5.

Question 1

bright-field scope

Answer 1

• specimens are visualized because of differences in contrast between specimen and surroundings
• two sets of lenses form the image
• objective lens (10x-100x mag.)
• ocular lens (10x-20x mag)
• total magnification= objective magnification X ocular magnification
• maximum mag. is ~2000x

Question 2

magnification light path

Answer 2

bottom to top

• light source
• condenser lens
• specimen
• objective lens
• intermediate image (inverted from that of the specimen)
• ocular lens
• eye
• visualized image

Question 3

magnification

Answer 3

ability to make an object larger

Question 4

resolution

Answer 4

• the ability to distinguish two adjacent objects as separate and distinct
• the ability of a lens to distinguish small objects that are close together
  ex. resolving power of 0.2um
  
  • two points can be distinguished if they are at least 0.2 um apart
• light must pass b/w two points for them to be viewed as separate objects
• as wavelength decreases resolution improves

Question 5

Effect of wavelength on resolution

Answer 5

• throw ink-covered objects at target E
• cannot fit fit between arms, poor resolution
• fit b/w arms, resolution improves
• as diameter of objects thrown decreases, greater numbers pass between the arms and the resolution increases

Question 6

improving contrast in light microscopy results in …?

Answer 6

a better final image

Question 7

staining improves CONTRAST

Answer 7

• dyes are organic compounds that bind to specific cellular materials
• examples of common stains are methylene blue, safranin, and crystal violet

Question 8

simple staining

Answer 8

one dye used to colour specimens

Question 9

chromophore

Answer 9

the coloured portion of a dye

Question 10

two types of dyes

Answer 10

basic

acidic

Question 11

basic dye

Answer 11

crystal violet

• positively charge chromophore
• binds to negatively charged molecules on cell surface

Question 12

acidic dye

Answer 12

• nigrosin
• negatively charged chromophore
• repelled by cell surface
• used to stain background
• negative stain

Question 13

preparing a specimen for microscopic examination

Answer 13

1. preparing a smear-spread culture in thin film over slide & air dry
2. heat fixing and staining-pass slide through flame to heat fix, flood slide with stain, rinse and dry
3. microscopy-place drop of oil on slide; examine with 100x objective lens

Question 14

gram stain

Answer 14

• separates bacteria into 2 groups based on cell wall structure
• gram positive
• gram negative

Question 15

gram positive

Answer 15

cells that retain a primary stain

-purple

Question 16

gram negative

Answer 16

cells that lose the primary stain

• take colour of counterstain
• red or pink

Question 17

gram stain steps

Answer 17

1. flood the heat fixed smear with crystal violet, all cells purple
2. add iodine solution, all cells remain purple
3. decolourize with alcohol briefly, gram +ive cells are purple; gram -ive are colourless
4. counterstain with safranin, gram+ive purple, gram -ive cells pink to red

Question 18

acid fast stain

Answer 18

• detects mycolic acid in the cell wall of the genus mycobacterium
• mycobacterium- retains primary stain (fuchsia) pink
• anything else on slide- colour of counterstain-blue

Question 19

endospore stain

Answer 19

• endospores retain primary green
• cells counterstained pink
  ex. bacillus anthracis spores

Question 20

phase contrast microscopy

Answer 20

• phase ring amplifies differences in the refractive index of cell and surroundings
• improves the contrast of a sample without the use of a stain
• allows for the visualization of live samples
• resulting image is dark cells on the light background

Question 21

dark field micrscopy

Answer 21

specimen is illuminated with a hollow cone of light

• only refracted light enters the objective
• specimen appears as a bright object on a dark background
• used to observe bacteria that dont stain well
  ex. treponema pallidum-the causative agent of syphilis

Question 22

fluorescence micropsopy

Answer 22

used to visualize specimens that fluoresce
-emit light of one colour when illuminated with another colour of light
*cells may fluoresce naturally
ex.photosynthetic cyanobacteria have chlorophyll
absorbs light at 430nm (blue violet)
emits at 670nm (red)
*or after staining with fluorescent dye
ex. DAPI specifically binds to DNA

Question 23

differential interference contrast (DIC) microscopy

Answer 23

• 3D
• uses a polarizer to create two distinct beams of polarized light
• gives structures such as endospores, vacuoles, and granules a #D appearance
• structures not visible by bright field microscopy are sometimes visible by DIC

Question 24

Confocal scanning laser microscopy (CSLM)

Answer 24

• 3D
• uses computerized microscope couple with a laser source to generate a 3D image
• computer can focus the laser on single layers of specimen
• different layers can then be compiled for 3D image
• resolution is 0.1 um for CSLM

Question 25

transmission electron microscope (TEM)

Answer 25

- electron beam focused on specimen by a condenser-magnets used as lenses - electrons that pass through the specimen are focused by two sets of lenses- compound microscope - electrons strike a fluorescent viewing screen

Question 26

TEM

Answer 26

high magnification and resolution (0.2nm) specimen must be very thin (20-60) unstained cells do a poor job of scattering electrons *must be stained with metals ->lead or uranium *bind to cell structures to make them more electron dense *enables visualization of structures at molecular level

Question 27

does light microscope or electron microscope have better resolution?

Answer 27

transmission electron microscope