microscopy Flashcards
chapter 2 (131 cards)
1
Q
what does visible light consist of
A
electromagnetic waves that behave like other waves
2
Q
what is a wavelength
A
the distance between one peak of a wave and the next peak
3
Q
what is amplitude
A
the height of each peak or depth of each trough
4
Q
what is frequency
A
the rate of vibration of the wave or the number of wavelengths within a specified time period
5
Q
when does reflection occur
A
when a wave bounces off of a material.
6
Q
what is absorbance
A
when material captures the energy of a light wave
7
Q
what is transmission
A
when a wave travels through a material, like light through glass
8
Q
what is interference
A
creating complex patterns of motion
9
Q
what is diffraction
A
when light waves interact with small objects or openings by bending or scattering
10
Q
when is diffraction larger
A
when the object is smaller relative to the wavelength of the light
11
Q
when does refraction occur
A
when light waves change direction as they enter a new medium
12
Q
what is refractive index
A
the extent to which a material slows transmission speed relative to empty space
13
Q
what does large differences between refractive indices of two material result in
A
a large amount of refraction when light passes from one material to the other
14
Q
what happens when light crosses a boundar
A
15
Q
into a material with a higher refractive index
A
its direction turns to be closer to perpendicular to the boundary
16
Q
what is the principle behind lenses
A
refractive index
17
Q
how can you think of a lense
A
an object with a curved boundary that collects the light that strikes it and refracts it so it all meets at the image point
18
Q
why can a convex lens be used to magnify
A
it can focus at a closer range than the human eye producing a larger image
19
Q
how are concave lenses and mirrors used in microscopes
A
to redirect light the light path to the focal point
20
Q
what is the focal length
A
distance to the focal point
21
Q
what is the relationship between higher wavelengths and shorter frequency
A
more oscillations per unit time than lower-freqeuncey waves
22
Q
how can white light be separated into its component colors
A
refraction
23
Q
what is dispersion
A
separation of colors in the rainbow like spectrum
24
Q
What is phosphorescence
A
when photons are emitted following a delay after absorption
25
what is magnification
the ability of a lens to enlarge the image of an object when compared to the real object
26
what is resolution
the ability to tell that two separate points or objects are spearate
27
what affects resolution
wavelength
numerical aperture
28
how does wavelength affect resolution
shorter wave lengths resolve smaller objects
29
what is numerical aperture
the measure of a lens ability to gather light the higher the better the resolution
30
what is leeuwenhoek referred to as
the father of microbiology
31
what type of microscope did Galileo use
compound to inspect insect parts
32
what kind of microscope did Leeuwenhoek use
simple microscope
33
how many lenses in a compound microscope
2
34
how many lenses in a simple microscope
1
35
what type of microscope did Hooke use
compound
36
what did Hooke look at through his microscope
cork cells
37
what did lister create
modern light microscope
38
what are examples of light microscopes
brightfieqld microscope
dark field microscope
phase contrast microscope
differential interference contrast microscopes
fluorescence microscope
confocal scanning laser microscopes
two photon microscopes
39
what is a brightfield microscope
a compound microscope with 2 or more lenses that produce a dark image on a bright background
40
where is the ocular lens
in each eyepiece
41
what is the magnification of the ocular lens
10
42
how do you calculate total magnification
the product of the ocular magnification times the objective magnification
43
what is the stage
where the specimen is placed
44
how is the specimen positioned over the light
x-y mechanical stage knobs
45
what is the coarse focusing knob used for
large scale movements
46
what is the fine focusing knob used for
small-scale movements
47
why do images become dimmer when magnified
less light per unit of area of image
48
what lights up a microscope
illuminator
49
what does light from the illuminator pass through
condenser lens
50
what does a condenser lens do
focuses all of the light onto the specimen to maximize illumination
51
how can the amount of light hitting specimen be adjusted
opening or closing a diaphragm
52
what are chromophores
pigments that absorb and reflect particular wavelengths of light
53
why is resolution compromised at higher magnification
the large difference between the refractive indices of air and glass, air scatter the light before they can be focused by the lens
54
how is refractive index fixed at large magnifications
using an oil immersion lens
55
how does an oil immersion lens help
oil has a refractive index similar to glass increasing maximum angle at which light can strike the lens
56
what is a dark field microscope
a brightfield microscope with an opaque disk between the illuminator and the condenser lens
57
what does the opaque light stop do
blocks most light from the illuminator producing a hollow cone of light that is focused on the specimen showing bright objects on a dark background
58
what are the benefits of darkfield microscopy
it can create high-contrast, high resolution images of specimens without the use of stains
59
What do phase-contrast microscopes do
use refraction and interference caused by structures in a specimen to create high contrast, high resolution images without staining.
60
how do phase-contrast microscopes work
by altering wavelengths of light rays passing through the specimen to create altered wavelength paths.
61
what is in the condenser of a phase contrast microscope
an annular stop
62
what does an annular stop do
produces a hollow cone of light that is focused on the specimen before reaching the objective lens
63
what does the objective lens contain in a phase contrast microscope
a phase plate with a phase ring
64
what does the phase rang cause
light traveling directly from the illuminator passes through the the place causing waves traveling through to be one-half of a wavelength out of phase with those passing through the plate.
65
what is a differential interference contrast microscope
similar to phase-contrast in that they use interference patterns to enhance contrast
66
what happens in a DIC microscope
two beams of light are created in which the direction of wave movement differs,
67
what are the benefits of a DIC microscope
it gives high contrast images of living organisms in a three-dimensional appearance, they allow you to structure in live unstained specimens
68
how does a fluorescence microscopework
it uses fluorochromes that are capable of absorbing energy from a light source and then emitting this energy as a visible light.
69
what does a fluorescence microscope transmit
an excitation light generally a form of EMR with a shot wavelength toward the specimen, chromophores absorb the excitation light and emit visible light with longer wavelengths
70
what are fluorescence microscopes useful for
identifying pathogens or the locations of particular molecules and structures within a cell and to determine living from dead cells
71
what is immunofluorescence used for
identifying certain disease causing microbes by observing if abx bind to them
72
what does a confocal microscope do
uses a laser to scan multiple z-plances successively
73
what does a confocal microscope produce
numerous two-dimensional images that can be constructed into a three dimensional image
74
what are confocal microscopes are useful for
examining thick specimens such as biofilms, which can be examined alive and unfixed.
75
what does a two-photon microscope do
uses a scanning technique, fluorochromes, and long-wavelength light to visualize specimens
76
what is a two-photon microscope useful for
examining living cells within intact tissues
77
what does an electron microscope do
uses short-wavelength electron beams rather than light to increase magnification and resolution
78
how much can an electron microscope magnify
up to 100,000x
79
what can an electron microscope be used for
sub cellular structures such as single strands of DNA
80
what are the two types of electron microscopes
transmission electron microscope
scanning electron microscope
81
can an electron microscope be used on living cells
no
82
what are the basic types of preparation for a light microscope
wet mount and fixed mount
83
what is a wet mount
when the specimen is placed on a slide in a single drop of liquid, includes urine
84
what is fixation
attaching cells to slide through heat or chemical treating.
85
what happens to a fixed specimen
the microorganism is killed while preserving the integrity of cellular components
86
how is a sample heat fixed
it is spread on the slide and briefly heated over a heat source
87
when are chemical fixatives preferred
for tissue specimens
88
what chemicals are used to chemically fix
a chemical agent such as acetic acid, ethanol, methanol, or formaldehyde
89
what happens to a chemically fixed specimen
it kills microorganisms in the specimen stopping degradation of tissues and preserving their structure
90
what do stains and dyes contain
salts with a positive ion or the negative ion
91
what is a positive ion classified as
basic dye
92
what is a negative ion classified as
acidic dye
93
what is a positive stain
a dye that will be absorbed by the cells or organisms being observed adding color to objects of interest to make them stand out against the background
94
what is a negative stain
absorbed by the background but not the cells producing an outline or silhouette of the organisms against a colorful backgroiund
95
what is the typical charge of a cell wall
negative
96
what normally sticks to cell walls
basic dyes making them positive stains
97
what are the commonly used basic dyes
basic fuchsin
crystal violet,
malachite green
methylene blue
safranin
98
what are common acidic dyes
acid fuchsin
eosin
rose bengal
99
what happens in simple staining
a single dye is used to emphasize a particular structure, all of the organism appear to be the same color even if more than one type of organism
100
what does differential staining do
distinguishes organisms based on interactions with multiple stains
101
what are types of differential stains
gram staining
acid-fast staining
endospore staining
flagella staining
capsule staining
102
what does gram staining do
differentiates between bacteria with different types of cell walls
103
what is the first step in a gram stain
crystal violet a primary stain is applied to a heat fixed smear giving it a purple color
104
what is the second step in a gram stain
grams iodine a mordant is added
105
what is a mordant
a substance used to set or stabilize stains or dyes
106
what does grams iodine do in gram staining
acts like a trapping agent that complexes with crystal violet making a complex clump and stay contained in thick layers of peptidoglycan in the cell walls
107
what is the 3rd step in gram staining
a decolorizing agent is added
108
what is the decolorizing agent
ethanol or acetone
109
what happens when decolorizing agent is applied
in cells with a thick peptidoglycan layers it stays purple
in thick walls the purple is washed away making them colorless
110
what is the last step in gram staining
secondary counterstain is aded staining decolorized cells pink
111
what is the counter stain
safranin
112
what are the purple, stains cells referred to as
gram positive
113
what are the red safranin dyed cells called
gram negative
114
what can affect gram stain results
older bacterial cells may have weak cell walls showing false gram negative
errors such as leaving decolorized on too long
115
what is the clinical significant of gram staining
helps classify bacterial pathogens to treat
116
what is acid fast staining able to do
differentiate between two types of gram positive cells, those with a waxy mycolci acid and those that do not
117
what are the methods for acid fast staining
ziehl-neelsen technique and the kinyoun technique
118
what is the primary stain in acid fast staining
carbolfuchsin
119
how do cells react to carbolfuchsin
waxy cells retain even after decolorizing
120
what is applied to non acid fast stains
a secondary counterstain methylene blue
121
what is the difference between acid fast staining techniques
whether heat is used during the primary staining process (ziehl-neelsen method)
122
what makes acid fast important
acid fast bacterial ill show red or pink on a blue background of surrounding tissue cells
123
what happens in capsule staining
a negative stain is used
124
does capsule staining need to be heat fixed
no
125
what are endospores
structures produced by bacterial cells that allow them to survive harsh conditions
126
how do endospores appear during gram staining
clear
127
what does endospore staining do
uses two stains to differentiate endospores from the rest of the cell.
128
what happens in endospore staining
heat is used to push the primary stain into the endospore, endospore retains the green stain after decolorization. it is then counterstained with safranin
129
what does flagella staining do
thickens flagella by applying mordant which Coates the flagella then is stained with pararosaniline
130
how are cells cut without damage
by being embedded in plastic resin and then dehydrated through a series of soaks in ethanol solutions
131
