Observing Microorganisms Through a Microscope Flashcards
Angstrom?
- 1 nm, 10^-10 m, 1 hydrogen atom= 1 Angstrom
Purpose of prism in light microscopy?
Direct image to the ocular lens.
Relationship between wavelength and resolution?
shorter the wavelength, the greater the resolution
Purpose of immersion oil?
To prevent bending of the light/image. Increases the refraction index
Brightfield illumination
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
Darkfield illumination
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
Two ways to increase contrast in light microscopy?
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.`
Phase contrast microscopy
Converts a difference in light wave phase into a difference in brightness. Allows for better contrast. Halo effect
Differential interference contrast microscopty (DIC)
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.
Advantages of phase contrast and DIC?
can be used with living cells, doesn’t require cell fixation or staining.
Fluorescence microscopy
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)
Confocal microscopy
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
2-photon
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.
Scanning acoustic microscopy
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
Electron microscopy
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.
Transmission electron microscope
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.
Scanning Electron microscopy
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.
Scanned probe microscopy
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
Atomic Force microscopy
Metal and diamond probe is forced onto a specimen and its movements are recorded as it moves along.
Basic and Acidic dyes
In a basic dye, chromophore = cation In an acidic dye, the chromophore is an anion (repelled by bacterial cell and stain the background instead)
Negative staining
Stains background, good for morphology and size, fixing isn’t necessary. Acidic dye used
Simple staining
staining with a basic dye to highlight entire organism to see shapes and basic structures
Differential staining
method that uses more than one chemical stain to differentiate between organisms (i.e. gram stain and acid-fast)
Endospore staining
Uses malachite green and counterstains with safranin (endospores appear green)
Flagella staining
Mordant used to coat flagella with stain until it’s thick enough to be seen (i.e. carbolfuchsin).
1 m
brown bear
1000 mm (10 cm)
squirrel
100 mm (1 cm)
ants
1 mm (1000 um)
nematode (light microscope)
0.2 mm (200 um)
human eye
0.1 mm (100 um)
mite (SEM)
10 um (microns)
eukaryotic cells, lymphocytes, macrophages (SEM)
1 um (1000 nm)
bacterial cells, prokaryotes (TEM)
100 nm
virus
10 nm
hemoglobin
1 nm (10 A)
DNA
- 1 nm (1 A)
H atom
to observe various stained specimens and to count microbes; does not resolve very small specimens, such as viruses
Brightfield light microscope
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
Darkfield
To facilitate detailed examination of the internal structures of living specimens
Phase-contrast
To provide three-dimensional images
DIC
For flourescent- antibody techniques (immunoflourescence) to rapidly detect and identify microbes in tissues or clinical specimens
Flourescence
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
confocal
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
two-photon
to examine living cells attached to another surface, such as cancer cells, artery plaque, and biofilms
scanning acoustic
to examine viruses or the internal ultrastructure in thin sections of cells (usually magnified 10k-100k X)
TEM
to study the surface features of cells and viruses (usually magnified 1000-10000 X)
SEM
Provides very detailed views of molecules inside cells
Scanning tunneling
Provides 3D images of biological specimens at high resolution in nearly atomic detail and can measure physical properties of biological specimens and molecular processes
atomic force
Tick (1mm)- observed with the unaided eye
DNA double helix (50 nm)
TEM
E.coli (1 um)
TEM
SEM
Light Microscope
Red Blood Cells (4 um)
unaided eye
light microscope
bacteriophage (50 nm, 0.1 um)
TEM
SEM
What is the order that light travels in a compound light microscope?
illuminator–> diaphragm–> condenser–> specimen–> objective lens–> ocular lens–> eyes
Equation for calculating total magnification?
objective lens x ocular lens
What is resolution?
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.
what microscope is used in this image? what are you looking at?
The distance between adjacent files of gold atoms in this TEM is 0.2 nm
what is the equation for determining resolution? In a light microscope, generally what is the resolution? In a electron microscope?
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
what is the refractive index?
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.
why does light bending not occur with immersion oil?
it has the same refractive index as glass
Protozoan
Paramecium
Brightfield microscopy
25 um
E.coli
Brighfield microscopy
Treponema pallidum
Darkfield illumination
Paramecium
Darkfield illumination
25 um
darkfield microscope without specimen on stage
darkfield microscope with specimen on stage
what are brightfield microscopes inadequete at visualizing?
transparent and colorless specimens
two waves in phase–> larger and brighter wave produced
two waves out of phase–> smaller, dimmer wave produced
what’s another name for DIC?
Nomarski
Phase contrast (background is grey)
Paramecium
25 um
A. Brightfield microscope
B. Phase contrast
(50 um)
DIC/ Nomanski
23 um
colored image/ 3D/ gray background
Top: Halos, visualized internal structures, phase contrast
Bottom: depth of field, DIC
In flourescent microscopy, what wavelength is absorbed initially & for what color, what wavelength is emitted at the end & for what color?
Wavelength absorbed: 450-490 (blue)
Wavelength emitted: 520-560 (green)
Treponema pallidum
2 um
Flourescence microscopy
testing for syphilis
standard vs. confocal flourescence
visualizing microtubules in the cell
in confocal flourescence each plane is individually accessed, hence a 3D depth appears
pointing to contractile vacuoles in phase contrast image
confocal image of pollen
what is photobleaching?
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
two-photon
20 um
pointing to food vacuoles
SEM
Biofilm
180 um
What does contrast in the electron microscope depends on?
the atomic number of the atoms in the sample
a colorized TEM of Paramecium
normally TEM’s are black and white
sliced internal structures
15 um
negative TEM stain of bacteriophage
3-dimensional reconstruction of a branched mitochondrion
used TEM
TEM micrograph showing metal shadowing of bacteria to demonstrate flagella.
TEM
Paramecium
15 um
scanning tunneling microscopy (STM)
resolution 100 pm
image of E. coli RecA protein
freeze-dried recA-DNA complexes coated with a conducting film
Top: DNA model
Bottom: STM model
Top: Pentacene model
Bottom: STM model
which has a better resolution, STM or TEM?
STM (100 pm vs. 2.5 nm)
Perfringolysin O toxin from Clostridium perfringens
atomic force microscopy (AFM)
ATM does not require special specimen prep.
Resolution similar to STM
Cocci that have been stained with crystal violet
what are the steps of the gram stain?
what is a mordant?
a substance added to a staining solution to hold the stain or coat the specimen. Coating is used to enlarge a specimen
what three bacteria are found in this picture?
- gram positive stained rods
- gram positive stained cocci
- gram negative stained vibrio
describe the procedure of acid fast staining
acid fast stain of M. leprae
5 um
light microscope
negative stain of Klebsiella pneumoniae
Light microscope
5 um
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?
Schaeffer-Fulton stain • Primary stain: Malachite green, steamed 5 min • Remove stain from all the cells structures except endospore: water/ 30 sec • Counterstain: Safranin
endospore staining
light microscope
5 um
endospores can be seen without staining, but are difficult to distinguish from other structures
what is the purpose of the mordant in flagella staining?
Mordant used to coat flagella with stain until thick enough to be seen • Carbolfuchsin simple stain
flagella staining
light microscope
5 um
done using a carbolfucshin stain
