Microbiology Chapter 2: Microscopy

Only seen directly by electron microscopes, ___ are long threadlike structures of locomotion found on some bacteria.

Flagella

In fluorescent microscopy, the ___ absorbs light energy from the excitation light and fluorescent brightly.

Fluorochrome

What compound is used to stain a specimen that will emit a different wavelength of light than it absorbs?

Fluorochromes

What term is used to describe the specific location where light rays pass through a lens and gain maximal intensity?

Focal Point

Because the cells are ___ quickly, freeze-etching minimizes the danger artifacts.

Frozen

The ___ stain was developed by ___ in ___.

Gram; Christian Gram; 1884

Which characteristics describe objectives that have a short walking distance?

Great resolving power Large numerical aperture

Match the cellular effects with the correct fixation technique.

Heat Fixation- inactivated proteins and destroys proteins of subcellular structures. Chemical fixation- inactivates proteins but protects fine cellular substructure.

Which types of microscopes are NOT able to create clear images of living specimens? A. Phase-contrast microscope B. Differential microscope C. Dark-field microscope D. Electron microscope E. Bright-field microscope

Electron microscope

Which bacterial cell structure is exceptionally resistant and produces to help a cell survive in harsh environments?

Endospore

True of False: The process of visualizing SEM images requires electrons to pass through the specimen and hit a detector on the other side.

False. The electrons bounce off the metal-coated specimen where detectors generate an image of the specimen's surface.

Which of the following must be done in order to prepare a specimen for SEM?

Fixed Dehydrated Dried Metal coated

TEM specimens are

Fixed in a plastic-like matrix and cut into thin slices

The differential interference contrast microscope is similar to the ___ microscope in that t creates an image by detecting the differences in refractive indices and thickness.

Phase-contrast

Capsules are networks composed mostly of ___ that surround many bacteria.

Polysaccharides

Green fluorescent gene encodes a ___ that naturally fluoresces green when exposed to light of a certain wavelength.

Protein

The direction of the magnification of bending of light are determined by the ___ indices of the two media forming the interface.

Refractive

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

What occurs when an objective produces not just a magnified image but a clear image?

Resolution

Match the objective with the correct magnification. Scanning; low power; high power; oil immersion

Scanning - 4X Low power - 10X High power - 40-45X Oil immersion - 90-100X

Which microscope allows a view of the atoms on the surface of a solid?

Scanning tunneling microscope

Which microscope has the greatest resolving power?

Scanning tunneling microscope

Which of the following is an example of a covalent dye?

Schiff's reagent

Match the type of covalent due with what it stains. Stains lipids or Stains DNA.

Schiff's reagent - stains DNA Sudan III - Stains Lipids

TEM specimens are coated with a thin film of platinum or other heavy metal in the ___.

Shadowing

Known for ease of use, ___ staining only uses one stain.

Simple

Scanning electron microscopy is most often used to reveal ___.

Surface structures

In the flagella stain, the thickness of the flagella is increased by coating them with mordants such as ___ acid and potassium alum.

Tannic acid

What is produced with rapid freezing of the sample prepared for electron cryotomography?

Vitreous ice

The maximum resolution of a light microscope is dependent on the shortest ___ of light able to be seen.

Wavelength

The minimal distance becomes smaller as the ___ of light used decreases and as the numerical aperture increases.

Wavelength

Objectives with large numerical apertures and great resolving power have short ___.

Working distances

As the resolution of a microscope system improves, the size of the smallest object that can be seen clearly ___.

is not affected

What is the total magnification if the ocular is 10x and the objective is 40x?

400

Our eye can minimally detect something that is about ___ in diameter.

0.2 mm

Correctly list the steps of the Gram stain.

1- Crystal violet, primary stain 2- Iodine, mordant 3- Acetone-alcohol, decolonized 4- Safranin, counterstain

Examine the short image. Starting from the light source and moving toward the image, put the parts of the phase-contrast microscope in the correct order.

1. Annular disk 2. Condenser 3. Slide 4. Phase plate

List the steps for the endosperm stain in the correct order.

1. Cells stained with malachite green 2. Slide with malachite green is placed over heat 3. Slide is rinsed with water 4. Counterstain slide with safranin

Using the image to help, list the steps in creating a SEM image in the correct order.

1. Electron beam back and forth over the specimen 2. Surface atoms discharge a tiny shower of electrons called secondary electrons 3. Secondary electrons are trapped by a detector 4. Secondary electrons strike a scintillator and light flashes 5. Photomultiplier converts to an electrical current and amplifies 6. The digitized signal is send to a computer to produce an image to be viewed

List the steps of the capsule stain in the correct order.

1. Mix cells with India ink 2. Spread out in a thin film on slide 3. Allow to air dry 4. View under microscope

List the steps of acid fast staining in the correct order

1. Primary stain with basic fuchsin 2. Heat to allow basic fuchsin to penetrate cells 3. Decolorize with acidic alcohol 4. Counterstain with methylene blue

Electron microscopes have a practical resolution roughly ___ times better than the light microscope.

1000

Which of the following describes vitreous ice?

A glass-like solid that preserves the native state of structure for use with an electron microscope.

How is lens strength related to focal length?

A lens with short length magnifies more than a lens with a longer focal length

In a confocal microscope, what removes stray light from parts of the specimen that lie above and below the plane of focus?

Aperture

The ability of the lens to gather light is called numerical ___.

Aperture

A second type of scanning probe microscope is called a(n) ___ force microscope.

Atomic

In negative staining of TEM specimens, the ___ appears dark.

Background

Which of the following are possible stains to use for flagella staining?

Basic fuchsin

Match the type of dye with the correct description. Acidic; Basic.

Basic- bind to negatively charged molecules Acidic- bind to positively charges molecules

A prism ___ light passing through it.

Bends

Confocal microscopy is used to study ___ which are clumps of bacterial cells that can form on many different types of surfaces, including medical devices.

Biofilms

Which of the following is considered to be a differential staining procedure?

Both Gram stain and acid-fast stain

Which type of microscope is used in microbiology labs to examine both stained and unstated specimens?

Bright-Field

What is the network usually made of polysaccharide, that surrounds many bacteria and some fungi?

Capsule

When performing a stain, which two things generally occur during the fixation step?

Cells are attached to slide Cells are killed

What usually happens to cells stained for viewing under a bright-field microscope?

Cells are killed

In dark-field microscopy, a dark-field stop is placed underneath the ___ lens system which is under the stage.

Condenser

In epifluorescent microscopes, what do objective lenses act as so the specimen is illuminated from above rather than below?

Condenser

The substage ___ focuses a cone of light on the slide.

Condenser

The instrument that produces a bright image of the specimen against a dark background is called a (n) ___ microscope.

Dark-field

The Gram stain is an example of ___ staining, distinguishing Gram negative and Gram positive bacteria.

Differential

The Gram-staining procedure is an example of ___.

Differential staining

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

Identify any of the following that are associated with electron microscopes.

Electron beams Viewing screen Electromagnets

In which process are specimens plunged into an extremely cold liquid?

Electron cryotomography

The use of ___ oil when viewing a slide prevents the refraction of light and allows the viewer to see the image much clearer (i.e., better resolution).

Immersion

The staining process used to prepare specimens for the TEM ___ the contrast in the material.

Increases

Basic and acidic dyes bind cells by what type of bond?

Ionic

Which type of lens magnifies an object more?

Lens with a short focal length

Match the microscope with the item at the limit of its resolution.

Light microscope- Mycoplasma bacteria Electron microscope- amino acid Scanning tunneling microscope- hydrogen atom

Which statements are limitations of the light microscope?

Light microscopes have a resolution limit of 0.2 micrometers. Light microscopes are not able to show detailed internal cellular structure.

Dried SEM specimens must be coated with a thin layer of ___ to prevent buildup of an electrical charge on the surface.

Metal

Acid-fast organisms have lipids constructed of ___ acid in their cell walls.

Mycolic

What unique characteristic exists in acid-fast bacterial cell walls?

Mycolic acid

In what type of staining is the background stained and not the cells?

Negative

TEM are spread out in a thin film with either phosphotungstic acid or uranyl acetate in ___ staining.

Negative

Match the negative staining and shadowing in TEM with the correct description.

Negative Staining - used to study virus particles and bacterial gas vacuoles. Shadowing - used to study viral particle morphology, bacterial and archaea flagella, and DNA

In the freeze-etching process, cells are rapidly frozen using liquid ___ which causes the cells to become brittle and break, exposing internal membranes.

Nitrogen

The ___ lens forms the magnified image that is further enlarged by one or more additional lenses.

Objective

The ___ lens further magnifies the image already enlarged by the objective lens.

Ocular

Match the microscope part to the description of its function. Ocular; Objective; Condenser.

Ocular: eyepiece used to view specimen Objective: lens closest to the specimen Condenser: focuses from the lamp to form a cone of light

When a microscope image stays in focus when the objective lens are changed, it is called ___.

Parfocal

An instrument that magnifies slight differences in the refractive index of cell structures is called a (n) ___ microscope.

Phase-contrast

Which part of the specimen scatters more electrons and therefor appears darker?

The most dense region

Which statement best describes specimen preparation for TEM?

The specimen is embedded into a matrix and cut into extremely thin slices

Which of the following are characteristics of many types of dyes used to stain microorganisms?

They are able to bind cells with ionic, covalent, or hydrophobic bonds. They have chromophore groups.

Specimens prepared for TEM must be cut to extremely ___ slices because electron beams are easily absorbed and scattered by solid matter.

Thin

Which type of electron microscope forms images from radiation that has passed through a specimen?

Transmission

Small internal cell structures are best visualized with a ___.

Transmission electron microscope

Which light rays strike the phase ring of the phase plate in a phase-contrast microscope?

Undeviated

As the magnification of a series of objective lenses increases, the working distance ___.

decreases