Microbiology lab 2

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Microbiology lab 2

  1. 1. MICROSCOPY A Brief Overview Dr Saleh M.Y. Practical Lab Experiment -1- MBBS-Phase II 19/10/2010 1
  2. 2. - Introduction - Definition - Historical Background - Varibles Used In Microscopy - Compound Microscope - Structure And Function - Use Of Microscope - Various Types Of Microscopes - Care Of Microscope 2
  3. 3. INTRODUCTION TO MICROSCOPY - Understanding the optical principles and construction of microscopes - Role of microscopy - Microscopic techniques and application 3
  4. 4. DEFINITION - A microscope (Greek: micron = small and scopos = to look) - MICROSCOPE: Is an instrument for viewing objects that are too small to be seen by the naked or unaided eye - MICROSCOPY: The science of investigating small objects using such an instrument is called microscopy 4
  5. 5. HISTORICAL BACKGROUND  1590 - Hans Janssen and his son Zacharias Janssen, developed first microscope.  1609 - Galileo Galilei - occhiolino or compound microscope.  1620 - Christian Huygens, another Dutchman, developed a simple 2-lens ocular system that was chromatically corrected. 5
  6. 6. Antony van Leeuwenhoek (1632-1723) - Anton van Leeuwenhoek is generally credited with bringing the microscope to the attention of biologists. - A tradesman of Delft, Holland.  1661 - He discovered bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells, microscopic nematodes etc. 6
  7. 7. Microscope used by Anton von Leeuwenhoek An old pocket Microscope 7
  8. 8. VARIABLES USED IN MICROSCOPY 8
  9. 9. MAGNIFICATION Degree of enlargement No of times the length, breadth or diameter, of an object is multiplied. MAGNIFICATION VS SHARPNESS USEFUL MAGNIFICATION AND EMPTY MAGNIFICATION 9
  10. 10. RESOLUTION: Ability to reveal closely adjacent structural details as separate and distinct LIMIT OF RESOLUTION (LR): The min distance between two visible bodies at which they can be seen as separate and not in contact with each other 10
  11. 11. LR = 0.61 x W W = Wavelength NA NA = Num aperture Types of microscope Resolving power Compound Microscope 200 nanometers Scanning Electron Microscope 10 nanometers Transmission Electron Microscope 0.2 nanometers 11
  12. 12. NUMERICAL APERTURE(NA) - Ratio of diameter of lens to its focal length - NA = n Sin θ/2 n = refractive index, θ = angle of aperture (CAD) B C D θ/2 n of air = 1 n of oil = 1.5 12 A
  13. 13. DEFINITION - Capacity of an objective to render outline of the image of an object clear and distinct - Depends of elimination of Spherical and Chromatic aberration 13
  14. 14. ABERRATION - Chromatic aberration - Correction of aberration – Achromatic objective and Apochromatic objectives. Incident light Blue focus Red focus 14
  15. 15. Spherical aberration Focus of axial rays Focus of marginal rays 15
  16. 16. TYPES OF MICROSCOPE - Simple microscope - Compound microscope - Phase Contrast Microscope - Dark Ground Microscope - Fluorescent Microscope - Electron Microscope - Others 16
  17. 17. COMPOUND MICROSCOPE Compound microscope made by John Cuff 1750 17
  18. 18. PARTS OF COMPOUND MICROSCOPE - Ocular (Eye piece) - Body or Tube - Coarse focusing knob - Fine focusing knob - Objective Lens - Movable stage - Condenser Lenses - Field (Iris) Diaphragm - Mirror and light source 18
  19. 19. OBJECTIVE LENS • Mounted on Nose piece  It forms magnified real image. • Magnification of objective = Optical Tube length Focal Length • Scan - 4X • Low Power - 10X TYPES • High Power - 40X • Oil immersion - 100X 19
  20. 20. OIL IMMERSION OBJECTIVE - Highest magnification - Oil prevents refraction of light outwards and allows it to pass straight in to objective G FBEG - OIL D ABCD - AIR E OIL C GLASS B 20 A
  21. 21. EYE PIECE - Forms magnified virtual & erect image - TYPES (a) Monocular (b) Binocular (c) Trinocular or (a) Huygenian (b) Ramsden (c) Compensating 21
  22. 22. What’s the power of this lens? To calculate the power of magnification, multiply the power of the ocular lens by the power of the objective, e..g.: 10x40=400 times What are the powers of magnification for each of the objectives we have on our microscopes? Fill in the table on your worksheet.
  23. 23. 23
  24. 24. Comparing Powers of Magnification We can see better details with higher the powers of magnification, but we cannot see as much of the image. 10x 40x Which of these images would be viewed at a higher power of magnification?
  25. 25. Let’s give it a try ... 1 – Turn on the microscope and then rotate the nosepiece to click the red-banded objective into place. 2 – Place a slide on the stage and secure it using the stage clips. Use the coarse adjustment knob (large knob) to get it the image into view and then use the fine adjustment knob (small knob) to make it clearer. 3 – Once you have the image in view, rotate the nosepiece to view it under different powers. Draw what you see on your worksheet! Be careful with the largest objective! Sometimes there is not enough room and you will not be able to use it! 4 – When you are done, turn off the microscope and put up the slides you used.
  26. 26. How to make a wet-mount slide … 1 – Get a clean slide and coverslip from your teacher. 2 – Place ONE drop of water in the middle of the slide. Don’t use too much or the water will run off the edge and make a mess! 3 – Place the edge of the cover slip on one side of the water drop. 4 - Slowly lower the cover slip on top of the drop. Cover Lower slowly Slip 5 – Place the slide on the stage and view it first with the red-banded objective. Once you see the image, you can rotate the nosepiece to view the slide with the different objectives. You do not need to use the stage clips when viewing wet-mount slides!
  27. 27. ILLUMINATION - Lamp, sunlight, battery operated lamp, 60 W bulb, Quartz halogen light. FILTERS - Blue, Green, Heat absorbing filters, Barrier filters. 27
  28. 28. KOHLER’S ILLUMINATION Multiple step operation employed to attain optimal illumination: 1. Remove any diffusing filter. 2. Put a slide on the stage and focus. 3. Completely close the field diaphragm. 4. Move the condenser until the border or the iris hexagon is neat and clear. 5. Center if necessary. 6. Open the field diaphragm until the tip of the hexagon touches the field limit 28
  29. 29. The Parts of a Microscope
  30. 30. The Parts of a Microscope Ocular Lens Body Tube Nose Piece Arm Objective Lenses Stage Stage Clips Coarse Adj Diaphragm Fine Adjustment Light Source Base
  31. 31. Body Tube The body tube holds the objective lenses and the ocular lens at the proper distance Diagram
  32. 32. Nose Piece The Nose Piece holds the objective lenses and can be turned to increase the magnification Diagram
  33. 33. Objective Lenses The Objective Lenses increase magnification (usually from 10x to 40x) Diagram
  34. 34. Stage Clips These 2 clips hold the slide/specimen in place on the stage. Diagram
  35. 35. Diaphragm The Diaphragm controls the amount of light on the slide/specimen Turn to let more light in or to make dimmer. Diagram
  36. 36. Light Source Projects light upwards through the diaphragm, the specimen and the lenses Some have lights, others have mirrors where you must move the mirror to reflect light Diagram
  37. 37. Ocular Lens/Eyepiece Magnifies the specimen image Diagram
  38. 38. Arm Used to support the microscope when carried. Holds the body tube, nose piece and objective lenses Diagram
  39. 39. Stage Supports the slide/specimen Diagram
  40. 40. Coarse Adjustment Knob Moves the stage up and down (quickly) for focusing your image Diagram
  41. 41. Fine Adjustment Knob This knob moves the stage SLIGHTLY to sharpen the image Diagram
  42. 42. Base Supports the microscope Diagram
  43. 43. Magnification To determine your magnification…you just multiply the ocular lens by the objective lens Ocular 10x Objective 40x:10 x 40 = 400 So the object is 400 times “larger” Objective Lens have their magnification written on them. Ocular lenses usually magnifies by 10x
  44. 44. HOW A MICROSCOPE WORKS ? 45
  45. 45. OPTICAL PATH IN COMPOUND MICROSCOPE 46
  46. 46. Method of using Compound Microscope 47
  47. 47. 1. Grasp the microscopes arm with one hand and place your other hand under the base. 2. Place the microscope on a bench. arm arm Adjust seat 3. Clean Lenses. 4. Turn the coarse adjustment knob to raise the body tube 48
  48. 48. 5. Revolve the nose piece to set low- power objective lens. 6. Adjust the Condenser lenses and diaphragm . 7. Place a slide on the stage and secure with stage clips. 8. Switch on the light at low intensity and then increase intensity. 49
  49. 49. 9. Turn the coarse adjustment knob to lower the body tube until the low power objective reaches its lowest point. 10. Looking through the eyepiece, very slowly move the coarse adjustment knob until the specimen comes into focus. 11. Adjust distance between eye piece. 50
  50. 50. 12. Switch to the high power objective lens only after adjusting condenser and iris diaphragm. 13. Place a drop of oil over specimen before using oil immersion objective. 14. Lower the objective until oil makes contact with objective. 15. Looking through the eyepiece, very slowly focus the objective away from the slide i.e by raising the objective lens. 51
  51. 51. How to observe a slide ? 52
  52. 52. Causes of Error in Focusing - Revolving Nose Piece is off centre - Preparation is upside down - Thick cover slip - Dirt or Dried oil over Lens - Air bubble in immersion oil - Poor illumination – Condenser not fully racked up 53
  53. 53. PHASE CONTRAST MICROSCOPE 54
  54. 54. Phase Contrast Microscope - First described in 1934 by Dutch physicist Frits Zernike - Produces high-contrast images of transparent specimens - Advantage - Living cells can be examined in their natural state 55
  55. 55. Principle Of Phase Contrast Microscopy - Unstained bacteria have constituents of different refractive index . - Diffraction of light - Phase contrast microscope employs an optical mechanism to translate minute variations in phase into corresponding changes in intensity of image. 56
  56. 56. 57
  57. 57. Requisite For Phase Contrast Microscopy - Annular Diaphragm - Phase Plate 58
  58. 58. Condenser Annulus - The condenser annulus or annular diaphragm is opaque flat-black (light absorbing) plate with a transparent annular ring. - Produces hollow cone of light. 59
  59. 59. Condenser annulus 60
  60. 60. Phase Plate - Placed in back focal plane of objective. - Function: 1. Enhances phase difference by retarding diffracted wave front by one quarter of wavelength . 2. Reduces intensity of direct rays and equalizes it with diffracted rays intensity. 61
  61. 61. Phase plate 62
  62. 62. Images of Phase Contrast Microscpy Spirilium volutans Clostridium botulinum 63
  63. 63. Comparision of Images of Bright Field and Phase Contrast Microscopy 64
  64. 64. Uses of Phase Contrast Microscopy - Phase contrast enables visualization of internal cellular components. - Diagnosis of tumor cells . - Examination of growth, dynamics, and behavior of a wide variety of living cells in cell culture 65
  65. 65. Dark Ground Microscope - Optical system to enhance the contrast of unstained bodies . - Specimen appears gleaming bright against dark background PRINCIPLE OF DGI 66
  66. 66. Optical path in Dark Ground Microscopy 67
  67. 67. Requisites for Dark Ground Microscopy - Dark ground condenser - High intensity lamp - Funnel stop 68
  68. 68. Uses of Dark Ground Microscopy Useful in demonstrating -Treponema pallidum - Leptospira - Campylobacter jejuni - Endospore Treponema pallidum 69
  69. 69. Fluorescence Microscopy PRINCIPLE Visible UV light radiation Fluorochrome FITC EX - 495 nm EM - 520nm TRITC EX – 540 nm EM – 590 nm Texas Red Ex – 600 nm EM – 615 nm 70
  70. 70. - UV rays passes through exciter filter - Dark ground condenser - Micro organisms stained with fluorescent dye, when examined under microscope with ultraviolet light are seen as bright object against dark background 71
  71. 71. Use of Fluorescence Microscopy - Auramine Rhodamine – Yellow Fl - Tubercle bacilli - Acridine Orange R - gives orange red Fl with RNA and yellow green Fl with DNA - QBC - IMMUNOFLUORESCENCE 72
  72. 72. Electron Microscope 73
  73. 73. ELECTRON MICROSCOPE -Electron Microscopes uses a beam of highly energetic electrons to examine objects on a very fine scale. This examination can yield the info about - Topography - Morphology - Composition - Crystallographic structure 74
  74. 74. TYPES OF EM - Transmission Electron Microscope (TEM) - Scanning Electron Microscope (SEM) 75
  75. 75. Transmission Electron Microscope (TEM) - Stream of electrons is formed - Accelerated using a positive electrical potential - Focused by metallic aperture and Electro magnets -Interactions occur inside the irradiated sample which are detected and transformed into an image . 76
  76. 76. TEM (Cont) -Projector Lens forms image on Fluorescent viewing screen - 2D Image - Magnification 10,000 X to 100,000 X 77
  77. 77. Scanning Electron Microscope - Scan a gold-plated specimen to give a 3-D view of the surface of an object which is black and white. - Used to study surface features of cells and viruses. - Scanning Electron microscope has resolution 1000 times better than Light microscope . 78
  78. 78. Working of SEM 79
  79. 79. SEM IMAGES Vibrio cholerae with Treponema pallidum polar flagella 80
  80. 80. OTHER MICROSCOPES INVERTED MICROSCOPE - Used in metallurgy - Examination of cultures in flat bottom dishes - Micro dissection - Examination of parasites - Observation of agglutination in serology 81
  81. 81. STEREO MICROSCOPE - Double Microscope - Produces 3D images POLARIZING MICROSCOPE - Uses two Polariser - Gives info about Birefringence of a body - Used in Crystallography, Urine examination - Apple Green Birefringerence in 82 AMYLODOSIS
  82. 82. CONFOCAL SCANNING LASER MICROSCOPE - Uses a laser beam to illuminate a specimen whose image is then digitally enhanced for viewing on a computer monitor. - Laser beam scans single plane of 1µm thickness. 83
  83. 83. Comparison of Depth of Light Collection and Image clarity Light Microscope Confocal Scanning Laser Microscope 84
  84. 84. PRINCIPLE OF CONFOCAL MICROSCOPY 85
  85. 85. USES OF CONFOCAL MICROSCOPE - Observing cellular morphology in multilayered specimen - Eg. used in diagnosing Ca cervix - Evaluation and diagnosis of basal cell carcinoma of skin 86
  86. 86. What is the advantage of using a confocal microscope? - By having a confocal pinhole, the microscope is really efficient at rejecting out of focus fluorescent light so that very thin section of a sample can be analyzed. - By scanning many thin sections through a sample, one can build up a very clean three-dimensional image . 87
  87. 87. NEWER MICROSCOPE - SCANNING PROBE MICROSCOPE - Class of Microscope that measures surface features by moving a sharp probe over object surface. Used to visualize atoms and molecules - Scanning Tunneling Microscope (STM) - Atomic Force Microscope (AFM) 88
  88. 88. CARE OF THE MICROSCOPE - Handling - Proper storage - Care of Lenses - Care of oil emersion objective - Care of lamp 89
  89. 89. 90

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