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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
osmium tetraoxide
fixative for TEM reacts with lipids gives density to structures in specimen
26
describe the 'dehydration' step
all water is removed through putting samples in ascending alcohol strength baths
27
what is the result of using ethanol baths with fat cells
dissolves neutral fats, so fat cells appear empty
28
why must we utilize dehydration when preparing slides?
all water must be removed because it will be embedded in hydrophobic materials
29
what is the next step after dehydration?
hydration | removal of alcohol used to dehydrate specimen
30
how do we removal ethanol from specimen?
replace alcohol with xylene or cedar oil
31
how does xylene effect specimen prep?
xylene removes alcohol but... | renders specimen transparent
32
why must we remove the alcohol from a specimen?
embedding materials, such a paraffin wax, will not mix with alcohol
33
what is the purpose of embedding a specimen?
turns it into a semi solid material that can easily be slicing for slide prep
34
describe the process of 'embedding'
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
35
what is used for embedding specimens for TEM?
infiltrate tissues with monomeric resin or epoxy resin resin is polymerized
36
what is the purpose for fixing tissues?
because most things are transparent
37
what is the purpose for embedding tissue before sectioning?
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
38
advantages of formalin fixative
can be used alone or in combination reacts with tissue proteins --- stabilizing structures cheap easy to use
39
examples of what formalin can be used in combination with
alcohol --- shrinks tissues | acetic acid ---- softens and counteracts alcohols effects
40
disadvantages of formalin fixative
not good if you want to see fine cytological details
41
what is the purpose of the dehydration and hydration cycles used for tissue processing
dehydration removes all water because embedding materials are hydrophobic hydration to keep specimen pliable and decrease deterioration ---- organic material is not solid
42
contraption that uses a sharp blade over a stage that can be raised or lowered per desired slice width
microtome
43
what are the two types of microtomes discussed in class?
rotary | hand-held
44
how does sectioning differ for TEM specimens?
must use diamond knives slices are 50 - 150nm thick a copper mesh is used
45
what is a copper mesh used for in tissue sectioning?
in TEM, slices are so thin, they are too fragile to be handled. slice must be floated on a coated copper mesh
46
how does a copper mesh affect the viewing ability of a specimen?
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
why is it preferred that tissues be stained for observation?
because animal tissues are mostly colorless | staining allows us to see the details
48
what are the steps involved in staining a paraffin embedded specimen?
``` paraffin removal from section dehydration stain application dehydration alcohol removal add drop of cement cover slip ```
49
how is paraffin removed from section?
via xylene
50
first dehydration step in staining
must remove xylene that was used on paraffin removal using series of alcohol to water --- baths then we can apply the stain
51
at what point in the steps of staining, is the actual stain applied?
after the first set of dehydration
52
what happens after the stain is applied to a specimen?
second round of dehydration | graded alcohol baths
53
after second dehydration phase of staining.....
alcohol is then removed with xylene xylene is now admissible with mounting agent
54
what is the purpose of the cement drop ?
causes the outside of specimen to be solid and let the inside remain liquid like
55
what are the components of the most common staining technique for tissues in general?
HnE stain | hematoxylin and eosin
56
why is HE stain so common?
because displays structural features not chemical
57
staining properties of hematoxylin
behaves like a basic dye helps stain stick to tissue capable of staining nuclear material and cytoplasmic components
58
staining properties of eosin
an acidic dye | stains most cytoplasmic components and extracellular material
59
compares colors of HE stain
H --- dark to light blue/purple E --- yellow to pinkish colors
60
react with anionic groups of tissue components
basic dyes
61
bind to tissue components by forming electrostatic links with cationic groups of tissue components
acidic dyes
62
any tissue component that reacts with a basic dye
basophilic
63
any tissue component that reacts with an acidic dye
acidophilic
64
examples of anionic groups of tissue components
phosphate, sulfate, carboxyl groups
65
examples of basic dyes
methyl green methylene blue pyronin G toluidine blue
66
can pH affect staining color hues?
yes
67
examples of cationic groups of tissue components
amino groups of proteins
68
what is the purpose of using acidic dyes in sequence?
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
examples of acidic dyes
aniline blue acid fuchsine orange G
70
aniline blue
acid dye | stains collagen
71
acid fuchsine
acid dye | stains ordinary cytoplasm
72
orange G
acid dye | stains red blood cells
73
what does metachromasia refer to?
the phenomenon where dye changes color after reacting with a tissue component
74
examples of metachromasia
toluidine blue | stains cartilage ground substances or mast cell granules
75
what is the defining factor of TEM stains?
stains are ions of heavy metals that electron dense
76
examples of TEM stains
osmium tetroxide uranyl nitrate uranyl acetate = lead citrate
77
what does SEM stand for?
scanning electron microscope
78
SEM stains
platinum | gold
79
used to study the chemistry of cells and tissues
histochemistry cannot use alcohol when viewing lipids frozen specimens work great for adipose tissues
80
used to study the presence of specific constitutes by using monoclonal antibodies
immunocytochemistry | constitutes = antigens
81
proteins, glycoproteins and proteoglycans
antigens
82
monoclonal antibodies are derived....?
from activated B cell clones that have been exposed to the specific antigen
83
a single immune response to an antigen
monoclonal
84
B lymphocytes can mutate into _____, resulting in a _____.
tumor cells | myeloma
85
the 2 ways to label in immunocytochemistry
direct labeling | indirect labeling
86
types of direct labeling
flurorescent microscopy -- fluorescent dye light - visible substance E-microscopy --- gold or ferritin
87
describe how direct labeling works
conjugation with a type of direct labels, produces a visible marker
88
describe how indirect labeling works
the marker is attached to a second antibody, which is specific to the antibody used to locate the antigen of interest
89
this reagent rxn depends on the formation of aldehyde groups following the exposure to HCl or periodic acid
Schiff reagent rxn
90
mild hydrolysis with HCl exposes aldehyde groups on deoxyribose, which allows ________ . what is this rxn called?
Schiff reagents to react with aldehyde groups resulting in deep pink color Feulgen rxn
91
periodic acid cleaves bonds between adjacent carbons of carbohydrates, thus forming aldehyde groups, which allows _______. what is this rxn called?
Schiff reagents can react with aldehyde groups resulting in deep pink color PAS = periodic acid-Schiff rxn
92
substances that can be displayed via PAS
``` polysaccharides glycosaminoglycans proteoglycans glycoproteins glycolipids ```
93
dye used to show glycogen deposits of the ____ .
best carmine | deposits in the liver
94
what stains are used for RNA staining?
basic dyes
95
in RNA staining ____ slides are needed for …..? what are these slides incubated with?
control slides --- to distinguish between other basophilic substances incubated with ribonuclease
96
used to study structure and function of macromolecules within a specimen
cytochemistry
97
agents used in cytochemistry
Schiff reagent rxn Feulgen rxn PAS best carmine
98
what does PAS stand for?
periodic acid Schiff rxn

Histology Exam 1 (8 decks)

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