Microscopy Flashcards
Microscopic diagnosis for bacteria
Bright-field M: +
Fluorescence M: +/-
Phase-contrast M: +
Dark-field M: +/- [spirochetes (borrelia, leptospira, treponema)]
Electron M: -
Microscopic Diagnosis for Fungi
Bright-field M: +
Fluorescence M: +/-
Phase-contrast M: +
Dark-field M: -
Electron M: +/-
Microscopic diagnosis for Parasites
Bright-field M: +
Fluorescence M: +/-
Phase-contrast M: +
Dark-field M: -
Electron M: +/-
Microscopic diagnosis for viruses
Bright-field M: -
Fluorescence M: +/-
Phase-contrast M: -
Dark-field M: -
Electron M: +/-
Used for stained and unstained samples
BRIGHT FIELD MICROSCOPE
Most common methods employed under bright field microscope
o Gram Stain
o Acid Fast Stain
o Potassium Hydroxide
Designed to provide maximum illumination and resolution when observing images using a microscope
Kohler Illumination
Scanning power
Objective lens magnification:
Ocular lens magnification:
Total Magnification:
Objective lens magnification: 4x
Ocular lens magnification: 10x
Total magnification: 40x
Low power objective lens
Objective lens magnification:
Ocular lens magnification:
Total magnification:
O bjective lens magnification: 10x
Ocular lens magnification: 10x
Total magnification: 100x
High-dry power objective lens
Objective lens magnification:
Ocular lens magnification:
Total magnification:
Objective lens magnification: 40x
Ocular lens magnification: 10x
Total magnification: 400x
Oil immwersion power objective lens
Objective lens magnification:
Ocular lens magnification:
Total magnification:
Objective lens magnification: 100x
Ocular lens magnification: 10x
Total magnification: 1000x
Observing microbial pathogens
Tools: eyes
Magnification: 0
Application:
Gross examination
Observing microbial pathogens
Tools: Magnifying glass
Magnification: 5
Application:
Gross examination
Observing microbial pathogens
Tools: dissecting microscope
Magnification: 2.5-30
Application:
Gross detailed examination & manipulation
Observing microbial pathogens
Tools: bright-field microscope
Magnification: 10-2000
Application:
Cells stained
Observing microbial pathogens
Tools: dark-field microscope
Magnification: 10-400
Application:
Cells not readily stained for bright-field microscopy
Observing microbial pathogens
Tools: phase-contrast microscope
Magnification: 10-400
Application:
Living or unstained cells
Observing microbial pathogens
Tools: fluorescence microscope
Magnification: 10-400
Application:
Preparations using fluorochrome stains, which can directly stain cells or be connected to antibodies that attach to cells
Observing microbial pathogens
Tools: transmission electron microscope
Magnification: 150-10Million
Application:
Determine ultra structure of cell organelles
Observing microbial pathogens
Tools: scanning electron microscope
Magnification: 20-10,000
Application:
Determine surface shapes and structures
• Motility of Spirochetes
• Also used for fluorescent stains – Substitute
• Higher resolving power than bright field
DARK FIELD MICROSCOPE
• Inclusion bodies seen on virus and Chlamydia, living cells/natural state
• Micro lymphocytotoxicity test for Human leukocyte antigen (inverted PCM)
PHASE-CONTRAST MICROSCOPE
• Requires fluorescent stain
• UVL – provided by mercury are lamp
• Substitute: Dark-field microscopy
FLUORESCENT MICROSCOPE
Stain cell wall of fungi (Chitin)
Calcofluor White
Stain high affinity with nucleic acid
Acridine Orange
Truant’s method; High affinity with mycolic acid (Mycobacterium spp.)
Auramine Rhodamine
For immunofluorescent techniques; 3 bacteria (Bordetella, Chlamydia, Legionella)
Fluorescein isothiocyanate (FITC)
• Viral morphology
• Resolution: 0.5 nm
• Fixatives: Glutaraldehyde and Osmium tetroxide
• Dehydrating agents: Alcohol
• Stain: Heavy metals (Lead citrate, uranyl acetate)
ELECTRON MICROSCOPE
• Transmission EM – _________ structure
• Scanning EM – ____________ structure
• Transmission EM – Internal structure
• Scanning EM – External structure
This is useful to obtain high resolution images and for three-dimensional reconstruction of biological models
Confocal Microscopy
This measures surface features by moving a sharp probe over the object’s surface.
Scanning Probe Microscopy
two types of scanning probe microscope
Scanning tunneling microscope
Atomic force microscope
Light microscope source
Visible light
Electron microscope source
Expensive
Light microscope vs electron microcope
Medium of transmission
Air - High vacuum
Light microscope vs electron microcope
Nature of Lens
Glass - Electromagnet
Light microscope vs electron microcope
Focusing mechanism
Lens position is adjusted mechanically
Current to the magnetic lens
Light M. vs. Electron M.
Source of Contrast
Differential light absorption
Scattering of electrons
Light M. vs. Electron M.
Specimen mount
Glass slide
Metal grid
Best resolution
Light M.
Electron M.
0.2um
0.5nm
Highest practical magnification
Light M.
Electron M.
1000 – 1500
Over 100,000
Affordability
Light M.
Electron M.
Cheaper
Expensive
