Which are smaller: eukaryotes or prokaryotes?
Why is the small size of prokaryotes important?
Small size allows nutrients to reach all parts of cell quickly which is important b/c prokaryotes don't have internal membrane structure.
How small is the smallest prokaryote?
How small is the smallest eukaryote?
Prokaryotes have many or few cell morphologies which help with______?
What are the 3 bacterial shapes?
1. Coccus (pl. cocci) (sphere)
2. Bacillus (pl. bacilli) (rod)
What are the 5 common arrangements of cocci bacteria?
1. Coccus- single cells
2. Diplococcus- 2 cells
3. Streptococcus- chains of more than 2 cells
4. Staphylococcus- grapelike cluster of cells
5. Tetrads- packets of 4 cells
What is the most common shape of bacteria?
How do bacilli bacteria occur in terms of arrangement and size of shape?
Can be short and fat or long and thin. Occur singularly or in chains.
What are the 3 types of spirals?
1. Rigid spiral-shaped bacteria are called spirilla (singular- spirillum).
2. Flexible spiral-shaped bacteria are called spirochetes (flexible corkscrew shaped cells with multiple curves)
3. Vibrio - curved rods
What diseases do some species of spirochetes cause?
Syphilis and Lyme disease.
Who developed the first simple microscope?
Antoni van Leewonhoek.
How many lens are in a simple microscope, and what is the magnification?
One lens. 50-300X.
How many lens are in a compound microscope, and what is the magnification?
Multiple. The ones in our lab magnify 1000X.
What is a compound microscope?
A system of multiple lens designed to correct or compensate for aberration.
What is the arrangement of a compound microscope?
The light source is placed at the bottom, shining upward through a series of lenses, including the condenser, objective, and ocular lenses.
Where is condenser located and what does it do?
Located between light source and specimen. Focuses light rays up through specimen (does not magnify) and improves resolution.
Where is the objective lens system located and what does it do?
Closest to specimen (typically on revolving nosepiece). Helps to increase magnification and makes up for imperfections if one lenses is scratched.
How many lenses are in an objective lens system and what magnifications are on a typical scope?
In each objective are multiple lenses.
Typical scope has 10, 40, & 100X objective lenses (we'll be using 10X and 100X).
Where is the ocular lens and what does it do and is there one or multiple lenses?
Closest to eye. Typically magnify 10X. Each ocular has multiple lenses
What is the magnification if using a 100X objective and a 10X ocular?
A microscope with a 100X objective and 10X ocular has a total magnification of 1000X.
(When viewing bacterial cells, will use 100X objective.) This means an object will 1000X larger than it is.
At 1000X magnification, can bacteria and viruses and their internal structures be seen?
Can see bacteria at this magnification, but not internal structures (sometimes can see endospores). Cannot see viruses.
What limits what we can see from a microscope: magnification or resolution?
What is resolution?
Ability to distinguish between 2 objects that are close together.
What is the highest resolution of a light microscope and what does it mean?
Highest resolution of a typical light microscope is 0.2 micrometers. This means that 2 objects closer than 0.2 µm to each other can't be distinguished.
What determines resolution?
Resolution is determined by wavelength of light and numerical aperture.
What four things increase resolution?
1. Multiple lenses
2. Shorter wavelength of light
3. Increase numerical aperture
4. Increase contrast
How does having multiple lenses increase resolution?
All lenses possess aberrations that detract from perfect parabolic curvature, so multiple lenses corrects this.
How does using shorter wavelengths of light increase resolution and which color is the shortest?
Blue light (~400 nm) is shortest wavelength so gives highest resolution b/c can travel b/n 2 bacteria.
What is numerical aperture and what is the equation?
Measure of light gathering ability of objective lens. NA = n sin (theta), where n is the product of the refractive index of the medium
How is numerical aperture increased?
Wider lens closer to specimen and immersion oil. Light bends as it passes through specimen into air and reduces resolving power. If space between specimen and objective is replaced with immersion oil, resolution increases. Oil (has same refractive index as glass, so basically extends lens) keeps light from scattering as much as in air.
How is contrast increased?
Contrast between bacteria & background is small so bacteria are very hard to see. Increase contrast by staining cells or using special type of microscope.
How do stains work?
Stain molecules (chromophore) absorb visible light and one or more positive charges from conjugated bonds or aromatic rings binds with negative charges in phospholipid membrane.
What are some examples of stains?
Crystal violet, methylene blue, safranin.
What is a disadvantage of fixation and staining?
These procedures kill the cell so live behavior cannot be observed.
What is a simple stain?
A stain that makes an object more opaque, increasing its contrast with the external medium or surrounding tissue.
What is a differential stain?
A stain that differentiates among objects by staining only particular types of cells or specific subcellular structures and also differentiates b/n Gram positive and Gram negative bacteria.
How many dye(s) do simple and differential stains use?
Simple: one. Differential: more than one.
What is a type of differential stain and what bacteria does it stain?
Acid-fast stain distinguishes Mycobacterium species from other bacteria.
What is a Gram stain?
A differential stain that distinguishes b/n cells that possess a thick cell wall and retain a positively charged stain (Gram-positive) from cells with a thin cell wall and out outer membrane that fail to retain the stain (Gram-negative).
Which microscope allows us to see bacteria against a white, bright background?
Bright-field light microscope.
Which 3 microscope allows us to see bacteria through increased contrast b/n cells and medium?
What is phase contrast microscopy?
Observation of a microscopic object based on the differences in the refractive index b/n cell components and the surrounding medium; contrast is generated as the difference b/n refracted light and transmitted light shifted out of phase.
What is dark field microscopy?
The detection of microbes too small to be resolved by light rays by observing the light they scatter.
How is the difference in phase in the phase contrast microscopy generated?
With the annular ring which stops light from passing directly through the center of the lens system and generates a hollow cone of light which is focused through the specimen and generates an inverted above it.
What does phase contrast microscopy let us see?
Can observe living cells in wet mount and organelles of eukaryotes.
What does dark field microscopy let us see?
Can observe living cell in a wet mount and bacterial motility and very narrow cells.
What is a disadvantage to dark field microscopy?
Dust particles also scatter light, interfering with visualization of specimen.
How do cells appear in dark field microscopy?
Cells appear bright against dark background.
What happens to light in a dark field microscope?
Dark-field stop (or spider condenser) in condenser doesn't let light pass directly through the specimen. Only light scattered by sample reaches objective.
What is interference microscopy?
Observation of two wave fronts. Interference can be additive (amplitudes in phase, constructive) or subtractive (amplitudes out of phase, destructive).
What does interference microscopy allow us to see?
Shape of cells in 3-D.
How does interference microscopy work?
1. Polarized light passes through specimen
2. Sample boundaries bend light
3. Second polarized lens blocks light
4. Bent light results in 3-D appearance
What is fluorescence microscopy?
Involves detection of specific cells or cell parts based on fluorescence by a fluorophobe.
What is electron microscopy?
A form of microscopy in which a beam of e- accelerated through a voltage potential is focused by magnetic lenses onto a specimen.
What are two types of electron microscopes?
Scanning and transmission.
What is scanning electron microscopy?
SEM is electron microscopy in which the e- beams scan across the specimen's surface to reveal the 3-D topology of the specimen.
What is transmission electron microscopy?
TEM is electron microscopy in which e- beams are transmitted through a thin specimen to reveal internal structure.
How do fluorophobes help in fluorescence microscopy?
Absorb high-energy light (short wavelength) then emit lower-energy light (longer wavelength). They stain the specimen.
For what fluorescence microscopy used?
Labeling molecules of interest of the cell and Used to view cells that make fluorescent compounds such as chlorophyll, or cells that have been stained with fluorescent dyes.
What kind of light do fluorescent microscopes use?
What do electron microscopes use instead of visible light and lenses?
Beams of e- and electromagnets.
What is the highest magnification of an electron microscope?
What is viewed with an electron microscope?
Viruses, proteins, and nucleic acids.
What must the sample do in an electron microscope in order to be seen?
Reflect the e-.
Where are the sample and e- beam in an electron microscope?
In a vacuum.
What disease was on the last slide in class?
In which microscope are samples sliced very thin?
Transmission e- microscope.
Which part of the transmission e- microscope contains the blade which is made of what?
What is used to stain samples in transmission electron microscopy?
Uranium and Osmium.
How do the e- react in TEM and SEM?
E- go through the specimen in TEM and reflected from the specimen in SEM.
Which microscope has high resolution?
For what is the TEM used?
Used to view cell structures, DNA & some protein molecules.
What is done to the sample in SEM?
Coated with metal and not sliced.
What sort of image does SEM transmit?
For what is the SEM used?
Visualize surface cell structures.