Lecture 3 - Microscopes Flashcards
1
Q
Who coined the term “cell”?
A
Robert Hooke
2
Q
The unaided eye can see things larger than ___ mircometers.
A
200 micrometers
3
Q
1 millimeter =
A
1/1000 of a meter
4
Q
1 micrometer =
A
1/1,000,000 of a meter
5
Q
1 nanometer =
A
1/1,000,000,000 of a meter
6
Q
Size of Red Blood Cells (RBCs)
A
5 micrometers
7
Q
What type of microscope is used to view viruses?
A
Electron Microscope
8
Q
Typical Size of a Virus
A
100 nm
9
Q
Typical Eukaryotic Cell Size
A
10 micrometers
10
Q
Typical Size of Prokaryotic Cell
A
1 micrometer
11
Q
Diameter of DNA Double Helix
A
~2 nanometers
12
Q
Which two Domains of Life are Prokaryotes?
A
Bacteria & Archaea
13
Q
Simple Light Microscope
A
uses only one lens to magnify specimen
14
Q
Compound Light Microscope
A
uses 2 or more lenses to magnify specimen
15
Q
Ocular vs. Objective Lenses
A
Ocular Lenses are the ones you look through with your eyes (#eyepiece) and the Objective Lenses are the lenses closest to the specimen being viewed.
16
Q
Who developed the first compound light microscope in 1595?
A
Zacharias Jansen
17
Q
Who Developed the first simple microscope in 1670?
A
Antony van Leeuwenhoek
18
Q
Illuminator
A
the light source
19
Q
diaphram on a microscope
A
controls how much light is allowed to reach the condenser below the specimen
20
Q
What role does the lens inside of the condneser of a microscope play?
A
it focuses the light that will shine up through the specimen
21
Q
Stage
A
where the specimen to be viewed is placed
22
Q
At which two points on a compound microscope is a specimen being magnified?
A
Maginified at the Objective & Ocular Lenses
23
Q
What is the standard magnification of the Ocular Lens on a compound microscope?
A
10x
24
Q
How to Determine the Total Magnification
A
multiply the magnification of the ocular lens by the magnification of the objective lens
Ocular Lens x Objective Lens
25
Name the 4 Standard Objective Lenses on a Compound Microscope and Their Magnifications
Scanning Lens 4x
Low Power Lens 10x
High Power Lens 40x
Oil Immersion Lens 100x
26
Which Lens on a Compound Microscope is the Shortest?
Scanning Lens
(4x magnification)
27
Which Lens on a Compound Microscope is the Longest?
Oil Immersion Lens
(100x magnification)
28
Resolving Power/Resolution
how far apart two adjacent points can be from each other and still be seen as two distinct points
#sharp/clear image
29
Refractive Index
how much the light waves bend when passing through a medium
30
Easiest Way to Change the Refractive Index of a Specimen
Staining
31
The refractive index of Immersion Oil is the same as ______.
Glass
32
Brightfield Microscopy
The background behind the specimen is bright.
33
Darkfield Microscopy
an opaque disk blocks some of the light from entering the condenser.
Specimen is seen on a dark bakground
34
Phase Contrast Microscopy
The background appears dark; Internal organelles are seen clearly and easily see cell surface features such as cilia.
35
Compare & Contrast Darkfield Miscroscopy vs Phase-Contrast Microscopy
Both have dark backgrounds, but organelles can be viewed easier with phase-contrast microscopy. Phase-contrast is also more expensive to use.
36
Two Types of Phase-Contrast Microscopy
Positive and Negative
37
Positive vs Negative Phase-Contrast Microscopy
Postive: the specimen appears darker than the background; lighter halos can also be seen around the specimen
Negative: the specimen shows up bright against the background.
38
DIC
Differential Interference Contrast Microscope
39
Explain How a DIC Microscope Works
Two beams of light are split by a prism which add contasting colors. The image is brightly colored (without staining the specimen) and looks 3D.
40
What type of Microscope Uses a Prism -- Making the Specimen Appear Brightly Colored & 3D?
DIC
(Differential Inference Contrast Microscope)
41
Explain How Fluorescent Microscopy Works
High energy UV light is aimed at the specimen which causes electrons in the specimen to jump to higher energy levels. When the electron falls back to its normal energy level, fluorescent light is emitted and then viewed through the microscope.
42
FA Technique AKA
Fluorescent-Antibody Technique
#immunofluorescence
43
Which microbe causes syphilis?
Treponema padillum
44
Explain How Fluorescent-Antibody (FA) Techniques Work
1. Fluorchrome is attached to antiboides.
2. The antibodies with Fluorochrome attached are added to the specimen and allowed to attach to the specimen.
3. The specimen with antibodies attached is hit with high energy UV light. The high energy causes the fluorochrome to fluoresce and the specimen will appear to glow under the microscope.
45
Confocal Micoscopy -
How Does it Work?
- Specimens are stained w/ fluorochromes
- Specimens are illuminated one plane at a time with SHORT wavelength light to excite the fluorochromes (one photon is needed to excite the fluorchrome) (illuminated in layers)
- Images of the different layers are reconstructed by a computer to produce a 3D image
46
Two-Photon Microscopy
- Specimens are stained w/ fluorochromes
- Specimens are illuminated one plane at a time with LONG wavelength light to excite the fluorochrome. (2 photons are needed to excite the fluorochrome)
47
Scanning Acoustic Microscopy is like ______
performing an ultrasound on a cell
48
SAM
Scanning Acoustic Microscopy
49
Hz
hertz AKA cycles per second
50
units of measurement for frequency
Hz
cycles per second
51
What bacteria causes TB (tuberculosis)?
Mycobacterium tuberculosis
52
Electron Microscope uses _______ instead of visible light.
an electron beam
53
Two Types of Electron Microscopes
Transmission Electron Microscope (TEM)
Scanning Electron Microscope (SEM)
54
TEM
Transmission Electron Microscope
(shows the inside of the specimen)
55
SEM
Scanning Electron Microscope
(shows the external surface of the specimen)
56
microtome
diamond blade that is used to cut microorganisms into thin slices in an electron microscope
57
Magnification of an Electron Microscope
10,000 - 100,000x
58
Do electron microscopes give you colored images?
No. The image is grayscale, then colored after the fact.
59
Scanned Probe Microscopy
A probe runs over the surface of the sample and the ridges and dips encountered are recorded and used to make an image of the specimen
60
Which microscopic technique is used to see living, moving microorganisms?
Hanging Drop Technique
61
Explain How the Hanging Drop Technique is Done
-The specimen is suspended in liquid then a drop is placed on a glass cover slip
-The cover slip is flipped upside down so the drop is hanging
-The cover slip with the hanging drop is carefully placed on top of glass slide with a depression in the center so the drop can continue to hang.
-The specimen is still viewed
62
What is a suspension?
a liquid containing microbes
63
Explain How Fixation of a Slide is Done
1. The culture is placed in a thin layer on the glass slide.
2. The culture is air dried on to the plate.
3. The slide (culture side up) is passed over a flame a few times to heat the sample enough to secure it in place on the glass.
64
Should you "fix a slide" if you want to view living organisms?
No, becasue fixing the slide kills the speciemen.
65
Mordant
used to intensify staining
66
Commonly used Mordant
Iodine
67
Differential Staining
different types of cells will look different even through they go though the same staining procedure
68
Gram-negative bacteria appears ________ in differential staining.
Pink
69
Gram-positive bacteria appears ________ in differential staining.
Purple
70
Explain the Gram Staining Procedure
1. Specimen is fixed to a glass slide.
2. Add Crystal Violet
3. Wash
4. Add Mordant (Iodine)
5. Decolor (alcohol wash)
6. Counterstain with Safranin
7. Wash
8. Look at under a microscope :)
71
What color is Gram Positive Bacteria in each step of differential staining?
purple
72
What color is Gram Negative Bacteria in each step of differential staining?
- purple after crystal violet is added
- purple after mordant (iodine) is added
- clear after decolorizer (alcohol) is added
- pink after safranin counterstain is added
73
Are Human Cells Gram-positive or Gram-negative?
Neither because human cells do not have a cell wall.
74
What are the 2 Main Types of Differential Staining?
Gram Staining
Acid-Fast Staining
75
Which bacteria produces mycolic acid?
mycobacterium spp.
76
Two species of Mycobacterium
Mycobacterium leprae
(causes lepracy)
Mycobacterium tuberculosis
(causes TB)
77
spp.
species
(refers to multiple species of a specific genus).
78
Explain the Acid-Fast Staining Procedures
1. Specimen is fixed to a glass slide.
2. Add Carbol Fuchsin
3. Wash
4. Decolor (wash with HCL in alcohol)
5. Wash
6. Counterstain with Methylene Blue
7. Wash
8. Look at under a microscope :)
79
What color is Acid-Fast Bacteria during each step of differential staining?
- pink after carbol fuchsin is added
- pink after decolorizer (HCL in alcohol) is added
- pink after methylene blue counterstain is added
80
What color is Non-Acid-Fast Bacteria during each step of differential staining?
- pink after carbol fuchsin is added
- colorless after decolorizer (HCL in alcohol) is added
- blue after methylene blue counterstain is added
81
3 Types of "Special Stains"
- Negative Stain
- Endospore AKA Shaeffer-Fulton Stain
- Flagella Stain
82
Negative Stain
The space between the bacteria is filled with dye rather than staining the bacteria itself, so it just pops out against the dye
Useful for viewing cell shape, size and CAPSULES
83
Shaeffer-Fulton Stain AKA
Endospore Stain
84
Name the 3 layers of cryptococcus neoformans
cell membrane
cell wall
capsule
85
Name the 3 layers of cryptococcus neoformans
plasma membrane
cell wall
capsule
86
What is the primary stain used in an Endospore (Shaeffer-Fulton) Stain?
malachite green
87
What is the counterstain used in an Endospore (Shaeffer-Fulton) Stain?
safranin
88
In an Endospore (Shaeffer-Fulton) Stain, what color will endospores stain?
Malachite Green
89
In an Endospore (Shaeffer-Fulton) Stain, what color will vegetative cells that are living and dividing stain?
Red (safranin)
90
Endospore
A protective coating formed around some bacteria which go dormant to survive harsh conditions.
91
Which 2 Genuses of Bacteria can form Endospores?
Bacillus & Clostridium
92
What is a Flagella Stain used for?
Stain and mordant are used to stain the flagella.
This allows one to view the presence of, number, and location of flagella on a bacteria and therefore identify the type of bacteria.
93
What is the only type of human cell that has a flagella?
Sperm
94
What is a flagella used for?
cell motility
95
What Two Types of Specimen Autofluoresce Well?
-chlorophyll
-carotenes
