Ch 3: Observic Microorganisms Through a Microscope Flashcards
One Angstrom (Å) is equivalent to ____ or ____.
10^-10 m
0.1 nm
What are the typical magnification ranges for objective and ocular lenses in a compound light microscope?
Objectives: 5 - 100x
Oculars: 10 - 20x
What does it mean when a microscope has a resolving power of 0.2 nm?
It can distinguish two points ≥0.2 nm
Total magnification = _____?
Objective lens magnification x ocular lens magnification
The ultimate limit to the resolution of a light microscope is set by __________?
The type of radiation it utilizes
Do shorter or longer wavelengths of light provide greater resolution?
Shorter
Resolution = ______
Resolution = (0.61*λ) / (n*sinθ)
λ = wavelength of light used
n = refractive index of medium
θ = half the angular width of the cone of rays by the objective lens from a typical point in the specimen
In an electron microscope, with an accelerating voltage of 100,000 V, the wavelength of an electron is ______?
0.004 nm
What is the refractive index of air?
1
For white light, the wavelength that is commonly assumed is _____.
0.53 μm
Define “refractive index”
A measure of the light-bending ability of a medium; dependent on how much the speed of light is reduced in the medium
Why does using immersion oil produce a better resolution in a microscope?
Immersion oil has the same refractive index as glass, thus does not bend the light
What are the six types of light microscopy?
- Brightfield
- Darkfield
- Phase-contrast
- Differential interference contrast (DIC/Nomarski)
- Fluorescence
- Confocal
In _____ microscopy, dark objects are visible against a bright background.
Brightfield
True or false. Brightfield microscopy is only useful for stained biological specimens.
True. Unstained cells are virtually invisible
What are some benefits of darkfield microscopy?
Useful for live organisms not visible with light, can’t be stained, or distorted by staining
Can be used to visualize very thin microorganisms (ex: Treponema pallidum)
Phase-contrast microscopy is useful because ___________
internal structures of a cell become more sharply defined, permitting detailed examination of living tissue
What advantage does DIC microscopy have over phase-contrast?
Allows for visualization of depth
Both phase-contrast and DIC microscopy _____. (3)
- Can be used with living cells
- Don’t require cell fixation/attachment slides
- Don’t require staining
True or false. Fluorescence microscopy takes advantage of fluorochromes which absorb long wavelengths of light (UV or near UV) and emit another, shorter wavelength (visible).
False. They absorb short and emit long
Describe the path of light in fluorescence microscopy
- Barrier filter lets only one wavelength of light through
- Beam-splitting mirror reflects light of below one wavelength but transmits light above another
- Second barrier filter cuts out unwanted noise before reaching eyepiece
Describe how immunofluorescence works
Antibodies conjugated to fluorochromes bind to specific antigens in the specimen of interest
What advantage does confocal microscopy have over standard fluorescence?
Illuminate individual planes of light in order to generate a 3D image and visualize depth
What two conditions can cause photobleaching of a specimen stained with a fluorochrome?
Exposure to high wavelengths of light
Long exposure times
What is the advantage of two-photon microscopy?
Study cells within live tissue up to 1mm deep (100 μm is the limit for confocals)
Super-resolution fluorescence can increase resolution from 200nm to ____?
20 nm
What is the resolution of electron microscopes?
0.002 nm
True or false. Transmission electron microscopy requires a special preparation that makes it possible to view live cells.
False. It is not possible to view live cells in TEM
Describe the fixation process for TEM
- Glutaraldehyde fixes proteins while osmium tetroxide binds and stabilizes proteins and lipids
- Dehydrate specimen and permeate it with monomeric resin to form a block of plastic
In TEM, elements with higher atomic numbers _____
scatter electrons better and produce greater contrast
Contrast in TEM can be increased by impregnation with ______
Salts of heavy metals
Ex: Ur [92], Pb [82], Os [76]
What is the difference between positive and negative staining?
Positive: stains sample
Negative: stains everything but the sample
When is negative staining preferred? (2)
- In living cells
- Visualization of VERY small samples
What is the major difference between SEM vs. TEM?
SEM allows for visualization of a whole specimen
How does SEM work?
An electron gun produces a beam of primary electrons that scans the surface of a whole specimen. A secondary electron is emitted from the surface of the specimen to produce an image
What are the advantages of SEM over TEM? (4)
- Sectioning not required (still must be fixed)
- Provides great depth of field
- 3D appearance
- Smaller, simpler, and cheaper than TEM
What are the limitations of SEM? (2)
- Only surface features can be examined
- Lower resolution than TEM (~10 nm)
How does scanning tunneling microscopy (STM) work?
Uses a sharp probe to scan over the surface of a sample
Electrons flow from surface to probe tip (tunneling)
Resolution of STM is not constrained by the wavelength of light or electrons, thus can resolve ______
at the atomic level
Atomic force microscopy was developed to overcome what drawback to STM?
STM can only image conducting or semiconducting surfaces
How does atomic force microscopy (AFM) work?
A probe is gently forced down onto a specimen. As the probe moves along the surface its movements are recorded
True or false. AFM does not require special specimen prep.
True
How are microbes fixed for brightfield microscopy?
Heat
In basic dyes, the chromophore is a ____ and typically neutralized by ____
Cation
Cl- ion
In acidic dyes, the chromophore is ______ and is typically neutralized by ______.
Anion
Na+ ion
Will bacteria stain with a more acidic dye or a more basic dye?
Basic dye
Bacteria are slightly negatively charged at pH 7, thus the colored positive ion of the basic dye is attracted to the bacterial cell
Negative staining is useful for determining _____.
Overall cell morphology size and shape, as they are highly visible against a contrasting background
What is simple staining? Examples?
Method of staining of microorganisms with a single basic dye
What are some examples of simple stains? (4)
Methylene blue
Carbolfusion
Crystal violet
Safranin
What is the primary goal by using a simple stain?
Highlight the entire microorganism to visualize cellular shapes and basic structures
What is differential staining?
Staining method that uses more than one chemical stain to differentiate between different organisms
What are some examples of differential stains?
Gram stain
Acid-fast stain
Who developed the Gram stain?
Danish bacteriologist Hans Christian Gram
Describe the process of Gram staining?
- Bacteria are fixed
- Crystal violet stains peptidoglycan
- wash
- Iodine treatment complexes with crystal violet
- wash
- Decolorization with alcohol
- Counter stain with safranin
Does cystal violet or safarin stain stronger?
Crystal violet
What is the role of the alcohol washings in both Gram-positive and Gram-negative stains? Do CV-I crystals remain in the sample?
Gram-positive: alcohol dehydrates thick peptidoglycan layer; CV-I crystals do not leave
Gram-negative: dissolves outer membrane, leaving behind a thin peptidoglycan layer; CV-I crystals wash out
What is mordant?
A substance added to a staining solution to hold the stain or coat the specimen to make it thicker and easier to see after staining
What are the structural differences between Gram-positive vs. Gram-negative bacteria?
Gram-positive: thicker peptidoglycan cell wall
Gram-negative: thinner peptidoglycan cell wall, but have a lipopolysaccharide (LPS) outer membrane
Acid-fast stain only binds strongly to ______. Examples?
bacteria that have waxy material in their cell wall.
Ex: Mycobacterium (M. tuberculosis, M. leprae) and Nocardia
Describe the process of an acid-fast stain.
- Fixation
- Carbol Fuschsin steam 5 min
- Cool water wash
- Acid alcohol wash
- Counterstain with methylene blue
What type of staining can reveal the presence of capsulated microbes? Stain examples?
Negative staining
Basic dye (Safranin)
What is the significance of being able to detect if a microbe is capsulated or not?
Capsulated microbes typically cause disease
Describe the process for Endospore staining.
- Stained with Malachite green
- steam 5 min
- Rinse 30 sec
- Counterstain Safranin
True or false. Endospores can be detected in a light microscope without staining.
True
How does a Flagella stain work? Stain example?
A mordant is used to coat flagella with stain until thick enough to be seen.
Ex: Carbolfuschin simple stain
