Chapter 1 lecture 3 Flashcards
Microscopy
microorganisms range in size from
microorganisms range in size from
Smallest=
nanometers (nm)
microorganisms range in size from
Largest=
protists (μm)
Colony is a ____ culture
pure culture
Magnification
ability to enlarge objects
Resolving power
ability to show detail
Magnification in most microscopes results from
an interaction between visible light waves and the curvature of a lens.
focal point
focus light rays at a specific
place
focal length
distance between center of
lens and focal point
strength of lens is related to the ____
the focal length
Refraction
bending of light when passing from one medium to another
Refractive index
measure of how greatly a substance slows the velocity of light
Direction and magnitude of bending is determined by
the refractive indices of the two media forming the interface (i.e., glass and air)
Glass has a _______ refractive index than air
higher
The Light Microscope Types
bright-field microscope
dark-field microscope
phase-contrast microscope
fluorescence microscope
confocal microscope
Modern microscopes are all _________ microscopes
compound (more than 1 lense)
The Bright-Field Microscope
Both stained and unstained
produces a dark image against a brighter background
has several objective lenses
parfocal microscopes
total magnification
- product of the magnifications of the ocular lenses and the objective lenses
Resolution
ability of lens to distinguish small objects that are close together
shorter wavelength –>
greater resolution
blue light 450 – 500 nm can not resolve structures smaller than ______
0.2 um
Numerical aperature
ability of the lens to gather light
Numerical aperture of lens ranges from
0.1 to 1.25
Shorter wavelength and larger numerical aperture will provide better ______
resolution
Refractive index
how much a substance bends a light ray
The refractive index of air is 1.00 if we increase this by using immersion oil, we can increase the ______________________
numerical aperture
Why can smaller working distances give better resolution?
A smaller working distance can better separate close objects because the light spreads out more
Immersion oil
Brings more light comes to the lense which increases numerical aperture
working distance
distance between the surface of lens and the surface of cover glass or specimen when it is in sharp focus
The Dark-Field Microscope
image is formed by light reflected or refracted by specimen
produces a bright image of the object against a dark background
used to observe living, unstained preparations
How to do Dark Field Microscopy
Uses a hollow cone of light so that only light that has been reflected or refracted by the specimen enters the lens
Examples of Dark-Field Microscopy
Treponema pallidum and Volvox
The Phase-Contrast Microscope
Uses slight differences in refractive index and cell density
Uses a hollow cone of light
Cone of light passes through a specimen some is retarded (out of phase).
Light passes through phase plate brining it back into phase excellent way to observe unstained, living cells
Examples of Phase-Contrast Microscopy
Pseudomonas sp., Amoeba, Paramecium
The Differential Interference Contrast Microscope (DIC)
Similar to phase-contrast - creates image by detecting differences in refractive indices and thickness of different parts of specimen
Uses two beams of polarized light to create a 3D image of specimen
excellent way to observe living cells
- live, unstained cells appear brightly colored and three-dimensional
Example of Differential Interference Contrast Microscopy
Amoeba proteus
The Fluorescence Microscope
developed by O. Shimomuram, M. Chalfie, and R. Tsien
exposes specimen to ultraviolet, violet, or blue light
specimens usually stained with fluorochromes (fluorescent dyes)
shows a bright image of the object resulting from the fluorescent light emitted by the specimen
has applications in medical microbiology and microbial ecology studies
Immuno-Fluorescence
antibody and fluorochrome combine
these then bind to the cell surface antigen molecules of a bacterial cell
Cells stained with fluorescent dyes
Can identify living, dead, population size, and strain
Confocal Microscopy
Uses lazer to focus on different depths of a sample, take photos of the depths to create a 3-D image
Specimen is usually fluorescently stained
Numerous applications including study of biofilms
Preparation for optical microscopes requires
wet mounts and hanging drop mounts which allow
examination of characteristics of live cells: size, motility, shape, and arrangement
Preparation and Staining of Specimens
Increases visibility of specimen.
Accentuates specific morphological features.
Preserves specimens.
Fixation/ Smear Preparation
Preserves internal and external structures and fixes them in position.
Organisms usually killed and firmly attached to microscope slide.
Heat fixation
Routinely used with bacteria and archaea.
Preserves overall morphology but not internal structures.
Chemical fixation
Used with larger, more delicate organisms.
Protects fine cellular substructure and morphology.
Basic dyes have ______ charges.
Positive
Acid dyes have ______ charges
Negative
Simple stains
A single stain is used.
Use can determine size, shape, and arrangement of bacteria.
Negative stain.
Differential Staining
Divides microorganisms into groups based on their staining properties.
For example, Gram stain.
For example, acid-fast stain.
Gram Staining
Most widely used differential staining procedure.
Divides bacteria into two groups, Gram-positive and Gram-negative, based on differences in cell wall structure.
Acid-Fast Staining
Particularly useful for staining members of the genus Mycobacterium.
For example, Mycobacterium tuberculosis—causes tuberculosis.
For example, Mycobacterium leprae—causes leprosy.
High lipid content in cell walls (mycolic acid) is responsible for their staining characteristics.
Capsule stain used to
visualize polysaccharide capsules surrounding bacteria
capsules may be colorless against a stained background
Flagella staining uses a
Mordant applied to increase thickness of flagella
Purple cells are gram ______
Positive
Red cells are gram ______
Negative
Endospore staining
To identify endospore which indicates which cells are producing them for survival purposes under stressful environments
