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Histology Exam 1 > Lecture 2: Microscopes, Prep, & Staining > Flashcards

Lecture 2: Microscopes, Prep, & Staining Flashcards

1
Q

major components of microscopes

A

light source
condenser
stage
objective and ocular lens

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2
Q

pros of compound microscopes

A

magnify up to 1000x
allows you to see structural detail in high resolution

resolution is defining factor of high magnifications

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3
Q

cons of compound microscopes

A

specimens must be very thin

if not stained, everything is the same color

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4
Q

converts phase shifts in light passing through a transparent specimen into brightness

A

phase contrast microscope

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5
Q

advantages of a phase contrast microscope

A

allows you to see features of a transparent specimen as if it weren’t transparent
can examine unstained specimens
useful for observing living specimens

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6
Q

detects molecules that emit light, in wavelengths that lie within the visible range when exposed to a UV light source

A

fluorescence microscope

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7
Q

a naturally occurring fluorescent molecule

A

vitamin A

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8
Q

through the addition of a pinhole, optical resolution and contrast are increased

A

confocal scanning microscope

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9
Q

how does a pinhole increase resolution and contrast?

A

by eliminating out of focus light

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10
Q

with a confocal scanning microscope we can use a computer to ______ ?

A

reconstruct 3D images

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11
Q

advantages of a confocal scanning microscope

A

computer can eliminate out of focus material
can create images of very thin specimens (1 micrometer)
computer creates 3D image

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12
Q

what does TEM stand for?

A

transmission electron microscope

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13
Q

how does a TEM work?

A

uses a beam of e- instead of light waves
e- are driven by electromagnets from source to specimen
and then image is projected onto a viewing screen and photo film

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14
Q

what is the major disadvantages to TEM?

A

very large

very expensive

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15
Q

list the basic steps in tissue fixing and embedding

A

fixing
dehydration
alcohol removal
embedding

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16
Q

describe ‘fixing’

A

prevents further deterioration and helps to harden the tissue prior to embedding but can radically distort the specimen.

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17
Q

what is the most common fixative?

A

formalin

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18
Q

acid fixatives are used to see…..?

A

fix chromatin, nucleoli, spindle fibers

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19
Q

basic fixatives are used to see…..?

A

mitochondria

chromatin are destroyed

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20
Q

Carnoy’s Fluid

A

acid fixative

for preserving glycogen

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21
Q

Zenker’s Fluid

A

for sharp histological details
but if not washed out black crystals will form

acid fixative

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22
Q

Bouin’s Fluid

A

cytological details
uses picric acid which is extremely volatile

acid fixative

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23
Q

Zirkle-Erliki

A

basic fixative

long fixing time ~2days

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24
Q

glutaraldehyde

A

fixative for TEM

preserves proteins

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25
Q

osmium tetraoxide

A

fixative for TEM
reacts with lipids
gives density to structures in specimen

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26
Q

describe the ‘dehydration’ step

A

all water is removed through putting samples in ascending alcohol strength baths

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27
Q

what is the result of using ethanol baths with fat cells

A

dissolves neutral fats, so fat cells appear empty

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28
Q

why must we utilize dehydration when preparing slides?

A

all water must be removed because it will be embedded in hydrophobic materials

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29
Q

what is the next step after dehydration?

A

hydration

removal of alcohol used to dehydrate specimen

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30
Q

how do we removal ethanol from specimen?

A

replace alcohol with xylene or cedar oil

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31
Q

how does xylene effect specimen prep?

A

xylene removes alcohol but…

renders specimen transparent

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32
Q

why must we remove the alcohol from a specimen?

A

embedding materials, such a paraffin wax, will not mix with alcohol

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33
Q

what is the purpose of embedding a specimen?

A

turns it into a semi solid material that can easily be slicing for slide prep

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34
Q

describe the process of ‘embedding’

A

moved through paraffin baths – usually 3
this removes xylene
then placed in a mold with molten paraffin wax
which rapidly hardens in a cold water batha

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35
Q

what is used for embedding specimens for TEM?

A

infiltrate tissues with monomeric resin or epoxy resin

resin is polymerized

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36
Q

what is the purpose for fixing tissues?

A

because most things are transparent

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37
Q

what is the purpose for embedding tissue before sectioning?

A

biological material is squishy and mostly water

we must harden and embed material in a solid substance before we are able to precisely cut and section the specimen

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38
Q

advantages of formalin fixative

A

can be used alone or in combination
reacts with tissue proteins — stabilizing structures
cheap
easy to use

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39
Q

examples of what formalin can be used in combination with

A

alcohol — shrinks tissues

acetic acid —- softens and counteracts alcohols effects

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40
Q

disadvantages of formalin fixative

A

not good if you want to see fine cytological details

41
Q

what is the purpose of the dehydration and hydration cycles used for tissue processing

A

dehydration removes all water because embedding materials are hydrophobic

hydration to keep specimen pliable and decrease deterioration —- organic material is not solid

42
Q

contraption that uses a sharp blade over a stage that can be raised or lowered per desired slice width

A

microtome

43
Q

what are the two types of microtomes discussed in class?

A

rotary

hand-held

44
Q

how does sectioning differ for TEM specimens?

A

must use diamond knives
slices are 50 - 150nm thick
a copper mesh is used

45
Q

what is a copper mesh used for in tissue sectioning?

A

in TEM, slices are so thin, they are too fragile to be handled. slice must be floated on a coated copper mesh

46
Q

how does a copper mesh affect the viewing ability of a specimen?

A

copper mesh is left in forever

but we are viewing such a small area that the mesh holes allow e- to easily pass thru

47
Q

why is it preferred that tissues be stained for observation?

A

because animal tissues are mostly colorless

staining allows us to see the details

48
Q

what are the steps involved in staining a paraffin embedded specimen?

A 
paraffin removal from section
dehydration
stain application
dehydration
alcohol removal
add drop of cement
cover slip
49
Q

how is paraffin removed from section?

A

via xylene

50
Q

first dehydration step in staining

A

must remove xylene that was used on paraffin removal
using series of alcohol to water — baths

then we can apply the stain

51
Q

at what point in the steps of staining, is the actual stain applied?

A

after the first set of dehydration

52
Q

what happens after the stain is applied to a specimen?

A

second round of dehydration

graded alcohol baths

53
Q

after second dehydration phase of staining…..

A

alcohol is then removed with xylene

xylene is now admissible with mounting agent

54
Q

what is the purpose of the cement drop ?

A

causes the outside of specimen to be solid and let the inside remain liquid like

55
Q

what are the components of the most common staining technique for tissues in general?

A

HnE stain

hematoxylin and eosin

56
Q

why is HE stain so common?

A

because displays structural features not chemical

57
Q

staining properties of hematoxylin

A

behaves like a basic dye
helps stain stick to tissue
capable of staining nuclear material and cytoplasmic components

58
Q

staining properties of eosin

A

an acidic dye

stains most cytoplasmic components and extracellular material

59
Q

compares colors of HE stain

A

H — dark to light blue/purple

E — yellow to pinkish colors

60
Q

react with anionic groups of tissue components

A

basic dyes

61
Q

bind to tissue components by forming electrostatic links with cationic groups of tissue components

A

acidic dyes

62
Q

any tissue component that reacts with a basic dye

A

basophilic

63
Q

any tissue component that reacts with an acidic dye

A

acidophilic

64
Q

examples of anionic groups of tissue components

A

phosphate, sulfate, carboxyl groups

65
Q

examples of basic dyes

A

methyl green
methylene blue
pyronin G
toluidine blue

66
Q

can pH affect staining color hues?

A

yes

67
Q

examples of cationic groups of tissue components

A

amino groups of proteins

68
Q

what is the purpose of using acidic dyes in sequence?

A

different acidic dyes can stains different cationic groups

specific sequences can be used to yield specific results pertaining to what you want to view

69
Q

examples of acidic dyes

A

aniline blue
acid fuchsine
orange G

70
Q

aniline blue

A

acid dye

stains collagen

71
Q

acid fuchsine

A

acid dye

stains ordinary cytoplasm

72
Q

orange G

A

acid dye

stains red blood cells

73
Q

what does metachromasia refer to?

A

the phenomenon where dye changes color after reacting with a tissue component

74
Q

examples of metachromasia

A

toluidine blue

stains cartilage ground substances or mast cell granules

75
Q

what is the defining factor of TEM stains?

A

stains are ions of heavy metals that electron dense

76
Q

examples of TEM stains

A

osmium tetroxide
uranyl nitrate
uranyl acetate = lead citrate

77
Q

what does SEM stand for?

A

scanning electron microscope

78
Q

SEM stains

A

platinum

gold

79
Q

used to study the chemistry of cells and tissues

A

histochemistry

cannot use alcohol when viewing lipids
frozen specimens work great for adipose tissues

80
Q

used to study the presence of specific constitutes by using monoclonal antibodies

A

immunocytochemistry

constitutes = antigens

81
Q

proteins, glycoproteins and proteoglycans

A

antigens

82
Q

monoclonal antibodies are derived….?

A

from activated B cell clones that have been exposed to the specific antigen

83
Q

a single immune response to an antigen

A

monoclonal

84
Q

B lymphocytes can mutate into _____, resulting in a _____.

A

tumor cells

myeloma

85
Q

the 2 ways to label in immunocytochemistry

A

direct labeling

indirect labeling

86
Q

types of direct labeling

A

flurorescent microscopy – fluorescent dye
light - visible substance
E-microscopy — gold or ferritin

87
Q

describe how direct labeling works

A

conjugation with a type of direct labels, produces a visible marker

88
Q

describe how indirect labeling works

A

the marker is attached to a second antibody, which is specific to the antibody used to locate the antigen of interest

89
Q

this reagent rxn depends on the formation of aldehyde groups following the exposure to HCl or periodic acid

A

Schiff reagent rxn

90
Q

mild hydrolysis with HCl exposes aldehyde groups on deoxyribose, which allows ________ . what is this rxn called?

A

Schiff reagents to react with aldehyde groups
resulting in deep pink color

Feulgen rxn

91
Q

periodic acid cleaves bonds between adjacent carbons of carbohydrates, thus forming aldehyde groups, which allows _______. what is this rxn called?

A

Schiff reagents can react with aldehyde groups
resulting in deep pink color

PAS = periodic acid-Schiff rxn

92
Q

substances that can be displayed via PAS

A 
polysaccharides
glycosaminoglycans
proteoglycans
glycoproteins
glycolipids
93
Q

dye used to show glycogen deposits of the ____ .

A

best carmine

deposits in the liver

94
Q

what stains are used for RNA staining?

A

basic dyes

95
Q

in RNA staining ____ slides are needed for …..? what are these slides incubated with?

A

control slides — to distinguish between other basophilic substances

incubated with ribonuclease

96
Q

used to study structure and function of macromolecules within a specimen

A

cytochemistry

97
Q

agents used in cytochemistry

A

Schiff reagent rxn
Feulgen rxn
PAS
best carmine

98
Q

what does PAS stand for?

A

periodic acid Schiff rxn

Histology Exam 1 (8 decks)

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