study of microbial structure (microscopes) Flashcards
who was the first person to observe/discover microbes
Antonie van Leeuwenhoek
how does the light microscope view objects
uses visible light
list the types of light microscopes
bright-field, dark-field, phase contrast, differential interference, fluorescence, confocal
which type of microscope is the most common
compound
define compound microscope
they make use of several lenses in combination to enlarge an image
what elements are needed to form an image regardless of the type of microscope used
source of illumination, specimen, system of lenses that focus the illumination on the specimen and form the image
what does bright field look like
a dark image against a brighter background
how does bright field work
light travels from the source of illumination through the condenser, through the specimen, through the objective lens, and through the eyepiece to the eye of the observer
how do we calculate total magnification
mag of objective x mag of eyepiece
define resolution
ability of a lens to distinguish between small objects that are close together
what is the term for a microscope that has good resolution
we say it has good resolving power
define what it means for a microscope to have high resolving power
it has a high resolution (ability to distinguish between two objects)
what is one factor that influences resolution
angular aperture
what is the angular aperture
the measure of how much light is leaving the specimen and enters the eyepiece
in terms of magnification, how do we get high angular aperture
increasing magnification increases the angular aperture = high resolution
what is the formula that determines the resolution of a microcope
d = 0.5(lambda)/n sin theta
what is theta in the resolution equation
angular aperture
what is lambda in the resolution equation
wavelength of light
what is n in the resolution equation
refractive index
what is n sin theta in the resolution equation
numerical aperture
in the resolution equation, what sizes do we want d, lambda, and n to be
d = small
lambda = small
n = big
how can we achieve maximum resolution on a bright field microscope
use the 100x objective lens with oil immersion
how does oil immersion increase resolution
it increases the refractive index
define refractive index
a measure of how a substance slows down the velocity of light
describe how oil immersion increases the refractive index
it bends more light towards the objective lens
what is the refractive index for oil? air?
oil = 1.5
air = 1.0
what is the max resolution using bright field without oil immersion
300 nm
what is the refractive index for bright field with oil immersion
200 nm
advantages of bright field
inexpensive, easy to use
disadvantages of bright field
specimen must have color
what is bright field useful for
viewing dead + stained cells
which light microscopes can be used to view living specimens
dark field, phase contrast, differential interference, confocal
how does a dark field image look
light specimen against a dark background
describe how internal structures appear with dark field vs bright field
dark field allows us to see internal structures more clearly
describe how dark field works
an opaque disk causes a hollow cone of light to pass through the specimen, so only light going through is what goes through the objective lens
what is dark field useful for
observing detailed images of live, unstained organisms
T or F: cells need to be stained for bright field
true
T or F: cells need to be stained for dark field
False: they can be unstained
what do phase contrast microscopes do
exploit differences between the thickness and refractive indices of various regions of a cell to increase contrast = dif levels of brightness
what are the extra components of a phase contrast microscope
annular diaphragm and a phase plate in the objective lense
uses of phase contrast?
looking at live, unstained cells, widely used to study eukaryotes and detect bacterial cell structures such as endospores and inclusions
describe differential interference microscope + how it works
similar to phase contrast but more sensitive. it makes use of prisms to split up light beams
what type of image do we get with differential interference
3D coloured images
describe fluorescence microscope + how it works
uses UV light as illumination source, and this is used to excite fluroescence in the specimen
uses of fluorescent microscope?
can be used to identify bacterial pathogens
how does confocal microscope work
makes use of lasers to illuminate a specimen, usually one that’s been fluorescently labelled. Lasers move plane by plane and then send this info to a computer
what type of image to we get with confocal
we get a stack of images to form a 3D image
describe electron microscopes + how they’re different from light microscopes
use electrons as a source of illumination instead of light
what are the two types of electron microscopes
transmission electron (TEM)
scanning electron (SEM)
describe TEM + what we look at with it
forms an image from electrons that are transmitted from the specimen. Used to look at internal cell structures
describe SEM + what we look at with it
forms an image from electrons that are deflected from the specimen’s outer surface. Used to look at surface topology of the cell = 3D image
list the strengths of an electron microscope compared to a light microscope
has better magnification (100,000x vs 1000x), has better resolution (0.2nm vs 200nm)
list the weaknesses of an electron microscope compared to a light microscope
cannot look at living specimens, time consuming + difficult to prepare specimens, requires technical expertise, machine is large and expensive
what are the two types of scanning probe microscopes
scanning tunneling
atomic force
describe scanning tunneling microscope
makes use of a probe to measure all the bumps and depressions of the surface
what can we see with a scanning tunneling mic.
can see the surface topology, individual atoms, specimens imersed in water
describe atomic force mic.
can be used to look at surfaces that do not conduct electricity very well, can look at proteins
