Tools of the Laboratory


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Tools of the Laboratory

  1. 1. Foundations in Microbiology Chapter 3 PowerPoint to accompany Fifth Edition Talaro Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
  2. 2. Tools of the Laboratory: The Methods for Studying Microorganisms Chapter 3
  3. 3. The 5 I’s of culturing microbes <ul><li>I noculation – introduction of a sample into a container of media </li></ul><ul><li>I ncubation – under conditions that allow growth </li></ul><ul><li>I solation –separating one species from another </li></ul><ul><li>I nspection </li></ul><ul><li>I dentification </li></ul>
  4. 4. Isolation <ul><li>If an individual bacterial cell is separated from other cells & has space on a nutrient surface, it will grow into a mound of cells- a colony </li></ul><ul><li>A colony consists of one species </li></ul>
  5. 5. Isolation technique
  6. 6. Media – providing nutrients in the laboratory <ul><li>Most commonly used: </li></ul><ul><ul><li>nutrient broth – liquid medium containing beef extract & peptone </li></ul></ul><ul><ul><li>nutrient agar – solid media containing beef extract, peptone & agar </li></ul></ul><ul><li>agar is a complex polysaccharide isolated from red algae </li></ul><ul><ul><li>solid at room temp, liquefies at boiling (100 o C), does not resolidify until it cools to 42 o C </li></ul></ul><ul><ul><li>provides framework to hold moisture & nutrients </li></ul></ul><ul><ul><li>not digestible for most microbes </li></ul></ul>
  7. 7. Types of media <ul><li>synthetic – contains pure organic & inorganic compounds in an exact chemical formula </li></ul><ul><li>complex or nonsynthetic – contains at least one ingredient that is not chemically definable </li></ul><ul><li>general purpose media - grows a broad range of microbes, usually nonsynthetic </li></ul><ul><li>enriched media - contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes </li></ul>
  8. 8. Enriched media
  9. 9. <ul><li>selective media - contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes </li></ul><ul><li>differential media – allows growth of several types of microbes and displays visible differences among desired and undesired microbes </li></ul>
  10. 10. selective & differential media
  11. 11. Selective media
  12. 12. Differential media
  13. 13. Miscellaneous media <ul><li>reducing medium – contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria </li></ul><ul><li>carbohydrate fermentation medium - contains sugars that can be fermented, converted to acids, and a pH indicator to show the reaction; basis for identifying bacteria and fungi </li></ul>
  14. 14. Carbohydrate fermentation media
  15. 15. <ul><li>magnification – ability to enlarge objects </li></ul><ul><li>resolving power – ability to show detail </li></ul>
  16. 16. compound light microscope
  17. 17. Pathway of light
  18. 18. Effect of wavelength on resolution
  19. 19. Oil immersion lens
  20. 20. Effect of magnification
  21. 21. Types of light microscopes <ul><li>Bright-field – most widely used, specimen is darker than surrounding field </li></ul><ul><li>Dark-field – brightly illuminated specimens surrounded by dark field </li></ul><ul><li>Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures </li></ul>
  22. 22. 3 views of a cell
  23. 23. Fluorescence Microscope <ul><li>Modified compound microscope with an ultraviolet radiation source and a filter that protects the viewer’s eye </li></ul><ul><li>Uses dyes that emit visible light when bombarded with shorter uv rays. </li></ul><ul><li>Useful in diagnosing infections </li></ul>
  24. 25. Electron microscopy <ul><li>Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds. </li></ul><ul><li>Electron waves are 100,000X shorter than the waves of visible light. </li></ul><ul><li>Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength. </li></ul><ul><li>Magnification between 5,000X and 1,000,000X </li></ul>
  25. 27. 2 types of electron microscopes <ul><li>Transmission electron microscopes (TEM) – transmits electrons through the specimen; darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts </li></ul><ul><li>Scanning electron microscopes (SEM)– provides detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it. </li></ul>
  26. 28. Transmission Electron Micrograph
  27. 29. Scanning Electron Micrograph
  28. 30. Specimen preparation <ul><li>wet mounts & hanging drop mounts – allow examination of characteristics of live cells: motility, shape, & arrangement </li></ul><ul><li>fixed mounts are made by drying & heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts. </li></ul>
  29. 31. Staining <ul><li>cationic dyes - basic, with positive charges on the chromophore </li></ul><ul><li>anionic dyes - acidic, with negative charges on the chromophore </li></ul><ul><li>surfaces of microbes are negatively charged and attract basic dyes – positive staining . </li></ul><ul><li>negative staining – microbe repels dye & it stains the background </li></ul>
  30. 32. Staining <ul><li>simple stains –one dye is used </li></ul><ul><li>differential stains – use a primary stain and a counterstain to distinguish cell types or parts. examples: Gram stain, acid-fast stain and endospore stain </li></ul><ul><li>special stains : capsule and flagellar stains </li></ul>
  31. 33. Types of stains