10731570.ppt
- 1. Chapter 3: Microscopic observation of microorganisms PHR- 110 (Pharmaceutical Microbiology-1) 1 Course Instructor- Md. Samiul Alam Rajib Senior Lecturer Department of Pharmacy BRAC University
- 2. In this chapter students will learn about- Different types of Microscopy such as Bright field, dark field, fluorescence and phase contrast microscopy, electron microscopy. Preparations of microscopic examinations, wet mount and hanging drop techniques, fixed and stained smears. microbiological stains: simple and differential staining methods
- 3. Scale 3
- 4. Discovery of Microorganisms Antony van Leeuwenhoek (1632-1723) first person to observe and describe micro- organisms accurately 4 Figure 1.1b
- 5. Lenses and the Bending of Light Light is refracted (bent) when passing from one medium to another Refractive index A measure of how greatly a substance slows the velocity of light Direction and magnitude of bending is determined by the refractive indexes of the two media forming the interface 5
- 6. Lenses Focus light rays at a specific place called the focal point Distance between center of lens and focal point is the focal length Strength of lens related to focal length short focal length more magnification 6
- 7. 7 Figure 2.2
- 8. The Light Microscope Different types Bright-field microscope Dark-field microscope Phase-contrast microscope Fluorescence microscopes Compound microscopes Image formed by action of 2 lenses 8
- 9. The Bright-Field Microscope Produces a dark image against a brighter background Has several objective lenses Parfocal microscopes remain in focus when objectives are changed Total magnification product of the magnifications of the ocular lens and the objective lens 9
- 10. 10 Figure 2.3
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- 12. Microscope Resolution Ability of a lens to separate or distinguish small objects that are close together Wavelength of light used is major factor in resolution shorter wavelength greater resolution 12 Which one is clearer image and why?
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- 14. 14 Working distance Distance between the front surface of lens and surface of cover glass or specimen
- 15. 15 Why oil is used in oil immersion microscopy?
- 16. The Dark-Field Microscope Produces a bright image of the object against a dark background Used to observe living, unstained preparations 16
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- 18. The Phase-Contrast Microscope Enhances the contrast between intracellular structures having slight differences in refractive index Excellent way to observe living cells 18
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- 21. The Differential Interference Contrast Microscope Creates image by detecting differences in refractive indices and thickness of different parts of specimen Excellent way to observe living cells 21
- 22. The Fluorescence Microscope Exposes specimen to ultraviolet, violet, or blue light Specimens usually stained with fluorochromes Shows a bright image of the object resulting from the fluorescent light emitted by the specimen 22
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- 25. Preparation and Staining of Specimens Increases visibility of specimen Accentuates specific morphological features Preserves specimens 25
- 26. Fixation Process by which internal and external structures are preserved and fixed in position Process by which organism is killed and firmly attached to microscope slide Heat fixing preserves overall morphology but not internal structures Chemical fixing protects fine cellular substructure and morphology of larger, more delicate organisms 26
- 27. Dyes and Simple Staining Dyes Make internal and external structures of cell more visible by increasing contrast with background Have two common features Chromophore groups Chemical groups with conjugated double bonds Give dye its color Ability to bind cells 27
- 28. Dyes and Simple Staining simple staining A single staining agent is used Basic dyes are frequently used dyes with positive charges e.g., crystal violet 28
- 29. Differential Staining Divides microorganisms into groups based on their staining properties e.g., Gram stain e.g., acid-fast stain 29
- 30. Gram staining Most widely used differential staining procedure Divides Bacteria into two groups based on differences in cell wall structure 30
- 32. 32 Escherichia coli – a gram-negative rod
- 33. Acid-fast staining particularly useful for staining members of the genus Mycobacterium e.g., Mycobacterium tuberculosis – causes tuberculosis e.g., Mycobacterium leprae – causes leprosy ◦ high lipid content in cell walls is responsible for their staining characteristics 33
- 34. Staining Specific Structures Negative staining Often used to visualize capsules surrounding bacteria Capsules are colorless against a stained background 34
- 35. Staining Specific Structures Spore staining double staining technique bacterial endospore is one color and vegetative cell is a different color Flagella staining mordant applied to increase thickness of flagella 35
- 36. Electron Microscopy beams of electrons are used to produce images wavelength of electron beam is much shorter than light, resulting in much higher resolution 36 Figure 2.20
- 37. The Scanning Electron Microscope (SEM) uses electrons reflected from the surface of a specimen to create image produces a 3-dimensional image of specimen’s surface features 37
- 38. 38 Figure 2.27
- 39. The Transmission Electron Microscope (TEM) electrons scatter when they pass through thin sections of a specimen transmitted electrons (those that do not scatter) are used to produce image denser regions in specimen, scatter more electrons and appear darker 39
- 40. 40 TEM
- 41. SEM Vs TEM SEM TEM
- 42. Specimen Preparation analogous to procedures used for light microscopy for transmission electron microscopy, specimens must be cut very thin specimens are chemically fixed and stained with electron dense material 42
- 43. Other preparation methods shadowing ◦ coating specimen with a thin film of a heavy metal freeze-etching ◦ freeze specimen then fracture along lines of greatest weakness (e.g., membranes) 43
- 44. 44 Figure 2.25
- 45. 45 Ebola
- 46. 46 Fly head
- 47. Newer Techniques in Microscopy confocal microscopy and scanning probe microscopy have extremely high resolution can be used to observe individual atoms 47 Figure 2.20
- 48. Confocal Microscopy confocal scanning laser microscope laser beam used to illuminate spots on specimen computer compiles images created from each point to generate a 3- dimensional image 48
- 49. 49 Figure 2.29
- 50. 50 Figure 2.30
- 51. Scanning Probe Microscopy scanning tunneling microscope ◦ steady current (tunneling current) maintained between microscope probe and specimen ◦ up and down movement of probe as it maintains current is detected and used to create image of surface of specimen 51
- 52. Scanning Probe Microscopy atomic force microscope ◦ sharp probe moves over surface of specimen at constant distance ◦ up and down movement of probe as it maintains constant distance is detected and used to create image 52
- 53. Magnification calculation of compound microscopy For tube length L = 10cm objective focal length fo = 40 mm and eyepiece focal length fe = 50 mm Linear magnification of the objective lens = ? Angular magnification of the eyepeiece= ? Total magnification = ?