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Microbio > Microscopy and Specimen Preparation > Flashcards

Microscopy and Specimen Preparation Flashcards

1
Q

type of microscope that use visible light to illuminate specimens

A

compound light microscope

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2
Q

types of light microscopy (4):

A
  • bright-field
  • dark-field
  • phase-contrast
  • fluorescence
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3
Q

type of light microscopy; what we use; image is dark w/ bright background

A

bright-field

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4
Q

how many lenses does a compound microscope require?

A

2

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5
Q

type of lens: focuses the light

A

condenser lens

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6
Q

process of light being refracted (bent) when passing from one medium to another

A

refraction

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7
Q

why does light refract/bend when passing from one medium to another?

A

it slows the velocity of the light = bend (towards the normal)

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8
Q

F =

A

focal point

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9
Q

f =

A

focal length

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10
Q

where light rays converge on the other side of the lens

A

focal point (F)

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11
Q

the ______ the focal length, the stronger the lens

A

shorter

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12
Q

the shorter the focal length, the ______ the magnification

A

greater

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13
Q

magnification has to do solely with the _______

A

lenses

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14
Q

using the bright-field microscope, specimens are visualized because of differences in _______ between the specimen and its surroundings

A

contrast (density)

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15
Q

total magnification =

A

magnification of OCULAR lens x magnification of OBJECTIVE lens

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16
Q

upper limit of magnification =

A

2000X

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17
Q

upper limit of resolution =

A

0.2 micrometers

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18
Q

ability to distinguish two objects as distinct and separate when viewed under a microscope; clarity of an image

A

resolution (d)

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19
Q

T/F: resolution and magnification are the same thing

A

false

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20
Q

d =

A

resolution

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21
Q

you want d, or resolution, to be extremely ________

A

small

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22
Q

formula for d (resolution) =

A

0.5 * wavelength / NA

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23
Q

NA =

A

numerical aperture

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24
Q

measure of light gathering ability

A

NA (numerical aperture)

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25
Q

formula for d (resolution) can ALSO be:

A

0.5 * wavelength / n (sin θ)

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26
Q

n =

A

refractive index

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27
Q

ways to increase resolution (aka make it smaller - 4):

A
  • decrease wavelength (ex: using blue light instead of white light)
  • decrease working distance
  • increase NA
  • increase n (refractive index)
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28
Q

_____ working distance to increase resolution

A

decrease

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29
Q

higher numerical aperture = _____ resolution

A

better

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30
Q

you should shorten the working distance to make θ ______ AND to make NA ______

A

increase; increase

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31
Q

how much a light ray bends

A

refractive index (n)

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32
Q

if you decrease n (refractive index), you lose _____ _____ and make resolution worse

A

light rays

33
Q

anything thicker than air will have a _____ n

A

hgiher

34
Q

refractive index (n) of air =

A

1

35
Q

refractive index (n) of oil =

A

1.25

36
Q

increasing n (refractive index) results in an increase in _____ which results in a better resolution

A

NA

37
Q

limit of resolution =

A

0.2 (~0.18) nanometers

38
Q

improving contrast in light microscopy (2 main steps):

A

1) fixation
2) staining

39
Q

preserves specimens and fixes them in position; organisms usually killed and firmly attached to microscope slide

A

fixation

40
Q

type of fixation: routine used for bacteria and archaeons; what we usually do; kills them

A

heat fixation

41
Q

type of fixation: use with larger, more delicate organisms

A

chemical fixation

42
Q

make internal and external structures of cell more visible by increasing contrast with background

A

staining

43
Q

2 common features of staining:

A

1) chromophore groups
2) ability to bind cells

44
Q

chemical gropus with conjugated double bonds; gives stain its color

A

chromophore groups

45
Q

stains ability to bind cells usually inovolves what types of bonds?

A

ionic (most are salts)

46
Q

types of stains (2):

A
  • basic stains (dyes)
  • acidic stains (dyes)
47
Q

type of stain: dyes with POSITIVE CHARGES; bind to negatively charged structures; when it ionizes, the chromophore group has a POSITIVE charge; attracted to surface of bacteria (mainly neg.)

A

basic stains

48
Q

type of stain: dyes with NEGATIVE charges; bind to positively charged structures; chromophore group is neg. charged when released/ionized

A

acidic stains (dyes)

49
Q

basic stains are colored _____ and acidic stains are colored ______

A

bases; acids

50
Q

stain the background to stain; negative stain; REPELS bacteria

A

acidic stain

51
Q

bacteria are mainly ____ charged

A

negatively

52
Q

types of staining techniques (2):

A

1) simple staining
2) differential staining

53
Q

type of staining technique: use of a single stain

A

simple staining

54
Q

type of staining technique: use of two contrasting stains separated by a decolorizing agent

A

differential staining

55
Q

purpose of simple staining =

A

for visualization of morphological shape + arrangement

56
Q

purpose of differential staining =

A

identification + visualization of structure

57
Q

examples of IDENTIFICATION differential staining (2):

A
  • Gram stain
  • Acid fast stain
58
Q

examples of VISUALIZATION differential staining (2):

A
  • spore stain
  • capsule stain
59
Q

a stimple stain looks at what 3 things of a cell?

A

1) size
2) shape
3) arrangement

60
Q

differential staining use more than one dye to ______ stain features

A

preferentially

61
Q

differential staining is used to detect the _____ or ______ of structures

A

presence or absence

62
Q

differential stains that divides microorganisms into GROUPS based on their staining properties (3)

A
  • Gram-stain
  • acid-fast stain
  • endospore stain
63
Q

most widely used differential staining procedure; divides bacteria into two groups based on differences in cell wall structure

A

gram staining

64
Q

when and by who was the Gram Stain discovered?

A

1884 by Christian Gram

65
Q

what was Christian Gram trying to distinguish between when disovering the Gram Stain?

A

eukaryotic nuclei and bacteria

66
Q

Procedure of Gram Staining (and color during steps):

A

1) flood the heat-fixed smear with crystal violet for 1 min (both purple)
2) add iodine solution for 1 min (both purple)
3) decolorize with alchohol briefly for 20 seconds (pos = purple; neg = colorless)
4) counterstain with safranin for 1-2 minutues (pos = purple; neg = pink)

67
Q

both Gram-postive and Gram-negative bacteria are _____ charged on their SURFACE

A

negatively

68
Q

useful for staining members of the genus Mycobacterium (ex: TB, leprisy); high lipid (mycolic acid) content in cell walls is repsonsible for their staining characteristicspi

A

acid-fast staining

69
Q

acid-fast cells stain ____

A

pink

70
Q

non-acid fast cells stain _____

A

blue

71
Q

why are cells like Mycobacterium hard to stain?

A

bc they have a really high lipid content, known as mycolic acid, in their cell walls

72
Q

“acid-fast if you hold on FAST to acidic conditions”

A
73
Q

acid-fast uses _______ and ____ ____ as two of the staining materials

A

methylene blue and acid-alcohol

74
Q

types of stains for SPECIFIC STRUCTURES (3):

A
  • endospore staining
  • capsule staining
  • flagella staining
75
Q

type of stain for a specific structure: heated, double staining technique; bacterial _____ is one color (GREEN) and vegetative cell is a dif. color (PINK)

A

endospore staining

76
Q

in endospore staining, bacterial endospores are ______ and vegetative cells are _____

A

green; pink

77
Q

type of stain for a specific structure: negative stain; _____ are colorless against a stained background

A

capsule staining

78
Q

in capsule staining, capsules are ______ against a stained background

A

colorless

Microbio (21 decks)

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