation: 1. Heat-fixation; not a standardized technique; excess heat will distort bacterial morphology 2. 36 Methanol-fixation; a standardized technique; the preferred method
  37. 37. Simple Bacterial Staining Technique 37
  38. 38. The Gram Staining Procedure • Divides bacteria into 2 major groups: – Gram-positive (bacteria are blue-to-purple) – Gram-negative (bacteria are pink-to-red) • The final Gram reaction (positive or negative) depends upon the organism’s cell wall structure. – The cell walls of Gram-positive bacteria have a thick layer of peptidoglycan, making it difficult to remove the crystal violet-iodine complex. – Gram-negative organisms have a thin layer of peptidoglycan, making it easier to remove the crystal violet; the cells are subsequently 38 stained with safranin.
  39. 39. Gram Staining 39
  40. 40. Gram-positive (bacteria are blue-to-purple) Gram-negative (bacteria are pink-to-red) 40
  41. 41. Various Gram-Positive Bacteria Chains of streptococci in smear from broth culture. 41 Streptococcus pneumoniae in blood culture.
  42. 42. Various Gram-Positive Bacteria Spores Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins A bacillus, Clostridium perfringens, in a smear from a broth culture. 42 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Clostridium tetani in a smear from a broth culture (note terminal spores on some cells).
  43. 43. Various Gram-Positive Bacteria Epithelial cells Many Gram-positive bacteria Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins 43
  44. 44. Gram-Negative Bacteria Gram-negative bacilli in a smear from a bacterial colony. 44 Loosely coiled Gram-negative spirochetes, Borrelia burgdorferi, the cause of Lyme disease.
  45. 45. The acid-fast stain • Some bacteria are not stained with Gram staining, because the cell walls of mycobacteria contain waxes : Mycobacterium spp. • Mycobacterium spp. are often identified using the acid-fast stain. • The acid-fast stain – – The heat is necessary because the cell walls of mycobacteria contain waxes, which prevent the stain from penetrating the cells. – – 45 Carbol fuchsin is the red dye that is driven through the bacterial cell wall Heat is used to soften the waxes in the cell wall Because mycobacteria are not decolorized by the acid-alcohol mixture, they are said to be acid-fast
  46. 46. Differential staining  The Gram and acid-fast staining procedures are referred to as differential staining procedures because they enable microbiologists to differentiate one group of bacteria from another. 46
  47. 47. Acid-Fast Mycobacteria Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Many acid-fast mycobacteria in a liver biopsy. 47 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Acid-fast bacilli in a digested sputum specimen.
  48. 48. Some Important Pathogenic Bacteria 48
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  50. 50. Motility • If a bacterium is able to “swim,” it is said to be motile. • Bacterial motility is most often associated with flagella; less often with axial filaments. • Most spiral-shaped bacteria and about 50% of bacilli are motile; cocci are generally nonmotile. • Motility can be demonstrated by stabbing the bacteria into a tube of semisolid medium or by using the hanging-drop technique. 50
  51. 51. Semisolid Agar Method for Determining Motility Nonmotile Motile Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Motile 51 semisolid agar produces turbidity (cloudiness).
  52. 52. Hanging-Drop Prep for Study of Living Bacteria Depression slide Depression slide with coverglass Side view of hanging-drop prep. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins When the preparation is examined microscopically, motile bacteria within the “hanging drop” will be seen darting around in every direction. 52
  53. 53. Colony Morphology • A bacterial colony contains millions of organisms. • Colony morphology (appearance of the colony) varies from one species to another. • Colony morphology includes: size, color, overall shape, elevation, and the appearance of the edge or margin of the colony. • Colony morphology also includes the results of enzymatic activity on various types of media. • As is true for cell morphology and staining characteristics, 53 colony morphology is an important “clue” to the identification of bacteria.
  54. 54. Colony Morphology 54
  55. 55. Colony Morphology Size of colonies is determined by the organism’s generation time and is another important characteristic of a particular bacterial species. Formation of a bacterial colony on solid growth medium; here, the generation time is assumed to be 30 minutes. 55
  56. 56. Atmospheric Requirements • Bacteria can be classified on the basis of their atmospheric requirements, including their relationship to O2 and CO2 56
  57. 57. With respect to O2, bacterial isolates can be classified as: • Obligate aerobes: require an atmosphere oxygen in concentrations comparable to 20%–21%. • Microaerophilic aerobes: that prefer an atmosphere containing about 5% oxygen. • Facultative anaerobes: are capable of surviving in either the presence or absence of oxygen • Aerotolerant anaerobes: does not require oxygen, grows better in the absence of oxygen, but can survive in O2 atmospheres • Obligate anaerobes: not require oxygen for life and reproduction. • Capnophilic organisms grow best in the presence of increased concentrations of CO2 (usually 5 to 10%) 57
  58. 58. Nutritional Requirements • All bacteria need some form of the elements carbon, hydrogen, oxygen, sulfur, phosphorus, and nitrogen for growth. • Some bacteria require special elements (e.g., calcium, iron, or zinc). • Organisms with especially demanding nutritional requirements are said to be fastidious (“fussy”). • The nutritional needs of a particular organism are usually characteristic for that species and are sometimes 58 important clues to its identity.
  59. 59. Biochemical and Metabolic Activities • As bacteria grow, they produce many waste products and secretions, some of which are enzymes. • Pathogenic strains of many bacteria, like staphylococci and streptococci, can be tentatively identified by the enzymes they secrete. • In particular environments, some bacteria produce gases such as carbon dioxide or hydrogen sulfide. 59
  60. 60. Pathogenicity • Many pathogens are able to cause disease because they possess capsules, pili, or endotoxins, or because they secrete exotoxins and exoenzymes that damage cells and tissues. • Frequently, pathogenicity is tested by injecting the organism into mice or cell cultures. • Examples of some common pathogenic bacteria: – Neisseria meningitidis, Salmonella typhi, Shigella spp., Vibrio cholerae, Yersina pestis, Treponema pallidum 60
  61. 61. Genetic Composition • Laboratory identification of bacteria is moving toward analyzing the organism’s DNA or RNA – techniques collectively referred to as molecular diagnostic procedures. – The composition of the genetic material (DNA) of an organism is unique to each species. – DNA probes make it possible to identify an isolate without relying on phenotypic characteristics. • Through the use of 16S rRNA sequencing, the degree of relatedness between 2 different bacteria can be 61 determined.
  62. 62. Unique Bacteria • Rickettsias, chlamydias, and mycoplasmas are bacteria, but they do not possess all the attributes of typical bacterial cells. • Rickettsias and chlamydias have a Gram-negative type of cell wall and are obligate intracellular pathogens (i.e., they must live within a host cell; they cannot grow on artificial culture media). – – 62 Rickettsias have “leaky membranes.” Chlamydias are “energy parasites,” meaning they prefer to use ATP molecules produced by their host cell.
  63. 63. Rickettsia prowazekii, the cause of epidemic typhus. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins 63
  64. 64. Mycoplasmas • Mycoplasmas – – Smallest of the cellular microbes Lack a cell wall and therefore assume many shapes (they are pleomorphic) – In humans, pathogenic mycoplasmas cause primary atypical pneumonia and genitourinary infections – Because they have no cell wall, they are resistant to drugs like penicillin that attack cell walls – 64 They produce tiny “fried egg” colonies on artificial media
  65. 65. Photosynthetic Bacteria • Photosynthetic bacteria include purple bacteria, green bacteria, and cyanobacteria; they all use light as an energy source, but not in the same way. 65
  66. 66. The Domain Archaea • Archaea (meaning ancient) were discovered in 1977; they are procaryotic organisms. • Genetically, archaea are more closely related to eucaryotes than they are to bacteria. • Archaea vary widely in shape; some live in extreme environments, such as extremely acidic, extremely hot, or extremely salty environments. • Archaea possess cell walls, but their cell walls donot contain peptidoglycan (in contrast, all bacterial 66 cell walls contain peptidoglycan).
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