Chapter 2 Flashcards
Resolution
- is the smallest distance by which two objects can be separated and still be distinguished
- limits the ability of what we can see with a microscope
Detection
is the ability to determine the presence of an object
Magnification
is the increase in the apparent size of an image to resolve smaller separations between objects
Microbial Shape
- Prokaryotic cell structures are generally simpler than those of eukaryotes
- Certain shapes of bacteria are common to many taxonomic groups
- Bacilli = rods
- Cocci = spheres
- Spiral forms = Spirochetes and Spirilla
For electromagnetic radiation to resolve an object, certain conditions must exist
- Contrast between object and its medium
- Wavelength smaller than the object
- Magnification
Absorption
means that the photon’s energy is acquired by the absorbing object
Reflection
means that the wavefront bounces off the surface of an object
Refraction
is the bending of light as it enters a substance that slows its speed
Scattering
occurs when the wavefront interacts with an object smaller than the wavelength of light
empty magnification
magnification without increasing detail is called
compound microscope
- a system of multiple lenses to increase magnification and produce an upright image
- Basic design contains 2 lenses = Ocular lens and Objective lens
- Total magnification = magnification of ocular multiplied by that of the objective
wet mount
-simple way to observe microbes is to place them in a drop of water on a slide with a coverslip
Advantages:
-Observation of live cells in natural state
Disadvantages:
- Little contrast between cell and background
- Sample may dry out quickly
Fixation
- cells are made to adhere to a slide in a fixed position with heat or chemicals
- usually kills the cells
Staining
-cells are given a distinct color with a dye, which produces increased contrast
simple stain
- adds dark color specifically to cells, but not to the external medium or surrounding tissue
- A commonly used stain is methylene blue
differential stain
- stains one kind of cell but not another
- The most common differential stain is the Gram stain
Gram stain
- differentiates between two types of bacteria
- Gram-positive bacteria retain the crystal violet stain because of their thicker cell wall
- Gram-negative bacteria do not
Acid-fast stain
-carbolfuchsin used to stain Mycobacterium species
Spore stain
malachite green used to detect spores of Bacillus and Clostridium
Negative stain
- like india ink
- leaves a white area around the cells which i the carbohydrate layer
- carbohydrate layer use to protect the cells from our immune system
- colors the background, which makes capsules more visible
fluorescence microscopy
- fluorescent molecules absorbs light of a defined wavelength, and then fluoresce by emitting light of lower energy, thus longer wavelength
- Can be used to stain specific cell types in complex samples or specific cellular components
- Can be used to follow expression of fluorescent reporter proteins such as GFP
excitation wavelength
specimen absorbs light of a specific wavelength
emission wavelength
emits light at a longer wavelength
filters
- To limit incident light to the wavelength of excitation and emitted light to the wavelength of emission
- high-intensity light source shines through an excitation filter that transmits only the range of wavelengths to excite the fluorophore
fluorophore
- is a fluorescent chemical compound
Its cell specificity can be determined in at least 3 ways:
1) Chemical affinity for a cellular component
2) Labeled antibodies that bind to specific cellular components 3) DNA hybridization to specific DNA sequences
Dark-Field Microscopy
- light stop is used to stop light from going directly through the sample to your eye
- enables microbes to be visualized as bright objects against a dark background
- Light shines on sample at an oblique angle
- Only light scattered by the sample reaches objective
- Makes visible objects below resolution limit
- Flagella and Very thin bacteria
Phase-Contrast Microscopy
- Superimposes refracted light and transmitted light shifted out of phase
- Reveals differences in refractive index of different cell components as patterns of light and dark resulting from constructive and destructive interference
- Can be used to view live unstained cells and internal cellular organelles of larger eukaryotic cells
Electron Microscopy
Electrons behave like light waves
- Very high frequency, which allows greater resolution
- A few nanometers
- Lenses are magnetic fields
Sample must absorb or reflect electrons
-Stained or coated with heavy metals (kills cells)
Electrons are easily affected by matter
- Electron beam and sample must be in a vacuum (cells must be dehydrated)
- Samples must be very thin
Transmission electron microscopy (TEM)
- Electrons pass through the specimen
- Reveals internal structures
- preparation- Embedded in a polymer for thin sections
- Microtome with a diamond knife is used to cut very thin slices
- Molecular samples can be sprayed onto a thin film supported by a copper grid
- closely parallels the design of the bright-field microscope
- light source is replaced by an electron source consisting of a high-voltage current applied to a tungsten filament, which gives off electrons when heated
Scanning electron microscopy (SEM)
- Electrons scan the specimen surface
- Reveals external features in 3D
- preparation- specimen is not sliced but is coated with heavy metal
- scattered and secondary electrons emitted from the sample are detected
- electron beam is scanned across a specimen coated in gold, which acts as a source of secondary electrons
X-ray crystallography
- For samples that can be crystallized, X-ray diffraction can determine the position of individual atoms in a molecule
- Many organic molecules and proteins can be crystallized, but some do not produce suitable crystals
- A beam of X-rays is shot at a crystallized sample
- Many molecules are in identical conformation in the crystal
- X-rays diffract according to position of atoms
- Compute position of atoms from pattern of scattered X-ray
- undergo mathematical analysis to generate molecular models
