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Microscopy
use of light and electrons to magnify objects
Magnification
the apparent increase in size of an object
Thickness of the lens
Curvature of the lens
Speed of light
the image enlarged depends of the following:
Resolution or Resolving Power
ability to distinguish objects as close as 0.2 micrometer
0.1 mm (100um)
human eye can see objects as small as about
shorter wavelength
greater numerical aperture (-the ability to gather light)
Better resolution are due to
Contrast
different intensity between two objects or its background
Par focal
ability of the microscope to stay in focus even you shift from different objectives
Working Distance
distance between the lens and the specimen
Total Magnification
the number of times the object is enlarge from its original size
Ocular lens
Remagnifies the image formed by the objective lens
Body
Transmits the image from the objective lens to the ocular lens using prisms.
Objective lenses
primary lenses that magnify the specimen
Stage
holds the microscope slide in position
Condenser
focuses light through the specimen
Diaphgram
controls the amount of light entering the condenser
Illuminator
light source for the microscope
Coarse focusing knob
move the stage up and down to focus the image
Fine focusing knob
Moves stage slowly for precise, final focusing
Bright-Field
Dark-Field
Phase-Contrast
Differential Interference Contrast (Nomarski)
Fluorescent
Confocal
Different type of light microscopes
Nomarski
Differential Interfence contrast mirscope is also called
Light microscopes
Useful magnification 1x to 200x; resolution to 200 nm
Light microscopes
Use visible light; shorter; blue wavelengths provide better resolution
Bright-field
Colored or clear specimens against bright background
Dark-field
Bright specimen against dark background
Phase-constrast
Specimen has light and dark areas
Differential interference contrast (Nomarski)
Image appears three-dimensional
Fluorescent
brightly colored fluorescent structures against dark background
Confocal
Single plane of structures or cells that have been specifically stained with fluorescent dyes
Bright-field
simple to use; relatively inexpensive, stained specimens often reuqired
Dark-field
use a special filter in the condenser that prevents light from directly passing through a specimen; only light scattered by the specimen is visible
Phase-contrast
use a special condenser that splits a polarized light into two beams
Phase-contrast
Use a special condenser that splits a polarized light beam into two beams, one of which passes through the specimen, and one of which bypasses the specimen; the beams are then rejoined before entering the oculars; contrast in the image results from the interactions of the two beams
Differential interference contrast (nomarski)
Use two separate beams instead of a split beam; false color and a three-dimensional effect result from interactions of light beams and lenses; no staining required
Fluorescent
An ultraviolet light source causes fluorescent natural chemicals or dyes to emit visible light
Confocal
Use a laser to fluoresce only one plane of the specimen at a time
Bright-field
Observation of killed stained specimens and naturally colored live ones
Bright-field
also used to count microorganisms
Dark-field
Observation of living; colorless; unstained organisms
Phase-contrast & Differential interference contrast (nomarski)
Observation of internal structures of living microbes
Fluorescent
Localization of specific chemicals or structures; used as an accurate and quick diagnostic tool for detection of pathogens
Confocal
Detailed observation of structures of cells within communities
Transmission
Scanning
Type of Electron Microscope
Electron microscope
Typical magnification 1000x to 100,000×: resolution to 0.001 nm
Electron microscope
Use electrons traveling as waves with short wavelengths; require specimens to be in a vacuum, so cannot be used to examine living microbes
Transmission
Monotone, two-dimensional, highly magnified images; may be color-enhanced
Scanning
Monotone, three-dimensional, sufrace images, may be color-enhanced
Transmission
produce a two-dimensional image of ultrastructure of cells
Scanning
Produce three-dimensional view of the surface of microbes and cellular structures
Transmission
Observation of internal ultrastructural detail of cells and observation of viruses and small bacteria
Scanning
Observation of the surface details of structures
Probe microscopes
Magnification greater than 100,000,000x with resolving power greater than that of electron microscopes
Scanning tunneling and Atomic force
Individual molecules and atoms visible
Scanning tunneling
Measures the flow of electrical current between the tip of a probe and the specimen to produce an image of the surface at atomic level
Atomic force
Measure the deflection of a laser beam aimed at the tip of a probe that travels across the surface of the specimen
Transmission
Observation of internal ultrastructural detail of cells and observation of viruses and small bacteria
Scanning
Observation of the surface details of structures
Scanning Tunneling
Observation of the sruface of objectsm provide extremely fine detail, high magnificatino and great resolution
Atomic force
Observation of living specimens at the molecular atomic levels
Staining
artificially coloring the organism with the use of different dyes and reagents
Simple stain
uses a signle dye
Crystal violet
Methylene blue
exampes under simple stain
Simple stain
reveals size, morphology, and arrangement of cells
Crystal violet
uniform purple stain
Methylene blue
uniform blue stain
Differential stains
use two or more dyes to differentiate between cells or structures
Gram stain
Ziehl-Neelsen acid-fast stain
Schaeffer-Fulton endospore stain
examples under differential stains
Gram stain
Gram-positive cells are purple; gram-negative cells are pink
Ziel-Neelsen acid-fast stain
Pink to red acid-fast cells and blue nonacid-fast cells
Schaeffer-Fulton endospore stain
Green endospores and pink to red vegetative cells
Gram stain
Differentiates between Gram-positive and Gram-negative bacteria, which is typically the first step in their identification
Ziehl-Neelsen acid-fast stain
Distinguishes the genera Mycobacterium and Nocardia from other bacteria
Schaeffer-Fulton endospore stain
Highlights the presence of endospores produced by species in the genera Bacillus and Clostridium
Negative stain for capsules
Flaggeral Stain
exmaples under special stains
Negative stain for capsules
Background is dark, cells unstained or stained with simple stain
Flagellar stain
Bacterial flagella become visible
Negative stain for capsules
Reveals bacterial capsules
Flagellar stain
Allows determination of number and location of bacterial flagella
MgRNA
GRAM (+) contains _______ which when combined to Crystal violet and gram's iodine will form an insoluble compound
decolorized
-GRAM (-) does not contain MgRNA, thus easily __
less; more
GRAM (+) are ____ permeable to the decolorizer
- GRAM (-) are ___permeable to the decolorizer
lower isoelectric pH
GRAM (+) have ______, thus they are more acidic
higher isoelectric pH
GRAM (-) have _______, thus, it does not easily combine with the basic dye (CV)
Reasons why Gram (+) becomes Gram (-)
1. Removal of MgRNAby reacting the MQS with bile salt solution- this will precipitate the MgRNA
2. Aging, dying, and autolyzing MQS
3. Using acidic Iodine or Lugol's Iodine 4. Technical error (e.g. over decolorization)
Neisseria
Veillonella
Moraxella (Branhamella)
1. ALL cocci are Gram(+) EXCEPT
Mycobacteria
Corynebacteria
Bacillus
Clostridium
Lactobacillus
Listeria monocytogenes
Erysipelotrix insidios
ALL bacilli are Gram(-) EXCEPT