Exam 1 Flashcards
(75 cards)
1
Q
what are some basic safety procedures to remember?
A
- blades go in sharps container
- xylene gets drained in a separate container
2
Q
what are some examples of infectious waste?
A
- microbiological or culture material
- pathological material
- blood
- sharps
3
Q
what are some examples of mechanical hazards?
A
- blades
- needles
- scalpels
- razors
- glassware
- electrical equipment
4
Q
what are examples of health hazards?
A
- infectious disease
- carcinogens
- cryogenic sprays
- toxins (reproductive)
5
Q
what are some examples of chemical hazards?
A
- corrosives
- irritants
- sensitizers
- carcinogens
6
Q
what are some examples of fire hazards?
A
- alcohol
- hydrocarbons
7
Q
what should you see on a properly processed & stained slide?
A
- nuclei should show a variety of chromatin patterns with crisp blue nuclear membrane
- cytoplasm should be well preserved & should stain well with eosin
- NO artifactual spaces between individual cells
- NO cell shrinkage
8
Q
what are some causes of delayed fixation?
A
- specimens obtained long after blood supply has been compromised
- specimen not open so that fixative can come into contact w/ all surfaces
- specimen not thinly cut
- inadequate volume of fixative
9
Q
what does delayed fixation look like?
A
- nuclei may show loss of chromatin
- blue halo
- fading nuclei
- disappearance of nuclei
- cell shrinkage
- disruption of cytoplasm
- some cells may completely disappear
10
Q
what does incomplete fixation look like?
A
- nuclei is muddy/smudgy
- tissue morphology not well maintained
11
Q
how is incomplete fixation caused?
A
- tissue sections not allowed enough time in fixative
- inadequate amount of fixative relative to tissue volume
- sections grossed too thick for good penetration
12
Q
what does excessively dehydrated tissue look like?
A
- microchatter most commonly seen at the edges of the tissue
- hairline cracks may also been seen
13
Q
how is excessively dehydrated tissue caused?
A
- too much time allowed in dehydrating solutions
- molecularly bound water is removed as well as free water
- processing biopsy tissues on same schedule as larger specimens
- processing biopsy tissues on a schedule that is too long
14
Q
what does poorly processed tissue look like?
A
- section may appear cloudy with nuclei varying in staining properties
- no chromatin definition in the nuclei
- some nuclei may appear very washed out
15
Q
what causes poorly processed tissue?
A
- residual water remaining in tissue when they are placed in clearing agent
- fixative left in tissues when placed in clearing agent
- incomplete paraffin infiltration
- too much clearing agent in paraffin
- too much heat during processing
16
Q
what does cell shrinkage look like?
A
- cells are shrunken and there are a lot of artifactual spaces around the cells
- nuclei appear pyknotic (a nucleus of a damaged cell that has decreased in volume & become darker due to some degree of condensation of nuclear chromatin)
17
Q
how is cell shrinkage caused?
A
- inadequate fixation followed by excessive dehydration
- drying before submersion in fixative
18
Q
what does formalin or mercury pigment in tissue look like?
A
- brown to black pigments in tissue
- formalin pigment is most prevalent in bloody tissues
- usually lie on top of cells (but rarely can occur within cells)
19
Q
how is formalin or mercury pigment caused?
A
- formalin solution has a pH below 5.5
- tissue has not been treated to remove mercury pigment
20
Q
what does nuclear bubbling look like?
A
- nuclei look as if they contain soapsuds
- chromatin pattern is disturbed and bubbly
21
Q
how is nuclear bubbling caused?
A
- incomplete fixation with formalin
22
Q
what does poor section orientation look like?
A
- all layers are not demonstrated on tissues that have walls or layers
- lumen cannot be seen in tubular structures such as fallopian tube or arteries
23
Q
what causes poor section orientation?
A
- poor specimen embedding technique
- tissue incompletely fixed and hardened before grossing, so layers have rolled
24
Q
what are the general steps of H & E staining?
A
- ) incubation at 60 degrees C
- ) xylene for deparaffination
- ) ethanol rinse for rehydration
- ) rinse w/ tap water
- ) hematoxylin stain & replace xylene vats
- ) rinse using hematoxylin rinsing vat
- ) ethanol rinse for differentiation
- ) eosin stain (counter-stain) & replace ethanol vats
- ) rinse with eosin rinsing vat
- ) repeat step 7 (dehydration)
- ) xylene rinse (clearing)
- ) mount with coverslip
25
what is the cause of nuclei not being crisp or appearing to be smudgy with no distinct chromatin patterns?
- fixation poor or incomplete
- water was not completely removed during dehydration & clearing
- slides were exposed to excessive heat during processing or drying
26
what causes the 3 shades of eosin to not be seen?
- inadequate fixation
- improper processing occurred
- poor differentiation of the eosin
- eosin solution is not at the correct pH
27
what causes poor contrast?
- nuclear stain is too dark for cytoplasmic stain
- nuclear stain is too pale for cytoplasmic stain
- cytoplasmic stain is too dark for nuclear stain
- cytoplasmic stain is too pale for nuclear stain
28
what causes the cytoplasmic stain to be too dark?
- exposure of sections to eosin solution is prolonged
- inadequate eosin differentiation in the alcohols that follow the eosin stain; aqueous formulations stain tissue darker then alcohol-based stains
- alcohol rinse is not performed properly after the eosin stain
- eosin may be too concentrated, especially if phloxine is present
- if using an alcoholic eosin product, water contamination may have interfered with complete differentiation
- isopropyl alcohol was used as the dehydrating solution (isopropyl does not differentiate eosin within tissue sections in the same manner as ethyl alcohol)
29
what causes the cytoplasmic stain to be too light?
- eosin solution is exhausted
- the pH of eosin staining solution is greater than 4.5
- bluing solution is not completely washed out before the slides were transferred to eosin solution
- differentiation in diluted alcohols is prolonged
- alcohol rinse after the eosin stain is incorrectly performed
- staining time in eosin is too brief
30
what causes the nuclear stain to be too dark?
- hematoxylin solution is too concentrated
- too much time was allowed in the hematoxylin solution
- pH of the hematoxylin solution is incorrect
31
what causes the nuclear stain to be too light?
- incomplete deparaffinization
- hematoxylin solution is exhausted or used beyond its shelf life
- hematoxylin solution is diluted by carryover from a previous water rinse
- sections are over-differentiated by using overly concentrated acid-alcohol solutions, or sections remained too long in acid-alcohol
- staining time is too short in the hematoxylin stain
- tap water is used in the water rinses before and after staining (can act as excess mordant & restrain hematoxylin)
- poor fixation and/or processing, resulting in tissues that are unable to bind stain
32
what causes uneven eosin or hematoxylin staining?
- section may be thick and thin
- some solutions are not high enough to cover the entire slide
- water rinse was not adequate after hematoxylin staining
- water rinse was not adequate after acid-alcohol (which is necessary to stop decolorization)
- water rinse was not adequate after bluing
- alcohol rinse (dehydration) was not adequate after eosin staining
33
what causes a mounting artifact to appear?
- mounting medium has retracted from the edges of the coverslip
- air bubbles are trapped under the coverslip
- mounting medium on top of the coverslip
34
what causes red-brown nuclei?
- sections have not been sufficiently blued
| - hematoxylin is breaking down
35
what causes blue staining of cellular elements (such as collagen or smooth muscle)?
- pH of hematoxylin solution is greater than 3.0
- inadequate differentiation
- slides have spent too much time in dehydrating/differentiation alcohols
- eosin is contaminated by carryover from the bluing solution
- acid-alcohol is too weak
- amount of time spent in acid-alcohol is too short
- hematoxylin stain formulation is not selective enough
36
what causes dark precipitate scattered throughout the section?
- deteriorated hematoxylin (hematoxylin beyond its expiration date or damaged from improper storage conditions)
- metallic film on surface solution due to the hematoxylin not being filtered before use
37
what causes bubbles on the tissue section or in tissue spaces?
section is not completely dehydrated
38
what causes sections with an overall hazy appearance, or eosin bleeding throughout the section?
- solutions that are contaminated with water from the previous solution
- sections that were not adequately dehydrated after eosin staining
39
what causes brown "pigment" throughout section & glossy black nuclei?
tissue section dried out before coverslip was applied
40
is acetic acid present in regressive H&E staining?
yes
41
is acetic acid present in progressive H&E staining?
no
42
what is the rate of stain uptake in regressive H&E staining?
rapid
43
what is the rate of stain uptake in progressive H&E staining?
slow
44
is regressive H&E staining easy to control?
no
45
is progressive H&E staining easy to control?
yes
46
is differentiation required in regressive H&E staining?
yes
47
is differentiation required in progressive H&E staining?
no
48
what are the components of hematoxylin?
- dye
- oxidizer
- mordant
- acidifier
- stabilizer
- glycerin
49
what is the active dye source in hematoxylin?
hematein
50
what is the dye precursor?
hematoxylin
51
what does hematoxylin stain?
basophilic (negatively charged) components such as nuclear material (nuclei, etc.)
52
what is the oxidizer in hematoxylin?
sodium iodate/mercuric oxide
53
what is the purpose of oxidizer in hematoxylin?
used to speed up the oxidation process to hematein
54
what is the mordant in hematoxylin?
ammonium alum/potassium alum
55
what is a mordant in general?
a metal with a valence of at least 2+ charge
56
what is the purpose of a mordant?
links the dye to the tissue by means of a covalent and coordinate bond
57
what is the acidifier in hematoxylin?
citric acid/acetic acid
58
what is the purpose of an acidifier?
adjust pH to extend shelf life
59
what is the stabilizer in hematoxylin?
chloral hydrate/absolute alcohol
60
what is the purpose of a stabilizer?
help control the rate of oxidation
61
what is the purpose of glycerin in hematoxylin?
delays over-oxidation & discourages fungal growth
62
what is eosin stain for?
used to demonstrate general structure of tissue & provides contrast to the now stained nuclei
63
what does eosin stain & what kind of dye is it?
- stains positively charged structures in the cytoplasm
| - is an acidophilic dye
64
is a mordant required for eosin stain?
no
65
what causes wrinkles?
not properly stretching tissue on waterbath
66
what causes folds?
picking up tissue incorrectly from waterbath onto slide or tissue not adhering properly to the slide & folding during staining
67
what causes Venetian blinding/chatter/wash-boarding?
loose or worn microtome parts
68
what causes parched earth?
incomplete fixation
69
what causes microchatter?
overdehydration or cutting too rapidly
70
what does OCT stand for & what is it for?
- Optimal Cutting Temperature compound
| - for cryostat
71
what acidifier is in progressive (Mayer) stain?
citric acid
72
what acidifier is in regressive (Harris) stain?
acetic acid
73
what causes knife line?
nicked/damaged blade
74
what causes muddy/faded nuclei?
poor fixation
75
what causes moth-eaten holes?
dull knife or poorly processed tissue
