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Microbiology > microscopy > Flashcards

microscopy Flashcards

chapter 2

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1
Q

what does visible light consist of

A

electromagnetic waves that behave like other waves

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2
Q

what is a wavelength

A

the distance between one peak of a wave and the next peak

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3
Q

what is amplitude

A

the height of each peak or depth of each trough

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4
Q

what is frequency

A

the rate of vibration of the wave or the number of wavelengths within a specified time period

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5
Q

when does reflection occur

A

when a wave bounces off of a material.

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6
Q

what is absorbance

A

when material captures the energy of a light wave

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7
Q

what is transmission

A

when a wave travels through a material, like light through glass

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8
Q

what is interference

A

creating complex patterns of motion

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9
Q

what is diffraction

A

when light waves interact with small objects or openings by bending or scattering

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10
Q

when is diffraction larger

A

when the object is smaller relative to the wavelength of the light

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11
Q

when does refraction occur

A

when light waves change direction as they enter a new medium

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12
Q

what is refractive index

A

the extent to which a material slows transmission speed relative to empty space

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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

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14
Q

what happens when light crosses a boundar

A
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15
Q

into a material with a higher refractive index

A

its direction turns to be closer to perpendicular to the boundary

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16
Q

what is the principle behind lenses

A

refractive index

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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

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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

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19
Q

how are concave lenses and mirrors used in microscopes

A

to redirect light the light path to the focal point

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20
Q

what is the focal length

A

distance to the focal point

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21
Q

what is the relationship between higher wavelengths and shorter frequency

A

more oscillations per unit time than lower-freqeuncey waves

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22
Q

how can white light be separated into its component colors

A

refraction

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23
Q

what is dispersion

A

separation of colors in the rainbow like spectrum

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24
Q

What is phosphorescence

A

when photons are emitted following a delay after absorption

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25
Q

what is magnification

A

the ability of a lens to enlarge the image of an object when compared to the real object

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26
Q

what is resolution

A

the ability to tell that two separate points or objects are spearate

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27
Q

what affects resolution

A

wavelength
numerical aperture

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28
Q

how does wavelength affect resolution

A

shorter wave lengths resolve smaller objects

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29
Q

what is numerical aperture

A

the measure of a lens ability to gather light the higher the better the resolution

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30
Q

what is leeuwenhoek referred to as

A

the father of microbiology

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31
Q

what type of microscope did Galileo use

A

compound to inspect insect parts

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32
Q

what kind of microscope did Leeuwenhoek use

A

simple microscope

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33
Q

how many lenses in a compound microscope

A

2

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34
Q

how many lenses in a simple microscope

A

1

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35
Q

what type of microscope did Hooke use

A

compound

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36
Q

what did Hooke look at through his microscope

A

cork cells

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37
Q

what did lister create

A

modern light microscope

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38
Q

what are examples of light microscopes

A

brightfieqld microscope
dark field microscope
phase contrast microscope
differential interference contrast microscopes
fluorescence microscope
confocal scanning laser microscopes
two photon microscopes

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39
Q

what is a brightfield microscope

A

a compound microscope with 2 or more lenses that produce a dark image on a bright background

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40
Q

where is the ocular lens

A

in each eyepiece

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41
Q

what is the magnification of the ocular lens

A

10

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42
Q

how do you calculate total magnification

A

the product of the ocular magnification times the objective magnification

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43
Q

what is the stage

A

where the specimen is placed

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44
Q

how is the specimen positioned over the light

A

x-y mechanical stage knobs

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45
Q

what is the coarse focusing knob used for

A

large scale movements

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46
Q

what is the fine focusing knob used for

A

small-scale movements

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47
Q

why do images become dimmer when magnified

A

less light per unit of area of image

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48
Q

what lights up a microscope

A

illuminator

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49
Q

what does light from the illuminator pass through

A

condenser lens

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50
Q

what does a condenser lens do

A

focuses all of the light onto the specimen to maximize illumination

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51
Q

how can the amount of light hitting specimen be adjusted

A

opening or closing a diaphragm

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52
Q

what are chromophores

A

pigments that absorb and reflect particular wavelengths of light

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53
Q

why is resolution compromised at higher magnification

A

the large difference between the refractive indices of air and glass, air scatter the light before they can be focused by the lens

54
Q

how is refractive index fixed at large magnifications

A

using an oil immersion lens

55
Q

how does an oil immersion lens help

A

oil has a refractive index similar to glass increasing maximum angle at which light can strike the lens

56
Q

what is a dark field microscope

A

a brightfield microscope with an opaque disk between the illuminator and the condenser lens

57
Q

what does the opaque light stop do

A

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
Q

what are the benefits of darkfield microscopy

A

it can create high-contrast, high resolution images of specimens without the use of stains

59
Q

What do phase-contrast microscopes do

A

use refraction and interference caused by structures in a specimen to create high contrast, high resolution images without staining.

60
Q

how do phase-contrast microscopes work

A

by altering wavelengths of light rays passing through the specimen to create altered wavelength paths.

61
Q

what is in the condenser of a phase contrast microscope

A

an annular stop

62
Q

what does an annular stop do

A

produces a hollow cone of light that is focused on the specimen before reaching the objective lens

63
Q

what does the objective lens contain in a phase contrast microscope

A

a phase plate with a phase ring

64
Q

what does the phase rang cause

A

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
Q

what is a differential interference contrast microscope

A

similar to phase-contrast in that they use interference patterns to enhance contrast

66
Q

what happens in a DIC microscope

A

two beams of light are created in which the direction of wave movement differs,

67
Q

what are the benefits of a DIC microscope

A

it gives high contrast images of living organisms in a three-dimensional appearance, they allow you to structure in live unstained specimens

68
Q

how does a fluorescence microscopework

A

it uses fluorochromes that are capable of absorbing energy from a light source and then emitting this energy as a visible light.

69
Q

what does a fluorescence microscope transmit

A

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
Q

what are fluorescence microscopes useful for

A

identifying pathogens or the locations of particular molecules and structures within a cell and to determine living from dead cells

71
Q

what is immunofluorescence used for

A

identifying certain disease causing microbes by observing if abx bind to them

72
Q

what does a confocal microscope do

A

uses a laser to scan multiple z-plances successively

73
Q

what does a confocal microscope produce

A

numerous two-dimensional images that can be constructed into a three dimensional image

74
Q

what are confocal microscopes are useful for

A

examining thick specimens such as biofilms, which can be examined alive and unfixed.

75
Q

what does a two-photon microscope do

A

uses a scanning technique, fluorochromes, and long-wavelength light to visualize specimens

76
Q

what is a two-photon microscope useful for

A

examining living cells within intact tissues

77
Q

what does an electron microscope do

A

uses short-wavelength electron beams rather than light to increase magnification and resolution

78
Q

how much can an electron microscope magnify

A

up to 100,000x

79
Q

what can an electron microscope be used for

A

sub cellular structures such as single strands of DNA

80
Q

what are the two types of electron microscopes

A

transmission electron microscope
scanning electron microscope

81
Q

can an electron microscope be used on living cells

A

no

82
Q

what are the basic types of preparation for a light microscope

A

wet mount and fixed mount

83
Q

what is a wet mount

A

when the specimen is placed on a slide in a single drop of liquid, includes urine

84
Q

what is fixation

A

attaching cells to slide through heat or chemical treating.

85
Q

what happens to a fixed specimen

A

the microorganism is killed while preserving the integrity of cellular components

86
Q

how is a sample heat fixed

A

it is spread on the slide and briefly heated over a heat source

87
Q

when are chemical fixatives preferred

A

for tissue specimens

88
Q

what chemicals are used to chemically fix

A

a chemical agent such as acetic acid, ethanol, methanol, or formaldehyde

89
Q

what happens to a chemically fixed specimen

A

it kills microorganisms in the specimen stopping degradation of tissues and preserving their structure

90
Q

what do stains and dyes contain

A

salts with a positive ion or the negative ion

91
Q

what is a positive ion classified as

A

basic dye

92
Q

what is a negative ion classified as

A

acidic dye

93
Q

what is a positive stain

A

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
Q

what is a negative stain

A

absorbed by the background but not the cells producing an outline or silhouette of the organisms against a colorful backgroiund

95
Q

what is the typical charge of a cell wall

A

negative

96
Q

what normally sticks to cell walls

A

basic dyes making them positive stains

97
Q

what are the commonly used basic dyes

A

basic fuchsin
crystal violet,
malachite green
methylene blue
safranin

98
Q

what are common acidic dyes

A

acid fuchsin
eosin
rose bengal

99
Q

what happens in simple staining

A

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
Q

what does differential staining do

A

distinguishes organisms based on interactions with multiple stains

101
Q

what are types of differential stains

A

gram staining
acid-fast staining
endospore staining
flagella staining
capsule staining

102
Q

what does gram staining do

A

differentiates between bacteria with different types of cell walls

103
Q

what is the first step in a gram stain

A

crystal violet a primary stain is applied to a heat fixed smear giving it a purple color

104
Q

what is the second step in a gram stain

A

grams iodine a mordant is added

105
Q

what is a mordant

A

a substance used to set or stabilize stains or dyes

106
Q

what does grams iodine do in gram staining

A

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
Q

what is the 3rd step in gram staining

A

a decolorizing agent is added

108
Q

what is the decolorizing agent

A

ethanol or acetone

109
Q

what happens when decolorizing agent is applied

A

in cells with a thick peptidoglycan layers it stays purple
in thick walls the purple is washed away making them colorless

110
Q

what is the last step in gram staining

A

secondary counterstain is aded staining decolorized cells pink

111
Q

what is the counter stain

A

safranin

112
Q

what are the purple, stains cells referred to as

A

gram positive

113
Q

what are the red safranin dyed cells called

A

gram negative

114
Q

what can affect gram stain results

A

older bacterial cells may have weak cell walls showing false gram negative

errors such as leaving decolorized on too long

115
Q

what is the clinical significant of gram staining

A

helps classify bacterial pathogens to treat

116
Q

what is acid fast staining able to do

A

differentiate between two types of gram positive cells, those with a waxy mycolci acid and those that do not

117
Q

what are the methods for acid fast staining

A

ziehl-neelsen technique and the kinyoun technique

118
Q

what is the primary stain in acid fast staining

A

carbolfuchsin

119
Q

how do cells react to carbolfuchsin

A

waxy cells retain even after decolorizing

120
Q

what is applied to non acid fast stains

A

a secondary counterstain methylene blue

121
Q

what is the difference between acid fast staining techniques

A

whether heat is used during the primary staining process (ziehl-neelsen method)

122
Q

what makes acid fast important

A

acid fast bacterial ill show red or pink on a blue background of surrounding tissue cells

123
Q

what happens in capsule staining

A

a negative stain is used

124
Q

does capsule staining need to be heat fixed

A

no

125
Q

what are endospores

A

structures produced by bacterial cells that allow them to survive harsh conditions

126
Q

how do endospores appear during gram staining

A

clear

127
Q

what does endospore staining do

A

uses two stains to differentiate endospores from the rest of the cell.

128
Q

what happens in endospore staining

A

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
Q

what does flagella staining do

A

thickens flagella by applying mordant which Coates the flagella then is stained with pararosaniline

130
Q

how are cells cut without damage

A

by being embedded in plastic resin and then dehydrated through a series of soaks in ethanol solutions

131
Q
A

Microbiology (15 decks)

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