Ch.4: Microscopy, staining and classification Flashcards
what are the general principles of microscopy?
Wavelength
magnification
resolution
contrast
what is wavelength of radiation?
distance between corresponding waves
what waves are very short
gamma rays 10-12 m
can humans see in the visible spectrum
yes
what waves are very long
Infrared rays
Microscopes can use different wavelengths to increase what?
resolving power
what is magnification?
-the apparent increase in size and object
-occurs when the beam of radiation refracts through a lens
what is clarity determined by
resolution and contrast
what is resolution?
the ability to distinguish objects that are close together
what is contrast
-the difference in intensity between two objects and the background
-important helps determine resolution
-staining helps to increase contrast
who created the simple microscope?
Van Leeuwenhoek
what are the types of light microscopy?
-bright field
-dark field
-phase contrast
-nomarski
what is bright field?
-Light passes through the specimen and into the lens.
-Dark specimen on a bright background.
-Created by the specimen absorbing or scattering light.
whats is total magnification?
objective lense X ocular lense
what is the ocular lens
magnifies the image formed by the objective lens seen by the human eye
what happens when immersion oil is used?
more light enters lens
what is the body
transmits the image from the objective lens to the ocular lens using prisms
what are objective lenses
primary lenses that magnify the specimen
what is the stage
holds the microscope slide in position
what is the condenser
focuses light through the specimen
what is the diaphragm
controls the amount of light entering the condenser
what is the illuminator
light source
what is the coarse focusing knob
moves the stage up and down to focus the image
what is the fine focusing knob
slightly moves the stage up and won for focusing
what is the dark field?
-The sample appears bright against a dark background, enhancing contrast without staining.
-best for pale objects
what is phase contrast?
used to examine living specimens and internal structures of living organisms
how is contrast created
light waves out of a phase
in phase
brighter
out of phase
darker
what is fluorescence in light microscope?
direct UV light source at specimen
-specimen radiates energy back at a longer visible wavelength
-UV light increases resolution and contrast
-some cells need to be stained and some are naturally fluorescent
what is an electron microscope
uses electron beams instead of light to magnify objects at much higher resolutions
-done under vacuum; not for living microbes
what is a Scanning electron microscope (SEM)
produce a 3-D view of the microbe’s surface and cellular structures
what is transmission electron microscopes (TEM)
produces a 2-D view to view internal ultra-cellular detail
what are acidic dyes
(negative charge) stain alkaline structures (+ charge)
ex: eosin binds + charges (amino acids)
what are basic dyes
(positive charge) stain acidic structure ( - charge)
what are the most common dyes
-crystal violet
-methylene blue
-safranin
how do you prepare a simple stain?
-a very thin film of the specimen on glass slide
-allow to air dry
-heat fix
-kills any live bacteria
-induces coagulation of proteins to “stick” the bacteria to the slide
-Helps prevent bacterial smear from washing off during procedure
-only uses one dye
gram positve cells stain what color?
purple
gram negative cells stain what color?
pink/red
what does a primary stain do?
stains all cells
what is mordant?
binds to the dye and makes it less soluble (stays in the cell wall)
