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Microbiology Lecture > Topic 3 - Microscopy > Flashcards

Topic 3 - Microscopy Flashcards

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0
Q

Microscopes produce ____ what?

A

Magnified images of objects

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1
Q

MO range in size from smallest viruses which are measured in _____, to the largest protists and bacteria, which can be about ______

A
  • Nanometers

- 500 micrometers

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2
Q

What three things do microscopes vary in?

A
  1. Illumination source: light, electrons, and others
  2. Focusing method: glass lenses, magnets, others
  3. Specimen preparation
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3
Q

Three characteristics of the light microscopes

A
  1. Use visible light to illuminate
  2. Use glass lenses to focus
  3. Have light pass through/around specimen
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4
Q

Two lenses on the light microscope and their meanings

A
  1. Simple = 1 focusing lens

2. Compound = 2 focusing lenses form the image

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5
Q

Two lenses on the compound microscope

A

Objective and ocular

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6
Q

Total magnification of a light microscope is determined how?

A

Product of the magnification of the two sets of lenses

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7
Q

What is the name of the total magnification in the light microscope?

A

Objective magnification X ocular magnification

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8
Q

What is the maximum magnification of the light microscope?

A

2,000 X

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9
Q

The ability to distinguish two close, adjacent objects as separate and distinct

A

Resolution

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10
Q

How is resolution determined?

A

By the wavelength of light used and the lenses used

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11
Q

Shorter wavelengths equal what?

A

Great resolution

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12
Q

Limit of resolution for best light microscopes is about ____

A

.2 um

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13
Q

Different kinds of light microscopes

A
  1. Bright field microscope
  2. Phase contrast microscope
  3. Phase contrast microscope
  4. Fluorescence microscope
  5. Confocal microscope
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14
Q
  • Very common microscope

- Specimens are visualized because of differences in contrast (density) between specimen and surroundings

A

Bright-field microscope

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15
Q

Bright field is due to what?

A

Specific condenser lens

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16
Q

What do the condenser lenses do?

A

Illuminate the entire field

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17
Q

Two sets of lenses that form the image in the bright field microscope

A

Objective lens and ocular lens

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18
Q

Total magnification = what?

A

Objective magnification x ocular magnification

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19
Q

Microscope that produces a dark image against a brighter background

A

The Bright-field microscope

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20
Q

The distance between the front surface of lens and surface of cover glass or specimen when it is in sharp focus

A

Working distance

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21
Q

What happens when air is replaced with immersion oil?

A

Many light rays that did not enter the objective due to reflection and refraction at the surfaces of the objective lens and slide will now do so
- This results in an increase in resolution and numerical aperture

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22
Q

Why do bright-field microscopes often require objects to be stained?

A

In order to achieve sufficient contrast between specimen and surrounding medium

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23
Q

Photos taken by bright-field microscopes

A

Photomicrographs

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24
Q

Three characteristics about the dark field microscope?

A
  1. Uses special condenser lens
  2. Produces a bright image of the object against a dark background
  3. Used to observe living, unstained preparations
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25
Q

What is the dark field microscope used to observe?

A
  1. Internal structures in eukaryotic MO

2. Identify bacteria such as Treponema Pallidum, the causative agent of syphilis

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26
Q

The six characteristics of the phase-contrast microscope

A
  1. Uses special condenser lenses
  2. Converts slight differences in refractive index and cell density into easily detected variations in light intensity
  3. Some light rays from hollow cone of light passing through an unstained cell are retarded and out of phase and dark compared to the bright background
  4. Used to observe living cells
  5. Studying microbial motility
  6. Detecting bacterial structures such as endospores and inclusion bodies that have refractive indices different from that of water
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27
Q
  • Used to visualize specimens that fluoresce after exposure specimen to ultraviolet, violet, or blue light
  • Shows a bright image of the object resulting from the fluorescent light emitted by the specimen
A

The Fluorescene Microscope

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28
Q

When some cells fluoresce naturally

A

Autofluorescence

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29
Q

How do cells that aren’t autofluorescent fluoresce?

A

They only fluoresce after they have been stained with a fluorescent dye

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30
Q

The fluorescene microscope has applications in what?

A

Medical microbiology and Microbial ecology studies

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31
Q

How does fluorescence microscopy work?

A
  • Fluorochrome-labeled probes, such as antibodies, or fluorochrome dyes tag specific cell constituents for identification of unknown pathogens
  • Localization of specific proteins in cells
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32
Q

Creates image by detecting differences in refractive indices and thickness of different parts of specimen

A

The differential interference contrast microscope

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33
Q

What is the differential interference contrast microscope best used for?

A
  • Excellent way to observe living cells
  • Live, unstained cells appear brightly colored and three dimensional
  • Cell walls, endospores, granules, vacuoles, and nuclei are clearly visible
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34
Q

Creates sharp, composite 3D image of specimens by using laser beam, aperture to eliminate stray light, and computer interface

A

Confocal Microscope

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35
Q

Application of confocal microscopy

A

Study of biofilms

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36
Q

Two types of slide preparations

A
  1. Unstained preparations

2. Stained preparations

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37
Q

What are wet mounts?

A
  • Drop of liquid with living organisms
  • Unstained
  • Hanging drop method; concave slides
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38
Q

Dye added to wetmount; stains living organisms; may eventually kill MO

A

Vital stains

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39
Q

The stained preparation that kills MO

A

Fixed and stained slides

40
Q

Improving contrast in a microscope results in _____

A

A better final image

41
Q

What does staining improve?

A

Contrast between specimen and surroundings

42
Q

Dyes or stains are compounds that do what?

A

Bind to specific cellular materials

43
Q

Examples of common stains

A
  1. Methylene blue
  2. Safranin
  3. Crystal violet
44
Q

Kills MO, preserves internal and external structures and adheres them to the slide

A

Fixation

45
Q

Any distortion in the true morphology of the MO due to the technique itself

A

Artifact

46
Q

Two types of fixations

A
  1. Heat fixation

2. Chemical fixation

47
Q

Two components to heat fixation

A
  1. Bunsen burner

2. Artifacts

48
Q

Characteristics of chemical fixation

A
  1. Chemicals
  2. More expense and more time
  3. Less artifacts
  4. Used with larger, more delicate organisms
49
Q

This technique protects fine cellular substructure and morphology

A

Chemical fixation

50
Q

Organic compounds or salts that have different affinities for specific cellular materials

A

Stains or dyes

51
Q

Most stains or dyes are what?

A

Salts of cations and anions

52
Q

Coloring ion

A

Chromophore

53
Q

Chromophore cation; binds to cells, most dyes

A

Basic dye

54
Q
  • Chromophore anion; repelled by cells; stains background
A

Acidic dye

55
Q

Make internal and external structures of cell more visible by increasing contrast with background

A

Dyes

56
Q

1 dye; few steps, all stained the same

A

Simple staining

57
Q

What is simple staining used for?

A

Used to determine size, shape, and arrangement of bactera

58
Q

2 or more dyes; several steps

A

Differential staining techniques

59
Q

Two groups of MO based on their staining properties

A
  1. Gram stain

2. Acid fast stain

60
Q

Technique used to presence or absence of structures

A

Differential stain

61
Q

Differential stains detect these things

A
  • endospores, flagella, capsules
62
Q

Most widely used differential staining procedure

A

Gram staining

63
Q

Gram staining divides bacteria into what two groups based on the differences in cell wall structure

A
  1. Gram positive

2. Gram negative

64
Q

Particularly useful for staining members of the genus Mycobacterium

A

Acid fast staining

65
Q

Causes tuberculosis

A

Mycobacterium tuberculosis

66
Q

Causes leprosy

A

Mycobacterium leprae

67
Q

What is responsible for Mycobacteriums staining characteristics?

A

High lipid content in cell walls (mycolic acid)

68
Q
  • Heated, double staining technique

- Bacterial endospore is one color and vegetative cell is a different color

A

Endospore staining

69
Q

Used to visualize capsules surrounding bacteria

A

Capsules

70
Q

Capsules may be colorless against a stained background

A

Negative stain

71
Q

Mordant applied to increase thickness of flagella

A

Flagella staining

72
Q

Uses electrons instead of light as the illuminating beam

A

Electron microscopy

73
Q

Instead of illumination, lenses, image, and stains….electron microscopy uses these things respectively

A

Illumination - electron beam
lenses - magnets
image - photograph or computer
stains - metals

74
Q

What is special about the wavelength of an electron beam?

A

Much shorter than light, resulting in much higher resolution

75
Q

What does the electron microscopy allow for?

A

Allows for the study of microbial morphology in great detail

76
Q

Seven characteristics of the transmission electron microscope

A
  1. Beam of electrons pass through specimen
  2. Electromagnets function as lenses
  3. Electrons scatter when they pass through thin sections of a specimen
  4. Transmitted electrons are under vacuum which reduces scatter and are used to produce clear image
  5. Denser regions in specimen, scatter more electrons and appear darker
  6. Enables visualization of structures at the molecular level
  7. Specimen must be ver thin (20-6-nm) and be stained with metalic stains
77
Q

Common specimen preparation method

A
  • Analogous to procedure used for light microscopy
  • For TEM, specimens must be cut very thin
  • Specimens are chemically fixed and stained with electron dense materials, such as heavy metals, that differentially scatter electrons
78
Q

Specialized specimen preparation methods

A
  • Negative stain
  • Shadowing
  • Freeze etching
79
Q
  • Heavy metals do not penetrate the specimen but render dark background
A

Negative stain

80
Q

What are negative stains used for?

A

Study of viruses, bacterial gas vacuoles

81
Q
  • Coating specimen with a thin film of a heavy metal on only one side
A

Shadowing

82
Q

What is shadowing used for?

A

Viral morphology, flagella, DNA

83
Q
  • Freeze specimen then fracture along lines of greatest weakness
  • Allows for 3D observation of shapes of intracellular structures
  • Reduces artifacts
A

Freeze etching

84
Q

Three facts about the Scanning Electron Microscope

A
  1. Uses electrons reflected from the surface of a specimen and collected by a detector to create detailed image
  2. Produces a realistic 3D image of specimen’s surface features
  3. Can determine actual in situ location of microogranisms in ecological niches
85
Q

The magnification of the scanning electron microscope

A

15x-100,ooox

86
Q

Three facts about the scanning probe microscopy

A
  1. Magnification 100 million times, can view atoms on surface of a solid
  2. Steady current (tunneling current) maintained between microscope probe and specimen
  3. Up/down movement of probe as it maintains current is detected, used to create image of surface of specimen
87
Q

The atomic force microscope is what kind of microscope?

A

Scanning probe microscopy

88
Q

The four steps steps on how the atomic force microscope works

A
  1. Sharp probe moves over surface of specimen at constant distance
  2. The stylus measures weak repulsive forces between it and the specimen
  3. Up and down movement of probe as it maintains constant distance is detected and sued to create image
  4. A computer generates an image based on the data
89
Q

To grow MO; the liquid or solid substance containing growing MO

A

Culture

90
Q

The nutrient material used to culture MO

A

Medium

91
Q

Different kinds of mediums

A
  • Liquid medium or broth medium

- Solid medium - contains solidifying agent

92
Q

Two solid mediums used for culturing MO

A

Agar or gelatin

93
Q

Two or more species for culturing MO

A

Mixed culture

94
Q

Only one species in culturing MO

A

Pure culture

95
Q

Free of all viable MO

A

Sterile

96
Q

Adding MO to culture media

A

Inoculation

97
Q

Any unwanted MO in the medium

A

Contaminant

98
Q

Anything done to minimize contamination

A

Asceptic (sterile) techniques

Microbiology Lecture (5 decks)

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