Viewing Bacterial Cells Flashcards
List 7 types of microscopes
1) compound
2) inverted
3) phase contrast
4) confocal
5) fluorescence
6) Electron (TEM & SEM)
7) Scanning probe ( SPM & STM)
Bright Field Microscopy
renders dark image against a lighter background
Dark Field Microscopy
increases contrast, renders brighter image against darker background
What are the 3 important parameters of microscopy
1) magnification
2) resolution
3) contrast
Total magnification=
Objective Magnification x Ocular Magnification
What is Resolution
ability to distinguish to points that are close together or
clarity in image
What is contrast?
difference between object and its background
or difference in intensity between 2 objects
What is Magnification?
ratio of image to its real size
what is path of light for a compound microscope?
illuminator-condenser lenses-specimen-objective lens-body-prism that separates light into ocular lens- line of vision
What does the addition of immersion oil do when using compound light microscope?
increases magnification and resolution
How does immersion oil work
light bends as it enter glass and as it leaves it into air. some of that scattered light does not go into objective lens. with immersion oil light travels at same speed of glass and does not refract which allows more light to enter objective lens
what is dark field microscope best used for?
live or unstained biological samples or single cell organism
What is a darkfield stop?
opaque light stop that is inserted into bright field microscope which is used to create a darkfield image
how does a darkfield stop work?
it blocks light traveling from the illuminator to the objective lens which allows only light that was reflected off sample to reach eye
How are the pieces placed in an inverted microscope?
inverted position compared to regular compound
What is an inverted microscope used for
viewing of biological material in tissue culture or petri dish
living cells or organism in more natural environment
How does Phase microscope work?
uses phase contrast in specimen to create high resolution images w/o staining
What are Phase Microscopes best used for?
viewing live unstained specimens and structures (i.e endospores and organelles)
how does fluorescence microscopy work
uses UV light to fluoresce objects. contrast is imporved
how does fluorescence microscopy work
uses UV light to fluoresce objects. contrast is improved
What is fluorescence microscopy best used for
1) ID Pathogens
2) distinguish between dead/living cells
3) find particular species
4) find location of specific molecule w/in a cell
5) immunofluorescence
How does direct immunofluorescence work
stain is absorbed by primary antibody which binds to antigen
How does indirect immunofluorescence work?
stain absorbed by 2ndary antibody which binds to primary antibody which in turns binds to antigen
How does a Confocal microscope work?
users laser to scan multiple Z-planes to produce multiple 2D images that can be constructed in 3D image
What are confocal microscopes best used for
viewing thick specimens i.e biofilms
How does Electron microscope works
uses electron beams focused with magnets to produce image
List 2 types of Electron Microscope
Transmission Electron Microscope
Scanning Electron Microscope
How does the Transmission Electron Microscope (TEM) work and what is it best used for
uses electron beam that passes through specimen to see small images
best used to see small/thin specimens and subcellular structures
How does the Scanning Electron Microscope (SEM) work and what is it best used for
uses electron beam to visualize surfaces
best used to observe 3D surface detail of specimens
What is an ultramicrotome
machine that slices specimen into thin slices
how does a scanning electron microscope work
uses sharp probes that are passed over surface of specimen and interact directly with it
scanning electron microscopes can produce magnification up to…
100,000,000x
what are the 2 types of scanning electron microscopes
Scanning Tunneling Microscope (STM)
Atomic Force Microscope (AFM)
Why do microorganisms need to be stained
they need to be stained in order to increase visibility and accentuate specific morphological features
Define a Dye and its composition
its organic contains Benzene ring + chromophore + auxochrome group
In a dye, what is Chromgen composed of?
Benzene ring + Chromophore
In a Dye, what is the Benzene ring
Organic, colorless substance
In a Dye what is. Chromophore
chemical that imparts color to Benzene (the coloring compound)
In a Dye, what is Auxochrome?
the ionization agent. allows salt to form to bind to tissues and fibre
list the 4 types of dye classification
Origin
Purpose
Activity
Based on charge
list the types of origin dyes (2)
1) naturual
2) Synthetic
List the types of dyes based on their purpose
1) direct
2) indirect
3) selective
4) differential
List type of dye-based on staining activity
1) nuclear
2) cytoplasmic
3) Histological
List dye types based on charge
1) Acid (-)
2) base (+)
3) Salts of acid and base
what is a Mordant
substance that forms insoluble compound w/stain to help fix color on cell
Explain purpose for Schaeffer-Fulton stain and whats it used for
- Used for endospore staining
- primary dye that is used is Melachite Green, then heat so it can fix dye so it acts like a mordant. 2ndary dye safranin that dyes pink or red
