Chap 3 Flashcards
What is the function of the diaphragm on a compound microscope?
Controls the amount of light entering the condenser
The ocular lens is used to
to remagnify the image formed by the objective lens.
The measure of the light-bending ability of a medium is the
refractive index
MOST ocular lenses magnify specimens by a factor of
10x
The ability of the lenses of a microscope to distinguish fine detail and structure is called
resolution
Which microscope uses an opaque disk to block light that would enter the objective lens directly?
Darkfield microscope
Which microscope uses an ultraviolet light source?
Fluorescence microscope
Which microscope illuminates specimens with blue light and produces three-dimensional images?
Confocal microscope
Which microscope forms an image from two sets of light rays, one from the light source and the other diffracted from a structure in the specimen?
Phase-contrast microscope
What microscope uses a metal-and-diamond probe to produce a three-dimensional image?
Atomic force microscope
Which microscope is especially useful in studying the surface structures of intact cells and viruses?
Scanning electron microscope
In transmission electron microscopy, the specimen is placed on a
copper mesh grid.
glass slide.
plastic slide.
magnesium mesh grid
Which microscope is extremely valuable for examining different layers of specimens and often involves the use of shadow casting?
Transmission electron microscope
With a scanning electron microscope, objects are generally magnified
1,000 to 500,000x
With a transmission electron microscope, objects are generally magnified
10,000 to 10,000,000x
Before microorganisms are stained, MOST appear ________ when viewed with brightfield microscopy
colorless
The chromophore of basic dyes is a ________ and is ________ to MOST types of bacteria.
cation; attracted
Which one of the following is NOT a kind of staining technique?
selective
The process of fixing a slide attaches the microorganisms while simultaneously
killing the organisms
A technique used for preparing a slide with colorless bacteria against a colored background is called
negative staining.
What is the mordant in the Gram stain?
Iodine
Flagella are stained with
carbolfuchsin
Capsules are stained using a(n)
negative stain
Endospores are stained using a(n)
Schaeffer-Fulton stain
What color are gram-negative bacteria at the completion of the Gram staining process?
Pink
The appearance of Mycobacterium leprae after they are stained with an acid-fast stain is
Red
Microorganisms are measured in
micrometers & and nanometers (nm)
How many nanometers is 10 micrometers?
10000 nm
A simple microscope has only one
lens
A microscope is similar to a magnifying glass, but with a much better
(higher magnification) lens
Any kind of microscope that uses visible light to observe specimens
Light Microscopy
In a _________ __________ the image from the objective lens is magnified again by the ocular lens
compound microscope
In a compound microscope, the image from the objective lens is magnified again by the
ocular lens
Total magnification
objective lens x ocular lens
Resolution
is the ability of the lenses to distinguish two points
A microscope with a resolving power of 0.4 n m can distinguish between two points at least . n m apart
0.4
________ wavelengths of light provide greater resolution
Shorter
refractive index
is a measure of the light-bending ability of a medium
Light may refract after passing through a specimen to an extent that it does not
pass through the objective lens
Immersion oil is used to
keep light from refracting
Brightfield illumination
- Dark objects are visible against a bright background
- Light reflected off the specimen does not enter the objective lens
Dark objects are visible against a bright background
Light reflected off the specimen does not enter the objective lens
Brightfield illumination
Light microscopes are useful for studying specimens ranging in size from about _mm to ___nm
1mm to 200 nm
Shortest wavelength of visible light
390 nm
most common form of light microscopy is called
brightfield microscopy
Shortest wavelength of light is
390 nm
What happens during brightfield microscopy?
the light beam passes through the sample and into the objective lens
lamp
Where the light beam is initiated
light lamp is divergent
Light spreads out at it approaches the condenser lens
Condenser lens
gathers the beam of light and focuses it so that it converges on the specimen with high intensity in a small area.
Specimen
Usually on a glass slide that rests on a platform called the stage
What happens when the light beam hits the sample
The light rays are reflected and refracted or absorbed by the sample
after the beam leaves the specimen, it passes through the
objective lens
objective lens
performs the bulk of the magnification of the specimen
eyepiece
receives the light beam from the objective lens and refocuses it to recreate the image in the viewer’s eye
ocular lenses
Lenses found within the eyepiece
Through what lenses does light pass in a compound microscope?
objective and ocular
What does it mean when a microscope has a resolution of 0.2 nanometer?
Two points can be distinguished if they are at least 0.2 nm apart.
Explain how electron microscopy differs from light microscopy.
The electron microscope uses electrons as the source of illumination instead of light. The beam has a short wavelength. The focusing element is electromagnets. It’s resolving power is 2nm instead of the light microscope’s 0.2um. It is used a lot for viruses and internal cell structures.
Light objects are visible against a dark background
Darkfield microscopy
Opaque disk placed in condenser
Darkfield microscopy
Only light reflected off the specimen enters the objective lens
Darkfield microscopy
Allows examination of living organisms and internal cell structures
Phase-contrast microscopy
Allows examination of living organisms and internal cell structures
Phase-Contrast Microscopy
How does phase-contrast microscopy work?
Brings together two sets of light rays, direct rays, and diffracted rays to form an image
Similar to phase-contrast
Differential Interference Contrast (D I C) Microscopy
Uses two light beams and prisms to split light beams, giving more contrast and color to the specimen
How does DIC microscopy work?
Uses two light beams and prisms to split light beams, giving more contrast and color to the specimen
How does fluorescence microscopy work?
- Uses U V (short wavelength) light
2.Fluorescent substances absorb U V light and emit longer wavelength (visible) light
3.Cells may be stained with fluorescent dyes (fluorochromes) if they do not naturally fluoresce
In confocal microscopy, cells are stained with
fluorochrome dyes
In confocal microscopy, short blue light is used to
excite a single plane of a specimen
In confocal microscopy, each plane of specimen is illuminated and
a three-dimensional image is constructed with a computer
Confocal microscopy can examine layers of cells to a depth of
100 micrometes
In Two-Photon Microscopy, cells are stained with
with fluorochrome dyes
In two-photon microscopy, Two photons of long-wavelength (red) light are used to
excite the dyes
Two-photon microscopy can study living cells up to
1mm deep
Super-Resolution Light Microscopy uses how many laser beams?
Uses two laser beams
Functions of the two laser beams in Super-Resolution Light Microscopy
- One wavelength stimulates fluorescent molecules to glow
2.Second wavelength cancels out all fluorescence except for that in one n m
In Super-Resolution Light Microscopy A computer scans the specimen nm by nm, then
puts the images together
Scanning Acoustic Microscopy measures
Measures sound waves that are reflected back from a specimen
Scanning Acoustic Microscopy is used to
Used to study cells attached to surfaces
resolution of Scanning Acoustic Microscopy
1 micrometer
How are brightfield, darkfield, phase-contrast, and fluorescence microscopy similar?
They all need a light source
Uses electrons instead of light
Electron microscopy
In electron microscopy, The shorter wavelength of electrons gives greater
resolution
When is electron microscopy used?
Used for images too small to be seen with light microscopes, such as viruses
How does Transmission Electron Microscopy work?
A beam of electrons passes through ultrathin sections of a specimen, then through an electromagnetic lens, then focused on a projector lens
in transmission electron microscopy specimens may be stained with
heavy-metal salts for contrast
Transmission Electron Microscopy magnification power
10,000 to 10,000,000 x; resolution of 10 p m
How does Scanning Electron Microscopy work?
An electron gun produces a beam of electrons that scans the surface of an entire specimen
In scanning electron microscopy, secondary electrons emitted from the specimen
produce a three-dimensional image
Magnification of Scanning electron microscopy
Magnifies objects 1,000 to 500,000 x;
Resolution of Scanning electron microscopy
10 nm
Use electrons to probe a sample
Electron microscope
Electron microscopes are useful for studying samples in the size range of
10 nm to 100 micrometer range
can see specimens are small as ribosomes and as large as RCS
electron microscope
Are used to produce two-dimensional images of internal structures of cells
transmission electron microscope
Produces three dimensional images of the surfaces of specimens
scanning electron microscopes
What can be used to stain specimens for electron microscopy?
Electron-dense compounds such as heavy metal salts
Electron gun
initiates a beam of electrons .
The electron gun beam is divergent, meaning that
it spreads out as it approaches the condenser lens
condenser lens of transmission electron microscope
gathers the beam of electrons and focuses it so that it converges on the specimen with high intensity in a small area
has electromagnetic lens
condenser in electron microscope
In transmission electron microscope the specimen is
a tiny slice of cells that rests on an equally tiny copper grid
In transmission electron microscopy, what happens when the electrons interact with the sample
they are either reflected and refracted, or absorbed by the sample.
In transmission electron microscopy, after hitting the specimen
the beam continues to diverge downward on its path away from the specimen and into the objective lens
Performs the bulk of magnification of the specimen
objective lens
Projector lens in transmission electron microscope
receives the electron beam from the objective lens and refocuses it to recreate the image that the viewer sees in the fluorescent screen
what captures the image in the transmission electron microscope
internal camera
similarities between a light microscope and a transmission electron microscope
- both have a source of light or electrons
-both have a condenser lens to focus the source
-both have an objective lens to refocus the image
differences between a light microscope and a transmission electron microscope
- ## the path of light is from bottom to top in the light microscope and opposite in the electron microscope
Ability to distinguish between objects that are close together
resolution
Resolution depends on the
wavelength of the source
smaller wavelength lead to
higher resolution
average wavelength of white light is
550 nm
wavelength of electron is
.1 nm
Why do electron microscopes have greater resolution than light microscopes?
because electrons have a shorter wavelength than light
Explain how Scanning Tunneling Microscopy works
Uses a tungsten probe to scan a specimen and reveal details of its surface
Resolution of scanning tunneling microscopy
1/100 of an atom
How does Atomic Force Microscopy work?
Uses a metal-and-diamond probe placed onto a specimen; movements are recorded
Atomic force microscopy produces
three-dimensional images at near atomic detail
For what is T E Meter used? S E Meter ? Scanned-probe microscopy?
TEM: the electrons pass through a thin section of the specimen
SEM: the electrons strike the surface of the specimen, secondary electrons leaving the surface are viewed on a screen.
Scanned Probe (SPM): family of tools used to make images of nanoscale surfaces and structures, including atoms.
Differentiate an acidic dye from a basic dye.
Acidic: negative ion (anion)
Basic: positive ion (cation)
Explain the purpose of simple staining
used to determine cell shape, size, and arrangement of microorganisms found in soil and water.
List Gram stain steps, and describe the appearance of gram-positive and gram-negative cells after each step
primary stain, mordant, decolorizer, counterstain
Compare and contrast the Gram stain and the acid-fast stain.
Gram Stain: Differential staining
G+ purple- staph, strep
G- red
Primary- crystal violet
Mordand- Iodine
Decolorizer- alcohol +purple -colorless
Counterstain- safranin (-) red
Acid Fast: TB, leprosy (hansens)
AF+ Red
AF- Blue
TB- waxy cell wall- mycelia acid
=if add primary stain and heat, melts wax. Cool wax- hardens
Primary- carbolfuchsin (red) (heat)
Decolorizer- AF-, colorless
Secondary- AF-, Blue
(if AF+, deep red instead)
Counterstain- Methylene Blue
Explain why each of the following is used: capsule stain, endospore stain, flagella stain`
Capsule: both the cell and background are stained. (negative stain)
Endospore: heat is required to drive a malachite green stain into endospores.
Flagella: requires a mordant to make the flagella wide enough to see.
Staining
coloring microorganisms with a dye that emphasizes certain structures
smear
a thin film of a material containing microorganisms spread over a slide
what does fixing do to microorganisms
Microorganisms are fixed (attached) to the slide, which kills the microorganisms
Live and/or unstained specimens have little contrast with the surrounding medium. Live specimens are used to
study cell behavior
very useful tools to study specimens smaller than about 1mm
microscopes
both light and electron microscopes use stains to
increase the contrast and visibility of the specimen under study and aid in classification
stains consist of
a positive and negative ion, one of which is colored (chromophore)
in a basic dye, the chromophore is a
cation
in an acidic dye
the chromophore is an anion
negative staining
Staining the background instead of the cell is called
simple staining
use of a single basic dye
Purpose of simple stains
Highlights the entire microorganism to visualize cell shapes and structures
Mordant purposes
may be used to hold the stain or coat the specimen to enlarge it
Why doesn’t a negative stain color a cell?
it stains the background instead of the cell
Why is fixing necessary for most staining procedures?
its purpose is to bind the specimen to the slide so that it does not wash off during staining. Killing the cells with heat fixation also increases their permeability to the dyes used in staining.
purpose of differential stains?
Used to distinguish between bacteria
Types of differential stains
Gram stain
Acid-fast stain
Gram-positive bacteria have
thick peptidoglycan cell walls
Gram-negative bacteria have
have thin peptidoglycan cell walls and a layer of lipopolysaccharides
The first synthetic drugs classified bacteria into
gram-positive or gram-negative
Steps of gram staining in my own words
- Application of crystal violet (purple dye)
- Application of iodine (mordant)
- Alcohol wash (decolorization)
- Application of safranin (counterstain)
Acid-fast stain binds only to
bacteria that have a waxy material in their cell walls, which is not decolorized by acid-alcohol
Acid-fast stain is used for the identification of
Mycobacterium
Nocardia
Primary stain: Carbolfuchsin
Color of acid-fast:
Color of non-acid-fast:
Primary stain: Carbolfuchsin
Color of acid-fast: Red
Color of non-acid-fast: Red
Decolorizing Agent: Acid-alcohol
Color of acid-fast:
Color of non-acid-fast:
Decolorizing Agent: Acid-alcohol
Color of acid-fast: Red
Color of non-acid-fast: Colorless
Counterstain: Methylene Blue
Color of acid-fast:
Color of non-acid-fast:
Counterstain: Methylene Blue
Color of acid-fast: Red
Color of non-acid-fast: Blue
Why is the Gram stain so useful?
Gram staining is useful since it allows us to distinguish whether microbial cells (bacteria) are gram-positive or gram-negative
Which stain would be used to identify microbes in the genera Mycobacterium and Nocardia?
Special stains used to
Used to distinguish parts of microorganisms
Structures that can be distinguished with special stains
Capsule stain
Endospore stain
Flagella stain
Capsules
are a gelatinous covering that do not accept most dyes
Suspension of India ink or nigrosin contrasts
the background with the capsule, which appears as a halo around the cell
Endospores
are resistant, dormant structures inside some cells that cannot be stained by ordinary methods
Primary stain of endospores
malachite green, usually with heat
What decolorizes endospores?
water
Counterstain for endospores
safranin
Spores appear green within
red or pink cells
Flagella
Structures of locomotion
Flagella staining uses a mordant and carbolfuchsin to thicken appearance of ________ making them visible under the light of a microscope
flagella
What thickens the appearance of flagella?
mordant and carbolsfuschin
How do unstained endospores appear? Stained endospores?
They appear contrasted and distinguishable from stored material. unstained appear under the light but are not distinguishable.
