Chapter 2-Microscopy
A 30 objective and a 20 ocular produce a total magnification of
600
Which of the following are best used for viewing unstained specimens?
-scanning tunneling -phase -atomic force -dark-field -dim
atomic force microscope
-sharp probe moves over surface of specimen at constant distance -up and down movement of probe as it maintains constant distance is detected and used to create image
Prior to staining, smears of microorganisms are heat-fixed in order to
attach it firmly to the slide
Compound microscope means there are at least sets of .
2 lenses
In order to view a specimen with a total magnification of 400, a __________ objective must be used if the ocular is 10.
40
A 45 objective and a 10 ocular produce a total magnification of
450
If you forgot the decolorization step while performing a gram stain, which outcome would you expect?
Gram negative would stain purple
chromophore groups
chemical groups with conjugated double bonds, give dye its color
This part of a microscope focuses a cone of light on the specimen.
condenser
Electron microscopes have much better resolution than light microscopes because
electrons have much shorter wavelengths than visible light
Scanning Probe Microscopy (SPM)
enables nanoscale observation of cell surfaces
Regions of a specimen with higher electron density scatter ___________ electrons and, therefore, appear __________ in the image projected onto the screen of a transmission electron microscope.
more, darker
In transmission electron microscopy, spreading a specimen out in a thin film with uranyl acetate, which does not penetrate the specimen is called
negative staining
Basic dyes such as methylene blue bind to cellular molecules that are: a. hydrophobic b. negatively charged c. positively charged d. aromatic
negatively charged
total magnification
ocular x objective
simple stains
one dye is used; reveals shape, size, and arrangement
If the objective lenses of a microscope can be changed without losing focus on the specimen, they are said to be
parfocal
Resolution
ability of a lens to separate small objects that are close together
If you forgot to apply safranin counterstain while performing a gram stain, what happens?
gram-negative bacteria would be unstained
An instrument that magnifies slight differences in the refractive index of cell structures is called a _____ microscope
phase-contrast
If the decolorizer is left on too long in the Gram-staining procedure, gram-positive organisms will be stained __________ and gram-negative organisms will be stained _____
pink, pink
Which of the following techniques would be best for viewing chromosome structure?
scanning tunneling
**Strength of lens related to focal length
short focal length=more magnification
The procedure in which a single stain is used to visualize microorganisms is called _____ staining
simple
Thin films of bacteria that have been air-dried onto a glass microscope slide are called __________.
smears
Scanning electron microscopy is most often used to reveal
surface structures
Small internal cell structures are best visualized with a
transmission electron microscope
In the Gram-staining procedure, the counterstain is
safranin
In the Gram-staining procedure, the mordant is
Iodine
Which type of microscopy-showing fine internal detail of eukaryotic organelles?
TEM
phase contrast microscope
-living organisms -differences in refractive indices among different subcellular structures
Electron Cryotomography
- Rapid freezing technique provides way to preserve native state of structures examined in vacuum - Images are recorded from many different directions to create 3-D structures - Provides extremely high resolution images of cytoskeletal elements, magnetosomes, inclusion bodies, flagellar motors, viral structures
Dark-Field Microscope
-Image is formed by light reflected or refracted by specimen -Produces a bright image of the object against a dark background -Used to observe living, unstained preparations
Differential stains distinguish between microbial groups by staining certain cell structures differently. Which of the following are differential stains?
-acid-fast -gram -capsule -flagella
Which of the following are examples of simple stains?
-capsule -flagella -methylene blue
shadowing
-coating specimen with a thin film of a heavy metal on only one side -useful for viral morphology, flagella, DNA
Scanning Electron Microscope (SEM)
-electrons excited from the surface of a specimen to create detailed image -produces 3D image of specimen's surface features
Transmission Electron Microscope (TEM)
-electrons scatter when they pass through thin sections of a specimen -under vacuum
Endospore staining
-heated, double staining technique -bacterial endospore is one color and vegetative cell is a different color
Negative stain
-heavy metals do not penetrate the specimen but render dark background -used for study of viruses, bacterial gas vacuoles
compound microscope
A light microscope that has more than one lens
Gram staining
A process by which components of bacterial cell walls are bound to Gram's stain. Depending on the amount of peptidoglycan in their cell walls, bacteria stain differently and are classified as Gram-negative or Gram-positive.
Differential Interference Contrast (DIC)
Accentuates diffraction of the light that passes through a specimen; uses two beams of light -live, unstained
Which of the following is considered to be a differential staining procedure?
Both Gram and Acid-fast stain
Fluorescene microscopy
Bright image of object resulting from fluorescent light emitted by specimen
focal length (f)
Distance between center of lens and focal point
The designer of the first transmission electron microscope, _________________, was awarded the 1986 Nobel Prize in physics.
Ernst Ruska
T/F: Because transmission electron microscopy uses electrons rather than light, it is not necessary to stain biological specimens before observing them.
FALSE
T/F: Immersion oil is used to prevent a specimen from drying out.
FALSE
T/F: Negative staining facilitates the visualization of bacterial capsules that are intensely stained by the procedure
FALSE
T/F: The Gram-staining procedure is widely used because it allow rapid identification of a microorganism with little additional testing
FALSE
_______ is the process by which internal and external structures of cells and organisms are preserved and maintained in position
Fixation
______ breaks frozen specimens along lines of greatest weakness, often down the middle of the lipid bilayer membranes so that they may be observed by transmission electron microscopy
Freeze-etching
Fluorochrome
Labeled probes, such as antibodies, dyes tag specific cell constituents for identification of unknown pathogens
Which type of microscopy would be preferred for creating a three dimensional view of the distribution and arrangement of flagella on a bacterial cell surface?
Scanning electron microscopy
The _____ microscope is capable of atomic resolution of specimens, even when they are immersed in water
Scanning tunneling
T/F: A light microscope with an objective lens numerical aperture of 0.65 is capable of allowing two objects 400nm apart to be distinguished when using light with a wavelength of 420nm
TRUE
T/F: A scanning electron microscope bombard specimens with a stream of electrons; however, the specimen image is produced by electrons that are derived from atoms of the specimen itself rather than by the electrons used to bombard the specimen
TRUE
T/F: A substage condenser is used to focus light onto the specimen, which increases the resolution of a light microscope
TRUE
T/F: Confocal microscopes, in combination with specialized computer software, can be used to create three-dimensional images of cell images of cell structures
TRUE
T/F: Gram-staining divides bacterial species into two groups based on differences in cell wall structure
TRUE
T/F: Immersion oil increases the amount of light entering the objective lens
TRUE
T/F: In order to stain flagella so that they may be readily observed by light microscopy, it is usually necessary to increase their thickness
TRUE
T/F: It is possible to build a light microscope capable of 10,000 magnification, but the image would not be sharp because resolution is independent of magnification
TRUE
T/F: It was possible to view viruses only after the invention of the electron microscope because they are too small to be seen with a light microscope.
TRUE
T/F: Light rays are refracted (bent) when they cross the interface between materials with different refractive indices.
TRUE
T/F: Mordants increase the binding between a stain and specimen.
TRUE
T/F: Negative staining with India ink can be used to reveal the presence of capsules that surround bacterial cells.
TRUE
T/F: Resolution improves when the wavelength of the illuminating light decreases
TRUE
T/F: Scanning tunneling electron microscopes creates a three-dimensional image of specimens of atomic level resolution
TRUE
If immersion oil was replaced with water, what would happen?
The refractive index of water would be greater than air but less than oil, improving resolution less than oil
If you forgot to heat fix a smear before doing a Gram stain, what will occur?
The smear may not adhere to the slide
parfocal microscope
allows for specimens to remain in focus when changing between magnification
Confocal microscopes exhibit improved contrast and resolution by
blocking out stray light with an aperture located above the objective lens
negative stain
capsules colorless against a stained background to determine how large capsule size is and if there are capsules
heat fixation
routinely used with bacteria and archaea -preserves overall morphology but not internal structures
confocal microscopy
creates sharp, composite 3D image of specimens by using laser beam, aperture to eliminate stray light, and computer interface
In the Gram-staining procedure, the primary stain is
crystal violet
The instrument that produces a bright image of the specimen against a dark background is called a ______ microscope
dark-field
As the magnification of a series of objective lenses increases, the working distance
decreases
A procedure that divides organisms into two or more groups depending on their individual reactions to the same staining procedure is referred to as ______ staining
differential
The gram-staining procedure is an example of
differential staining
working distance
distance between objective lens and specimen
Differential staining
divides microorganisms into groups based on their staining properties
Atomic force microscopes use a scanning probe that maintains a fixed distance from the surface of the specimen. It is useful for specimens that
do not conduct electricity well
In the Gram-staining procedure, the decolorizer is
ethanol/acetone
A microscope that exposes specimens to ultraviolet, violet, or blue light and forms an image with the light emitted at a different wavelength is called a ____ microscope
fluorescene (bruh i can't spell this )
The special dyes used in florescence microscopy that absorb light at one wavelength and emit light at a different wavelength are called ____
fluorochromes
The _____ is the distance between the center of a lens and the point at which it focuses parallel beams of light
focal length
Lenses focus light rays at a
focal point
The ______ is the point at which a lens focuses parallel beams of light
focal point
focal point (F)
focus light rays at a specific place
Freeze-etching
freeze specimen then fracture along lines of greatest weakness
As the resolution of a microscope system improves, the size of the smallest object that can be seen clearly
is smaller
refracted
light is refracted (bent) when passing through one medium to another
Acid-fast organisms such as Mycobacterium tuberculosis resist decolorization by an acid -alcohol wash because of the high concentration of _____ in their cell walls. a. proteins b. carbohydrates c. lipids d. peptidoglycan
lipids
An electron microscope uses ______ lenses to focus beams of electrons onto a specimen
magnetic
Refractive index
measure of how greatly a substance slows the velocity of light
Flagella staining
mordant applied to increase thickness of flagella
A specimen has been prepared for viewing with a TEM using uranyl acetate as a negative stain. The area stained by the uranyl acetate will be ____ electron dense compared to the specimen itself.
more
Fixation
preserves internal and external structures and fixes them in position
Bright-Field Microscope
produces a dark image against a brighter background
After the decolorizer has been added, gram-positive organisms are stained __________ and gram-negative organisms are stained __________.
purple, colorless
After the secondary stain has been added, gram-positive organisms are stained __________ and gram-negative organisms are stained __________.
purple, pink
After the primary stain has been added but before the decolorizer has been used, gram-positive organisms are stained __________ and gram-negative organisms are stained __________.
purple, purple
If the decolorizer is not left on long enough in the Gram-staining procedure, gram-positive organisms will be stained __________ and gram-negative organisms will be stained _____
purple, purple
A light ray moving from air to glass, or vice versa, is a process known as
refraction
Immersion oil can be used to increase the resolution achieved with some microscope lenses because it increases the __________ between the specimen and the objective lens.
refractive index
Acid-Fast Staining
used to detect mycobacterium
Capsule stain
used to visualize polysaccharide capsules surrounding bacteria
chemical fixation
used with larger, more delicate organisms -protects fine cellular substructure and morphology
The useful magnification of a light microscope is limited by the ___________ of the light source being utilized.
wavelength
The ____ is the distance between the specimen and the objective lens when the specimen is in focus
working distance
Electron microscopy
• Electrons replace *light* as the illuminating beam • *wavelength* of electron beam is much *shorter* than light, resulting *much higher resolution* • allows for study of microbial morphology in *great* detail
