Microscopy and Staining

Question 1

what is one micrometer

Answer 1

one millionth of a meter

Question 2

what is a nanometer

Answer 2

one billionth of a meter

Question 3

how small can naked eye see

Answer 3

bigger than 100 micrometer

Question 4

What is the resolution

Answer 4

distance between 2 objects at which object still seen as sepearte

Question 5

what is contrast in microscopy

Answer 5

difference in light absorbance between 2 objects

Question 6

what is relationship between contrast and backround

Answer 6

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

Question 7

What is a bright field microscope

Answer 7

simplest form of light microscope

Question 8

How does a bright field microscope work

Answer 8

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

Question 9

What is the condensor

Answer 9

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

Question 10

What is the Iris diaphram

Answer 10

controls amount of light passes through sample and into objective lens

Question 11

What is objective lens

Answer 11

closes to sample and yields greatest amount of magnification

Question 12

what is magnification in relationship to light needed

Answer 12

directly proportional to amount of light needed

to see image clearly @ increased magnification= need more light

Question 13

What is the ocular lens

Answer 13

the eye piece

Question 14

How does eyepiece work

Answer 14

light passes through sample and objective before coming to eye piece

Question 15

What is the most common power of ocular lens

Answer 15

10x

Question 16

How do you get total magnification

Answer 16

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

Question 17

What is staining used for

Answer 17

used to visualize cells more clearly

Question 18

How does staining process work

Answer 18

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

Question 19

what is phase contrast microscope

Answer 19

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

Question 20

What is special about condenser and objective in phase contrast microscope

Answer 20

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

Question 21

What is a dark field microscope

Answer 21

used to increase contrast between specimen and background

results in dark background with bright objects

Question 22

How does a dark field microscope work

Answer 22

reflects light off specimen @ an angle

does not allow to see intracellular structures

Question 23

What is a fluorescence microscope

Answer 23

uses fluorescent molecules to see cells on dark background

Question 24

What form of light is in fluorescence microscope

Answer 24

energy of incoming light is in UV form

Question 25

How does UV light work in fluorescent microscope

Answer 25

UV excites flurophor @ different wave length

allows scientists to use many colors during imaging

Question 26

What are GFP, YFP, RFP proteins in fluorescent microscope

Answer 26

GFP- green
YFP- yellow
RFP- red

Question 27

How can the proteins be expressed

Answer 27

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

Question 28

When looking at the proteins how does fluorescent present

Answer 28

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

Question 29

What is a confocal microscope-

Answer 29

lasar scanning

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

Question 30

How does confocal microscope work

Answer 30

uses lasers to focus on single object with increased accuracy

Question 31

How does the 3-D process work

Answer 31

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

Question 32

What is an electron microscope TEM and SEM

Answer 32

used to see small specimen

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

Question 33

What does TEM mean

Answer 33

transmission electron microscope

Question 34

How does TEM work

Answer 34

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

Question 35

How does scanning microscope work

Answer 35

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

Question 36

What is STEHM stand for

Answer 36

scanning, transmission electron holography microscope

Question 37

How does STEHM work

Answer 37

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

Question 38

define PM

Answer 38

pico meter: 1 trillionth of a meter

Question 39

How are stains used in sample staining

Answer 39

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

Question 40

What is gram stain

Answer 40

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 (-)

Question 41

Describe gram (+) bacteria

Answer 41

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

Question 42

Describe gram (-) bacteria

Answer 42

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

Question 43

What is decolorization

Answer 43

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

Question 44

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

Answer 44

secondary stain( counterstain) called safranin is used

Question 45

What color does gram (-) hold after counterstain

Answer 45

retained pink color

Question 46

What is a differential stain

Answer 46

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