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