2) Observing The Microbial Cell Flashcards
Resolution
the smallest distance by which two objects can be separated and still be distinguished
Which part of the human eye can the finest resolution of two separate point be perceived
Fovea
Fovea
The portion of the retina where the photoreceptors are packed at the highest density
Detection
The ability to determine the presence of an object
Magnification
The increase in the apparent size of an image to resolve smaller separations between objects
Eukaryotic Microbes
Protozoa, algae, fungi
10 - 100 um
Can be seen under a light microscope
Prokaryotes
Bacteria, archaea
0.4 - 10 um
Subcellular structures too small to resolve by light microscopy
Wavelength of Visible Light
400 - 750 nm
For electromagnetic radiation to resolve an object 3 conditions must exist
1) Contrast between object and its medium
2) Wavelength smaller than the object
3) Magnification
Absorption means
The photon’s energy is acquired by the absorbing object
Reflection means
That the wavefront bounces off the surface of an object
Refraction means
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
Magnification requires the
Bending of light rays (refraction)
How does refraction accomplish magnification
Refraction magnifies an image when light passes through a refractive material shaped so as to spread its rays
Bright-Field Microscopy
Generates a dark image of an object over a light background
To increase resolution
- Use shorter wavelength light
- Reduce contrast
- Use immersion oil
- Use wider lens closer to specimen
- Higher numerical aperture (NA)
Compound microscope
A system of multiple lenses designed to correct or compensate for aberration
Total magnification
Magnification of the ocular multiplied by that of the objective
Wet mount
Placing a drop of water on a slide with coverslip
Advantages - Observation of 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
Staining
- Cells are given a distinct color
- Most stains have conjugated double bonds or aromatic rings, as well as one or more positive charges
The detection and resolution of cells under a microscope are enhanced by
Fixation and Staining
Different kinds of stains
Simple stain and differential stain
Simple Stain
Adds dark color specifically to cells, but not to the external medium or surrounding tissue
(Methylene blue is the most commonly used stain)
Differential Stain
Stains one kind of cell but not another
(Most famous differential stain is the Gram stain)
Gram Stain devised
In 1884 by Hans Christian Gram
Gram-positive bacteria
Gram-negative bacteria
- retain the crystal violet stain because of their thicker cell wall
- bacteria do not
Fluorescence microscopy
The specimen absorb light of a defined wavelength and then emits light of lower energy, thus the longer wavelength
Excitation wavelength
The specimen absorbs light of a specific wavelength
Emission wavelength
Emits light at a longer wavelength
First to demonstrate the possibly of single-molecule tracking of fluorescent proteins in bacteria
William Moerner
Dark-field Microscopy
Enables microbes to be visualized as halos of bright light against darkness
Phase-contract microscopy (PCM)
Exploits difference in refractive index between the cytoplasm and the surrounding medium or between different organelles
Differential Interference Contrast Microscopy (DIC)
Enhances contrast by superimposing an image of the specimen onto a second beam of light that generates interference fringes
Electron microscopy (EM)
The foremost tool for observing the shapes of macro molecular structures
Scanning probe microscopy
Images the contours of live bacteria
X-ray crystallography
The tool of choice for atom-level detail of a macromolecule
Two major types of electron microscopy
Transmission Electron Microscopy (TEM)
Scanning Electron Microscopy (SEM)
Transmission Electron Microscopy
- Electrons pass through the specimen
- Reveals internal structures
Scanning Electron Microscopy (SEM)
- Electrons scan the specimen surface
- Reveals external features in 3D
Cryo-electron microscopy (cryo-EM)
High-strength electron beams now permit low-temperature
-Specimen does not require staining
- Specimen must be flash-frozen
Scanning probe microscopy (SPM)
Enables nanoscale observation of cell surfaces
Atomic force microscope (AFM)
(example if an SPM)
- It measures the van der Waals forces between electron shells of adjacent atoms of the cell surface and the sharp tip
- It can be used to observe live bacteria in water or exposed to air
Bright-field:
Fluorescence:
Dark-field:
Phase-contrast:
- employs various stains
- employs fluorophores for labeling
- detects unresolved objects
- exploits differences in refractive indices
TEM: provides
SEM: provides
- Internal details in 2D
- External details in 3D
Scanning probe microscopes (SPMs) include the atomic force microscope (AFM)
- Allow observation of living cells in water or in air
Molecules can be visualized by
X-ray crystallography
Which of the microscopes allows the best view of bacterial flagella during motility
Dark-field microscope
Wavelength is the
Distance between one peak of a wave and the peak
The height of each peak (or depth)
Amplitude
Frequency of the wave
The rate of vibration of the wave or the number of wavelengths within a specified time period
The variety of staining techniques (used for light microscopy)
Gram staining, acid-fast staining, capsule staining, endospores staining and flagella staining
Commonly used light microscopes
Brightfield, darkfield, phase-contrast, differential interference contrasts fluorescence, confocal and two-photon microscopes
