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Microbiology > Microscopy and Staining > Flashcards

Microscopy and Staining Flashcards

1
Q

what is one micrometer

A

one millionth of a meter

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2
Q

what is a nanometer

A

one billionth of a meter

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3
Q

how small can naked eye see

A

bigger than 100 micrometer

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4
Q

What is the resolution

A

distance between 2 objects at which object still seen as sepearte

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5
Q

what is contrast in microscopy

A

difference in light absorbance between 2 objects

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6
Q

what is relationship between contrast and backround

A

decrease contrast between object and back round- har to see object
increased contrast to back round- easier to visualize

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7
Q

What is a bright field microscope

A

simplest form of light microscope

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8
Q

How does a bright field microscope work

A

light is emitted from the bulb, enters microscope from the base and is reflected via mirrors toward the sample

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9
Q

What is the condensor

A

light passes through converging light beams into focused area on the sample

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10
Q

What is the Iris diaphram

A

controls amount of light passes through sample and into objective lens

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11
Q

What is objective lens

A

closes to sample and yields greatest amount of magnification

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12
Q

what is magnification in relationship to light needed

A

directly proportional to amount of light needed

to see image clearly @ increased magnification= need more light

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13
Q

What is the ocular lens

A

the eye piece

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14
Q

How does eyepiece work

A

light passes through sample and objective before coming to eye piece

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15
Q

What is the most common power of ocular lens

A

10x

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16
Q

How do you get total magnification

A

when microscope uses 2 lenses- objective and ocular- multiple objective x ocular
40x objective x 10x ocular= 400 mag

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17
Q

What is staining used for

A

used to visualize cells more clearly

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18
Q

How does staining process work

A

stain uses dyes
often required because of limited resolution
to stain may need to heat which could kill sample

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19
Q

what is phase contrast microscope

A

has advances over light microscopes- able to see structures otherwise invisible
provide detailed images of cells without staining

20
Q

What is special about condenser and objective in phase contrast microscope

A

Increased differences between cell and background to make visible
used to see cell movements without altering cell charge brought from treating cell with an agent to help see it

21
Q

What is a dark field microscope

A

used to increase contrast between specimen and background

results in dark background with bright objects

22
Q

How does a dark field microscope work

A

reflects light off specimen @ an angle

does not allow to see intracellular structures

23
Q

What is a fluorescence microscope

A

uses fluorescent molecules to see cells on dark background

24
Q

What form of light is in fluorescence microscope

A

energy of incoming light is in UV form

25
Q

How does UV light work in fluorescent microscope

A

UV excites flurophor @ different wave length

allows scientists to use many colors during imaging

26
Q

What are GFP, YFP, RFP proteins in fluorescent microscope

A

GFP- green
YFP- yellow
RFP- red

27
Q

How can the proteins be expressed

A

fluorescent illuminate cell as a whole
linked to normal cellular protein while color is showing protein movement and localization
used as tags on molecular antibodies to designate prescence or absence of specific protein

28
Q

When looking at the proteins how does fluorescent present

A

if sees the proteins fluorescent microscope detects and reflects the light
if no proteins then does not reflect the light

29
Q

What is a confocal microscope-

A

lasar scanning

combine fluorescent microscope with ability to see cells in 3-D

30
Q

How does confocal microscope work

A

uses lasers to focus on single object with increased accuracy

31
Q

How does the 3-D process work

A

takes the image
laser moves to adjacent plane and capture image repeating process until desired depth of sample is covered
basically many 2-D layers layered together

32
Q

What is an electron microscope TEM and SEM

A

used to see small specimen

labor intensive and requires sample to be fixed( killed) and process may alter cell structure

33
Q

What does TEM mean

A

transmission electron microscope

34
Q

How does TEM work

A

use a thin slice of the sample
usually heavily treated with many preservatives
placed between beam source and decor
image formed between interaction of electrons passing’s through thin section: process to see sub cellular organelles, substructures and viral particles

35
Q

How does scanning microscope work

A

electrons don’t go through this model
use beam of electrons which reflect off specimen
coated with gold or palladium to increase the electrons reflected
can only be used to generate detailed 3D shell model of surface of specimen- not live samples

36
Q

What is STEHM stand for

A

scanning, transmission electron holography microscope

37
Q

How does STEHM work

A

uses electron beam but also uses holography techniques
to study surface of protein and sub cellular structures
ability to magnify subatomic structure up to 20 million x larger
can resolve 35 pm and possible smaller

38
Q

define PM

A

pico meter: 1 trillionth of a meter

39
Q

How are stains used in sample staining

A

used to examine tissues, specific types of cells, blood, bacteria organelles etc within individual cell

40
Q

What is gram stain

A

developed by Hans christian gram in 1884
bacteria react differently to various dyes
distinction noted by color: 2 categories: pink and purple
Gram (+) and gram (-)

41
Q

Describe gram (+) bacteria

A

have thick cell wall w/overlap strands of peptidoglycan
thick peptidoglycan provides protective barrier to environment
layers: not immpermeable- water can still pass through to intercellular space
shows this by crystal violet dye and iodine
peptidoglycan traps dye and iodine in the cell
appear purple

42
Q

Describe gram (-) bacteria

A

has thin peptidoglycan followed by outer membrane of lipopolysaccharides- sets apart from gram (+)
during process of staining gram(-) will retain initially crystal violet dye
when decolorized, lipopolysaccharide and peptidoglycan unable to retain dye and outer membrane depleted of color

43
Q

What is decolorization

A

by wash cell with alcohol- affects the membrane and with gram(+) no effect, gram(-) will wash violet stain away

44
Q

What needs to happen to see gram(-) after decolorization

A

secondary stain( counterstain) called safranin is used

45
Q

What color does gram (-) hold after counterstain

A

retained pink color

46
Q

What is a differential stain

A

gram stain distinguish between bacteria with thick peptidoglycan wall and those that don’t have thick wall or have a very thin wall
generalized term for stain techniques separate specimen into further subgroup- usually at least 2 dyes

Microbiology (7 decks)

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