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Microbiology > Observing Microorganisms Through a Microscope > Flashcards

Observing Microorganisms Through a Microscope Flashcards

1
Q

Angstrom?

A
  1. 1 nm, 10^-10 m, 1 hydrogen atom= 1 Angstrom
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2
Q

Purpose of prism in light microscopy?

A

Direct image to the ocular lens.

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3
Q

Relationship between wavelength and resolution?

A

shorter the wavelength, the greater the resolution

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4
Q

Purpose of immersion oil?

A

To prevent bending of the light/image. Increases the refraction index

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5
Q

Brightfield illumination

A

Dark objects are visible against a bright background light absorbed by specimen doesn’t enter the objective lens (areas that scatter more light are darker) This is only useful for stained biological specimens

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6
Q

Darkfield illumination

A

Light objects are visible against a dark background light reflected off specimen enters objective useful for live organisms not visible with a light microscope/ unstainable; this is commonly used to see spirochetes

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7
Q

Two ways to increase contrast in light microscopy?

A

Incident light (white) - amplitude of light is decreased Incident light (green) - out of phase waves traveling through specimen and into the objectives in phase once they pass through.`

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8
Q

Phase contrast microscopy

A

Converts a difference in light wave phase into a difference in brightness. Allows for better contrast. Halo effect

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9
Q

Differential interference contrast microscopty (DIC)

A

Uses two polarized light beams, a reference beam goes through the background, and both the specimen and ref. beams recombine where wave interference occurs, resulting in the image beam. Gives 3D gray appearance.

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10
Q

Advantages of phase contrast and DIC?

A

can be used with living cells, doesn’t require cell fixation or staining.

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11
Q

Fluorescence microscopy

A

Uses fluorescent dyes that absorb short wavelengths and emit light at longer wavelengths that are visible (this can also be done using immunofluorescence or fusing fluorescent compounds to proteins)

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12
Q

Confocal microscopy

A

The cells must be stained with fluorochromes, the light illuminates each plane in a specimen to produce a 3D image up to 100 um deep

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13
Q

2-photon

A

Alternative to confocal (but still stained with fluorochromes), needs long (red) wavelengths to excite dyes, no pinholes are used (unlike confocal), used to study cells within live tissues up to 1mm deep. An issue with this though is that photobleaching can occur.

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14
Q

Scanning acoustic microscopy

A

measures sound waves that are reflected back from specimen. This is used to study live cells attached to a surface noninvasively. The resolution is 1 um

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15
Q

Electron microscopy

A

Use electrons instead of light which will allow for better resolution (0.002, but with a more realistic resolution of 2.5 nm due to problems of specimen prep, contrast and radiation damage). Uses magnets.

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16
Q

Transmission electron microscope

A

Similar to light microscope, but uses magnetic coils instead of lenses and usually much larger (~2m in height). Cells must be dead, fixed with glutaraldehyde and stained with a heavy metal salt, as well as dehydrated and permeated with a resin. The specimen must be ultra thin because the electrons have poor penetration power.

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17
Q

Scanning Electron microscopy

A

Electron gun fires beam of primary electrons that scan the surface of the whole specimen and the secondary emitted electrons are knocked out from the surface to produce the image. Pros: sectioning isn’t required, provides good depth, image has 3D appearance, and smaller/cheaper than TEM Cons: only surface can be examined, and the resolution is less.

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18
Q

Scanned probe microscopy

A

Uses a sharp tungesten or platinum probe to scan over the surface; resolution is not constrained by wavelength, so it can resolve at the atomic level without any special prep. Again, only surface features are observed. Resolution of 100 pm

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19
Q

Atomic Force microscopy

A

Metal and diamond probe is forced onto a specimen and its movements are recorded as it moves along.

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20
Q

Basic and Acidic dyes

A

In a basic dye, chromophore = cation In an acidic dye, the chromophore is an anion (repelled by bacterial cell and stain the background instead)

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21
Q

Negative staining

A

Stains background, good for morphology and size, fixing isn’t necessary. Acidic dye used

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22
Q

Simple staining

A

staining with a basic dye to highlight entire organism to see shapes and basic structures

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23
Q

Differential staining

A

method that uses more than one chemical stain to differentiate between organisms (i.e. gram stain and acid-fast)

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24
Q

Endospore staining

A

Uses malachite green and counterstains with safranin (endospores appear green)

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25
Q

Flagella staining

A

Mordant used to coat flagella with stain until it’s thick enough to be seen (i.e. carbolfuchsin).

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26
Q

1 m

A

brown bear

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27
Q

1000 mm (10 cm)

A

squirrel

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28
Q

100 mm (1 cm)

A

ants

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29
Q

1 mm (1000 um)

A

nematode (light microscope)

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30
Q

0.2 mm (200 um)

A

human eye

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31
Q

0.1 mm (100 um)

A

mite (SEM)

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32
Q

10 um (microns)

A

eukaryotic cells, lymphocytes, macrophages (SEM)

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33
Q

1 um (1000 nm)

A

bacterial cells, prokaryotes (TEM)

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34
Q

100 nm

A

virus

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35
Q

10 nm

A

hemoglobin

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36
Q

1 nm (10 A)

A

DNA

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37
Q
  1. 1 nm (1 A)
A

H atom

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38
Q

to observe various stained specimens and to count microbes; does not resolve very small specimens, such as viruses

A

Brightfield light microscope

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39
Q

to examine living microorganisms that are invisible in brightfield microscopy, do not stain easily, or are distorted by staining; frequently used to detect Treponema pallidum in the diagnosis of syphilis

A

Darkfield

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40
Q

To facilitate detailed examination of the internal structures of living specimens

A

Phase-contrast

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41
Q

To provide three-dimensional images

A

DIC

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42
Q

For flourescent- antibody techniques (immunoflourescence) to rapidly detect and identify microbes in tissues or clinical specimens

A

Flourescence

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43
Q

Uses a single photon to illuminate one plane of a specimen at a time. To obtain two- and three dimensional images of cells for biomedical applications

A

confocal

44
Q

Uses two photons to illuminate a specimen. To image living cells, up to depth of 1 mm, reduce phototoxicity, and observe cell activity in real time

A

two-photon

45
Q

to examine living cells attached to another surface, such as cancer cells, artery plaque, and biofilms

A

scanning acoustic

46
Q

to examine viruses or the internal ultrastructure in thin sections of cells (usually magnified 10k-100k X)

A

TEM

47
Q

to study the surface features of cells and viruses (usually magnified 1000-10000 X)

A

SEM

48
Q

Provides very detailed views of molecules inside cells

A

Scanning tunneling

49
Q

Provides 3D images of biological specimens at high resolution in nearly atomic detail and can measure physical properties of biological specimens and molecular processes

A

atomic force

50
Q
A

Tick (1mm)- observed with the unaided eye

51
Q
A

DNA double helix (50 nm)

TEM

52
Q
A

E.coli (1 um)
TEM
SEM

Light Microscope

53
Q
A

Red Blood Cells (4 um)
unaided eye
light microscope

54
Q
A

bacteriophage (50 nm, 0.1 um)

TEM

SEM

55
Q

What is the order that light travels in a compound light microscope?

A

illuminator–> diaphragm–> condenser–> specimen–> objective lens–> ocular lens–> eyes

56
Q

Equation for calculating total magnification?

A

objective lens x ocular lens

57
Q

What is resolution?

A

is the ability of the lenses to distinguish two points a specific distance apart. A microscope with a resolving power of 0.2 nm can distinguish two points ≥ 0.2 nm.

58
Q

what microscope is used in this image? what are you looking at?

A

The distance between adjacent files of gold atoms in this TEM is 0.2 nm

59
Q

what is the equation for determining resolution? In a light microscope, generally what is the resolution? In a electron microscope?

A

resolution= .61(lamda)/ nsin(theta)

light microscope- 0.32 bc sin theta= 1, n= refractive index of air= 1, lambda=.5

electron microscope has a smaller wavelegth, so the resolution will be greater

60
Q

what is the refractive index?

A

a measure of the lightbending ability of a medium (dependent on how much the speed of light is reduced in the medium).

For example, Light rays change direction when they cross the interface from glass to the air (and vice versa) The light may bend so much that most misses highmagnification objective lens.

61
Q

why does light bending not occur with immersion oil?

A

it has the same refractive index as glass

62
Q
A

Protozoan

Paramecium

Brightfield microscopy

25 um

63
Q

E.coli

Brighfield microscopy

A
64
Q
A

Treponema pallidum

Darkfield illumination

65
Q
A

Paramecium

Darkfield illumination

25 um

66
Q
A

darkfield microscope without specimen on stage

67
Q
A

darkfield microscope with specimen on stage

68
Q

what are brightfield microscopes inadequete at visualizing?

A

transparent and colorless specimens

69
Q
A

two waves in phase–> larger and brighter wave produced

70
Q
A

two waves out of phase–> smaller, dimmer wave produced

71
Q

what’s another name for DIC?

A

Nomarski

72
Q
A

Phase contrast (background is grey)

Paramecium

25 um

73
Q
A

A. Brightfield microscope

B. Phase contrast

(50 um)

74
Q
A

DIC/ Nomanski

23 um

colored image/ 3D/ gray background

75
Q
A

Top: Halos, visualized internal structures, phase contrast

Bottom: depth of field, DIC

76
Q

In flourescent microscopy, what wavelength is absorbed initially & for what color, what wavelength is emitted at the end & for what color?

A

Wavelength absorbed: 450-490 (blue)

Wavelength emitted: 520-560 (green)

77
Q
A

Treponema pallidum

2 um

Flourescence microscopy

testing for syphilis

78
Q
A

standard vs. confocal flourescence

visualizing microtubules in the cell

in confocal flourescence each plane is individually accessed, hence a 3D depth appears

79
Q
A

pointing to contractile vacuoles in phase contrast image

80
Q
A

confocal image of pollen

81
Q
A
82
Q

what is photobleaching?

A

Fluorochrome loses ability to fluoresce, absorb and emit light, due to photon-induced damage. Covalent bond cleavage or non-specific reactions between the flurochrome and surrounding molecules. It is a problem when

83
Q
A

two-photon

20 um

pointing to food vacuoles

84
Q
A

SEM

Biofilm

180 um

85
Q

What does contrast in the electron microscope depends on?

A

the atomic number of the atoms in the sample

86
Q
A

a colorized TEM of Paramecium

normally TEM’s are black and white

sliced internal structures

15 um

87
Q
A

negative TEM stain of bacteriophage

88
Q
A

3-dimensional reconstruction of a branched mitochondrion

used TEM

89
Q
A

TEM micrograph showing metal shadowing of bacteria to demonstrate flagella.

90
Q
A

TEM

Paramecium

15 um

91
Q
A

scanning tunneling microscopy (STM)

resolution 100 pm

image of E. coli RecA protein

freeze-dried recA-DNA complexes coated with a conducting film

92
Q
A

Top: DNA model

Bottom: STM model

93
Q
A

Top: Pentacene model

Bottom: STM model

94
Q

which has a better resolution, STM or TEM?

A

STM (100 pm vs. 2.5 nm)

95
Q
A

Perfringolysin O toxin from Clostridium perfringens

atomic force microscopy (AFM)

ATM does not require special specimen prep.

Resolution similar to STM

96
Q
A

Cocci that have been stained with crystal violet

97
Q

what are the steps of the gram stain?

A
98
Q

what is a mordant?

A

a substance added to a staining solution to hold the stain or coat the specimen. Coating is used to enlarge a specimen

99
Q

what three bacteria are found in this picture?

A
  1. gram positive stained rods
  2. gram positive stained cocci
  3. gram negative stained vibrio
100
Q

describe the procedure of acid fast staining

A
101
Q
A

acid fast stain of M. leprae

5 um

light microscope

102
Q
A

negative stain of Klebsiella pneumoniae

Light microscope

5 um

103
Q

what’s another name for endospore staining? what is the primary stain used? What is used to wash out the primary stain, and for how long is the wash? what is the counterstain?

A

Schaeffer-Fulton stain • Primary stain: Malachite green, steamed 5 min • Remove stain from all the cells structures except endospore: water/ 30 sec • Counterstain: Safranin

104
Q
A

endospore staining

light microscope

5 um

endospores can be seen without staining, but are difficult to distinguish from other structures

105
Q

what is the purpose of the mordant in flagella staining?

A

Mordant used to coat flagella with stain until thick enough to be seen • Carbolfuchsin simple stain

106
Q
A

flagella staining

light microscope

5 um

done using a carbolfucshin stain

Microbiology (15 decks)

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