Ch. 2 - Microscopy

Question 1

Objective lens

Answer 1

• The lens closest to the specimen

* it forms a magnified image that is further enlarged by one or more additional lenses

Question 2

Bright-field microscope

Answer 2

• routinely use in microbiology labs to examine both stained and unstained specimens
•Cells are usually killed
• called this because it forms a dark image against a brighter background
• has several objective lenses
• total magnification
-product of the magnification of the ocular lenses and the objective lenses

Question 3

Substage condenser

Answer 3

• mounted within or beneath the stage and focuses a cone of light on the slide

Question 4

Ocular lenses (aka eyepieces)

Answer 4

• The curved part of the arm holds the body assembly, to which a nose piece and one or more ocular lenses are attached

Question 5

Parfocal

Answer 5

• ideally what a microscope should be which is it should remain in focus when objective lenses are changed

Question 6

Microscope resolution

Answer 6

• The ability of a lens to separate or distinguish between small objects that are close together
• wavelength of light used is major factor in resolution
• shorter wavelength➡️ greater resolution

Question 7

Working distance

Answer 7

• distance between the front surface of lens and surface of color glass or specimen when it is in sharp focus

Question 8

Focal length

Answer 8

• distance between Center of lens and focal point

* short focal length➡️ more magnification

Question 9

Immersion oil

Answer 9

• Will replace air that kept many light rays from entering the objective due to reflection and refraction at the surfaces of the objective lens
• results in an increase in resolution and numerical aperture

Question 10

Dark-field microscope

Answer 10

• 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

Question 11

Dark-field microscope uses

Answer 11

• used to observe internal structures in eukaryotic microorganisms
• used to identify bacteria such as Treponema pallidum, the causative agent of syphilis

Question 12

Phase-contrast microscope

Answer 12

• converts differences in refractive index/cell density into detected variations in light intensity
• some light rays from hollow cone of light passing through unstained sell slowed/out of phase (dark against bright background)
• excellent way to observe living cells

Question 13

Differential interference contrast microscope (DIC)

Answer 13

• creates image by detecting differences in reflective indices and thickness of different parts of specimen
• excellent way to observe living cells
-Live, unstained cells appear brightly colored and 3-D
-cell walls, endospores, granules, vacuoles, and nuclei are clearly visible

Question 14

Florescence microscope

Answer 14

• exposes specimen to ultraviolet, violet, or blue light
• specimens usually stained with fluorochromes
• shows bright image of object resulting from fluorescent light emitted by the specimen
• has applications in medical microbiology and microbial ecology studies

Question 15

fluorescent microscopy

Answer 15

•Essential tool in microbiology

• fluorochrome-labeled probes, such as antibodies, or fluorochrome dyes tag specific cell constituents for identification of unknown pathogens
• localization of specific protein in cells

Question 16

Confocal microscopy

Answer 16

• confocal scanning laser microscopy (CLSM) creates sharp, composite 3-D image of specimens by using laser beam, aperture to eliminate Straylight, and computer interface
• numerous applications including study of biofilms

Question 17

Purpose of staining specimens

Answer 17

• increases visibility of specimen
• accentuates specific morphological features
• preserves specimen

Question 18

Fixation

Answer 18

• preserves internal and external structures and fixes them and position
• it inactivates enzymes that might disrupt cell morphology and toughens cell structures so they do not change during staining and observation
• Microorganism usually is killed and attached firmly to slide
• cells are not stained nor distorted during this process
• heat fixation
-routinely used with bacteria and archaea
-preserves overall morphology but not internal structures
• Chemical fixation
-used with larger, more delicate organisms
-protects fine cellular substructure and morphology

Question 19

Dyes

Answer 19

• make internal and External structures of cell more visible by increasing contrast with background
• Basic dyes have positive charges
• acid dyes have negative charges

Question 20

Simple stains

Answer 20

• A single stain is used

* can determine size, shape, and arrangement of bacteria

Question 21

Differential staining

Answer 21

• divides micro organisms into groups based on their staining properties
• used to detect presence or absence of structures such as endospores, flagella, and capsules

Question 22

Gram staining

Answer 22

• most widely used differential staining procedure
• divides bacteria into two groups based on differences in cell wall structure
1) gram-positive
2) gram-negative

Question 23

Acid fast staining

Answer 23

• mostly used for staining members of the genus mycobacterium
- examples: mycobacterium tuberculosis, mycobacterium leprae
• high lipid content in cell walls (mycolic acid) is responsible for their staining characteristics

Question 24

Endospore staining

Answer 24

• heated, double staining technique

* bacterial endospores is one color and vegetative cell is a different color

Question 25

Capsule staining

Answer 25

* used to visualize polysaccharide capsules surrounding bacteria * example of negative staining -capsules may be colorless against a stained bacteria

Question 26

Flagella staining

Answer 26

• mordant applied to increase thickness of flagella

Question 27

Electron microscopy

Answer 27

* electrons replace light as the illuminating beam * wavelength of electron beam is much shorter than light, resulting in much higher resolution * allows for study of microbial morphology in great detail

Question 28

Transmission electron microscope (TEM)

Answer 28

* electrons scatter when they pass through thin sections of a specimen * transmitted electrons are under vacuum which reduces scatter and are used to produce clear image * denser regions in specimen scatter more electrons and appear darker * form an image from radiation that has passed through the specimen

Question 29

Electron microscopy specimen preparation

Answer 29

* analogous to procedures used for light microscopy * specimens must be cut very thin * specimens are chemically fixed and stained with electron dance materials, such as heavy metals, that differentially scatter electrons

Question 30

Negative stain for electron microscopy

Answer 30

* heavy metals do not penetrate the specimen but render dark background * used for study of viruses, bacterial gas vacuoles * background is stained but not the cells * unstained cells appear as bright objects against a dark background

Question 31

Shadowing for electron microscopy

Answer 31

* coating specimen with a thin film of a heavy metal on only one side * useful for viral morphology, flagella, DNA

Question 32

Freeze etching for electron microscopy

Answer 32

* freeze specimen then fracture along lines of greatest weakness (I.e., membranes) * allows for 3-D observation of shapes of intracellular structures * reduces artifacts * allows us to observe the shapes of organelles within the cells

Question 33

Scanning electron microscope

Answer 33

* uses electrons excited from the surface of a specimen to create detailed image * produces a realistic 3-D image of specimens surface features * can determine actually in situ location of microorganisms in ecological niches * form image from electrons released from atoms on the object surface

Question 34

Electron cryotomography

Answer 34

•Rapid freezing technique provides a 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

Question 35

Scanning probe microscopy

Answer 35

• among the most powerful microscopes which measure surface features of an object by moving a sharp probe over object surface • scanning tunneling microscope -magnification 100 times, can view atoms on surface of a solid -steady current maintained between microscope probe and specimen -up down movements of probe as it maintains current is detected, used to create image of surface of specimen • 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

Question 36

Refractive index

Answer 36

* A measure of how greatly a substance slows the velocity of light * The direction and magnitude of bending are determined by the refractive indices of the two media forming the interface

Question 37

Refractive index

Answer 37

• The direction and magnitude of bending of light are determined by theses of the two media forming the interface

Question 38

What size object would be the smallest size our eye can successfully detect

Answer 38

0.2 mm

Question 39

Heat fixation

Answer 39

- routinely used with bacteria and archaea | - preserves overall morphology but not internal structures

Question 40

Chemical fixation

Answer 40

- used with larger, more delicate organisms | - protects fine cellular substructure and morphology

Question 41

Acid-fast staining steps

Answer 41

1) primary staying with basic fuchsin 2) heat to allow basic fuchsin to penetrate cells 3) decolorize with acidic alcohol 4) counterstain with methylene blue

Question 42

Scanning tunneling microscope

Answer 42

* magnification 100 times, can view atoms on surface of a solid * steady current maintained between microscope probe and specimen * up down movements of probe as it maintains current is detected, used to create image of surface of specimen

Question 43

Atomic force microscope

Answer 43

* The second type of scanning probe microscope to be developed * 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

Question 44

Steps of gram stain process

Answer 44

1) primary stain (crystal violet) 2) treatment with iodine solution - increases interaction between Cell and dye so cell is stained better 3) smear is decolorized by washing with alcohol or acetone 4) counterstain usually with safranin - Colors gram-negative bacteria pink to red and leaves gram-positive bacteria dark purple

Question 45

What are two characteristics of many types of dyes used to stain microorganisms

Answer 45

* they are able to bind cells with ionic, covalent, or hydrophobic bonds * They have chromatophore groups - contain double bonds that give the dye its color

Question 46

Limitations of light microscope

Answer 46

* Not able to show detailed internal cellular structure | * have a resolution limit of 0.2 micrometers

Question 47

Three types of light microscopes that create images of living specimens

Answer 47

* dark field microscopes * phase contrast microscopes * differential interference contrast microscopes