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Microbiology Chapter 3.1
Microscopic Techniques: The Instruments
Terms in this set (39)
Name the microscopes used to study microorganisms?
-The Light Microscope (Most Important)
-The Electron Microscope
-The Atomic Force Microscope
What is the purpose of the Light Microscope?
The light microscope uses visible light to observe objects.
-The light microscope can magnify images up to 1,000x
-The light Microscope makes it easier to view prokaryotic cells size, shape and motility.
What is the purpose of the Electron Microscope?
The Electron Microscope magnifies images to reveal many fine details of cell structure.
-The electron microscope magnifies images up to 100,000x
What is the purpose of the Atomic Force Microscope?
The atomic force microscope allows scientist to produce images of individual atoms on a surface.
What is the principles of Light Microscopy?
In light microscopy light typically passes through a specimen and then through a series of magnifying lenses.
-Light microscope uses glass lenses, visible light and eyes to observe
What is the most common type of light microscopy?
The bright-field microscope which evenly illuminates the field of view. It is also the easiest to use.
Name the 2 magnifying lenses of the light microscope?
Objective Lens & Ocular Lens (Together they are called compound microscope)
How is the objective lens & Ocular lens used?
These lenses enlarge an object by a factor equal to the product of each lens magnification. example, object is magnified 1,000 fold when viewed through 10x ocular lens along with a 100x objective lens.
What are the selections for compound microscope lenses?
The power level start with 4x, 10x, 40x and 100x which allows different magnifications.
What is the purpose of the Condenser Lens?
The condenser lens is positioned between the light source and the speciment which is used to focus light on the specimen. The condenser lens does not affect the magnification it's used to focus light.
The usefulness of a microscope depends on?
It depends on the degree of magnification and its ability to separate or resolve 2 objects that are very close together.
What is the resovling power defined as?
The resolving power is defined as the minimum distance existing bt 2 objects when those objects can still be observed as separate oranisms. The resolving power determines how much detail can be seen in a specimen.
The resolving power of a microscope depends on?
Resolving power depends on:
-Quality & type of lens
-Wavelength of light
-How specimen under observation have been prepared
What is the maximum resolving power of light microscope?
0.2um which is good to observe the general morphology of a prokaryotic cell but it's too low to tell the size of most viruses.
What should be used to obtain maximum resolution when using high power lens?
When using high power objectives such as 100x lens, oil should be used to displace the air bt the lens & the specimen. This avoids the bending of light rays.
Refraction is the bending of light rays when light passes from glass to air.
What does refraction prevents?
Refractions prevent rays from entering the relatively small openings of higher-power objective lenses.
Oil has the same refractive index as?
Oil has the same refractive index as glass.
Define Refractive Index.
Refractive index is a measure of the relative velocity (Speed) of light as it passes through a medium. As light travels from a medium of one refractive index to another, those rays are bent.
What happens when oil displaces air?
When oil displaces air at the interface of the glass slide and glass lens, light rays pass with little refraction occurring.
Name something contrast reflects?
Contrast reflects the # of visible shades in a speciment-high contrast is 2 shades (Black & White). Different specimen require different degrees of contrast in order to see more details of the specimen. Example bacteria are transparent against a bright colorless background.
How can we solve the lack of contrast problem when viewing objects?
The lack of contrast problem when viewing objects can be solved by staining the bacteria with a dye.
Light Microscope that Increase Contrast
Light microscopes that increase contrast overcome some difficulties of observing unstained bacteria. The light microscope increases the contrast bt microorganisms and their surrondings.
Phase Contrast Microscope
The phase-contrast microscope enlarges the slight difference bt the refractive-index of cells & the surrounding medium, resulting in a darker appearance of the denser material.
How does light pass through the Phase-Contrast Microscope?
As light passes through cells, it is refracted slightly different than when it passes through its surroundings.
The Interference Microscope
The interference Microscope causes the speciment to appear as a three-dimensional image.
-This mircrocope & the phase contrast microscope depends on differences in refractive index as light passes through different materials.
What is the most requently usind microscope of Interference?
The Nomarski Differential Interference Contrast (DIC) Microscope.
-DIC separates light into 2 beams that pass through the speciment & then recombine.
-Light waves are out of phase as they recombine, this causes the 3 dimensional appearance of the specimen.
The Dark-Field Microscope
Organisms viewed through a dark-field microscope stand out as bright objects against a dark background.
Dark-Field Macroscopy can detect?
Treponema Pallidum the causative agent of syphilis which stains poorly & is difficult to see under bright-field micrscopy.
The Fluorescence Microscope
The fluorescence microscope is used to view cells or other materials that are naturally fluorescent or have been stained or tagged wtih fluorescent dyes.
-The fluorescence microscope projects light through a specimen
-Then only captures only the light send out by the fluorescence molecule to form the image. This allows Fluorescence cells to stand out as illuminated objects against a dark-background.
Fluorescent molecule absorbs light at?
Fluorescence microscope absorbs light at 1 wavelength (Normally ultraviolet light) then send out light of a longer wavelength.
Name a commomn fluorescence microscope?
-This microscope projects the ultraviolet light through the objective lens & onto the speciment.
-Since light is not transmitted through the speciment, cells attached to soil particles or other opaque materials can be seen.
The Confocal Scanning Laser Microscope
The confocal scanning laser microscope is used to construct a 3-dimensional image of a thick structure such as a community of microorganisms.
-Confocal microscopy, lenses focus a laser beam to illuminate a given point on one vertical plane of a specimen.
-Mirrors then scan the laser beam across the speciment, illuminating successive regions & planes until the entire specimen have been scanned.
-A computer construct the data & create 3-dimensional image.
-This microscope is a miniature CAT scan for cells.
Specimens are 1st stained or tagged with?
A Fluorescent dye.
-Fluorescent tags binds to a given protein or other compound
-The cellular location of the compound can be determined
The electron microcope uses electromagnetic lenses, electrons and a fluorescent screen to produce the magnified image.
-Drawbacks= Lenses and specimen must be in a vacuum, if not the molecules that make up air would interfere with the path of the electrons.
Image that is captured on a photographic flim is called?
-Electrons have a wavelength of 1,000x shorter than visible light therefore, the resolving power increases to 1,000 fold to about 0.3 nm or 0.3x10-3um.
-More details can be observed due to higher resolution which can be magnified to 100,000x.
The Transmission Electron Microscope (TEM)
Transmission Electron Microscope
-Used to observe fine details of cell structure
-It directs a beam of electrons at a speciment
-To view details of internal structure a speciment preparation called thin sectioning.
-Thin sectioning is when cells are carefully treated with a preservative & dehydrated in an organic solvent before being embedded in a plastic resin. After that the cells must be sliced and stained with heavy metals.
-A process called freeze fracturing is used to observe the shap of structures within the cell. The specimen is frozen then fractured with a knife blade.
-A variation of freeze fracturing is freeze etching. This process is when the frozen surface exposed by fracturing is dried slightly under vacuum, which allows different parts to be exposed.
The Scanning Electron Microscope
The scanning Electron Microscope (SEM) is used for observing surface details of cells.
-A beam of electrons scan back and forth over the surface of a specimen coated with a thin film of metal.
-3 dimensional images can be viewd with this microscope
Atomic Froce Microscopy
The Atomic Force Microscopy (AFM) produces detailed images of surfaces.
-The resolving power is greater than electron microscope.
-No special preparation is required
-This microscope can inspect samples either in the air or submerged in liquid.
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