Block One Lecture 3
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. What would you like to do?
Which measurements do we use?
microns (1 micrometer= 10^-6 m=10^-3 mm)
A simple microscope has __.
There is also the __.
What is magnification?
What are the magnifications in terms of lens?
only one lens
binocular compound microscope
magnification= optical lens x objective
4X scanning lens
10X low power
40X high power
What is the resolution?
To describe structure and arrangement, we need __
What is the relationship between magnification and resolution?
0.4 nm on a good light microscope
the higher the magnification, the less the resolution, the shorter the distance, and the less aperture there is
What is aperture?
The numerical aperture of a microscope objective is the measure of its ability to gather light and to resolve fine specimen detail while working at a fixed object (or specimen) distance.
All __ have a __, causing light.
How many condenser lenses are there?
three types to bend at a minimum of 45-ish degrees
When light is scattered, what happens?
Less magnification means what?
- less light goes up the tube
- more light to collect
How to combat the fact that light is scattered and less magnification occurs?
Explain it in terms of a, b, and c.
immersion oil lens
A= has a certain RI
B= shorter R.I. depending on viscosity
C= 50:50 mixture of both
Light goes through lens and through teh oil, therefore collecting more light
Brightfield Illumination (Light)
- background light?
- opaque disk? dark?
- - cells, for the most part, are clear
- - We go from red to violet (ROYGBIV) for stains except for green
- - All the background light enters the lens
- - Looking at max 1000 times magnification
- - With opaque disk, it removes/ dampens background light; only light reflected by specimen
- - Dark: used to look at structures in relation to cell wall or plasma membrane
Phase Contrast Microscopy
based on principle that, if you keep the wavelength the same, but make them out of phase, to get finer detail/ structure
--> in phase means moving identically (can't tell it apart)
1400x: more magnification but not tremendous
- out of phase
- two light sources
- possibly two prisms to split the light
Fluorescence Microscopy (ungodly important)
- tremendous enormous diagnostic tool
- - we combine microbio, immuno, and physics
With Fluorescent Microscopy, what is used?
antibodies are used that can take up a fluorescent molecule; the arms are bound to the organism; reacts with rest of immune system
uses lasers to excite the fluorophores
eight different fluorescent molecules work
EM is the __ and __.
What must occur with this?
TEM and SEM
the specimen must be thin with heavy salts
- scans whole surface
- electrons bounce off the specimen and activate the specimen--> surface topography seen
Prep of Specimen for the Light Microscope
Stains, such as methylene blue and Safranin
India ink: used to stain a slide containing micro-organisms. The background is stained while the organisms remain clear. This is called a negative stain. India ink, along with other stains, can be used to determine if a cell has a gelatinous capsule.
What is a mordant?
used to hold a stain because stains themselves don't stick to the organism
it is an ion that binds a chemical dye and holds it down such that the dye remains stuck on teh organism
What is differential staining?
staining processes which use more than one chemical stain.
Ex: gram stains
Explain gram stains
- Positive: multilayers thck that have alot of polypeptides
- Negative: thin cell wall; no fancy polypeptides, weaving it through
Gram stains exploint differences between strong and weak cell walls; two stains distinguish between positive and negative
Iodine as a stain
Iodine is also used as a mordant in Gram's staining, it enhances dye to enter through the pore present in the cell wall/membrane
It is dark and does not really emit light so it doesn't manipulate the color
It physically reacts with it, but doesn't change the color
Third step in gram staining?
- gram + and negative
dehydrate but strip cell wall
Gram positive should stay purple because of large cell wall. CV-I complex get in and can't get back out--> color remains
Gram negative should not maintain purple because the CV-I complex gets washed out due to thin wall--> washed away-->counterstain
What would you like to do?
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