microscopy and staining


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This is a series of lectures on microbiology useful for undergraduate medical and paramedical students

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  • R.P. = 0.5 (lambda) / (η sinØ)
  • Uses of darkfield and Phase contrast microscopy
    View unstained cells
    Not subject to shrinkage, artefacts
    Some bacteria do not stain easily, e.g., spirochetes (Borrelia, Leptospira, Treponema)
    View living cells
    Can observe processes, e.g., motility, predation
  • Acid fast stain
    Acid fast stain (Ziehl-Neelson) for identifying mycobacteria
    The lipid mycolic acid (from mycobacteria) is the determinant of retaining the basic fuchsin in the acid-fast stain
  • microscopy and staining

    1. 1. Fundamental Techniques in Micr obiolog y Micr oscopy and Staining Dr. Ashish V. Jawarkar (M.D.) Parul Sevashram Hospital
    2. 2. Fundamental Techniques Microscopy Staining Aseptic technique Sterilization and waste disposal
    3. 3. Microscopy Measurement  Microorganisms are very small  Use metric system  Metre (m) : standard unit  Micrometre (µm) = 1 x10-6 m  Nanometre (nm) = 1 x10-9 m  Angstrom (Å) = 1 x10-10 m
    4. 4. Terms Relevant to Microscopy Total Magnification  Eyepiece x objective lens Resolution  Ability of the lens to distinguish two points as separate  Theoretical limit for light microscope is 0.2 µm
    5. 5. Types of Microscopes Simple: one lens Compound: more than one lens
    6. 6. The Compound Microscope READ BOTTOM TO TOP! enters the eye  sees virtual, inverted image further magnif. by ocular forms magnified real image enters objective focuses light on object light enters condenser ocular objective object condenser
    7. 7. Objectives 10X Scanning object Find the (Course focus) 40X High-Dry (Fine the object Focus focus) 100X Oil immersion Fine focus
    8. 8. Bright-field Microscope Contains two lens systems for magnifying specimens Specimens illuminated directly from above or below Advantages: convenient, relatively inexpensive, available Disadvantages: R.P 0.2 µm at best; can recognize cells but not fine details Needs contrast. Easiest way to view cells is to fix and stain.
    9. 9. Different magnifications
    10. 10. Special Microscopy Applications Dark Field Phase Contrast Fluorescence Electron Microscope
    11. 11. Dark Field Microscopy special condenser diaphragm  occludes direct light, passes wide angle light  angle too wide to enter objective diffracted light scattered enters objective objects light on dark background diffracted light
    12. 12. Phase Contrast Microscopy light rays through objects of different η → change in phase, not intensity special ring-shaped condenser diaphragm special glass disc in objective change phase differences to intensity differences  can view transparent objects as dark on light background (without staining)  Right; human brain glial cells
    13. 13. Fluorescence Microscopy Illuminate specimen with UV → visible fluorescence (filter removes harmful UV) View auto-fluorescent objects (e.g., chloroplasts) Stain with specific fluorescent dyes, which absorb in region 230-350 nm & emit orange, yellow or greenish light Images appear coloured against a dark background
    14. 14. Electron Microscopy
    15. 15. Stains and Staining Staining produces contrast
    16. 16. Types of stains Simple stains Negative staining Differential staining Special stains
    17. 17. Simple stains Simple stain  Aqueous or alcohol solution of single basic dye  Stains bacteria  Background unstained
    18. 18. Simple Stains
    19. 19. Negative staining background is stained, leaving the actual specimen untouched
    20. 20. Differential Stains Stains both background and bacteria
    21. 21. Differential Stains Acid-fast stain  Used to detect Mycobacterium species
    22. 22. Special Stains Capsule stain  Klebsiella pneumonia
    23. 23. Special Stains Flagella stain
    24. 24. Gram stain procedure Gram positive – violet Gram negative – red Primary staining Decolorisation Counter staining
    25. 25. Acid fast stain / Ziehl Neelsen Stain
    26. 26. Albert’s stain For C. Diphtheria Granules black Body green