Introduction to Microbiology


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Introduction to Microbiology

  1. 1. Observing Microorganisms through a Microscope Chapter 3
  2. 2. Basic techniques needed to study Bacteria <ul><li>1. Grow Bacteria </li></ul><ul><li>2. Isolate Bacteria </li></ul><ul><li>3. Grow Bacteria in pure culture </li></ul><ul><li>4. Observe Bacteria </li></ul><ul><li>5. Identify Bacteria </li></ul>
  4. 5. <ul><li>Zacharias Janssen - compound microscope, 1590. </li></ul><ul><li>uses lenses and light to enlarge the image and is also called an optical or light microscope </li></ul><ul><li>magnifying glass = simplest form </li></ul><ul><ul><ul><li>(10X) magnification. </li></ul></ul></ul><ul><li>compound microscope has two systems of lenses for greater magnification: </li></ul><ul><ul><li>1) the ocular, or eyepiece lens that one looks into </li></ul></ul><ul><ul><li>2) the objective lens, or the lens closest to the object </li></ul></ul>MICROSCOPE
  5. 6. Microscope <ul><li>Resolving Power - ability to distinguish two distinct points </li></ul><ul><ul><li>absolute limit of the Resolving Power is about 1/2 the wavelength of light that is used to illuminate the specimen </li></ul></ul>
  6. 7. 10X or 15X power.  Connects the eyepiece to the objective lenses Supports the tube and connects it to the base used to reflect light from an external light source up throug
  7. 8. <ul><li>Objective Lenses :  </li></ul><ul><li>- 3 or 4 objective lenses  </li></ul><ul><li>- consist of 4X, 10X, 40X and 100X powers.  </li></ul><ul><li>- with a 10X (most common) eyepiece lens, magnifications of </li></ul><ul><li> 40X (4X times 10X), 100X , 400X and 1000X.  </li></ul><ul><li>LPO - shortest lens </li></ul><ul><li>OIO - longest one with the greatest power.  </li></ul><ul><li>- Lenses are color coded and if built to DIN standards are interchangeable between microscopes.  </li></ul><ul><li>- The high power objective lenses are retractable (i.e. 40XR).  ( if they hit a slide, the end of the lens will push in (spring loaded) thereby protecting the lens and the slide.  </li></ul>
  8. 9. <ul><li>Rack Stop :  </li></ul><ul><li>- an adjustment that determines how close the objective lens can get to the slide.  </li></ul><ul><li>- only need to adjust if you are using very thin slides and you weren't able to focus on the specimen at high power. </li></ul>
  9. 11. <ul><li>Condenser Lens :  </li></ul><ul><li>- purpose to focus the light onto the specimen .  </li></ul><ul><li>- most useful at the highest powers (400X and above).  </li></ul><ul><li>Diaphragm or Iris:  </li></ul><ul><li>- most microscopes have a rotating disk under the stage.  </li></ul><ul><li>- this diaphragm has different sized holes and is used to vary the intensity and size of the cone of light that is projected upward into the slide.  </li></ul>
  10. 12. <ul><li>Bright-field microscopy </li></ul><ul><ul><li>Most common </li></ul></ul><ul><ul><li>Visible light </li></ul></ul><ul><ul><li>Max magnification is about 1,000x </li></ul></ul><ul><li>Phase contrast </li></ul><ul><ul><li>Use high-contrast objectives </li></ul></ul>3.1 Microscopic Techniques: The Instrument
  11. 13. <ul><li>Other microscopes </li></ul><ul><ul><li>Dark-field </li></ul></ul><ul><ul><ul><li>side illumination </li></ul></ul></ul><ul><ul><ul><li>dark background, light specimen </li></ul></ul></ul><ul><ul><li>Fluorescence microscope </li></ul></ul><ul><ul><ul><li>Requires staining of specimen with fluorescent dye </li></ul></ul></ul><ul><ul><ul><li>Higher magnification than bright field </li></ul></ul></ul><ul><ul><li>Confocal scanning laser microscope </li></ul></ul><ul><ul><ul><li>Computer controlled </li></ul></ul></ul><ul><ul><ul><li>Laser light source </li></ul></ul></ul><ul><ul><ul><li>Fluorescent </li></ul></ul></ul><ul><ul><ul><li>Can sequentially image multiple planes of a sample and provide 3D images </li></ul></ul></ul>
  12. 14. <ul><li>Electron microscope </li></ul><ul><ul><li>transmission (TEM) </li></ul></ul><ul><ul><li>scanning (SEM) </li></ul></ul>
  13. 15. <ul><li>Stains </li></ul><ul><ul><li>All stains must have one or more dyes </li></ul></ul><ul><ul><ul><li>Basic dyes have a positive charge </li></ul></ul></ul><ul><ul><ul><li>Acidic dyes have a negative charge </li></ul></ul></ul><ul><ul><li>Most stains are performed on glass slides </li></ul></ul><ul><li>Simple stains </li></ul><ul><ul><li>Usually use a basic dye that provides color and contrast to a colorless microbe </li></ul></ul>3.2 Microscopic Techniques: Dyes and Staining
  14. 16. Preparing smears for staining <ul><li>1. Bacteria on slide </li></ul><ul><li>2. Air Dry </li></ul><ul><li>3. Bacteria are HEAT FIXED to the slide </li></ul><ul><li>4. Stain is applied </li></ul>
  15. 17. Staining Reaction <ul><li>Stains - salts composed of a positive and negative ion, one of which is colored (chromophore) </li></ul><ul><li>Basic Dyes - chromophore is the positive ion </li></ul><ul><ul><li>dye+ Cl- </li></ul></ul><ul><li>Acid Dyes - chromophore is the negative ion </li></ul><ul><ul><li>Na+ dye- </li></ul></ul>
  16. 18. Bacteria are slightly negative, so are attracted to the positive chromophore of the BASIC DYE <ul><li>Common Basic Dyes </li></ul><ul><ul><li>crystal violet </li></ul></ul><ul><ul><li>methylene blue </li></ul></ul><ul><ul><li>safranin </li></ul></ul><ul><ul><li>basic fuchsin </li></ul></ul>
  17. 19. Acid Dyes - used for Negative Staining (background is stained) Mordant - intensifies the stain or coats a structure to make it thicker and easier to see after it is stained Example: Flagella - can not normally be seen, but a mordant can be used to increase the diameter of the flagella before it is stained Salmonella typhosa
  18. 20. Differential Stains <ul><li>React differently with different types of bacteria </li></ul><ul><li>2 Most Common </li></ul><ul><ul><li>Gram Stain </li></ul></ul><ul><ul><li>Acid-Fast Stain </li></ul></ul>
  19. 21. Gram Stain <ul><li>1884 Hans Christian Gram </li></ul><ul><li>most important stain used in Bacteriology </li></ul><ul><li>Divides all Bacteria into 2 groups: </li></ul><ul><ul><li>Gram (+) </li></ul></ul><ul><ul><li>Gram (-) </li></ul></ul>
  20. 22. <ul><li>Differential stains distinguish groups of microbes </li></ul><ul><ul><li>Gram stain separates bacteria into Gram positive (purple) or negative( red) </li></ul></ul><ul><ul><ul><li>Primary stain is crystal violet (purple) </li></ul></ul></ul><ul><ul><ul><li>Secondary stain is safranin (red dye) </li></ul></ul></ul>
  21. 23. Gram Stain 1. Crystal violet
  22. 24. Gram Stain 2. Grams Iodine (mordant)
  23. 25. Gram Stain 3. Alcohol
  24. 26. Gram Stain 4. Safranin (Counterstain)
  25. 27. Results <ul><li>Gram (+) Purple </li></ul><ul><li>Gram (-) Red </li></ul><ul><li>Difference - due to structure of cell wall </li></ul><ul><ul><li>Gram (+) Thick cell wall </li></ul></ul><ul><ul><li>Gram (-) Thin cell wall </li></ul></ul>
  26. 28. Identification of a Bacteria Unknown <ul><li>1. Gram Reaction </li></ul><ul><li>2. Morphology </li></ul>
  27. 29. Acid - Fast Stain <ul><li>Differential Stain - divides bacteria into 2 groups </li></ul><ul><li>Acid - Fast </li></ul><ul><li>Non Acid - Fast </li></ul><ul><li>Used to identify organisms in the Genera Mycobacterium (high lipid and wax content in cell wall) </li></ul>
  28. 30. <ul><li>Acid-fast stain is commonly used for microbes that do not readily stain with Gram’s stain </li></ul><ul><ul><li>The bacterial cells are flooded with carbol fuchsin (red) and heat is applied </li></ul></ul><ul><ul><li>The cells are rinsed with acid alcohol , which removes carbol fuchsin from cells that are not acid-fast </li></ul></ul><ul><ul><li>Methylene blue is used as a counterstain </li></ul></ul>
  29. 31. 2 Important Pathogens Mycobacterium tuberculosis
  30. 32. Mycobacterium leprae
  31. 33. Acid - Fast Stain <ul><li>1. Carbolfuchsin (Red) </li></ul><ul><li>2. Acid Alcohol </li></ul><ul><li>3. Counterstain with Methylene Blue </li></ul><ul><li>Acid - Fast Cells Red </li></ul><ul><li>Non Acid - Fast Blue </li></ul>
  32. 34. <ul><li>Special stains are used to observe cell structures </li></ul><ul><ul><li>Malachite green is used to stain endospores </li></ul></ul><ul><ul><li>India ink is used to stain capsular bacteria </li></ul></ul>
  33. 35. Special Stains Capsule Stain Klebsiella pneumoniae
  34. 36. Flagella Stain Spirillum volutans
  35. 37. Endospore Stain Bacillus cereus
  36. 38. Clostridium botulinum
  37. 39. <ul><li>Fluorescent dyes </li></ul><ul><ul><li>DNA-binding dyes </li></ul></ul><ul><ul><ul><li>acridine orange </li></ul></ul></ul><ul><ul><ul><li>ethidium bromide </li></ul></ul></ul><ul><ul><li>Immunofluorescence relies upon a fluorescently-labeled antibody specific to a cellular component </li></ul></ul>
  38. 40. 5 basic groups of microbes
  39. 41. <ul><ul><ul><ul><ul><li>a. Bacteria </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>b. fungi: yeasts and molds </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>c. Viruses </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>d. protozoa </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>e. algae </li></ul></ul></ul></ul></ul>5 basic groups of microbes
  40. 42. a. bacteria <ul><li>Bacteria are typically unicellular, microscopic, prokaryotic organisms that reproduce by binary fission </li></ul>
  41. 43. <ul><li>Note gram-positive (purple) cocci in clusters. </li></ul>
  42. 44. Staphylococcus aureus -Gram-positive, MRSA, coccus prokaryote (dividing); causes food poisoning, toxic shock syndrome and skin and wound infections such as scalded skin syndrome, scarlet fever, erysipelas and impetigo. Magnification:-- x3,025--(Based on an image size of 1 inch in the narrow dimension)
  43. 45. b. fungi: yeasts and molds <ul><li>Yeasts are typically unicellular, microscopic, eukaryotic fungi that reproduce asexually by budding </li></ul><ul><li>Molds are typically filamentous, eukaryotic fungi that reproduce by producing asexual reproductive spores </li></ul>
  44. 46. Electron Micrograph of Saccharomyces Saccharomyces cerevisiae Note budding yeast (arrows). Wine yeast with bud and bud scars ( Sacchromyces spp.). Magnification:-- x2,270--(Based on an image size of 1 inch in the narrow dimension)
  45. 47. c. viruses <ul><li>typically submicroscopic, acellular infectious particles that can only replicate inside a living host cell. </li></ul><ul><li>The vast majority of viruses possess either DNA or RNA but not both. </li></ul>Transmission Electron Micrograph of Adenovirus
  46. 48. d. protozoa <ul><li>are typically unicellular, microscopic, eukaryotic organisms that lack a cell wall </li></ul>Photomicrograph of Amoeba proteus Note the numerous pseudopodia.
  47. 49. e. algae <ul><li>are typically eukaryotic microorganisms that carry out photosynthesis </li></ul>