Chapter 3 Study Guide

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Microscopy

use of light or electrons to magnify objects

wavelength

the distance between two corresponding parts of a wave

Magnification

the apparent increase in size of an object example 40x

When does Magnification occur?

when a beam of radiation refracts(bends) as it passes through a lens.

What are the properties that determine the clarity of an image?

resolution and contrast

Resolution

(resolving power) is the ability to distinguish between objects that are close together

Contrast

refers to differences in intensity between two objects or between an object and its background

Contrast is important in determining what?

resolution

Empty magnification

the combining of lenses to obtain an image magnified millions of times but the image is faint and blurry

How do you increase the contrast between microorganisms and their background?

stain them

What is the most common microscope?

bright field

What are the two types of bright field microscopes?

simple and compound

Who first reported his observations of microorganisms using a simple microscope?

Leeuwenhoek

How many magnifying lens does a simple microscope have ?

one and example is a magnify glass

How many magnifying lens does a compound microscope have?

2 lenses,ocular and objective

total magnification

magnification of the objective lens x the magnification of the ocular lens

micrographs

photographs of a microscopic image

Dark-Field Microscope

pale objects are best observed with this microscope, so the specimen appears light against a dark background.(small and colorless cells) in order to increase the contrast

Phase Microscope

examine living organisms or specimens that would be damaged or altered by attaching them to slides or staining them

What are the two types of Phase microscopes?

phase contrast and differential interference contrast

Phase-Contrast Microscope

the simplest phase, produce sharply defined images in which fine structures can be seen in living cells such as cilia and flagella

Differential Interference Contrast Microscope

(Nomarski microscope) increases contrast and gives an image a 3 dimensional or shadowed appearance, this techniques produces unnatural colors which enhance contrast (like light is hitting on side of specimen)

Fluorescent Microscopes

uses UV light to increase resolution because it has a shorter wavelength than visible light and contrast is improved because fluorescent structures are visible against a black background in order to get a quick diagnosis

Confocal MIcroscopes

use fluorescent dyes or antibodies, and use ultraviolet lasers to illuminate the fluorescent chemicals in only a single plane the rest of the specimen remains dark and out of focus

Electron Microscopy

uses a beam of electrons and have a shorter wavelength than visible light and provides greater resolution at a higher magnification (10,000x100,000) provide detailed views of the smallest bacteria, viruses, internal cellular structures and even molecules and large atoms

What are the two types of Electron MIcroscopy?

Transmission and Scanning

Transmission Electron Microscope (TEM)

transmits a beam of electrons through the specimen that produces an image on a fluorescent screen.

Scanning Electron Microscope (SEM)

uses a magnetic field to manipulate a beam of electrons called primary electrons, so that the beam is bounced off the surface of the specimen to give it a 3 dimensional view of the cell and its surface and they do this by coating the surface with heavy metals such as platinum and gold to reflect the beam(shadowcasting)

Staining increases ____and ____?

contrast and resolution

How do you prepare a specimen for staining?

spread culture in a circular motion over slide, air dry, then pass slide through a bunsen burner to fix the cells to the slide

Who developed heat fixation?

Robert Koch

chemical fixation

applying methyl alcohol or formalin (a solution of fomalde-hyde in water)to the smear for one minute.

What is Desiccation?

drying

What kills a microorganism?

Desiccation and fixation in order to preserve their shape and size

What is a the colored portion of a dye called?

chromophore

What are the two types of stains?

Simple and Differential

Simple Stain

composed of a basic dye such as crystal violet, safranin, or methylene blue. They are simple because you only have to soak the smear in dye for 30-60 seconds and are used to observe size, shape and arrangement of cells.

Differential Stain

the most stain used, which use one or more dyes so that different cells, chemicals, or structures can be disguinished when examined. determines morphology and arrangement

What are some common differential stains used?

Gram stain, acid-fast stain, and endospore stain

Who developed the Gram stain?

Hans Christian Gram

Gram Stain

differentiates between two large gorups of microorganisms by using purple staining gram-positive cells and pink-staining, gram-negative cells in order to identify bacterial pathogens.

What is a mordant?

Iodine, a substance that binds to a dye and mekes it less soluble (keeping cells purple)

What does a decolorizing agent do?

breaks down the thin cell wall of gram-negative cells allowing the stain and the mordant to wash away leaving the cell colorless, but the gram positive cells remain purple

What does a counterstain do?

Safranin(red), provides a contrasting color to the primary stain. so making gram negative cells a pink color because it absorbs the safranin and keeps the gram positive cells purple.

Acid-Fast Stain

stains cells of the genera Mycobacterium and Nocardia which cause many human diseases(TB,leprosy and other lung and skin infections). These bacteria have cells that contain large amounts of waxy lipid in their cell walls and do not readily stain with the gram stain. This stain results in pink acid fast cells that can be differentiated from blue non acid fast cells including human cells and tissue.

Endospore Stain

bacteria those of the genera Bacillus an Costridium contain species that cause diseases such as anthrax,gangrene and tetanus that produce endospores. Endospores cannot be stained by normal staining because their walls are impermeable to all chemicals. so the endospore stain uses heat to drive the primary stain, malachite green into the endospore and then decolorized and the conterstained with safaranin and results in green stained endospores and red(pink) colored vegetative cells.

Negative (Capsule) Stain

used to primarily to reveal the presence of negatively charged bacterial capsules. acidic dyes are repulsed by negative charges on the surface of cells and do not stain them leaving the cells colorless and the background stained.

Flagellar Stain

allows determination of number and location of bacterial flagella

Taxonomy

is the science of classifying and naming orgainisms and consists of classification, nomenclature, and identification.

What is the order of Linnaean taxonomic scheme?

Domain, Kingdom, Phylum, Class, Order, Family, Genus, Species

Binomial Nomenclature

the Genus and Species Names of an organism
Genus- always capitalized and underlined (italicized)
Species- always lower case and underlined (italicized)
Ex. Streptococcus pyogenes

What are the three domains?

Eukarya, Bacteria, and Archaea based on ribosomal nucleotide sequences

Which of the following groups of organisms does NOT belong in the Domain Eukarya?

A archaea
B protozoa
C both bacteria and archaea
D bacteria
answer is C

Fluorescent microscopy is used primarily for:

A determining the presence of bacterial endospres
B determining morphology and arrangemnt of bacteria
C diagnosis of certain bacteria
D determining cell wall type of bacteria
answer is C

Which of the following are prokaryotic, heterotrophic extremophiles?

A Cyanobacteria
B Archaea
C Eubacteria
D all of the above
answer is B

How are negative stains different from other types of stains?

A They stain the desired structure or specimen black.
B They stain the background, leaving the cells colorless.
C They stain more than one type of specimen.
answer is B

What is the fundamental purpose of staining in light microscopy?

A To make the specimen appear larger in the microscope
B To kill the specimen
C To see the specimen without the aid of a microscope
D To increase the contrast and visibility of the specimen
answer is D

Which of the following interactions with visible light is needed to view a specimen?

A reflection
B transmission
C absorption
D refraction
answer is B

Why is visualization not sufficient to properly identify bacteria?

A identification is only needed in clinical specimens.
B Many unrelated bacteria can share the same shape.
C Bacteria have a limited set of shapes.
D Bacteria have a limited set of shapes and many unrelated bacteria share the same shape.
E Not all bacteria can be seen with a light microscope.
answer is D

What is the hallmark of dichotomous keys?

A They only relate to the shape of the cell.
B They consist of a series of paired statements, in which only one statement of each pair applies to a given organism.
C They only relate to biochemical processes of the cell.
D They are open-ended questions.
answer is B

Biochemical tests _________________.

A are the main methods used to identify unknown bacteria
B are visualized using microscopes
C are the most effective way to determine bacterial shape
D are used to determine rate of growth
answer is A

Which of the following is a lens found on electron microscopes but not on light microscopes?

A Projector lens
B Condenser lens
C Eyepiece lens
D Objective lens
answer is A

Which type of microscope would allow the viewer to see ribosomes inside a cell?

A A light microscope, transmission electron microscope, and scanning electron microscope can all view ribosomes inside a cell.
B A transmission electron microscope
C A scanning electron microscope
D A light microscope
the answer is B

Which of the following is a characteristic shared by both electron and light microscopes?

A They both employ the use of objective lenses.
B Both microscopes require an internal camera to visualize the specimen.
C They both utilize white light to magnify the specimen.
D Both microscopes have a resolving power of 0.01 nm.
answer is A

What is the fate of the electrons that interact with a specimen in an electron microscope?

A They are reflected by the specimen.
B They are refracted by the specimen.
C They are absorbed by the specimen.
D They may be absorbed, reflected, or refracted by the specimen.
answer is D

Staining is an important way to improve which of the following aspects of microscopy?

A numerical aperture
B contrast
C magnification
D resolution and contrast
answer is D

Which of the following is used to stain the background rather than the cell?

A methylene blue
B anionic dye
C crystal violet
D cationic dye
answer is B

What happens to the light rays when they hit the specimen?

A They are diverted to the ocular lens.
B They are focused into a small area towards the objective lens.
C They are reflected, refracted, or absorbed by the specimen.
D They are absorbed by the stage.
answer is C

In a typical brightfield microscope (seen in the animation), at which point does magnification begin?

A The ocular lens
B The stage
C The lamp
D The condenser lens
E The objective lens
answer is E

Which of the following types of microscopes can magnify more than 2000X?

A a phase-contrast microscope
B a transmission electron microscope
C a dark-field microscope
D a bright-field microscope
answer is B

If a microbiologist omitted the decolorizer from a Gram-stained smear, which of the following would happen?

A All cells in the smear would be purple.
B All cells in the smear would be pink.
C All cells in the smear would be colorless.
D Gram-positive cells would be pink and Gram-negative cells would be purple.
answer is A

Which of the following is used to stain living unstained cells?

A brightfield microscopy
B phase contrast microscopy
C scanning electron microscopy
D fluorescent microscopy
answer is B

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