Microscopy Flashcards
Simple microscope
Principles: contains a single lens to examine various natural substances for microorganisms
Applications: can see general structures of microbes
Limitations: Crude, hard to use, cannot see much
compound/bright field microscope
Principles: has a magnification of 100-1,500x and resolution of 200nm
Applications: can view live or non alive cells
Limitations: Light refraction of images and resolution will lessen at increased magnifications
Magnification
Enlarge the image (lens)
Resolution
Ability to distinguish between two adjacent items
Contrast
Ability to distinguish items from background
Numerical aperture (NA)
A measure of light-capturing ability
increased NA= increased resoltuion
Physical properties of resolution
Wavelength of light/electrons
shorter wavelength =increased resolution
Oil immersion technique
Increases resolution by decreasing light refraction (increases numerical aperture)
Basic Dyes from Staining
Crystal violet which binds to DNA
Stains all types of cells
Differential Dyes from staining
Gram stain
2 different colors based on characteristics of the cell wall
Gram (+) = purple
Gram (-) = pink
Staining A/D
Advantages: increase contrast and differentiate cell types
Disadvantages: kills the cells and can distort their features
Phase contrast Microscopy
Principles: refraction index, phase ring, and improved contrast
Applications: viewing live cells
Limitations: can only see so much inside the cell
Dark-field microscopy
Principles: light specimen from the side and results in dark background
Applications: viewing live cells, cell motility, and flagella viewing
Limitations: Image is not always clear and cannot see inside cells well
Fluorescence microscopy
Principles: visualize light emitting from cells and autofluorescence (from cells) v stained (DAPI stain)
Applications: viewing live and fixed cells, quantifying cell numbers
Limitations: staining kills cells, no clear resolution
Differential Interference Contrast (DIC) microscopy
Principles: Polarized light, two beams to one, and enhanced cell structures
Applications: view cellular structures (nucleus, endospores, and vacuoles)
Limitations: can only be used for larger microbial cells
Atomic force microscopy
Principles: cantilever, laser detector, and repulsive forces
Applications: viewing live specimens and no fixative or coating
Limitations: cannot see inside cells, only outside of them and image can be distorted easily by fast-moving microbes
Confocal scanning laser microscopy
Principles: laser + fluorescence and focusees on different layers
Applications: Thick specimens (ex biofilm) and multilayer image
Limitations: cannot see inside cells very well
Electron microscopy
Principles: electron beam, electromagnetic lens, and operates in a vacuum
Applications: increased magnification and resolution, used for viewing inside microbial cells
Limitations: expensive and a lot of prep
Transmission electron microscope
Higher resolution, 2D images, and internal features
Thin sectioning
Scanning Electron microscope
Lower resolution, 3D images, and external features
heavy metal coating (gold)
