Topic 3 - Microscopy Flashcards
(99 cards)
0
Q
Microscopes produce ____ what?
A
Magnified images of objects
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
2
Q
What three things do microscopes vary in?
A
- Illumination source: light, electrons, and others
- Focusing method: glass lenses, magnets, others
- Specimen preparation
3
Q
Three characteristics of the light microscopes
A
- Use visible light to illuminate
- Use glass lenses to focus
- Have light pass through/around specimen
4
Q
Two lenses on the light microscope and their meanings
A
- Simple = 1 focusing lens
2. Compound = 2 focusing lenses form the image
5
Q
Two lenses on the compound microscope
A
Objective and ocular
6
Q
Total magnification of a light microscope is determined how?
A
Product of the magnification of the two sets of lenses
7
Q
What is the name of the total magnification in the light microscope?
A
Objective magnification X ocular magnification
8
Q
What is the maximum magnification of the light microscope?
A
2,000 X
9
Q
The ability to distinguish two close, adjacent objects as separate and distinct
A
Resolution
10
Q
How is resolution determined?
A
By the wavelength of light used and the lenses used
11
Q
Shorter wavelengths equal what?
A
Great resolution
12
Q
Limit of resolution for best light microscopes is about ____
A
.2 um
13
Q
Different kinds of light microscopes
A
- Bright field microscope
- Phase contrast microscope
- Phase contrast microscope
- Fluorescence microscope
- Confocal microscope
14
Q
- Very common microscope
- Specimens are visualized because of differences in contrast (density) between specimen and surroundings
A
Bright-field microscope
15
Q
Bright field is due to what?
A
Specific condenser lens
16
Q
What do the condenser lenses do?
A
Illuminate the entire field
17
Q
Two sets of lenses that form the image in the bright field microscope
A
Objective lens and ocular lens
18
Q
Total magnification = what?
A
Objective magnification x ocular magnification
19
Q
Microscope that produces a dark image against a brighter background
A
The Bright-field microscope
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
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
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
23
Q
Photos taken by bright-field microscopes
A
Photomicrographs
24
Three characteristics about the dark field microscope?
1. Uses special condenser lens
2. Produces a bright image of the object against a dark background
3. Used to observe living, unstained preparations
25
What is the dark field microscope used to observe?
1. Internal structures in eukaryotic MO
| 2. Identify bacteria such as Treponema Pallidum, the causative agent of syphilis
26
The six characteristics of the phase-contrast microscope
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
27
- 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
The Fluorescene Microscope
28
When some cells fluoresce naturally
Autofluorescence
29
How do cells that aren't autofluorescent fluoresce?
They only fluoresce after they have been stained with a fluorescent dye
30
The fluorescene microscope has applications in what?
Medical microbiology and Microbial ecology studies
31
How does fluorescence microscopy work?
- Fluorochrome-labeled probes, such as antibodies, or fluorochrome dyes tag specific cell constituents for identification of unknown pathogens
- Localization of specific proteins in cells
32
Creates image by detecting differences in refractive indices and thickness of different parts of specimen
The differential interference contrast microscope
33
What is the differential interference contrast microscope best used for?
- 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
34
Creates sharp, composite 3D image of specimens by using laser beam, aperture to eliminate stray light, and computer interface
Confocal Microscope
35
Application of confocal microscopy
Study of biofilms
36
Two types of slide preparations
1. Unstained preparations
| 2. Stained preparations
37
What are wet mounts?
- Drop of liquid with living organisms
- Unstained
- Hanging drop method; concave slides
38
Dye added to wetmount; stains living organisms; may eventually kill MO
Vital stains
39
The stained preparation that kills MO
Fixed and stained slides
40
Improving contrast in a microscope results in _____
A better final image
41
What does staining improve?
Contrast between specimen and surroundings
42
Dyes or stains are compounds that do what?
Bind to specific cellular materials
43
Examples of common stains
1. Methylene blue
2. Safranin
3. Crystal violet
44
Kills MO, preserves internal and external structures and adheres them to the slide
Fixation
45
Any distortion in the true morphology of the MO due to the technique itself
Artifact
46
Two types of fixations
1. Heat fixation
| 2. Chemical fixation
47
Two components to heat fixation
1. Bunsen burner
| 2. Artifacts
48
Characteristics of chemical fixation
1. Chemicals
2. More expense and more time
3. Less artifacts
4. Used with larger, more delicate organisms
49
This technique protects fine cellular substructure and morphology
Chemical fixation
50
Organic compounds or salts that have different affinities for specific cellular materials
Stains or dyes
51
Most stains or dyes are what?
Salts of cations and anions
52
Coloring ion
Chromophore
53
Chromophore cation; binds to cells, most dyes
Basic dye
54
- Chromophore anion; repelled by cells; stains background
Acidic dye
55
Make internal and external structures of cell more visible by increasing contrast with background
Dyes
56
1 dye; few steps, all stained the same
Simple staining
57
What is simple staining used for?
Used to determine size, shape, and arrangement of bactera
58
2 or more dyes; several steps
Differential staining techniques
59
Two groups of MO based on their staining properties
1. Gram stain
| 2. Acid fast stain
60
Technique used to presence or absence of structures
Differential stain
61
Differential stains detect these things
- endospores, flagella, capsules
62
Most widely used differential staining procedure
Gram staining
63
Gram staining divides bacteria into what two groups based on the differences in cell wall structure
1. Gram positive
| 2. Gram negative
64
Particularly useful for staining members of the genus Mycobacterium
Acid fast staining
65
Causes tuberculosis
Mycobacterium tuberculosis
66
Causes leprosy
Mycobacterium leprae
67
What is responsible for Mycobacteriums staining characteristics?
High lipid content in cell walls (mycolic acid)
68
- Heated, double staining technique
| - Bacterial endospore is one color and vegetative cell is a different color
Endospore staining
69
Used to visualize capsules surrounding bacteria
Capsules
70
Capsules may be colorless against a stained background
Negative stain
71
Mordant applied to increase thickness of flagella
Flagella staining
72
Uses electrons instead of light as the illuminating beam
Electron microscopy
73
Instead of illumination, lenses, image, and stains....electron microscopy uses these things respectively
Illumination - electron beam
lenses - magnets
image - photograph or computer
stains - metals
74
What is special about the wavelength of an electron beam?
Much shorter than light, resulting in much higher resolution
75
What does the electron microscopy allow for?
Allows for the study of microbial morphology in great detail
76
Seven characteristics of the transmission electron microscope
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
Common specimen preparation method
- 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
Specialized specimen preparation methods
- Negative stain
- Shadowing
- Freeze etching
79
- Heavy metals do not penetrate the specimen but render dark background
Negative stain
80
What are negative stains used for?
Study of viruses, bacterial gas vacuoles
81
- Coating specimen with a thin film of a heavy metal on only one side
Shadowing
82
What is shadowing used for?
Viral morphology, flagella, DNA
83
- Freeze specimen then fracture along lines of greatest weakness
- Allows for 3D observation of shapes of intracellular structures
- Reduces artifacts
Freeze etching
84
Three facts about the Scanning Electron Microscope
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
The magnification of the scanning electron microscope
15x-100,ooox
86
Three facts about the scanning probe microscopy
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
The atomic force microscope is what kind of microscope?
Scanning probe microscopy
88
The four steps steps on how the atomic force microscope works
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
To grow MO; the liquid or solid substance containing growing MO
Culture
90
The nutrient material used to culture MO
Medium
91
Different kinds of mediums
- Liquid medium or broth medium
| - Solid medium - contains solidifying agent
92
Two solid mediums used for culturing MO
Agar or gelatin
93
Two or more species for culturing MO
Mixed culture
94
Only one species in culturing MO
Pure culture
95
Free of all viable MO
Sterile
96
Adding MO to culture media
Inoculation
97
Any unwanted MO in the medium
Contaminant
98
Anything done to minimize contamination
Asceptic (sterile) techniques
