2-TISSUE PROCESSING, FIXATIVES Flashcards
1
Q
What is the goal of tissue processing?
A
Toproducequalityslidesformicroscopicevaluation
2
Q
What is the first and most critical step of tissue processing?
A
Fixation
3
Q
What is the step where free water and unbound fixative are removed from tissue?
A
Dehydration
4
Q
What is the step where dehydrating solutions are displaced to make tissues receptive to infiltrating medium?
A
Clearing
5
Q
What is the process where tissue is permeated with a support medium like paraffin wax?
A
Infiltration
6
Q
What is the process where tissue samples are oriented in a support medium to create a block?
A
Embedding
7
Q
What is the process that aims to expose paraffin-embedded tissue for representative sectioning?
A
Trimming
8
Q
What is the process where embedded tissues are cut into thin sections using a microtome?
A
Sectioncutting(microtomy)
9
Q
What is the process where tissues are deparaffinized and stained with dyes like hematoxylin and eosin?
A
Staining
10
Q
What is the process where tissues are impregnated with a transparent medium and sealed with a coverslip?
A
Mounting
11
Q
What is the final step of tissue processing involving automated or manual identification?
A
Labelling
12
Q
What is the goal of tissue processing?
A
To produce quality slides for microscopic evaluation
13
Q
What is the first and most critical step of tissue processing?
A
Fixation
14
Q
What is the step where free water and unbound fixative are removed from tissue?
A
Dehydration
15
Q
What is the step where dehydrating solutions are displaced to make tissues receptive to infiltrating medium?
A
Clearing
16
Q
What is the process where tissue is permeated with a support medium like paraffin wax?
A
Infiltration
17
Q
What is the process where tissue samples are oriented in a support medium to create a block?
A
Embedding
18
Q
What is the process that aims to expose paraffin-embedded tissue for representative sectioning?
A
Trimming
19
Q
What is the process where embedded tissues are cut into thin sections using a microtome?
A
Section cutting (microtomy)
20
Q
What is the process where tissues are deparaffinized and stained with dyes like hematoxylin and eosin?
A
Staining
21
Q
What is the process where tissues are impregnated with a transparent medium and sealed with a coverslip?
A
Mounting
22
Q
What is the final step of tissue processing involving automated or manual identification?
A
Labelling
23
Q
What happens if fixation is inadequate?
A
Zonal fixation+altered morphology+affected staining characteristics
24
Q
What is the most common fixative used in tissue processing?
A
10% neutral buffered formalin
25
What happens during over-processing in dehydration?
Shrinkage+parched earth effect+dry brittle tissue+abnormal staining
26
What happens during incomplete dehydration?
Specimen remains soft+non-receptive to paraffin wax infiltration+impaired clearing agent penetration
27
What clearing agent is most widely used during clearing?
Xylene
28
What alternative clearing agents are less hazardous than xylene?
Citrus-based clearants+aliphatic hydrocarbons
29
What happens during incomplete infiltration?
Tissues become soft and crumbly due to insufficient paraffin wax penetration
30
What happens if tissues spend too much time in high-temperature wax during infiltration?
Excessive shrinkage+dry brittle tissues
31
What embedding technique involves first embedding with agar or nitrocellulose before paraffin wax infiltration?
Double embedding
32
What is the purpose of a warm bath during section cutting?
To stretch or flatten the tissue ribbon for slide mounting
33
What is automated tissue processing?
A method that is more convenient and efficient allowing specimens to be infiltrated with a sequence of different solvents finishing in molten paraffin wax
34
What mechanism agitates the cage vertically in each solution during automated tissue processing?
Carousel
35
What is the starting solution used in automated tissue processing?
10% neutral buffered formaldehyde
36
What chamber feature in modern processors shortens turnaround time?
Specimens held in a chamber with solutions pumped in and out
37
What factors impact the duration of tissue processing and extent of infiltration?
Tissue density+thickness+agitation+temperature+vacuum+pressure
38
What happens at high temperatures during tissue processing?
Tissue shrinks+becomes hard and brittle
39
What happens at low temperatures during tissue processing?
Increased viscosity of reagents reducing diffusion rate
40
How does reduced pressure affect tissue processing?
Increases infiltration rate+decreases processing time
41
How does vacuum improve tissue processing quality?
Aids removal of trapped air in porous tissues
42
What are common artifacts caused by under-processing?
Fragmented fibro-fatty tissue+lack of nuclear definition+peculiar staining due to retained solvent (H&E)
43
What are common artifacts caused by over-processing?
Brittle sections+fine cracks+exacerbation by poor microtomy technique (H&E)
44
What is the process of preserving tissues from decay by preventing autolysis or putrefaction?
Fixation
45
What is self-digestion caused by enzymes produced by the cell itself?
Autolysis
46
What is tissue decay caused by microorganisms destroying cellular structures?
Putrefaction
47
What process aims to prevent degenerative processes
terminate biochemical reactions
48
What changes occur in tissues during fixation (shrinkage
swelling
49
How does fixation prevent self-digestion by inactivating lysosomal enzymes?
Fixation
50
How does fixation inhibit microbial growth that causes tissue decay?
Fixation
51
What is the primary goal of preserving cellular morphology and chemical integrity in a life-like manner?
Fixation
52
What process prevents tissue degeneration and distortion after removal from the body?
Fixation
53
How does fixation protect tissues during handling by hardening them?
Fixation
54
What are the objectives of preserving tissues
preventing cellular breakdown
55
What process allows thin sectioning
prevents autolysis
56
What practical steps ensure proper fixation (timing
volume
57
What is the ratio of fixative volume to tissue volume required for proper fixation?
20:1 or 10:1
58
What enhances fixation by changing the solution at intervals and agitation?
Volume management
59
What is the optimal pH range for fixation to avoid artifacts like formalin heme pigment?
37050
60
What buffers are commonly used in fixation?
Phosphate+bicarbonate+cacodylate+veronal
61
What temperature is used in tissue processors for fixation?
40°C
62
What temperature range is used for electron microscopy and histochemistry fixation?
0-4°C
63
What thickness of tissue section is ideal for adequate fixation?
2 cm² wide and 0.4 cm thin
64
What is the rate of aldehyde fixative penetration per hour?
2-3 mm per hour
65
What concentration of formaldehyde is commonly used in fixation?
0.1
66
What duration of fixation is generally recommended?
2-6 hours
67
What facilitates fixation by reducing processing time and improving quality?
Heat+vacuum+agitation+microwave techniques
68
What happens if fixation is delayed for small biopsies or incisional specimens?
Artifacts+loss of cellular organelles+nuclear clumping due to drying
69
What are the general effects of fixatives on tissues?
Reduce infection risk+harden tissues+make cells resistant to damage+inhibit bacterial decomposition+increase optical differentiation+act as mordants for staining
70
What characteristics define a good fixative?
Stable+safe to handle+minimum distortion of cells+inhibit bacterial decomposition+permit rapid penetration+harden tissues+isotonic+facilitate staining procedures
71
What method uses heat to fix tissues by dehydration and attachment to slides?
Heat fixation
72
What method uses glyoxal-based fixatives heated at 55°C to avoid toxic vapors?
Microwave fixation
73
What method involves freezing tissues in liquid nitrogen and removing water in a vacuum chamber at -40°C?
Freeze drying
74
What method substitutes water with fixatives like acetone or alcohol at -40°C?
Freeze substitution
75
What chemical fixative group includes formaldehyde and glutaraldehyde?
Aldehydes
76
What chemical fixative group includes osmium tetroxide and potassium permanganate?
Oxidizing agents
77
What chemical fixative group includes methyl alcohol
ethyl alcohol
78
What chemical fixative group includes mercuric chloride and picric acid?
Metallic fixatives
79
What type of fixatives add covalent reactive groups to induce cross-links between proteins and nucleic acids?
Cross-linking fixatives
80
What type of fixatives remove free water and coagulate proteins by breaking hydrophobic bonds?
Dehydrant coagulant fixatives
81
What type of fixatives coagulate proteins but result in poor preservation of mitochondria and secretory granules?
Coagulant fixatives
82
What are examples of coagulant fixatives that act by denaturing proteins through changes in pH or salt formation?
Acetic acid+trichloroacetic acid+mercuric chloride+zinc acetate
83
What are simple fixatives composed of a single component like formaldehyde or acetone?
Simple fixatives
84
What are compound fixatives that combine agents like alcoholic formalin for specific effects?
Compound fixatives
85
What type of fixatives preserve tissue structures without altering intercellular relationships?
Microanatomical fixatives
86
What are examples of microanatomical fixatives?
10% formal saline+10% neutral buffered formalin+Heidenhain’s Susa+Zenker’s solution+Bouin’s solution
87
What type of fixatives preserve specific parts of cells?
Cytological fixatives
88
What type of fixatives preserve nuclear structures with a pH of 4.6 or less?
Nuclear fixatives
89
What are examples of nuclear fixatives?
Flemming’s fluid+Carnoy’s fluid+Bouin’s fluid+Newcomer’s fluid
90
What type of fixatives preserve cytoplasmic structures with a pH greater than 4.6?
Cytoplasmic fixatives
91
What are examples of cytoplasmic fixatives?
Flemming’s fluid without acetic acid+Kelly’s fluid+Regaud’s fluid (Muller’s fluid)+Orth’s fluid
92
What type of fixatives preserve chemical constituents of cells and tissues?
Histochemical fixatives
93
What are examples of histochemical fixatives?
Formalin saline 10%+Absolute ethyl alcohol+Acetone+Newcomer’s fluid
94
What is the process of placing already fixed tissue in a second fixative to improve staining and hardening?
Secondary fixation
95
What is the process where tissue is placed in potassium dichromate for 24 hours to act as a mordant?
Post-chromatization
96
What is the process of removing excess fixative from tissues after fixation?
Washing out
97
What substance is used to remove excess chromates from tissues fixed in Zenker’s or Flemming’s solutions?
Tap water
98
What substance is used to remove excess picric acid from Bouin's solution-fixed tissues?
50-70% alcohol
99
What substance is used to remove excess mercuric fixatives from tissues?
Alcoholic iodine
100
What problem arises from failure to fix tissues immediately or insufficient fixative volume?
Failure to arrest early autolysis of cells
101
What causes the loss of soluble substances (e.g.
glycogen) during fixation?
102
What artifact occurs due to incomplete washing of fixative?
Presence of artifact pigments (e.g.
103
What indicates incomplete fixation in tissue consistency?
Tissues are soft and feather-like
104
What issue arises when enzymes needed for study are inactivated?
Wrong choice of fixative
105
What tissue changes result from overfixation?
Shrinkage and swelling of cells
106
What problem occurs with prolonged fixation?
Brittle and hard tissue blocks
107
What eliminates artifacts like crush artifacts or formalin pigment?
Fixation in phenol-formalin or neutral buffered formalin (NBF)
108
What causes intense eosinophilic staining at tissue centers due to partially fixed proteins?
Crush artifact
109
What pigment appears black in poorly buffered formalin-fixed tissues?
Formalin heme pigment
110
What example shows zonal fixation (intact RBCs vs. hemolyzed areas)?
Marrow aspirate with uneven fixation
111
What liver section artifact demonstrates poor stain uptake in outer segments?
Incomplete fixation causing pale
112
What fixatives preserve lipids (e.g.
phospholipids)?
113
What method preserves cholesterol in tissues?
Digitonin fixation
114
How are lipids best demonstrated without dissolving in alcohol?
Cryostat/frozen sections+general lipid stains
115
What fixatives preserve glycogen?
Rossman’s fluid or cold absolute alcohol
116
What fixatives are used for amino acid histochemistry?
Neutral buffered formal saline or formaldehyde vapor
117
What fixative is most commonly used for ultrastructural preservation?
Glutaraldehyde
118
What fixative provides electron-dense contrast for membranes?
Osmium tetroxide
119
What fixative is used as a vapor for specific protein studies?
Paraformaldehyde
120
What fixative concentration preserves enzyme activity?
4% formaldehyde
121
What method retains enzyme integrity for staining?
Formal-saline overnight fixation
122
How are enzymes preserved in cryostat sections?
Brief acetone/formaldehyde fixation+distilled water washing
123
What fixation method is used for paraffin-embedded sections?
Formalin fixation
124
How are cryostat sections fixed for immunofluorescence?
Brief methanol/acetone immersion
125
What preserves antigenicity for fluorescence studies?
Cryopreservation or limited cross-linking reagents
126
What process uses a second fixative to improve staining/hardening?
Secondary fixation
127
What step uses potassium dichromate as a mordant?
Post-chromatization
128
What removes excess chromate/osmic acid from tissues?
Tap water washing
129
What removes picric acid from Bouin’s-fixed tissues?
50-70% alcohol
130
What removes mercuric chloride residues?
Alcoholic iodine treatment
