Microscopy and Staining Flashcards
What is magnification?
The capacity of a microscope to enlarge an image uising the lenses in the microscope.
What is resolution?
It allows to distinguish between two adjacent objects and separate them into two objects.
What is resolution limit?
The limit depends on the numerical aperture. It is highly dependent on light and power.
What is contrast?
It allows to differentiate between the cell and surroundings in absorbing or scattering light.
Oragnisms are (blank) so there is a need to create (blank).
tranpsarent, contrast
How is contrast achieved?
Through various stains and dyes
This microscope uses illumination from visible light.
Bright Field Microscope
Light in BFM may come from (blank) or (blank).
external, microscope
How do we see images in BFM?
Light source concentrates light into the condenser, projecting it to the objective lens
How does the condenser work in Dark Field Microscope?
Condenser prevents light from passing through. Light is directed to the sides of the specimen.
What is the Dark Field Microscope used for?
To observe motility
Phase Contrast Microscope relies on?
The refractive indices of cells (ability of cells to alter the speed of light) and the background
True or false. Cells have unique refractive indices, resulting to different brightness.
True
How does Phase Contrast Microscope work?
Amplified by the phase ring in the condenser
What is Phase Contrast Microscope used for?
Used for living organisms that cannot be stained
The Nomarski Microscope is also called?
Differential Interference Contrast [DIC]
This microscope uses a polarizer in the condenser to produce polarized light.
Nomarski
In Nomarski, light passes through a (blank) generating (blank) with different refractive indices.
prism, 2 beams
How does Nomarski work?
Light interfere w/ one another, creating a 3D optical effect, enhancing the subtle differences in cell structures
True or false. Nomarski cannot show internal cell structures and is used to observe motility.
False. Nomarski is used for unstained cells, revealing internal cell structures invisible by BFM without staining.
Fluorescence Microscope uses (blank) that can activate fluorescence molecules in cells.
UV light
Example of naturally occuring activated fluorescence
chlorophylls
Examples of stain that can activate fluorescence?
fluorochromes, DAPI
How does Fluorescence Microscope work?
Filters eliminate other wavelengths of light other than the fluorescent light
What is the use of Fluorescence Microscope?
Visualizing certain cell structures
Computer assembles multiple layers to create a single high-resolution, 3D image.
Confocal Scanning Laser
Best for thick specimens.
Confocal Scanning Laser
In Confocal Scanning Laser, dead cells are stained (blank) and viable are stained (blank).
red, green
Electron microscopy uses (blank) as lenses.
electromagnets
Electron microscope is opened in a (blank).
vaccum
- allows you to visualize the internal cell structures
- magnification up to 500,000x
proteins & nucleic acids are visible
Transmission Electron Microscope
Samples in TEM are stained using (blank) with (blank) to improve contrast.
shadow-casted
atom, high atomic weights
- used to visualize external structures or surfaces of the cell
- magnification up to 100,000x
Scanning Electron Microscope
Specimen in SEM is usually coated in (blank).
thin sheet of heavy metal
How does SEM work?
Electron beams scan the surface then direct it back to the computer
Tiny stylus positioned extremely close to the specimen surface
Atomic Force Microscopy
Which microscope can be used at the surface of live specimens?
Atomic Force Microscopy
Wet mounts don’t need to be stained with dyes. What is used to see the specimen?
hay infusion, pond water
Addition of (blank) can create a viscous environment to slow down movement.
carboxymethylcellulose
If carboxymethylcellulose is not avaiable, what can we use to create “tents” to trap organisms incone area?
cotton fiber
True or false. E. coli is motile.
True
What will the cell arrangement look like if you smeared too much?
Cell arrangement won’t be too visible
cells will be on top of each other
What will the cell arrangement look like if your smear is too thin?
Arrangement will be scattered everywhere
Examples of positively charged dyes:
crystal violet, methylene blue, safranin
Why do we need to heat fix?
To attach bacteria on slide
True or false. Positive staining include acidic dyes.
False. Positive staining dyes are basic/cationic.
methylene blue, crystal violet, safranin
Example of acidic dyes:
nigrosine black, India ink, and Congo red
True or false. We need to heat fix everytime.
False. No need for heat fixing if dye is negative.
Organisms are heat-sensitive
Three different types of staining:
simple, structural, differential
Highlights certain structures of the cell
structural staining
Schaeffer-Fulton Method is also called?
Endospore staining
Primary stain for endospore staining
Malachite green
Endospore wall becomes permeable to malachite green due to (blank).
steaming
Gram staining differentiate betwen (blank) and (blank).
positive, negative
Acid fast staining is also called?
Ziehl-neelsen Stain Method
You can see the presence of (blank) in Ziehl-neelsen Stain Method
mycolic acids
retains primary stain: carbolfuchsin
Primary stain for Ziehl-Neelsen
carbolfuchsin
Counter stain of Ziehl-Neelsen
methylene blue
