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30 Cards in this Set

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What is Resolution?
-The ability of a lenses to distinguish fine detail and stucture.
-A microscope with the resolving power of 0.4nm can distinguish between two points great than or equal to 0.4nm
-Shorter wavelengths of light provide greater resolution
Principles of Light Microscopy
-Refractive index is the light bending ability of a medium
-Immersion oil is used to keep light from bending
Brightfield(light)Microscopy
-Dark objects are visible agaisnt a bright background.
-Light reflected off the specimen does not enter the objective lens.
Darkfield Microscopy
-Light objects are visible against a dark background
-Light reflected off the specimen
-Special condenser
-Useful in diagnonsis of SYPHILLIS( T. pallidum)
-Spirocette
-Used to examine microbes that are invisble in brightfeild microscopy, cannont be stained by standard methods, or are distorted by staining
Phase Contrast Microscopy
-Detailed examination of internal structures in living microbes
-NO STAINING REQUIRED
-Accentuates diffraction of the light that passes through a specimen
Fluorescence Microscopy
-Uses UV light
-Fluorescent substances absorb UV light and emit visible light
-Cells are stained with Fluorescent dyes
-Rapidly detect and identify microbes in tissues or clinical specimens
-Principle use is for immunoflurescence
Fluorescent Micrscopy
-Used to rapidly detect and identify fluorescent(yellow or green-colored)microbes:
Treponema pallidum-Syphillis

Mycobacterium tuberculosis- TB

Legionella pneumophilla- legionaires disease
Electron Microscopy
- Uses a beam of electrons
- Uses elctromagnetic lenses
-The shorter wave length of electrons give greater resolution
- less than 0.2 micrometer such as VIRUSES and RIBOSOMES
Transmission Electron Microscopy
-ultrathin sections of specimens
-Light passes through specimen, then an electromagnetic lens, to a screen or film
-Specimen may be stained ith heavy metal salts
-10,000-100,000x; resolution 2.5nm
Scanning Electron Microscopy(SEC)
-An electron gun produces a beam of electrons that scans the surface of the whole specimen
-Secondary electrons emmited from the specimen produce the image
-1000-10,000x; resolution 20nm
Scanning-Probe Microscopy
-Scanning tunneling microscoy uses a metal probe to scan the specimen
-Resolution 1/100 of an atom
-Detailed view of molecules inside cell(DNA)
Atomic Force Microscopy
-Uses a metal and diamond probe inserted into the specimen
-Produces 3-d images of bacterial surface layer protein or toxin
- Holes because the organism gives off gas Bacillus cases Gas gangrene
Preparation of Specimens for Light Microscopy
-A thin film of solution of microbes on a slide is a smear
-A smear is fixed by heat flame of the Bunsen burner or a slide warmer to attach and kill the microbes
- A smear is stained with a basic or acidic dye
Unstained Specimens
-Live or unstainded cells have little contrast
-Researchers make discoveries about cell behavior looking at live specimens
Wet Mounts
-A stain is rarely used
- Live organisms are seen under the high power objective
-Motility of an organism can be viewed on a wet mount
-Motile bacteris include Escherichia coli, Pseudomonas, and Proteus
Simple Stains
-Using a single basic dye
-Used to observe the shape and size of a cell
-cell morphology is shown
Differential Stains-Gram Stains
-Classifies bacteria into positive and negative
-Gram-postive tend to be killed by detergents and penicillin
-Gram-negative bacteria are more resistant to antibiotics
Gram Stain Procedures
1)Apply crystal Violet(primary stain colors all cells purple)2min rinse H20
2)Apply Iodine(mordant forms a complex with the crystal violet and holds the primary stainCV-1)1min rinse H20
3)Ethyl alcohol or Acetone(decolorizes and removes crystal violet-iodide complex from some bacteria)rinse H20 1min
4)Apply safranin9counterstain has contrasting color to the primary stain)rinse 30 sec
Results of Gram Stain
-Gram-positive bacteria resist decolorization and retain the crystal violet dye and stain purple
-gram-negative bacteria having much lipid in their cell wall are dcolorized and take up the safranin dye and stain pink
Importance of Gram Stain
1)1st step in id of bacteria
2)Useful in clinical diagnosis
3)Treatment
Differential Stains: Acid Fast
-Useful in diagnosis of tuberculosis caused by Myobacterium tuberculosis
Steps in Acid Fast stain
1) Ad Carbolfuchsin Dye(red)5min
2)Acid/Alcohol(decolorizer)20sec
3)Methylene Blue(counterstain)2min
Acid Fast Stain result
1)Cells that retain a carbolfuchsin stain in the presence of acid-alcohol are call acid fast such as myobacterium stain red
2)Non-acid fast cells lose the carbolfuchsin stain whne rinsed with acid-alcohol and take up the counter stain and appear blue
What disease can be diagnosed using the acid fast stain?
T.B. and Leprocy
Special Stains
-Staining the background instead of the cell
-Used to color or isolate a specific part of the microbe such as capsules,endospores or flagella
Negative Staining
-Is used to make capsules visible
-Capsules serve as virulence factor
-Capsule appears as a clear "halo"(Klebsiella pneumoniae, Streptococcus pneumoniae)
Cryptococcus neoformans
-a yeast
-has a thick capsule
-Capsule visible in India Ink
What is the value of capsule to bacteria and yeast?
Antiphagocytic
Speacial Stains
-Endospores are usually dormant, resting structures that allow bacteris to survive in harsh environments
-Endospores are usually produced by gram positive bacteria(Bacillus species and Clostridum species)
SPecial Staining
-Flagella are whiplike appendages that allow for bacterial movement
-Flagella Staining requires mordant to make th flagella wide enough to see
-number and arrangment of flagella can be observed