Lectures 5, 6, 7 (Chapter 3) Flashcards
The smallest object the human eye can see is about
0.2mm (half a grain of salt)
Light microscopes can visualize a range of sizes from
200nm or .2um to 10mm
1,000nm = ______ um
1
1nm = _____ um
.001
1 bacterium length
about 1 um long
10mm = ______ cm
1
Microscope with only one lens who was it used by?
simple microscope Anton Van Leeuwenhoek
Light Microscopy and types?
-use of any kind of microscope that uses visible light to see specimens -brightfield -darkfield -phase-contrast -fluorescence -confocal
Compound light microscope
-First used by Hooke -has 2 lenses
Compound microscope parts
….add pic
In a compound microscope, the image from the ________ lens is magnified again by the ________ lens
objective, ocular
Total magnification =
objective lens x ocular lens
resolution compound light microscope resolution?
ability to distinguish two adjacent objects as separate and distance -0.2um
resolving power limited by wavelength. to improve resolving power a ______ wavelength of light is needed
shorter
refraction in compound light microscope
the bending of light as it travels through substances with different densities
what does immersion oil do
a drop of immersion oil that is placed on the slide, which has the same refractive index as the glass slide allows the light to continue in the correct pathway (prevents refraction) -oil immersion lenses (usually 100x) are used to minimize refraction -light rays are collected to increase clarity
Bright Field Microscopy
•Dark objects are visible against a bright background •Light reflected off the specimen does not enter the objective lens •Best for fixed, stained specimens, specimens that absorb light •Image is dark and/or highly colored against a bright, often light gray/white background •Specimens are visualized because of differences in contrast (density) between specimen and surroundings •Compound microscope used
Darkfield illumination
uses darkfield condenser that contains an opaque disk to examine unstained live specimens
Phase contrast
way to observe live specimens, permits more detail to view internal structures
Fluorescence
takes advantage of fluorescence, the ability of substances to absorb short wavelengths of light (UV) and give off a light at a longer wavelength(visible).
Confocal
microscopy illuminates each plane in a specimen to produce a three-dimensional image up to 100 µm deep and also uses fluorescence.
Staining improves ______
contrast
Positively charged dyes bind to negatively charged cell components:
membranes, nucleic acids (ribosomes), proteins, cell wall
Examples of common stains are:
methylene blue, safranin, and crystal violet
Types of stains:
simple, differential, special
What do you use for a simple stain and Steps for simple stain:
-Use a single basic dye -A mordant may be used to hold the stain or coat the specimen to enlarge it I. preparing a smear - spread culture on thin film over slide, and allow to air dry II. Heat fix and III. then stain - flood slide with stain; rinse and dry III. view
Separate bacteria into groups and examples
differential stain -acid fast stain -gram stain
Differential Stain: The Gram Stain
Gram (+) vs Gram (-) Gram rxn can provide valuable info for treatment downsides: kills cells, distorts features
Because peptidoglycan wall is negatively charged - gram positive stains what color
Gram positive: pepidoglycan wall stains blue/purple (dye will bind to all negatively charged areas in the cell)
Differential Acid Fast Stain
-acid fast stain – for identification of mycobacteria and pathogenic Nocardia, which have a waxy lipid cell wall (mycolic acid). ♣Acid fast stains retain red/pink ♣Non-acid bacteria cells will be colorless – counterstain stains them blue
Special Stains
♣Special stains – are used to color and isolate a specific part of a microbe, such as endospores or flagella. Stain special structures -Flagella: carbolfuschin -Capsules: Colloids (India Ink) – presence of a capsule is a means of determing an organisms’ virulence (note colorless capsules that surround the purple stained rods) -Endospores: Malachite Green
How do unstained endospores appear?
refractive under the light (white hollow hole)
Addition of alcohol is the critical step in gram staining b/c
-alcohol will cause the outer part of cell membrane of gram - bacteria to pull off (dehydrate) -gram (+) it will cause cell wall to shrink and will hold purple dye a lot tighter
EM can be used to examine
objects smaller than about 0.2 um (which is the limit of resolution for a light microscope), such as viruses and macromolecules which are in the nm range.
EM vs. Light Microscope
The source of illumination is visible light for a light microscope and an electron beam for an EM. Instead of using glass lenses, an EM uses electromagnetic lenses to focus a bean of electrons onto a specimen. Like light, free electrons travel in waves, however in EM, the waves travel in a vacuum as opposed to air.
TEM
Transmission Electron Microscope (TEM) was the first type of Electron Microscope to be developed and is patterned exactly on the Light Microscope except that a focused beam of electrons is used instead of light to “see through” the specimen. Specimen must be prepared in ultra thin slices since electrons must pass through the sample (electron transparency). Incredible internal detail. Images produced by EM are always black and white, but they may be colored artificially to accentuate certain details.
SEM
Scanning the beam of electrons across the sample. The SEM overcomes the sectioning/slicing problem associated with the TEM and produces a 3D-image surface of the specimen, and especially useful in studying the surface structures of intact cells and viruses. Best for viewing external structures.
We saw in lecture 5 that the compound light microscope will increase out ability to see by about _______ fold. An EM gives us approximately another ______ fold magnification.
1,000, 1,000 - EM’s show us viruses and macromolecular structures
EM vs. Light Microscopes
